RANBP9 and RANBP10 cooperate in regulating non-small cell lung cancer proliferation

RANBP9 and RANBP10 cooperate in regulating non-small cell lung cancer proliferation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article RANBP9 and RANBP10 cooperate in regulating non-small cell lung cancer proliferation Arturo Orlacchio, Yasuko Kajimura, Lara Rizzotto, Anna Tessari, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5707591/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Aug, 2025 Read the published version in Journal of Experimental & Clinical Cancer Research → Version 1 posted 7 You are reading this latest preprint version Abstract Background RANBP9 and RANBP10, also called Scorpins, are essential components of the C-terminal to LisH (CTLH) complex, an evolutionarily conserved poorly investigated multisubunit E3 ligase. Their role in non-small cell lung cancer (NSCLC) is unknown. Methods In this study, first we used stable loss-of function and overexpression inducible cell lines to investigate the ability of either RANBP9 or RANBP10 to form their own functional CTLH complex. Then, we probed lysates from patient tumors and analyzed data from publicly available repositories to investigate the expression of RANBP9 and RANBP10. Finally, we used inducible cell lines in vitro to recapitulate the expression observed in patients and investigate the changes of the proteome and the ubiquitylome associated with either RANBP9 or RANBP10 in NSCLC. Results Here, we show that the two Scorpins are both expressed in NSCLC cells and that either of them can independently support the formation of the CTLH complex. Short-term experiments revealed that the RANBP9 and RANBP10 proteins balance each other in terms of expression, and the acute overexpression of one or the other results in significant reshaping of the NSCLC cell proteome and ubiquitylome. A higher RANBP9/RANBP10 ratio is associated with greater proliferation in both NSCLC cell lines and patients. Acute increased expression of RANBP10 slows NSCLC cell proliferation and decreases the level of proliferation-associated proteins, including key players in DNA replication. Conclusions We present evidence that the Scorpins act as partial antagonists and work together as one sophisticated rheostat to modulate the CTLH complex ubiquitylation output, which regulates cell proliferation and other key biological processes in NSCLC. These results suggest that the two Scorpins can be considered as targets for the treatment of NSCLC. Lung cancer non-small cell lung cancer NSCLC CTLH complex GID complex RANBP9 RANBPM SCORPIN ARMC8 GID4 GID8 TWA1 MAEA MKLN1 RANBP10 RMND5A RMND5B WDR26 YPEL5 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction The scaffold protein containing a C-terminal to LisH (CTLH) domain named RAN Binding Protein 9 (RANBP9; also known as RANBPM) is involved in cell homeostasis, survival, proliferation, adhesion, and migration 1 – 5 . Its increased expression promotes the signaling of several receptor tyrosine kinases (RTKs), including major players in non-small cell lung cancer (NSCLC) tumorigenesis 6 – 11 . In NSCLC cells subjected to DNA damage, RANBP9 is not only a target but also an enabler of the ATM (Ataxia Telangiectasia Mutated) protein kinase 12 , 13 . RANBP9 protein expression is high in advanced NSCLC, and patients expressing higher levels of RANBP9 protein have worse outcomes when treated with platinum-based regimens, which is in line with the hypothesis that RanBP9 is protective of cancer cells when subjected to DNA damage 14 . Hence, targeting RANBP9 could be a valid strategy to treat NSCLC in combination with other therapeutic modalities 15 . RANBP9 operates in the context of the ubiquitously expressed unconventional multisubunit E3 ligase CTLH complex, where it constitutes the inner core and tightly binds to GID8 (Glucose-Induced degradation Deficient 8) 3 , 16 – 18 . This multisubunit enzyme is emerging as a central regulator of mammalian cell metabolism and regulates key bioenergetic signaling nodes, such as AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) 19 – 21 . Currently, there are 11 known members of the CTLH complex. In addition to RANBP9 and GID8, other established core members are ARMC8 (Armadillo Repeat Containing protein 8), MAEA (Macrophage Erythroblast Attacher E3 ligase), RMND5A and RMND5B (Required in Meiotic Division 5 A and B). On the other hand, GID4 (Glucose-Induced degradation Deficient 4), WDR26 (WD Repeat containing protein 26), and YPEL5 (Yippee Like protein 5) are peripheral members 2 , 22 . Recent biochemical and structural studies have shown that MAEA and RMND5 bind to a RANBP9-GID8-ARMC8 core and, together, provide E3 enzymatic activity 23 . Finally, MKLN1 (Muskelin 1) has been shown to be not only a peripheral member but also a substrate of the complex, and its levels can be used as a proxy for CTLH enzymatic activity 17 , 20 . Although the CTLH complex was initially characterized as a heterodecameric aggregate, it is becoming increasingly clear that its proteins can assemble in different configurations that include or exclude selected core and peripheral members. For example, RMND5A and RMND5B are mutually exclusive 3 . In addition, the two splicing isoforms of ARMC8, ARMC8α and ARMC8β, can determine the inclusion (ARMC8α) or exclusion (ARMC8β) of GID4, the first and only known substrate receptor until recently 19 , 24 . The presence of RANBP9, GID8, RMND5A, and WDR26 together has been shown to promote proliferation 3 , 24 . The topological features of the CTLH E3 ligase have been inferred from studies in S. cerevisiae , where its counterpart, the GID (Glucose-Induced degradation Deficient) complex, responds to metabolic stress, modulates mitochondrial functions and participates in the disposal of enzymes necessary for gluconeogenesis 5 , 25 – 27 . Elegant structural work has shown that the GID complex can assemble into “supra-molecular” oval-shaped rings with a diameter equivalent to the length of the proteasome and quickly ubiquitylate multimeric metabolic enzymes in the central cavity 28 . Yeast Gid1 is essential for the assembly of the complex and tightly binds Gid8. Together, Gid1 and Gid8 provide the inner core on which the structure is built, and their proteins stabilize each other 29 . Deletion of Gid1 disrupts the formation of the complex 25 , 29 . Therefore, it has been hypothesized that ablation of RANBP9 disrupts the formation of the whole CTLH complex 30 . However, the eleventh and understudied member of the CTLH complex is a highly similar RANBP9 paralog called RANBP10, which presents the same domains that are essential for aggregation into the CTLH complex 3 , 13 , 31 – 33 . RANBP9 and RANBP10 are also called Scorpins (Spry-containing Ran-binding proteins) to distinguish them from other RAN-binding proteins that are involved mainly in nuclear‒cytoplasmic shuttling 33 . RANBP9 is markedly expressed in highly proliferative precursors, whereas RANBP10 expression becomes predominant in more differentiated cells during erythroid maturation. Moreover, both RANBP9 and RANBP10 can form their own CTLH complex 34 . However, the reciprocal dynamics governing the expression of Scorpins in the same cell type are not known. Although RANBP10 is ubiquitously coexpressed with RANBP9 and consistently coimmunoprecipitates with other CTLH proteins both in humans and in mice, the function of RANBP10 is not well understood in general 3 , 22 , 31 , 35 . Apart from a potential tumor-promoting role in glioblastoma 36 , the relevance of RANBP10 in cancer has not been addressed. Taking into consideration evidence from different model systems, we reasoned that RANBP9 and RANBP10 need to be studied together to fully understand their biological roles because they are both the result of duplication of the ancestral yeast Gid1 gene 13 , 22 . Phenotypic and structural similarities, as well as differences, indicate that these genes might have only partially overlapping functions and are likely to cross-regulate each other 13 . Here, we present data that show how the two Scorpins work in concert and how their modulatory role on the CTLH complex ubiquitylation output depends on the ratio of their amount. The predominant expression of RANBP9 over RANBP10 in NSCLC correlates with increased proliferation in both NSCLC cell lines and patients, where increased expression of RANBP10 resulted in a reduction in cell proliferation and in the level of proteins involved in cell proliferation. Here, we reveal that the two Scorpins constitute a unique sophisticated rheostat that modulates the expression and ubiquitylation of a variety of proteins that participate in fundamental NSCLC oncogenic processes, such as cell proliferation. Materials and methods Generation of cell lines and KOs A549 and H460 NSCLC cell lines (see supplementary info) were purchased from ATCC and cultured in RPMI 1640 medium (Gibco™) supplemented with 10% FBS (Gibco™) and MycoZap™ Plus-CL (Lonza). sgRNAs were designed at http://crispr.mit.edu (see supplementary info). Briefly, for each guide, two DNA primers were designed and then annealed and phosphorylated using T4 polynucleotide kinase (NEB). Phosphorylated sgRNAs were then cloned and inserted into the vectors pSpCas9(BB)-2A-GFP (5’ prime sgRNA) and pSpCas9(BB)-2A-Puro (3’ prime sgRNA) via the BbsI restriction enzyme (NEB) and the Rapid DNA Dephos & Ligation Kit (ROCHE). Following the transformation of the ligase reaction plasmid DNA into DH5α competent cells (Thermo Fisher Scientific), the plasmid DNA was purified via endotoxin-free Maxiprep kits (QIAGEN). NSCLC cell lines (~ 1x10 6 ) were transfected with both sgRNA-containing vectors via Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s specifications, and after 48 hours, single cells were sorted for GFP expression via a BD FACSAria™ II Cell Sorter. Scorpin WT or Scorpin DKO A549 cells were transduced via a 4D-Nucleofector X Unit (Lonza) according to the manufacturer’s optimized protocol. Briefly, 2 × 10^5 cells were resuspended in 20 µl of SF buffer and electroporated via the nucleofection program CM-130 with 0.1 µg of AAVS1-TRE3G-EGFP, AAVS1-TRE3G-RANBP9 or AAVS1-TRE3G-RANBP10 in the presence of 0.1 µg of pX330-U6-Chimeric_BB-CBh-hSpCas9-hGem (1/110)-AAVS1 gRNA. The latter allows CRISPR/Cas9-mediated integration of inducible plasmids into the AAVS1 safe harbor site of the human genome. Forty-eight hours post-transfection, puromycin (1 µg/mL) was applied to enrich the cells with successful transgene integration. The AAVS1-TRE3G and pX330-U6-Chimeric_BB-CBh-hSpCas9-hGem (1/110) base plasmids for safe-harbor integration of the inducible system were acquired from Addgene (see also Extended Material) 37 , 38 . Preparation of MEF cell cultures All the animal studies were conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Ohio State University. Embryos from RanPBP9, RanBP10 KO and Double KO mice (see also Extended Material) were isolated at E12.5. After the heads, tails, limbs, and most of the internal organs were removed, the embryo carcasses were minced, passed through a 70 µm cell strainer, and then seeded into 10 cm cell culture dishes in 10 mL of high-glucose DMEM supplemented with 15% FBS, 1% penicillin‒streptomycin (Gibco™) and 0.01% 2-mercaptoethanol (Sigma-Aldrich). Primary MEFs were cultured in 3% oxygen and passaged two or three times to obtain a morphologically homogenous culture. Immortalized MEFs were generated by transfecting early-passage primary cells with 2 µg of Large-T antigen-expressing plasmid via Lipofectamine™ 3000. The cells were then cultured in normal oxygen (20%) and passaged for an additional 5 generations to obtain a stable population. Western blot Western blot The cells and tumor tissues were homogenized on ice in NP-40 buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein concentration was determined via the use of the Bio-Rad protein assay dye (Bio-Rad). Western blot analysis was performed using 30 to 50 µg of protein run on Mini-PROTEAN TGX precast gels (Bio-Rad). Primary antibodies (see also Extended Material) were used at a dilution of 1:1,000 in 5% milk in TBS-T. Signals were detected with HRP-conjugated secondary antibodies and the chemiluminescence substrate SuperSignal West Pico PLUS or Femto (Thermo Fisher Scientific). Equivalent loading among samples was confirmed with an anti-vinculin antibody (Cell Signaling). Images were acquired via the KwikQuant Digital Western Blot Detection System (Kindle Biosciences, LLC). Protein expression levels were quantified via optical densitometry via ImageJ software version 1.53t ( https://imagej.nih.gov/ij/ ) and KwikQuant Image Analyzer 5.4 ( https://kindlebio.com/12-downloads ). RNA extraction and real-time qPCR RNA extraction and real-time qPCR The cells and tumor tissues were homogenized on ice in TRIzol™ reagent (Invitrogen™), and RNA was extracted according to the manufacturer’s guidelines. Following extraction, 1 µg of total RNA was then treated with DNase and converted into cDNA via the Maxima First Strand cDNA Synthesis Kit for RT‒qPCR with dsDNase (Thermo Fisher Scientific). For real-time qPCR, 10–15 ng of cDNA was amplified via TaqMan™ Fast Advanced Master Mix. Samples were amplified simultaneously in triplicate in one assay run via TaqMan probes specific for (RanBP9, RanBP10, GID8, WDR26, ARMC8, RMND5A, RMND5B, YPEL5, MAEA and MKLN1). OAZ1 and GAPDH were used as endogenous controls for human tumor samples and cell lines, respectively. Analysis was performed with GraphPad Prism 10.0 software via the Δ-ct method (see also Extended Material). Proteomics Cell Lysis The cell pellets were washed with PBS three times before being resuspended in 5% SDS buffer in 50 mM TEAB (triethylammonium bicarbonate) solution. The samples were then vortexed briefly before sonication by using a Bioruptor® Pico (Diagenode, Denville, NJ) following the manufacturer’s suggested protocol. Briefly, the sonication temperature was set at 4°C; the sonication cycle was set at 30 sec on and 30 sec off. A total of 20 cycles were performed for the cell pellets. After sonication, the samples were centrifuged at 20000 × g for 15 min at 4°C to remove any remaining insoluble material. The protein concentrations were measured via a Qubit fluorometer (Thermo Fisher Scientific). S-trap Digestion Proteins were digested with trypsin via S traps (Protifi, Fairport, NY). For the TMT-labeled samples, 200 µg of each protein sample was subjected to trypsin digestion on an S-trap microcolumn (K02-micro); for the ubiquitylation enrichment samples, 2 mg of each protein sample was digested on an S-trap midi column (C02-Midi). Briefly, samples were reduced with DTT and alkylated with iodoacetamide before the addition of 12% phosphoric acid (to a final volume of 1.2%). The proteins were then diluted with S-Trap binding buffer (MeOH:TEAB, 90:10 v/v) at a 1:6 (sample:S-Trap binding buffer, v/v) ratio. The sample was then applied to the S-Trap column and washed three times with S-Trap binding buffer 4 times. Sequencing grade trypsin (Promega) dissolved in 50 mM TEAB was added to the sample at a 1:50 (trypsin:protein, w:w) ratio for TMT-labeled samples or a 1:200 ratio for ubiquitylated enrichment samples. The sample was incubated overnight at 37°C and eluted sequentially with 50 mM TEAB, 0.2% formic acid, and 0.2% formic acid in 50% acetonitrile. The samples were pooled and dried in a centrifuge concentrator for further use. TMT labeling One hundred micrograms of peptides in 50 mM TEAB solution were labeled per the manufacturer’s instructions (A44522, Thermo Fisher Scientific). The TMT-labeled peptides were then pooled together and fractionated via a Pierce high-pH reversed-phase peptide fractionation kit (Cat# 84868, Thermo Fisher Scientific). Peptides eluted with 5, 10, 12.5, 15, 17.5, 20, 22.5, 25 and 50% acetonitrile/triethylamine (0.1%) solutions were collected and analyzed on a Fusion Orbitrap mass spectrometer. Ubiquitylation Enrichment The dried peptides were resuspended in PTMScan HS IAP Bind Buffer and placed on ice. PTMScan HS Magnetic Immunoaffinity Beads were washed four times with ice-cold 1X PBS. The dissolved peptides were combined with the washed magnetic beads and incubated on a tabletop mixer at 4°C for six hours. After incubation, magnetic beads with bound peptides were placed on a magnetic separator, and unmodified peptides were removed. The magnetic beads were washed four times with chilled HS IAP Wash Buffer, followed by two washes with chilled LC-MS Grade H 2 O. Ubiquitylated peptides were eluted twice by the addition of 0.15% TFA with gentle agitation for 10 minutes. Ubiquitylated peptides were placed into glass vials and dried prior to LC-MS analysis. Nano-LC/MS/MS analysis Nanoliquid chromatography-nanospray tandem mass spectrometry (nano-LC‒MS/MS) for protein identification was performed on a Thermo Scientific orbitrap Fusion mass spectrometer equipped with an EASY-Spray™ Sources operated in positive ion mode. The samples were separated on an easy spray nanocolumn (Pepmap™ RSLC, C18 3 µ 100A, 75 µm X250 mm Thermo Scientific) via a 2D RSLC HPLC system from Thermo Scientific. Mobile phase A was 0.1% formic acid in water, and acetonitrile (with 0.1% formic acid) was used as mobile phase B. The flow rate was set at 300 nL/min. For the TMT samples, a 3-hour gradient was used after the samples were desalted via a trap column. For ubiquitylated peptides, samples were directly loaded and separated on an easy spray nanocolumn bypassing the desalting column, and a 1-hour gradient was used for analysis. MS/MS data were acquired with a spray voltage of 1.95 kV, and a capillary temperature of 305°C was used. The scan sequence of the mass spectrometer was based on the preview mode data-dependent TopSpeed™ method. To achieve high mass accuracy MS determination, the full scan was performed in FT mode, and the resolution was set at 120,000 with internal mass calibration. For TMT-labeled samples, FT mode (resolution set at 50000) was used for MS2 data acquisition to ensure that mass tags that differ by only one N15 and C13 can be well resolved for accurate quantitation. For ubiquitinated samples, MSn was performed via HCD in ion trap (IT) mode to ensure the highest signal intensity of the MSn spectra. Three FAIMS compensation voltages (cv=-50, -65 and − 80v) were used for data acquisition. The AGC target ion number for the FT full scan was set at 4 × 10 5 ions, the maximum ion injection time was set at 50 ms, and the microscan number was set at 1. The HCD collision energy was set at 32%. The AGC target ion number for the ion trap MSn scan was set at 3.0E4 ions, the maximum ion injection time was set at 35 ms, and the microscan number was set at 1. Dynamic exclusion is enabled with a repeat count of 1 within 60 s and a low mass width and high mass width of 10 ppm. Data analysis and quantitation were performed via MASCOT on Proteome Discoverer following workflows recommended by Thermo. Results The combined deletion of Scorpins causes the disappearance of GID8 and the functional inactivation of the CTLH complex . We previously generated RANBP9 shRNA-knockdown and complete knockout (KO) NSCLC cells 12 , 39 , 40 . However, we did not assess the levels of RANBP10, which is reported to be expressed at lower levels than its paralog. For example, in HEK293T cells, the number of RANBP10 protein copies (7.6x10 4 ) is estimated to be approximately one-third that of RANBP9 (2.3x10 5 ) 41 . To better characterize the role of both Scorpins in NSCLC in the context of the CTLH complex, we used CRISPR/Cas9 technology to generate RANBP10 knockout (KO) A549 and H460 cell lines. We also generated NSCLC cells in which both Scorpins were genetically inactivated (double-KO [DKO] cells) (Fig. 1 A- 1 B). When tested for other CTLH members, DKO cells presented nearly complete ablation of GID8 and MAEA, whereas the levels of ARMC8 and WDR26 did not appear to consistently change among the two cell lines and different genotypes. On the other hand, the levels of MKLN1 were consistently elevated in DKO cells (Fig. 1 A- 1 B). The combined Scorpin deletion had similar effects on the levels of GID8 and MAEA in mouse embryonic fibroblasts (MEFs) ( Supplementary Fig. 1A ). These cells were derived from embryonic stem cell-generated mice deficient in RANBP9, RANBP10, or both Scorpins together (DKO), indicating that the disappearance of GID8 and MAEA is not dependent on cell type or species and is not caused by the use of CRISPR/Cas9 42, 43 . MKLN1 levels were increased in DKO MEFs, which was consistent with observations in NSCLC cells, whereas WDR26 levels appeared to be unchanged in Scorpin-mutant MEFs ( Supplementary Fig. 1A ). RT‒PCR evaluation of GID8 and MAEA mRNA levels in both A549 and H460 DKO cells generally revealed a modest increase (less than 25%). The exception was GID8 mRNA, whose expression modestly but significantly decreased in H460 cells. These results indicated that changes in GID8 and MAEA protein levels were not due to changes in the corresponding transcripts ( Supplementary Fig. 1B-C ), which is in agreement with previous observations in cell lines 17 , 34 , 44 . Overall, both RANBP9 and RANBP10 can independently stabilize GID8 in mammalian cells, and only their stable combined deletion disrupts the formation of a functional CTLH complex via protein-mediated mechanisms. RANBP9 or RANBP10 acute re-expression is sufficient to stabilize GID8 and MAEA and restore CTLH complex formation . To further prove that both RANBP9 and RANBP10 can independently stabilize GID8 and enable the formation of a canonical CTLH complex, we generated A549 DKO cells in which either RANBP9 alone or RANBP10 alone can be re-expressed by a doxycycline (Doxy)-inducible system ( Supplementary Fig. 2A ). The re-expression of RANBP9 or RANBP10 resulted in the clear reappearance of GID8 and MAEA, whereas the amount of MKLN1, which is also a substrate of the complex, decreased to levels similar to those of the WT (Fig. 2 , Supplementary Fig. 2B ) 17 , 20 . The de novo expression of GFP had no appreciable effects on the Scorpins or other CTLH proteins. We concluded that in NSCLC cells, the expression of either one of the two Scorpins is sufficient to stabilize its assembly and restore the formation and function of the CTLH complex. RANBP9 and RANBP10 cross-regulate each other’s expression . When either RANBP9 shRNA-knockdown or CRISPR-KO NSCLC cells were generated, we previously reported that, in general, the expression of RANBP10 was greater than that in parental cells (Fig. 1 A- 1 B, Supplementary Fig. 1C ) 8 , 12 , 40 . This observation, together with the high similarity between the two proteins, led us to hypothesize that the two paralogs may functionally compensate for each other’s absence. To further investigate this phenomenon in a short period of time, we engineered A549 parental (Scorpin WT) cells bearing Doxy-inducible safe-harbor gene integration of either RANBP9 or RANBP10, similar to what we previously reported in A549 Scorpin DKO cells ( Supplementary Fig. 2A ). Time course experiments revealed that the protein expression of RANBP9 or RANBP10 was consistently upregulated from 6 to 48 hours (Fig. 3 A-D). When RANBP9 was induced, the amount of RANBP10 protein decreased accordingly (Fig. 3 A-B). The reverse was also true. When the expression of RANBP10 was induced, the expression of RANBP9 was proportionally downregulated (Fig. 3 C-D). GID8, MAEA, and WDR26 trended toward increased expression with the induction of either RANBP9 or RANBP10 (Fig. 3 A-D; Supplementary Fig. 3A ). At the indicated times, Doxy treatment did not cause any appreciable change in Scorpin expression in the A549 parental line (Fig. 3 E-F). The strong artificial increase in either the RANBP9 or RANBP10 transcript did not have a significant effect on the transcripts of their paralog or other CTLH complex members ( Supplementary Fig. 3C ). Together with previous observations, these results demonstrate that the expression of the RANBP9 and RANBP10 proteins is cross regulated in NSCLC cells and that their dynamic changes reciprocally affect each other’s protein expression in the short term. Overall, CTLH BP 9 and CTLH BP 10 are subject to balanced expression at the protein level. GID8 and RANBP9 are overexpressed, whereas RANBP10 is downregulated in NSCLC at both the RNA and protein levels . The results above demonstrated that RANBP9 and RANBP10 acutely cross-regulate each other’s protein expression in vitro , without being correlated with their transcript levels. Next, we proceeded to establish the relevance of these findings in NSCLC patients. We previously reported that the protein levels of RANBP9 are higher both in NSCLC cells and in patient samples than in their normal counterparts 12 , 40 . However, we could not unequivocally determine how pervasive the overexpression of the RANBP9 protein was because of the small number of NSCLC samples we had available at the time 40 . For this study, we acquired a collection of fifty (50) frozen NSCLC samples (T) with matched normal adjacent tissue (N), which included 16 adenocarcinoma (LUAD), 8 squamous carcinoma (LUSQ), 6 carcinoid, and other histotypes (OSU collection; Supplementary Table 1 ) samples. We measured the expression levels of the Scorpins and their binding partner GID8 via WB and RT‒PCR (Fig. 4 A ‒E ; Supplementary Fig. 4A ; Supplementary Materials ). We found that the RANBP9 and GID8 proteins were significantly more abundant in T vs. matched N samples (Fig. 4 A-B; Supplementary Fig. 4A ). We also detected significant overexpression of GID8 and RANBP9 transcripts in T compared with N (Fig. 4 C-D). On the other hand, we did not find a significant difference in the level of RANBP10 mRNA between the N and T stages (Fig. 4 E). Regrettably, the low affinity/specificity of the commercially available antibody did not allow reliable quantitation of the RANBP10 protein. To corroborate our findings concerning the overexpression of RANBP9 and GID8 in NSCLC, we analyzed the level of expression of the CTLH genes in the publicly available TCGA collection ( www.cbioportal.org ) of lung cancer samples (T) with paired normal adjacent tissue (N) both squamous cell carcinoma (LUSQ) and adenocarcinoma (LUAD) samples. As shown in Supplementary Fig. 4B-C , RANBP9 and GID8 (a.k.a. C20orf11) were significantly higher in T than in N both in LUAD and LUSQ. Interestingly, RANBP10 mRNA levels were significantly lower in the T dataset than in the N dataset in both the LUAD and LUSQ datasets. Overall, the TCGA collection of NSCLC data revealed that RANBP9 and GID8 mRNAs were overexpressed, whereas RANBP10 transcript levels were decreased in both LUAD and LUSQ samples compared with those in T vs N samples. Collectively, these results show that both the transcripts and proteins of GID8 and RANBP9 are upregulated in tumors of various histotypes in NSCLC patients. On the other hand, RANBP10 mRNA is expressed at low levels. To further investigate Scorpin expression in NSCLC, we sought to analyze the transcriptomic and proteomic data characterized by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) for lung adenocarcinoma (LUAD) and squamous lung cancer (LUSQ) recently published and available at kb.linkedomics.org 45 – 47 . Consistent with our observations in the OSU collection of NSCLC samples analyzed by WB (Fig. 4 A-B; Supplementary Fig. 4A ) , the RANBP9 and GID8 proteins were significantly upregulated in both LUAD and LUSQ samples (Fig. 5 A, B, D, E ) . On the other hand, the RANBP10 protein was significantly underexpressed compared with that in normal matched controls (Fig. 5 C, F). The expression of mRNAs exhibited highly similar patterns ( Supplementary Fig. 5G-L ). Although RANBP10 is expressed at lower levels on average, some tumors presented relatively high RANBP10 expression compared with normal controls. These latter cases appear to have correspondingly higher levels of GID8, as indicated by the significant positive correlation between GID8 and RANBP10 in both the LUAD and LUSQ datasets ( Supplementary Fig. 5A, B, D, E ). We also determined that in both LUAD and LUSQ, the maximum values of either RANBP9 or RANBP10 were much more strongly correlated with GID8 protein levels than either of the two Scorpins alone were ( Supplementary Fig. 5C, F ). The latter result is concordant with the stoichiometric relationship observed in our cell line experiments, suggesting that the predominant expression of one member suppresses the expression of its counterpart and that both RANBP9 and RANBP10 can stabilize the CTLH, protecting GID8 from degradation. Taken together, these data show that RANBP9 expression dominates RANBP10 expression at both the mRNA and protein levels, indicating that the chronic changes in CTLH protein levels are mediated by adjustments in transcript levels differently from those observed in cell lines in short-term experiments. Moreover, the expression of both RANBP9 and RANBP10 was positively correlated with the GID8 level. RANBP9 and RANBP10 correlate with significantly different proteomes in NSCLC patient tumors . Having established that RANBP9 and GID8 are upregulated while RANBP10 is downregulated or expressed at low levels in NSCLC patients, we next proceeded to assess their correlations with the global proteome in the CPTAC LUAD and LUSQ datasets. The regression analysis of the absolute expression of both Scorpins and their differential expression (RANBP9 minus RANBP10 = delta [DRANBP] expression) revealed that RANBP9 and RANBP10 expression correlated negatively and positively with hundreds of other proteins both in LUAD (Fig. 6 A ‒C ) and LUSQ (Fig. 6 D ‒F ; Supplementary Material ). As mentioned, both RANBP9 and RANBP10 were positively correlated with GID8 (Fig. 6 A, B, D, E; Supplementary Fig. 6A-B ) but negatively correlated with each other (Fig. 6 A-F). In both LUAD and LUSQ, the list of proteins positively and negatively correlated with RANBP9 was significantly different from that associated with RANBP10. A selection of the top 200 proteins expressed in correlation with one of the three CTLH members in both types of tumors revealed that the overlapping proteins favor the comparisons RANBP10 LUAD vs RANBP10 LUSQ, RANBP9 LUAD vs RANBP9 LUSQ, and GID8 LUAD vs GID8 LUSQ, suggesting strongly concordant effects on tumor biology across distinct tumor histologies (Fig. 6 G, I). RANBP9-associated proteins highly overlap with GID8-associated proteins (Fig. 6 G, blue boxes), and relatively fewer proteins are unique to one group ( Supplementary Material ). Conversely, only limited similarity was observed when comparing RANBP10 vs GID8, especially when comparing RANBP9 to RANBP10. Collectively, these results indicate that RANBP9 and RANBP10 expression in NSCLC correlates with different proteins because they both positively correlate with GID8, thus suggesting a partially different functional role for the two paralogs. RANBP9 expression is associated with increased proliferation in NSCLC . To gain biological insights into the proteomes potentially regulated by RANBP9 and RANBP10 in NSCLC, we performed two independent analyses to search for gene sets, pathways, and biological processes. First, we performed gene set enrichment analysis (GSEA) via the LinkedOmics website (kb.linkedomics.org) 45 – 47 , which queries associations with the publicly available WEB-basedGEne SeT AnaLysis Toolkit (WebGestalt: webgestalt.org) ( Supplementary Fig. 7A-H ). Second, we used the list of proteins that were positively or negatively associated with RANBP9 or RANBP10 in the CPTAC LUSQ and LUAD collections ( Supplementary Material ) to perform Metascape analyses ( https://metascape.org ) ( Supplementary Fig. 7I‒P ) 48 . Both the WebGestalt and the Metascape results indicated that RANBP9 and RANBP10 have strong positive associations with gene sets related to all the different steps of RNA metabolism in both LUSQ and LUAD ( Supplementary Fig. 7A-P ). The results also revealed that RANBP9 and RANBP10 were negatively correlated with processes related to different aspects of the immune response, both innate and adaptive, together with endocytosis, vesicle and membrane trafficking, and cell adhesion processes ( Supplementary Fig. 7I-P ). However, both the GSEA and the Metascape analysis also revealed differences between RANBP9 and RANBP10. GSEA revealed that RANBP9 was positively associated with “DNA replication” in both LUSQ and LUAD, whereas RANBP10 was not ( Supplementary Fig. 7A-B ). In the Metascape analysis, RANBP9 expression was strongly associated with terms related to cell proliferation, such as “cell cycle” (R-HSA-1640170), “DNA metabolic process” (GO:0051052), “mitotic cell cycle” (GO:0000278) or “mitotic cell cycle process” (GO:1903047) or “mitotic G2-G2M phases” (R-HSA-453274), “cell cycle checkpoints” (R-HSA-69620), “regulation of cell cycle process” (GO:0010564), “DNA replication” (GO:0006260 and WP466), “regulation of DNA replication” (GO:0007265), and “S-phase” (R-HSA-69242) ( Supplementary Fig. 7I-J ). RANBP10 was associated with “regulation of cell cycle process” (GO:0010564) in LUAD and with “cell cycle” (R-HSA-1640170) in LUSQ, although the statistical significance of these latter associations was markedly lower. Collectively, these results show that in NSCLC tumors, both the CTLH BP 9 and the CTLH BP 10 configurations are potentially involved in the regulation of biological processes such as multiple steps of RNA metabolism, but they also have distinct preferential associations with other biological processes such as cell proliferation, which was found to be strongly associated with the increased protein ratio RANBP9/RANBP10. To corroborate the preferential association between cell proliferation and RANBP9 expression in comparison with RANBP10, we selected a recently published protein proliferation signature 49 and analyzed its correlation with the two Scorpins and GID8 in both the CPTAC LUAD and LUSQ datasets (Fig. 7 A-F). Even if both RANBP9 and RANBP10 expression was positively correlated with GID8 expression (Fig. 6 A, B, D, E; Supplementary Fig. 6A-B ), the results clearly revealed that RANBP9 and GID8 expression was positively correlated with the proliferation signature in both LUAD and LUSQ, whereas RANBP10 expression was not (Fig. 7 A-F). Taken together, these results indicate that the CTLH complexes formed by RANBP9 or RANBP10 are associated with a variety of fundamental biological processes and proteins, where a relatively high RANBP9/RANBP10 ratio is positively correlated with proliferation in NSCLC tumors. Compared with RANBP9, the acute overexpression of RANBP10 causes different changes in the NSCLC proteome, downregulating several proliferation-associated proteins . Next, to gain mechanistic insight into the effects of RANBP9 overexpression versus RANBP10 overexpression, we aimed to establish whether the increase in RANBP9 or RANBP10 levels differentially affected iA549 cell proliferation. We previously reported that ablation of RANBP9 in NSCLC cells caused a modest but consistent reduction in cell proliferation 12 . In contrast, the downregulation of RANBP9 in HEK293 cells increased cell proliferation, and the silencing of RANBP10 was found to reduce glioblastoma cell proliferation 36 , 50 . However, our A549 WT cell lines, where either RANBP9 (Scorpin WT A549 iBP9) or RANBP10 (Scorpin WT A549 iBP10) are induced without the constitutive ablation of endogenous genes, provide a tool that avoids artifacts due to in vitro cell adaptation previously observed when manipulating CTLH proteins and better mimics human NSCLC tumors 2 , 3 (Fig. 3 ). We treated Scorpin WT iBP9 and iBP10 cells with Doxy for 24 hours. Quadruplicate samples were collected and analyzed via tandem mass spectrometry via an isotopic labeling approach ( Supplementary Fig. 8A ). For the CPTAC data collection, we considered the effects of RANBP9 induction, RANBP10 induction, and DRANBP separately (Fig. 8 A-C). The overexpression of the two Scorpins caused significant global proteome changes (Fig. 8 A-C; Supplementary Material ). RANBP9 can affect the expression levels of other proteins both negatively and positively 8 , 17 , 22 , 51 – 54 . After the induction of RANBP9 in A549 iBP9 cells, 396 and 394 proteins correlated positively and negatively with RANBP9 expression ( log10 p≤1.3), respectively (Fig. 8 A). When RANBP10 was induced in A549 iBP10 cells, 229 and 404 proteins correlated positively and negatively with RANBP10 levels, respectively ( log10 p≤ 1.3) (Fig. 8 B). In both induced cell lines, the amounts of GID8 and MAEA were positively correlated with either RANBP9 (Fig. 8 A) or RANBP10 (Fig. 8 B), which is in line with our previous results. When considering the expression of DRANBP, 288 proteins were found to be positively correlated with a higher DRANBP, and 293 proteins were positively correlated with a lower DRANBP (log10 p≤ 1.3) (Fig. 8 C; Supplementary Material ). These results are in line with the observations in the NSCLC CPTAC data, where the expression of the two paralogs correlated with only partially overlapping enriched proteomes, while both correlated positively with GID8 and other CTLH members (Fig. 6 A-I). Proteins that were differentially expressed when RANBP9 was overexpressed were significantly similar to RANBP9-associated proteins in both LUSQ and LUAD (Fig. 8 D). This similarity was in part due to an enrichment of proliferation-associated proteins (Fig. 8 A-C, E-G), in agreement with the positive association of RANBP9 expression with proliferation observed in CPTAC patient tumors. However, our experiments also revealed that the overexpression of RANBP10 clearly downregulated the expression of proliferation-associated proteins (Fig. 8 B; Supplementary Fig. 8B-C ). RANBP9 loss of function or downregulation in NSCLC cells causes a modest but consistent reduction in cell proliferation 12 , 40 . Therefore, we investigated the effect of RANBP10 overexpression on iA549 cell proliferation. We again used iA549 WT iBP9 and iBP10 cells treated with or without doxycycline and monitored cell growth for three days. While the overexpression of RANBP9 did not significantly affect cell growth, the overexpression of RANBP10 caused a modest but significant decrease in the growth rate, which became evident after 24 hours (Fig. 8 H). These results indicate that in iA549 cells, an increase in RANBP10 (a lower RANBP9/RANBP10 ratio) has a “braking effect” on cell proliferation. Collectively, these results show that the artificial overexpression of the two Scorpins modulates distinct groups of proteins. Moreover, the overexpression of the CTLH BP 10 complex downregulates proliferation-associated proteins, leading to a measurable decrease in the growth rate. These observations are also consistent with the association of RANBP9 with an increased proliferative phenotype in CPTAC NSCLC tumors and decreased proliferation when RANBP9 is silenced or ablated. Compared with RANBP9, the acute overexpression of RANBP10 causes different changes in the A549 ubiquitylome, which includes proliferation-associated proteins . All observations made thus far in NSCLC patients and cell lines have indicated that the CTLH BP 9 and CTLH BP 10 complexes coexist in a tightly regulated balance and that the functional effects of these two CTLH configurations are different, especially when considering cell proliferation. Next, we aimed to identify potential mechanistic candidates to guide future studies by assessing the effects of Scorpin manipulation on the A549 ubiquitylome. We repeated the Scorpin WT iA549 iBP9 and iBP10 cell induction and collected samples after proteasomal inhibition with MG132. Quadruplicate samples were collected and processed to enrich for ubiquitylated peptides with KGG remnants as proxies for ubiquitylation ( Supplementary Fig. 9A ). We found that RANBP9 overexpression was positively correlated with 453 KGGs and negatively correlated with 436 KGGs (log10 p≤1.3). On the other hand, the induction of RANBP10 was positively correlated with 1,765 KGGs and negatively correlated with 598 peptides with KGGs (Fig. 9 A-C; Supplementary Material ). Considering the expression of DRANBP, 1559 genes were positively correlated with it, and 1611 genes were negatively correlated with it (Fig. 9 C). These results clearly demonstrated that the upregulation of RANBP9 has significantly different effects on reshaping the ubiquitylome of iA549 cells than the increase in RANBP10 does. We found that proteins displaying more than one KGG peptide, including RANBP9 and RANBP10, were significantly enriched upon induction with the Scorpins themselves ( Supplementary Material ). We constructed lists of ubiquitylated proteins associated either positively or negatively with RANBP9 or RANBP10 ( Supplementary Material ) and performed a Metascape analysis to assess which gene sets, pathways, and biological processes were potentially perturbed upon manipulation of the Scorpin expression levels ( Supplementary Fig. 9B-E ). “Metabolism of RNA” (R-HSA-8953854) was one of the most significantly enriched terms in all 4 different groups, further confirming that the two Scorpins participate together in the regulation of the transcriptome. The term “cell cycle” (R-HSA-164070) was among the top two enriched terms in the list compiled with ubiquitylated proteins associated either positively or negatively with RANBP10 ( Supplementary Fig. 9D-E ). Importantly, the same “cell cycle” (R-HSA-164070) term was not significantly enriched in the Metascape analysis of ubiquitylated proteins positively or negatively associated with RANBP9 ( Supplementary Fig. 9B-C ). However, other terms related to cell proliferation, such as “cell cycle, mitotic” (R-HSA-69278”) and “mitotic cell cycle process”, were significantly enriched with the proteins negatively associated with RANBP9. Overall, these results indicate that a change in the balance between CTLH BP 9 and CTLH BP 10 at 24 hours results in hundreds of ubiquitylation changes in NSCLC cells, potentially fine-tuning a wide variety of key biological processes, including RNA processing and cell proliferation. Analysis of the ubiquitylome revealed that several proliferation-associated proteins had significantly different ubiquitylations when RANBP9 or RANBP10 was overexpressed in iA549 cells (Fig. 9 A-C). To prioritize candidates most likely to be functionally relevant, we focused on the intersection of differentially ubiquitylated peptides whose total protein expression was also significantly altered by altered Scorpin expression. To broaden the list of putative candidates, we used a less stringent statistical cutoff for the total protein associations, requiring both a difference in Doxy induction with p RANBP10 effect with P < 0.1; for the ubiquitylation effect, p RANBP10 remained significantly enriched for proliferation-associated proteins with the use of relaxed statistical cutoffs ( Supplementary Fig. 9F; 27 of 337; odds ratio 1.8, p < 0.01). Among the 337 RANBP9-associated proteins, 43 had at least one ubiquitylation site whose expression was increased by RANBP10 induction, and this subset presented the greatest enrichment of proliferation-associated proteins (8 of 43; odds ratio 4.9, P < 0.001). These differentially expressed proteins and their corresponding ubiquitylation sites putatively represent candidate substrates that may link CTLH E3 ubiquitin ligase activity with the regulation of cell growth and proliferation. Ultimately, we found that seven proliferation-associated proteins displayed significantly different specific ubiquitylation events that could explain the different levels of expression observed in the analysis of the proteome (Fig. 9 D). This restricted list included the two members of the replisome MCM5 and MCM7, the calcium binding protein CACYBP1, which regulates replisome functions, the cohesin SMC1A, the core component of the RNA polymerase II POLR2B, the splicing factor SF3B3, and XAB2. Among these, CACYBP1, MCM5, MCM7, SMC1A, and SF3B3 were previously reported to be bona fide CTLH interactors in at least two different studies 35 , 51 , 55 – 57 . These results demonstrate that the overexpression of RANBP9 or RANBP10 changes the total amount and ubiquitylation pattern of proteins critically involved in cell proliferation, such as members of the replisome and cohesins 58 , 59 , with prioritized candidates that warrant further mechanistic validation in future studies. Discussion Despite significant advances in patient survival, NSCLC remains the deadliest cancer in the United States 60 , 61 . A better understanding of the pathogenesis of the disease is needed to design more efficacious biology-driven treatments 62 – 64 . The present work illustrates the role of two variants of the CTLH complex, an E3 ligase that is emerging as a central node connecting cell signaling and metabolism, in NSCLC 2 , 19 – 22 . Here, we show that RANBP9 and RANBP10, also called Scorpins 33 , work in concert to modulate the ubiquitylation output of the CTLH complex in NSCLC. First, we demonstrated that the CTLH complex coexists in two different configurations, one based on the scaffold provided by RANBP9 (CTLH BP 9 ) and the other built on RANBP10 (CTLH BP 10 ). Both complexes are expressed in normal lung and NSCLC tumors (Fig. 1 A-C, Supplementary Fig. 1B-C , Fig. 4 A-E, Fig. 5 A-F, Supplementary Fig. 5A-L , Fig. 6 A-F). In agreement with previous observations demonstrating the existence of both CTLH BP 9 and CTLH BP 10 during erythroid maturation, RANBP9 or RANBP10 were independently sufficient to protect their binding partner GID8 from proteolysis and stabilize the core on which the complex is formed (Fig. 1 A-C, Supplementary Fig. 1A , Fig. 2 , Supplementary Fig. 2A, B ) 34 . CTLH BP 9 and CTLH BP 10 complexes acutely cross-regulate each other at the protein level. An artificial increase in the amount of RANBP9 caused a decrease in RANBP10, and vice versa , the forced increase in RANBP10 caused a proportional decrease in RANBP9 (Fig. 3 A-D; Supplementary Fig. 3A, B ). Notably, upon acute induction of the expression of RANBP9 or RANBP10, the transcripts of the uninduced paralog did not appreciably change within the short period we analyzed ( Supplementary Fig. 3C ). Similarly, in single-KO NSCLC cells, the paralog transcript level did not significantly increase, whereas the protein level did ( Supplementary Fig. 1B-C ). In Scorpin double-KO (DKO) NSCLC cells, the disappearance of GID8 and MAEA, on the one hand, and the increase in MKLN1, on the other hand, are not in line with the levels of their relative transcripts ( Supplementary Fig. 1B-C ). It is not uncommon to find a poor correlation between the mRNA and protein levels of CTLH family members and proteins involved in proteostasis in general 12 , 22 , 65 – 68 . However, albeit not in all tumors taken singularly, the overall increase in RANBP9 protein in NSCLC masses corresponded to an overall increase in the RANBP9 transcript, and the overall decrease in RANBP10 was also consistently observed at both the protein and mRNA levels (Fig. 4 A-D, Fig. 5 A-F; Supplementary Fig. 5G-L ). Therefore, we can conclude that while acute changes in RANBP9 and RANBP10 cause changes in protein expression, long-term changes in Scorpin mRNA expression are involved in NSCLC tumorigenesis. Existing evidence indicates that RANBP9 and RANBP10 originated from the duplication of the ancestral yeast Gid1 gene and that they can partially compensate for each other’s absence 22 69 70 . In contrast, there is evidence that these two proteins may have opposite functions 9 , 71 . Hence, the two paralogs should be considered partially antagonistic, similar to other proteins that originated from evolutionary duplications 72 . In line with this concept, here, we show that the combined targeting of both Scorpins in NSCLC impairs the formation of functional CTLH complexes (Fig. 1 A-C; Supplementary Fig. 1A ) 23 , 28 . We also showed that, compared with RANBP10 induction, the controlled overexpression of RANBP9 in iA549 cells caused changes in the expression and ubiquitylation of a significantly different number of proteins (Fig. 8 A-D, Supplementary Fig. 8A-C , Fig. 9 A-C, Supplementary Fig. 9B-F ). The dynamics observed in vitro (Fig. 3 A) were consistent with the observations in CPTAC patients, where RANBP9 was overexpressed, whereas RANBP10 was maintained at low levels (Fig. 6 A-F). In vivo and in vitro , the expression of either RANBP9 or RANBP10 was positively correlated with that of other members of the CTLH complex, but the two paralogs were negatively correlated with each other (Fig. 6 A-C, Fig. 8 A-B). We identified proteins affected by the overexpression of RANBP9 and/or RANBP10 in both iA549 (Fig. 8 A-B) and CPTAC patients (Fig. 6 A-F). Collectively, our data suggest that, in NSCLC, the increase in RANBP9, together with the decrease in RANBP10, works in concert to obtain proteome and ubiquitylome changes that potentially directly and indirectly modulate many other proteins ( Supplementary Fig. 7A-P ). The lists of proteins whose total amount or ubiquitylation was altered depending on the expression of the two Scorpins in CPTAC NSCLC patients and in iA549 cells indicate that CTLH BP 9 and CTLH BP 10 have the theoretical ability to regulate all aspects of cellular life ( Supplementary Fig. 7A-P ; Fig. 8 A-D; Supplementary Fig. 9B-E ) 48 . The iA549 proteome and ubiquitylome consistently showed a strong association of “RNA metabolism” with both RANBP9 and RANBP10 expression, which is in line with the CPTAC data. Both proteins appeared to be involved in the regulation of the transcriptome in vitro ( Supplementary Fig. 7A-P ; Fig. 8 A-C; Supplementary Fig. 9B-E ). This finding is in line with previous evidence indicating that RANBP9 is involved in RNA transcription and splicing 55 , 73 , 74 . However, we also showed that RANBP10 can also modulate the RNAome and that the regulation exerted by Scorpins could be much more pervasive than previously thought ( Supplementary Fig. 7A-P ). The GO term “cell cycle” was also strongly associated with a higher BP9/BP10 ratio in the CPTAC LUAD and LUSQ proteomic data ( Supplementary Fig. 7I-P ). We used an unbiased protein proliferation signature to corroborate this initial finding (Fig. 7 A-F ) 49 . Given its elevated expression in NSCLC tumors, RANBP9 is expected to be protumorigenic and proproliferative compared with RANBP10. This assumption is consistent with the fact that RANBP9 enhances signaling through receptor tyrosine kinases such as MET (hepatocyte growth factor receptor), whereas RANBP10 ablates that effect and predominantly expresses RANBP9 in highly proliferative progenitor cells, in contrast with the predominance of RANBP10 in differentiated erythroid cells 9 , 34 , 71 . Therefore, we can hypothesize that increased expression of the CTLH BP 9 complex favors the stability of proteins that are associated with cell proliferation in NSCLC 3 . However, since RANBP10 has been reported to promote glioblastoma cell growth and RANBP9 silencing can increase proliferation, we can speculate that these effects are likely cell type dependent. In iA549 cells, the preferential association of the proliferation-associated protein signature with increased RANBP9 expression was consistent with the increased BP9/BP10 ratio observed in CPTAC NSCLC patients (Fig. 8 E-G ). However, we found that high levels of RANBP10 caused a significant decrease in the expression of selected proliferation proteins (Fig. 8 B-C; Supplementary Fig. 8B-C ) . Moreover, Doxy-treated iBP10 cells exhibited decreased proliferation, which became significant at 48 h, whereas Doxy-treated iBP9 cells were indistinguishable from their noninduced controls (Fig. 8 H). These results showed that a decreased BP9/BP10 ratio decelerates cell proliferation (Fig. 8 H). We also found that the acute overexpression of RANBP9 or RANBP10 significantly changed the ubiquitylome landscape of A549 cells, which included specific ubiquitylations of proliferation-associated proteins (Fig. 9 A-B). Therefore, while the decrease in proliferation observed upon RANBP10 overexpression could be explained by the concomitant decrease in RANBP9, the ubiquitylation and decrease in specific proliferation-associated proteins such as MCM5, MCM7, and SMC1A, for example, cannot be easily explained by the decrease in RANBP9. The specific RANBP9-associated lysine ubiquitylations that we observed are largely different from those found to be positively associated with RANBP9 expression in HEK293 cells 51 . However, in that study, RANBP9 was stably silenced, the expression of RANBP10 was not investigated, and differences can be caused by technical and/or cell type-specific factors 51 . An in-depth analysis of the observed lysine ubiquitylations suggests a model in which CTLH BP 9 and CTLH BP 10 are likely to act on proteins/targets that are CTLH BP 9 specific, CTLH BP 10 specific, or common between the two. Several ubiquitylations showed positive or negative associations only with RANBP9 or RANBP10 expression (Fig. 9 A-B; Supplementary Material ). Interestingly, we found that the ubiquitylation of one or more lysines in some proteins changed when RANBP9 or RANBP10 expression was induced. However, in some cases, the correlation was positive or negative for both Scorpins (concordant), but in other cases, the correlation was opposite (discordant), where the same ubiquitylated lysine was positively correlated with one Scorpin expression but negatively correlated with the other. We also identified cases in which the same protein presented two different ubiquitylated lysine residues, one correlating positively and the other with RANBP9 or RANBP10 negatively (Fig. 9 A-C; Supplementary Material ). Therefore, we can hypothesize that these proteins whose ubiquitylation changed in more than one residue likely represent proteins on which Scorpins converge to functionally regulate them either in the same or opposite functional direction (Fig. 10 ). This model can explain how Scorpins have only partially overlapping functions, sometimes having a concordant final effect, and some other times an opposite outcome. This model is also in agreement with previously reported observations showing that the overexpression of RANBP9 increased the stabilization of several binding partners and not their degradation, as expected for an E3 ligase protein. We hypothesize that some proteins undergo degradation upon RANBP9 expression, whereas others are stabilized, and vice versa . A clear example is the proteins that we found to be differentially ubiquitylated on specific lysines that were also previously reported as both putative targets of the CTLH complex and part of the proliferation signature, such as members of the replisome (Fig. 9 D). In this context, our findings indicate that RANBP9 and RANBP10 might finely regulate major complexes involved in DNA replication 75 – 78 . These observations are supported by previous studies in which proteins involved in DNA replication were reported as putative interactors, including our recent study, which demonstrated that RanBP9 interacts with members of the replisome in normal lungs 35 , 51 , 55 – 57 . Our study has several limitations. For example, we cannot conclude that the changes in ubiquitylation observed following altered expression of the CTLH complex are directly caused by changes in the abundance of proteins such as CACYBP, MCM7, and SMC1A. Moreover, we cannot completely exclude the possibility that the effects on protein abundance and ubiquitylation were indirect or that deubiquitylation could be involved 53 . More than one-third of the modified iA549 ubiquitylome that we identified in this study upon Scorpin induction has been previously reported as a putative CTLH complex interactant ( Supplementary Material ). Therefore, many proteins subject to changes in ubiquitylation are likely not direct targets. In this context, several proteins whose ubiquitylation changes upon RANBP9 or RANBP10 overexpression are E2 or E3 ligases themselves ( Supplementary Tables ). These findings suggest that the CTLH complex may act indirectly through other ubiquitylation machineries. Evidence supporting this indirect model already exists in yeast, where it was shown that the GID complex can ubiquitylate rsp5 (Reverse Spt-phenotype 5), which is an E3 ligase of the NEDD4 family that in turn regulates vesicular trafficking 79 . If confirmed in mammalian cells, this model would indicate that the CTLH complex has the ability to fine-tune all aspects of cellular life through protein-mediated mechanisms. Conclusions The two Scorpins should be considered as one functional unit that acts as a sophisticated rheostat to modulate the enzymatic output of the CTLH complex. Due to the substantial number of ubiquitylation events that changes when their expression changes, RANBP9 and RANBP10 significantly impact NSCLC pathogenesis, including tumor cell proliferation. Since their deletion disrupts the formation of a functional CTLH complex, they should be considered as potential targets for therapy. Abbreviations NSCLC Non-Small Cell Lung Cancer RANBP9 Ran Binding Protein 9 RANBP10 Ran Binding Protein 10 GID8 Glucose-induced degradation Deficient 8 CTLH C-Terminal to LisH CTLH BP9 CTLH complex built on RANBP9 CTLH BP10 CTLH complex built on RANBP10 SCORPIN Spry-COntaing Ran binding ProteIN ARMC8 Armadillo Repeat Containing protein 8 GID4 Glucose-Induced degradation Deficient 4 MAEA Macrophage Erythroblast Attacher E3 ligase MKLN1 Muskelin 1 RMND5A Required in Meiotic Division 5 A RMND5B Required in Meiotic Division 5 B WDR26 WD Repeat containing protein 26 YPEL5 Yippee Like protein 5 KO Knockout DKO Double KO MEFs Mouse Embryonic Fibroblasts Doxy doxycycline LUAD Lung Adenocarcinoma LUSQ Lung Squamous carcinoma T Tumor tissue N Normal adjacent tissue WB Western blot Co-IP Co-immunoprecipitation CPTAC Clinical Proteomic Tumor Analysis Consortium GSEA Gene Set Enrichment Analysis GO Gene ontology iA549 DKO Scorpin DKO A549 Doxy-inducible controls A549 DKO iBP9 Scorpin DKO A549 with Doxy-inducible RANBP9 A549 DKO iBP10 Scorpin DKO A549 with Doxy-inducible RANBP10 iA549 WT parental A549 Doxy-inducible controls A549 WT iBP9 parental A549 with Doxy-inducible RANBP9 A549 WT iBP10 parental A549 with Doxy-inducible RANBP10 Declarations Ethics approval and consent to participate Not Applicable. Consent for publication All authors approved publication. Competing interests The authors declare that they have no competing interests. Funding This work is partially supported by the NIH R03CA259389 to V.C. and the Pelotonia Idea Award GR123713 “Establishing the role of CTLH proteins in NSCLC” to V.C. Author Contribution Designed the research study: A.O., Y.K., A.L., G.F., D.P., J.K., and V.C.; conducted the experiments: A.O., Y.K., L.R., A.T., S.H.S., L.Z., B.F., D.P., and V.C.; provided resources: L.T., R.V., D.P.C., V.C.; acquired the data: A.O., Y.K., L.Z.; analyzed the data: A.O., Y.K., L.Z., M.F., J.K., and V.C.; wrote and edited the manuscript: A.O., Y.K., A.T., L.T., J.A., R.V., D.P.C., A.A., A.L., G.F., M.F., D.P., J.K., and V.C.; project conception: V.C. Acknowledgement The authors are extremely grateful to Laura Monovich of the Biospecimen Shared Resource (Director N. Single) of the Ohio State University and James Comprehensive Cancer Center (OSU-CCC) for the procurement of the 50 NSCLC patient samples through the Total Cancer Care Program. The authors would also like to thank the Flow Cytometry Shared Resource, the Genome Editing Shared Resource, the Genome Sequencing Shared Resource, and the Comparative Pathology and Digital Imaging Shared Resource of OSU-CCC, which were all supported by the P30 CA016058 grant to R. Pollock. This work was also supported by the Pelotonia Institute of Immuno-Oncology (PIIO). The content is solely the responsibility of the authors and does not necessarily represent the official views of the PIIO. Data Availability “Data is provided within the manuscript or supplementary information files”. Data is also available upon request. References Das S, Suresh B, Kim HH, Ramakrishna S. RanBPM: a potential therapeutic target for modulating diverse physiological disorders. Drug Discov Today (2017). 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Calcyclin-binding protein contributes to cholangiocarcinoma progression by inhibiting ubiquitination of MCM2. Oncol Res. 2023;31:317–31. Rankin BD, Rankin S. The MCM2-7 Complex: Roles beyond DNA Unwinding. Biology (Basel) 13 (2024). Moreno SP, Bailey R, Campion N, Herron S, Gambus A. Polyubiquitylation drives replisome disassembly at the termination of DNA replication. Science. 2014;346:477–81. Bailey R, Priego Moreno S, Gambus A. Termination of DNA replication forks: Breaking up is hard to do. Nucleus. 2015;6:187–96. Langlois CR, et al. A GID E3 ligase assembly ubiquitinates an Rsp5 E3 adaptor and regulates plasma membrane transporters. EMBO Rep. 2022;23:e53835. Additional Declarations No competing interests reported. Supplementary Files THEPAPERFINALSupplementaryFigure1DEC2024.pdf THEPAPERFINALSupplementaryFigure2DEC2024.pdf THEPAPERFINALGraphicalAbstracthighresolutionDEC2024.pdf THEPAPERFINALSupplementaryFigure3DEC2024.pdf THEPAPERFINALSupplementaryFigure4DEC2024.pdf THEPAPERFINALSupplementaryFigure5DEC2024.pdf THEPAPERFINALSupplementaryFigure6DEC2024.pdf THEPAPERFINALSupplementaryFigure7AHDEC2024.pdf THEPAPERFINALSupplementaryFigure7IPDEC2024.pdf THEPAPERFINALSupplementaryFigure8DEC2024.pdf THEPAPERFINALSupplementaryFigure9DEC2024.pdf THEPAPERSupplementaryTable150NSCLCOSUcollection.xlsx THEPAPERSupplementaryTable2Ubiquitylationassociations.xlsx THEPAPERSupplementaryTableFigure3A549itimecourseWBquantification.xlsx THEPAPERSupplementaryTableFigure450OSUQuantificationandpatientsorder.xlsx THEPAPERSupplementaryTableFigureS1BA549RTPCR.xlsx THEPAPERSupplementaryTableFigureS2BA549DKOWBquantification.xlsx THEPAPERSupplementaryTableFigureS3BWBquantification.xlsx THEPAPERsupplementaryTableFigureS3CRTPCR.xlsx THEPAPERFINALEXTENDEDMATERIALDEC2024.pdf Cite Share Download PDF Status: Published Journal Publication published 29 Aug, 2025 Read the published version in Journal of Experimental & Clinical Cancer Research → Version 1 posted Editorial decision: Revision requested 18 Jan, 2025 Reviews received at journal 18 Jan, 2025 Reviewers agreed at journal 06 Jan, 2025 Reviewers invited by journal 03 Jan, 2025 Editor assigned by journal 03 Jan, 2025 Submission checks completed at journal 03 Jan, 2025 First submitted to journal 24 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5707591","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":403834338,"identity":"3cc15a1a-f416-4557-9b5a-8d6846986434","order_by":0,"name":"Arturo Orlacchio","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Arturo","middleName":"","lastName":"Orlacchio","suffix":""},{"id":403834339,"identity":"01f6c5e5-ad89-47d3-acb5-19c54ba07d68","order_by":1,"name":"Yasuko Kajimura","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Yasuko","middleName":"","lastName":"Kajimura","suffix":""},{"id":403834340,"identity":"ee63e966-1c91-4259-81f4-d6ee96356203","order_by":2,"name":"Lara Rizzotto","email":"","orcid":"","institution":"The Ohio State University and OSUCCC","correspondingAuthor":false,"prefix":"","firstName":"Lara","middleName":"","lastName":"Rizzotto","suffix":""},{"id":403834341,"identity":"d95f78f8-bc7c-4a36-a2b3-7308d7e693d1","order_by":3,"name":"Anna Tessari","email":"","orcid":"","institution":"Ohio State Wexner Medical Center, The Ohio State University and OSUCCC","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Tessari","suffix":""},{"id":403834342,"identity":"cb3219ac-e9e7-47fa-bad0-eaef7d48530b","order_by":4,"name":"Shimaa H.A. Soliman","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Shimaa","middleName":"H.A.","lastName":"Soliman","suffix":""},{"id":403834343,"identity":"cd3fd22b-4802-4686-8c1c-b3d738d3cb5f","order_by":5,"name":"Rosa Visone","email":"","orcid":"","institution":"G. d'Annunzio University","correspondingAuthor":false,"prefix":"","firstName":"Rosa","middleName":"","lastName":"Visone","suffix":""},{"id":403834344,"identity":"01a91fb4-9aba-4000-9861-d9668a48dccd","order_by":6,"name":"Liwen Zhang","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Liwen","middleName":"","lastName":"Zhang","suffix":""},{"id":403834345,"identity":"33df0121-515e-40c4-a9cd-d679f1ddb650","order_by":7,"name":"Brian Fries","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Brian","middleName":"","lastName":"Fries","suffix":""},{"id":403834346,"identity":"a39f7d40-93c8-43ef-93d4-4ab6502ecea0","order_by":8,"name":"Lino Tessarollo","email":"","orcid":"","institution":"NCI/Center for Cancer Research, NIH","correspondingAuthor":false,"prefix":"","firstName":"Lino","middleName":"","lastName":"Tessarollo","suffix":""},{"id":403834347,"identity":"61c6db11-7247-4a60-a0c5-b84e9b1bd57e","order_by":9,"name":"Joseph Amann","email":"","orcid":"","institution":"Ohio State Wexner Medical Center, The Ohio State University and OSUCCC","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"","lastName":"Amann","suffix":""},{"id":403834348,"identity":"33c4e301-19d6-4527-a6e3-33f535c8336a","order_by":10,"name":"David P. Carbone","email":"","orcid":"","institution":"Ohio State Wexner Medical Center, The Ohio State University and OSUCCC","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"P.","lastName":"Carbone","suffix":""},{"id":403834349,"identity":"e61b4da5-cade-4b6b-91ab-c19cd9d1bb4f","order_by":11,"name":"Alessia Lodi","email":"","orcid":"","institution":"University of Texas at Austin","correspondingAuthor":false,"prefix":"","firstName":"Alessia","middleName":"","lastName":"Lodi","suffix":""},{"id":403834350,"identity":"22720f02-ace4-4cea-909f-2bc15345ff64","order_by":12,"name":"Amer Ahmed","email":"","orcid":"","institution":"University of Bari Aldo Moro","correspondingAuthor":false,"prefix":"","firstName":"Amer","middleName":"","lastName":"Ahmed","suffix":""},{"id":403834351,"identity":"50868d46-8e07-4494-833d-b7473d1ca7fc","order_by":13,"name":"Giuseppe Fiermonte","email":"","orcid":"","institution":"University of Bari Aldo Moro","correspondingAuthor":false,"prefix":"","firstName":"Giuseppe","middleName":"","lastName":"Fiermonte","suffix":""},{"id":403834352,"identity":"fb106613-55b0-42aa-b8e2-378233dd5e0a","order_by":14,"name":"Mike Freitas","email":"","orcid":"","institution":"The Ohio State University","correspondingAuthor":false,"prefix":"","firstName":"Mike","middleName":"","lastName":"Freitas","suffix":""},{"id":403834353,"identity":"0a787f8a-e8c1-4e35-9485-f5f161152602","order_by":15,"name":"Dario Palmieri","email":"","orcid":"","institution":"The Ohio State University and OSUCCC","correspondingAuthor":false,"prefix":"","firstName":"Dario","middleName":"","lastName":"Palmieri","suffix":""},{"id":403834354,"identity":"2c1f2403-1315-4c20-bff3-d080fbdbef50","order_by":16,"name":"Jacob Kaufman","email":"","orcid":"","institution":"Ohio State Wexner Medical Center, The Ohio State University and OSUCCC","correspondingAuthor":false,"prefix":"","firstName":"Jacob","middleName":"","lastName":"Kaufman","suffix":""},{"id":403834355,"identity":"b5dbd96e-b44e-447b-adc8-f3de9108b5db","order_by":17,"name":"Vincenzo Coppola","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYDACZlSuBQM/nH2AOC0SDJINhLSgAQkGA7hKHFoMjvMe/PBxR50cA/vhY48LKiTkjY+3P5O6UcMgx3cjAbuWw3zJkjPPsBkz8KSlG884I2G47cwZM+mcYwzGkji0SDbzmDHztvEkNkjwmEnztkkwbruRwyad28CQuAGflr9tEnAt9ptnpD8DaanHpYWfGaiFsc0AriVxg0SCGUhLggFuLcaSvW0JxmxQvyTPOHPG2DrnmIThzDMPsGph4z9j+OFnW50cPyTEbGz729sf3s6psZHnO47dFoReIEKOVgn8ymG6mAmrGQWjYBSMgpEIAHfHUckOxu+CAAAAAElFTkSuQmCC","orcid":"","institution":"Ohio State Wexner Medical Center, The Ohio State University and OSUCCC","correspondingAuthor":true,"prefix":"","firstName":"Vincenzo","middleName":"","lastName":"Coppola","suffix":""}],"badges":[],"createdAt":"2024-12-24 17:23:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5707591/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5707591/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13046-025-03491-8","type":"published","date":"2025-08-29T15:56:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75308108,"identity":"920ed472-9315-4bd2-b7c1-9ce8b216ff54","added_by":"auto","created_at":"2025-02-03 08:44:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1673175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe combined deletion of Scorpins disrupts the CTLH complex in NSCLC cells\u003c/strong\u003e\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) CRISPR/Cas9 was used to generate A549 (\u003cstrong\u003eA\u003c/strong\u003e) and H460 (\u003cstrong\u003eB\u003c/strong\u003e) cell lines lacking RANBP9 (9KO), RANBP10 (10KO), or both (DKO). Total cell lysates were analyzed by WB for the presence of the indicated CTLH proteins. The vertical lines on the right side of the panels represent blots from the same gel. Vinculin was used as a loading control foreach blot. (\u003cstrong\u003eC\u003c/strong\u003e) Illustrations of CTLH complex formation and disruption in Scorpin WT, 9KO, 10KO, and DKO NSCLC cells generated via Biorender (www.biorender.com).\u003c/p\u003e","description":"","filename":"F1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/5ae01bbedc510f3b3a4ec195.jpg"},{"id":75308145,"identity":"66485e29-188f-4324-ac80-f61b1c1490bf","added_by":"auto","created_at":"2025-02-03 08:44:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1577574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRANBP9 or RANBP10 re-expression is sufficient to stabilize GID8 and MAEA and restore CTLH complex formation\u003c/strong\u003e\u003c/em\u003e. Scorpin DKO A549 cells bearing doxycycline (Doxy)-inducible enhanced GFP (A549 iGFP), RANBP9 (DKO iBP9), or RANBP10 (iBP10) cDNA, together with Scorpin WT A549 cells, were left untreated or exposed to Doxy at 1 mg/mL for 24 hours before harvesting. Total cell lysates were probed by WB for the presence of the indicated CTLH proteins. The vertical lines on the right side of the panels group together blots from the same gel. Vinculin was used as a loading control for each blot.\u003c/p\u003e","description":"","filename":"F2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/349415348a236be63ac6038d.jpg"},{"id":75308116,"identity":"eefc9a57-55cd-43dd-97db-0d4a43dbd32a","added_by":"auto","created_at":"2025-02-03 08:44:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1333845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRANBP9 and RANBP10 cross-regulate each other’s expression\u003c/strong\u003e\u003c/em\u003e. \u003cem\u003e\u003cstrong\u003eTime course of inducible RANBP9 and RANBP10 expression in Scorpin WT A549 iBP9 and iBP10 cells\u003c/strong\u003e\u003c/em\u003e. Total cell lysates from Scorpin WT A549 (\u003cstrong\u003eE\u003c/strong\u003e), iBP9 (\u003cstrong\u003eA\u003c/strong\u003e), and iBP10 (\u003cstrong\u003eC\u003c/strong\u003e) cells were probed by WB for the expression of RANBP9 (red), RANBP10 (blue), or GID8 before and after exposure to Doxy at 1 mg/mL for the indicated times. A549 Scorpin DKO cells were used as negative controls for RANBP9 and RANBP10 expression. The vertical lines on the right side of the panels represent blots from the same gel. Vinculin was used as a loading control for each blot. ImageJ version 1.53t (\u003ca href=\"https://imagej.nih.gov/ij/\"\u003ehttps://imagej.nih.gov/ij/\u003c/a\u003e) was used for the quantitation of the RANBP9 and RANBP10 band intensities normalized to the corresponding Vinculin intensity results, as shown in panel (\u003cstrong\u003eB\u003c/strong\u003e) for A549 iBP9 cells, in panel (\u003cstrong\u003eD\u003c/strong\u003e) for A549 iBP10 cells, and in panel (\u003cstrong\u003eF\u003c/strong\u003e) for A549 WT cells.\u003c/p\u003e","description":"","filename":"F3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/3ea556aa7a432f00f374fc7f.jpg"},{"id":75308139,"identity":"1a050b8b-4e6e-423e-99ce-8867235818b7","added_by":"auto","created_at":"2025-02-03 08:44:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1092226,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGID8 and RANBP9 are overexpressed, whereas RANBP10 is downregulated in NSCLC\u003c/strong\u003e\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003e\u003cstrong\u003eGID8 and RANBP9 proteins are overexpressed in 50 NSCLC samples (OSU NSCLC collection)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e RANBP9 and GID8 protein expression was quantified by assessing the WB band intensity normalized to that of vinculin in 50 NSCLC tumors with matched normal adjacent tissues, as shown in Supplementary Figure 4A. (\u003cstrong\u003eC\u003c/strong\u003e-\u003cstrong\u003eE\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGID8 and RANBP9 transcripts are overexpressed in 50 NSCLC samples (OSU NSCLC collection)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. \u003c/em\u003eRNA was extracted from the same 50 frozen NSCLC tumors, and RT‒PCR was performed to measure the amount of RANBP9, GID8, and RANBP10 mRNA. The statistical significance of the differences was assessed via two-way ANOVA using RStudio. **** p\u0026lt;0.001; *** p=0.001; ** p=0.01.\u003c/p\u003e","description":"","filename":"F4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/edd99ca39da42df2724dc891.jpg"},{"id":75308964,"identity":"77355a31-4cdb-4d5a-a2ff-1a4f76748e00","added_by":"auto","created_at":"2025-02-03 08:52:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1173547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGID8 and RANBP9 are overexpressed, whereas RanBP10 is downregulated in the CPTAC NSCLC collection of NSCLC tumors compared with matched normal adjacent tissue\u003c/strong\u003e\u003c/em\u003e. Quantitation of protein expression from the CPTAC LUAD dataset revealed that both the RANBP9 (\u003cstrong\u003eA\u003c/strong\u003e) and GID8 (\u003cstrong\u003eB\u003c/strong\u003e) proteins are significantly overexpressed, whereas the RANBP10 (\u003cstrong\u003eC\u003c/strong\u003e) protein is significantly downregulated in LUAD tumors compared with normal adjacent tissues. The quantification of protein expression from the LUSQ data collection revealed that both the RANBP9 (\u003cstrong\u003eD\u003c/strong\u003e) and GID8 (\u003cstrong\u003eE\u003c/strong\u003e) proteins were significantly overexpressed, whereas theRANBP10 (\u003cstrong\u003eF\u003c/strong\u003e) protein was significantly downregulated in LUSQ tumors compared withnormal adjacent tissues. The reported data were downloaded from \u003ca href=\"https://kb.linkedomics.org/\"\u003ehttps://kb.linkedomics.org/\u003c/a\u003e. The statistical analysis was performed viaRStudio.\u003cstrong\u003e \u003c/strong\u003eThe statistical significance of the differences was assessed via two-way ANOVA using RStudio. **** p\u0026lt;0.001; *** p=0.001; ** p=0.01.\u003c/p\u003e","description":"","filename":"F5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/7bc502287ff369d059a898f4.jpg"},{"id":75308114,"identity":"dc4b4d41-1dd0-48fe-951d-38e723ce1691","added_by":"auto","created_at":"2025-02-03 08:44:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1486777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRANBP9 and RANBP10 correlate with significantly different proteomes in NSCLC patient tumors\u003c/strong\u003e\u003c/em\u003e. Volcano plots illustrating regression analysis of proteins related to RANBP9 (\u003cstrong\u003eA\u003c/strong\u003e,\u003cstrong\u003e D\u003c/strong\u003e), RANBP10 (\u003cstrong\u003eB\u003c/strong\u003e,\u003cstrong\u003e E\u003c/strong\u003e) or DBP expression (differences in Z scores for RANBP9 minus RANBP10) (\u003cstrong\u003eC, F\u003c/strong\u003e) in the LUAD and LUSQ CPTAC collection, respectively. Protein expression was converted to a Z score, and regression analysis was performed in RStudio. Unadjusted log10-transformed p values on the y-axis are plotted against regression estimates. Values exceeding the plotted ranges are shown at the corresponding maximum or minimum values. The CTLH family members RANBP9, RANBP10, GID8, MAEA, and WRD26 are labeled. Venn diagrams showing the overlap between the top 200 proteins for associations with RANBP9 vs GID8 (\u003cstrong\u003eG\u003c/strong\u003e), RANBP9 vs RANBP10 (\u003cstrong\u003eH\u003c/strong\u003e), and RANBP10 vs GID8 (\u003cstrong\u003eI\u003c/strong\u003e) for the CPTAC LUAD and LUSQ datasets. For RANBP10 (R10) vs RANBP9 (R9) (\u003cstrong\u003eH\u003c/strong\u003e) and RANBP10 (R10) vs GID8 (G8) (\u003cstrong\u003eI\u003c/strong\u003e), the overlapping proteins favor R10.AD~R10. SQ(where AD=LUAD and SQ=LUSQ), R9.AD~R9. SQ, and G8.AD~G8.SQ. The similarity is minimal when R10~G8 or especially R10~R9 are considered. The opposite is observed for RANBP9 vs GID8 (\u003cstrong\u003eG\u003c/strong\u003e), in which manyoverlapping proteins are shared between R9~G8 (in blue boxes)both in LUAD and LUSQ, and relatively fewer proteins are unique to R9.AD~R9. SQ or G8.AD~G8.SQ.In red boxes, proteins that are correlated with each of the three proteins common between the LUAD and LUSQ collections are shown. The statistical analysis was performed via RStudio; the top 200 associated proteins were ranked by p value from linear regression with the indicated CTLH member in the respective tumor cohort. The data were downloaded from \u003ca href=\"https://kb.linkedomics.org/\"\u003ehttps://kb.linkedomics.org/\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"F6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/a4290d557d4515403386bca1.jpg"},{"id":75308150,"identity":"40688ff6-9c66-4770-a3fe-acd8c40d5f68","added_by":"auto","created_at":"2025-02-03 08:44:12","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1168485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRANBP9 expression is associated with cell proliferation-related proteins in NSCLC\u003c/strong\u003e\u003c/em\u003e. A 282-protein proliferation signature was used to establish the correlation between RANBP9 and proliferation in LUAD (\u003cstrong\u003eA\u003c/strong\u003e) and between LUSQ (\u003cstrong\u003eD\u003c/strong\u003e), RANBP10 in LUAD (\u003cstrong\u003eB\u003c/strong\u003e) and LUSQ (\u003cstrong\u003eE\u003c/strong\u003e), and GID8 in LUAD (\u003cstrong\u003eC\u003c/strong\u003e) and LUSQ (\u003cstrong\u003eF\u003c/strong\u003e). The statistical analysis was performed via RStudio; the proliferation score was calculated as the mean Z score for the corresponding 282 proteins and was plotted against the CTLH member as indicated. P value results from linear regression. The data were downloaded from https://kb.linkedomics.org/.\u003c/p\u003e","description":"","filename":"F7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/a6c022191fd96583422d158f.jpg"},{"id":75308954,"identity":"8b5689e2-3dcc-4073-869c-d96e15da0853","added_by":"auto","created_at":"2025-02-03 08:52:11","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1712191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCompared with RANBP9, the acute overexpression of RANBP10 causes different changes in the NSCLC proteome, downregulating several proliferation-associated proteins and reducing cell proliferation\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eVolcano plots illustrating the proteins whose expression significantly changed in relation to that of RANBP9 (\u003cstrong\u003eA\u003c/strong\u003e), RANBP10 (\u003cstrong\u003eB\u003c/strong\u003e), and DBP (\u003cstrong\u003eC\u003c/strong\u003e) in iA549 cells. Red indicates proteins whose statistical significance is \u003csub\u003elog10\u003c/sub\u003e p £ 1.300. CTLH complex members and proliferation-associated proteins are labeled. (\u003cstrong\u003eD\u003c/strong\u003e) Bar graph reporting the number of proliferation-associated proteins (PAPs \u003csup\u003e49\u003c/sup\u003e) found in total (gray bar), in the list of proteins positively associated with RANBP10 (blue bar), and in the list of proteins positively associated with RANBP9 (red bar). The results of Fisher’s test revealed a statistically significant increase in the number of PAPs positively associated with RANBP9 (p=0.0001), with an odds ratio (OR)=3.1. Proteins were considered to be positively associated with RANBP9 or RANBP10 if they were significantly different between the Doxy-treated and control conditions and significantly different in expression vs the opposite Scorpin member in the Doxy-treated condition, each with P\u0026lt;0.05 according to Student’s t test. Proteins were ranked for differential expression of RANBP9 vs RANBP10 induction in A549 cells after Doxy treatment for 24 hours, and gene set enrichment analysis was performed for the indicated gene set: (\u003cstrong\u003eE\u003c/strong\u003e) PAP \u003csup\u003e49\u003c/sup\u003e signature; (\u003cstrong\u003eF\u003c/strong\u003e) top 200 proteins associated with RANBP9 in CPTAC LUSQ; (\u003cstrong\u003eG\u003c/strong\u003e) top 200 proteins associated with RANBP9 in CPTAC LUAD. P=p value; FDR=false discovery rate; NES=normalized enrichment score. Plots generated via GSEA_4.3.2. \u003cstrong\u003eH\u003c/strong\u003e) Scorpin WT A549 iBP9 (round dots) and iBP10 (square dots) cells were seeded onto 96-well plates and exposed to 1 mg/mL Doxy (red) or not (iBP9 blue and iBP10 green). Cell growth was measured via microscopy-based technology with an IncuCyteÔ instrument. Cell growth was normalized to that on day 0, and each dot represents the average of 4 wells.\u003c/p\u003e","description":"","filename":"F8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/1af4861e1b912a57c425839c.jpg"},{"id":75308112,"identity":"a03177b4-f8dd-4fe3-8a84-1114551c4aba","added_by":"auto","created_at":"2025-02-03 08:44:11","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1815874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCompared with RANBP9, the overexpression of RANBP10 causes different changes in the NSCLC ubiquitylome, which includes proliferation-associated proteins\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e Volcano plots illustrating the proteins whose expression significantly changed in relation to that of RANBP9 (\u003cstrong\u003eA\u003c/strong\u003e) or RANBP10 (\u003cstrong\u003eB\u003c/strong\u003e). Red indicates proteins whose statistical significance is \u003csub\u003elog10\u003c/sub\u003e p£1.300. Specific ubiquitylations of PPAs of interest are labeled. (\u003cstrong\u003eC\u003c/strong\u003e) Heatmap depicting ubiquitylations (p=0.05) observed in proteins differentially expressed between iA549 iBP9 and iBP10 cells (DBP p=0.05 and increased under RANBP9 conditions) upon treatment with Doxy for 12 hours. Peptides corresponding to proliferation-associated proteins (PAPs \u003csup\u003e49\u003c/sup\u003e) are labeled. \u003cstrong\u003eD\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eIllustrative plots of total protein changes (top panels) and their specific ubiquitylations (bottom panels) observed with the opposite pattern of seven selected PPAs. Plots were generated via RStudio.\u003c/p\u003e","description":"","filename":"F9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/642e569761c71010c8d1ea05.jpg"},{"id":75308956,"identity":"06b067fc-591a-4b13-b100-cfb4c1c68ef0","added_by":"auto","created_at":"2025-02-03 08:52:11","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":890189,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eScorpins cooperate in regulating the CTLH complex ubiquitylation output\u003c/strong\u003e\u003c/em\u003e. Proposed model of the action of the rheostat formed by the two Scorpins. The CTLH\u003cstrong\u003eBP9\u003c/strong\u003e and CTLH\u003cstrong\u003eBP10\u003c/strong\u003e configurations have both common and selective substrates. The net functional result depends on the molecular role of the specific lysine that is ubiquitylated and/or the cumulative effect of multiple ubiquitylations. The illustration was generated via Biorender (www.biorender.com).\u003c/p\u003e","description":"","filename":"F10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/18ce2fcb17c86864f3fb27d7.jpg"},{"id":90344815,"identity":"5f9d5ead-bcec-4686-8f47-f03728d52153","added_by":"auto","created_at":"2025-09-01 16:03:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16533009,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/d77ab1cf-f77b-4193-b98a-6d0170d5e551.pdf"},{"id":75308109,"identity":"e962d4e6-c3ee-4c7b-a90d-73af3c354739","added_by":"auto","created_at":"2025-02-03 08:44:10","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1244376,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERFINALSupplementaryFigure1DEC2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/474294f0bcc637c6c7f1227f.pdf"},{"id":75308111,"identity":"3f8ff670-950c-4091-86a2-f1973d90be9e","added_by":"auto","created_at":"2025-02-03 08:44:11","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":225504,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERFINALSupplementaryFigure2DEC2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/e01eae6e5387e96e6afcc808.pdf"},{"id":75308141,"identity":"a594a751-b8b2-402f-bd71-4178d7071f81","added_by":"auto","created_at":"2025-02-03 08:44:12","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1847393,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERFINALGraphicalAbstracthighresolutionDEC2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/dca678ff7a8719a429302e02.pdf"},{"id":75308119,"identity":"965c92f4-6b0b-4f39-bb45-7287f8cb7105","added_by":"auto","created_at":"2025-02-03 08:44:11","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3056818,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERFINALSupplementaryFigure3DEC2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/b4062755fca33069cdfb45f3.pdf"},{"id":75308147,"identity":"8bf07c46-03c9-4022-905b-b896c992248e","added_by":"auto","created_at":"2025-02-03 08:44:12","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":434182,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERFINALSupplementaryFigure4DEC2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/d535741f2ed80127f893cb6c.pdf"},{"id":75308126,"identity":"e16b05dd-59f4-4367-be67-4541e5408a04","added_by":"auto","created_at":"2025-02-03 08:44:11","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1328656,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERFINALSupplementaryFigure5DEC2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/489538bb60d66933255c0389.pdf"},{"id":75308967,"identity":"390c7e7e-7df7-4d7b-918c-08e40c443991","added_by":"auto","created_at":"2025-02-03 08:52:13","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":143208,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERFINALSupplementaryFigure6DEC2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/1430a0f9b6ae675c817506e9.pdf"},{"id":75308117,"identity":"3a68abfa-2fac-485b-8b4f-39df4abfafff","added_by":"auto","created_at":"2025-02-03 08:44:11","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":223497,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERFINALSupplementaryFigure7AHDEC2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/bd54cbe76162052a4bb5b00a.pdf"},{"id":75308123,"identity":"d43e4443-f0b2-4672-8944-0f1bcaff4eab","added_by":"auto","created_at":"2025-02-03 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08:44:11","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":158831,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERFINALSupplementaryFigure9DEC2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/3871c7db085b8f9743109c8d.pdf"},{"id":75308125,"identity":"cb4b54a3-e028-45f3-87ff-de8f1f53ff2f","added_by":"auto","created_at":"2025-02-03 08:44:11","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":15979,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERSupplementaryTable150NSCLCOSUcollection.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/8a0e3d13a36b59dba774388b.xlsx"},{"id":75308146,"identity":"59a84cb1-cd5d-4303-8243-068ade512ec6","added_by":"auto","created_at":"2025-02-03 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08:52:11","extension":"xlsx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":18903,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERSupplementaryTableFigure450OSUQuantificationandpatientsorder.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/7cd8690296aefbdabd0faefa.xlsx"},{"id":75308135,"identity":"1735fdcd-108d-43aa-919c-bf0461972456","added_by":"auto","created_at":"2025-02-03 08:44:12","extension":"xlsx","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":123838,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERSupplementaryTableFigureS1BA549RTPCR.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/3970c780943b72f486025274.xlsx"},{"id":75308137,"identity":"731d0f12-e9ba-4ea0-932b-5fba76d5be9a","added_by":"auto","created_at":"2025-02-03 08:44:12","extension":"xlsx","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":263220,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERSupplementaryTableFigureS2BA549DKOWBquantification.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/e0a6bca72ccd2796a5d4b740.xlsx"},{"id":75308165,"identity":"d1eea5b7-9144-403f-ba37-f5202c2f8061","added_by":"auto","created_at":"2025-02-03 08:44:13","extension":"xlsx","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":165286,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERSupplementaryTableFigureS3BWBquantification.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/46fbea73a422d00f81bd2d26.xlsx"},{"id":75308128,"identity":"6807f549-6b41-4bbe-9700-0474b1d54384","added_by":"auto","created_at":"2025-02-03 08:44:11","extension":"xlsx","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":140717,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERsupplementaryTableFigureS3CRTPCR.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/780d1309e40205ff2e0cebac.xlsx"},{"id":75308955,"identity":"482fbd4c-5f14-40cd-99e5-f349945a8d77","added_by":"auto","created_at":"2025-02-03 08:52:11","extension":"pdf","order_by":20,"title":"","display":"","copyAsset":false,"role":"supplement","size":104841,"visible":true,"origin":"","legend":"","description":"","filename":"THEPAPERFINALEXTENDEDMATERIALDEC2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5707591/v1/24d98775cdfbe31b98573733.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"RANBP9 and RANBP10 cooperate in regulating non-small cell lung cancer proliferation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe scaffold protein containing a C-terminal to LisH (CTLH) domain named RAN Binding Protein 9 (RANBP9; also known as RANBPM) is involved in cell homeostasis, survival, proliferation, adhesion, and migration \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Its increased expression promotes the signaling of several receptor tyrosine kinases (RTKs), including major players in non-small cell lung cancer (NSCLC) tumorigenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn NSCLC cells subjected to DNA damage, RANBP9 is not only a target but also an enabler of the ATM (Ataxia Telangiectasia Mutated) protein kinase \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. RANBP9 protein expression is high in advanced NSCLC, and patients expressing higher levels of RANBP9 protein have worse outcomes when treated with platinum-based regimens, which is in line with the hypothesis that RanBP9 is protective of cancer cells when subjected to DNA damage \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Hence, targeting RANBP9 could be a valid strategy to treat NSCLC in combination with other therapeutic modalities \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRANBP9 operates in the context of the ubiquitously expressed unconventional multisubunit E3 ligase CTLH complex, where it constitutes the inner core and tightly binds to GID8 (Glucose-Induced degradation Deficient 8) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. This multisubunit enzyme is emerging as a central regulator of mammalian cell metabolism and regulates key bioenergetic signaling nodes, such as AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) \u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Currently, there are 11 known members of the CTLH complex. In addition to RANBP9 and GID8, other established core members are ARMC8 (Armadillo Repeat Containing protein 8), MAEA (Macrophage Erythroblast Attacher E3 ligase), RMND5A and RMND5B (Required in Meiotic Division 5 A and B). On the other hand, GID4 (Glucose-Induced degradation Deficient 4), WDR26 (WD Repeat containing protein 26), and YPEL5 (Yippee Like protein 5) are peripheral members \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Recent biochemical and structural studies have shown that MAEA and RMND5 bind to a RANBP9-GID8-ARMC8 core and, together, provide E3 enzymatic activity \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Finally, MKLN1 (Muskelin 1) has been shown to be not only a peripheral member but also a substrate of the complex, and its levels can be used as a proxy for CTLH enzymatic activity \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough the CTLH complex was initially characterized as a heterodecameric aggregate, it is becoming increasingly clear that its proteins can assemble in different configurations that include or exclude selected core and peripheral members. For example, RMND5A and RMND5B are mutually exclusive \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In addition, the two splicing isoforms of ARMC8, ARMC8α and ARMC8β, can determine the inclusion (ARMC8α) or exclusion (ARMC8β) of GID4, the first and only known substrate receptor until recently \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The presence of RANBP9, GID8, RMND5A, and WDR26 together has been shown to promote proliferation \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe topological features of the CTLH E3 ligase have been inferred from studies in \u003cem\u003eS. cerevisiae\u003c/em\u003e, where its counterpart, the GID (Glucose-Induced degradation Deficient) complex, responds to metabolic stress, modulates mitochondrial functions and participates in the disposal of enzymes necessary for gluconeogenesis \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Elegant structural work has shown that the GID complex can assemble into \u0026ldquo;supra-molecular\u0026rdquo; oval-shaped rings with a diameter equivalent to the length of the proteasome and quickly ubiquitylate multimeric metabolic enzymes in the central cavity \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Yeast Gid1 is essential for the assembly of the complex and tightly binds Gid8. Together, Gid1 and Gid8 provide the inner core on which the structure is built, and their proteins stabilize each other \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Deletion of Gid1 disrupts the formation of the complex \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Therefore, it has been hypothesized that ablation of RANBP9 disrupts the formation of the whole CTLH complex \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, the eleventh and understudied member of the CTLH complex is a highly similar RANBP9 paralog called RANBP10, which presents the same domains that are essential for aggregation into the CTLH complex \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. RANBP9 and RANBP10 are also called Scorpins (Spry-containing Ran-binding proteins) to distinguish them from other RAN-binding proteins that are involved mainly in nuclear‒cytoplasmic shuttling \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. RANBP9 is markedly expressed in highly proliferative precursors, whereas RANBP10 expression becomes predominant in more differentiated cells during erythroid maturation. Moreover, both RANBP9 and RANBP10 can form their own CTLH complex \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, the reciprocal dynamics governing the expression of Scorpins in the same cell type are not known. Although RANBP10 is ubiquitously coexpressed with RANBP9 and consistently coimmunoprecipitates with other CTLH proteins both in humans and in mice, the function of RANBP10 is not well understood in general \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Apart from a potential tumor-promoting role in glioblastoma \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, the relevance of RANBP10 in cancer has not been addressed.\u003c/p\u003e \u003cp\u003eTaking into consideration evidence from different model systems, we reasoned that RANBP9 and RANBP10 need to be studied together to fully understand their biological roles because they are both the result of duplication of the ancestral yeast Gid1 gene \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Phenotypic and structural similarities, as well as differences, indicate that these genes might have only partially overlapping functions and are likely to cross-regulate each other \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we present data that show how the two Scorpins work in concert and how their modulatory role on the CTLH complex ubiquitylation output depends on the ratio of their amount. The predominant expression of RANBP9 over RANBP10 in NSCLC correlates with increased proliferation in both NSCLC cell lines and patients, where increased expression of RANBP10 resulted in a reduction in cell proliferation and in the level of proteins involved in cell proliferation.\u003c/p\u003e \u003cp\u003eHere, we reveal that the two Scorpins constitute a unique sophisticated rheostat that modulates the expression and ubiquitylation of a variety of proteins that participate in fundamental NSCLC oncogenic processes, such as cell proliferation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of cell lines and KOs\u003c/h2\u003e \u003cp\u003eA549 and H460 NSCLC cell lines (see supplementary info) were purchased from ATCC and cultured in RPMI 1640 medium (Gibco\u0026trade;) supplemented with 10% FBS (Gibco\u0026trade;) and MycoZap\u0026trade; Plus-CL (Lonza).\u003c/p\u003e \u003cp\u003esgRNAs were designed at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.mit.edu\u003c/span\u003e\u003cspan address=\"http://crispr.mit.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (see supplementary info). Briefly, for each guide, two DNA primers were designed and then annealed and phosphorylated using T4 polynucleotide kinase (NEB). Phosphorylated sgRNAs were then cloned and inserted into the vectors pSpCas9(BB)-2A-GFP (5\u0026rsquo; prime sgRNA) and pSpCas9(BB)-2A-Puro (3\u0026rsquo; prime sgRNA) via the BbsI restriction enzyme (NEB) and the Rapid DNA Dephos \u0026amp; Ligation Kit (ROCHE). Following the transformation of the ligase reaction plasmid DNA into DH5α competent cells (Thermo Fisher Scientific), the plasmid DNA was purified via endotoxin-free Maxiprep kits (QIAGEN). NSCLC cell lines (~\u0026thinsp;1x10\u003csup\u003e6\u003c/sup\u003e) were transfected with both sgRNA-containing vectors via Lipofectamine\u0026trade; 3000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s specifications, and after 48 hours, single cells were sorted for GFP expression via a BD FACSAria\u0026trade; II Cell Sorter. Scorpin WT or Scorpin DKO A549 cells were transduced via a 4D-Nucleofector X Unit (Lonza) according to the manufacturer\u0026rsquo;s optimized protocol. Briefly, 2 \u0026times; 10^5 cells were resuspended in 20 \u0026micro;l of SF buffer and electroporated via the nucleofection program CM-130 with 0.1 \u0026micro;g of AAVS1-TRE3G-EGFP, AAVS1-TRE3G-RANBP9 or AAVS1-TRE3G-RANBP10 in the presence of 0.1 \u0026micro;g of pX330-U6-Chimeric_BB-CBh-hSpCas9-hGem (1/110)-AAVS1 gRNA. The latter allows CRISPR/Cas9-mediated integration of inducible plasmids into the AAVS1 safe harbor site of the human genome. Forty-eight hours post-transfection, puromycin (1 \u0026micro;g/mL) was applied to enrich the cells with successful transgene integration. The AAVS1-TRE3G and pX330-U6-Chimeric_BB-CBh-hSpCas9-hGem (1/110) base plasmids for safe-harbor integration of the inducible system were acquired from Addgene (see also Extended Material) \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of MEF cell cultures\u003c/h3\u003e\n\u003cp\u003eAll the animal studies were conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Ohio State University. Embryos from RanPBP9, RanBP10 KO and Double KO mice (see also Extended Material) were isolated at E12.5. After the heads, tails, limbs, and most of the internal organs were removed, the embryo carcasses were minced, passed through a 70 \u0026micro;m cell strainer, and then seeded into 10 cm cell culture dishes in 10 mL of high-glucose DMEM supplemented with 15% FBS, 1% penicillin‒streptomycin (Gibco\u0026trade;) and 0.01% 2-mercaptoethanol (Sigma-Aldrich). Primary MEFs were cultured in 3% oxygen and passaged two or three times to obtain a morphologically homogenous culture. Immortalized MEFs were generated by transfecting early-passage primary cells with 2 \u0026micro;g of Large-T antigen-expressing plasmid via Lipofectamine\u0026trade; 3000. The cells were then cultured in normal oxygen (20%) and passaged for an additional 5 generations to obtain a stable population.\u003c/p\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot\u003c/div\u003e \u003cp\u003eThe cells and tumor tissues were homogenized on ice in NP-40 buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein concentration was determined via the use of the Bio-Rad protein assay dye (Bio-Rad). Western blot analysis was performed using 30 to 50 \u0026micro;g of protein run on Mini-PROTEAN TGX precast gels (Bio-Rad).\u003c/p\u003e \u003cp\u003ePrimary antibodies (see also Extended Material) were used at a dilution of 1:1,000 in 5% milk in TBS-T. Signals were detected with HRP-conjugated secondary antibodies and the chemiluminescence substrate SuperSignal West Pico PLUS or Femto (Thermo Fisher Scientific). Equivalent loading among samples was confirmed with an anti-vinculin antibody (Cell Signaling). Images were acquired via the KwikQuant Digital Western Blot Detection System (Kindle Biosciences, LLC). Protein expression levels were quantified via optical densitometry via ImageJ software version 1.53t (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and KwikQuant Image Analyzer 5.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kindlebio.com/12-downloads\u003c/span\u003e\u003cspan address=\"https://kindlebio.com/12-downloads\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eRNA extraction and real-time qPCR\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eRNA extraction and real-time qPCR\u003c/div\u003e \u003cp\u003eThe cells and tumor tissues were homogenized on ice in TRIzol\u0026trade; reagent (Invitrogen\u0026trade;), and RNA was extracted according to the manufacturer\u0026rsquo;s guidelines. Following extraction, 1 \u0026micro;g of total RNA was then treated with DNase and converted into cDNA via the Maxima First Strand cDNA Synthesis Kit for RT‒qPCR with dsDNase (Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003eFor real-time qPCR, 10\u0026ndash;15 ng of cDNA was amplified via TaqMan\u0026trade; Fast Advanced Master Mix. Samples were amplified simultaneously in triplicate in one assay run via TaqMan probes specific for (RanBP9, RanBP10, GID8, WDR26, ARMC8, RMND5A, RMND5B, YPEL5, MAEA and MKLN1). OAZ1 and GAPDH were used as endogenous controls for human tumor samples and cell lines, respectively. Analysis was performed with GraphPad Prism 10.0 software via the Δ-ct method (see also Extended Material).\u003c/p\u003e\n\u003ch3\u003eProteomics\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell Lysis\u003c/h2\u003e \u003cp\u003eThe cell pellets were washed with PBS three times before being resuspended in 5% SDS buffer in 50 mM TEAB (triethylammonium bicarbonate) solution. The samples were then vortexed briefly before sonication by using a Bioruptor\u0026reg; Pico (Diagenode, Denville, NJ) following the manufacturer\u0026rsquo;s suggested protocol. Briefly, the sonication temperature was set at 4\u0026deg;C; the sonication cycle was set at 30 sec on and 30 sec off. A total of 20 cycles were performed for the cell pellets. After sonication, the samples were centrifuged at 20000 \u0026times; g for 15 min at 4\u0026deg;C to remove any remaining insoluble material. The protein concentrations were measured via a Qubit fluorometer (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eS-trap Digestion\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProteins were digested with trypsin via S traps (Protifi, Fairport, NY). For the TMT-labeled samples, 200 \u0026micro;g of each protein sample was subjected to trypsin digestion on an S-trap microcolumn (K02-micro); for the ubiquitylation enrichment samples, 2 mg of each protein sample was digested on an S-trap midi column (C02-Midi). Briefly, samples were reduced with DTT and alkylated with iodoacetamide before the addition of 12% phosphoric acid (to a final volume of 1.2%). The proteins were then diluted with S-Trap binding buffer (MeOH:TEAB, 90:10 v/v) at a 1:6 (sample:S-Trap binding buffer, v/v) ratio. The sample was then applied to the S-Trap column and washed three times with S-Trap binding buffer 4 times. Sequencing grade trypsin (Promega) dissolved in 50 mM TEAB was added to the sample at a 1:50 (trypsin:protein, w:w) ratio for TMT-labeled samples or a 1:200 ratio for ubiquitylated enrichment samples. The sample was incubated overnight at 37\u0026deg;C and eluted sequentially with 50 mM TEAB, 0.2% formic acid, and 0.2% formic acid in 50% acetonitrile. The samples were pooled and dried in a centrifuge concentrator for further use.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eTMT labeling\u003c/h3\u003e\n\u003cp\u003eOne hundred micrograms of peptides in 50 mM TEAB solution were labeled per the manufacturer\u0026rsquo;s instructions (A44522, Thermo Fisher Scientific). The TMT-labeled peptides were then pooled together and fractionated via a Pierce high-pH reversed-phase peptide fractionation kit (Cat# 84868, Thermo Fisher Scientific). Peptides eluted with 5, 10, 12.5, 15, 17.5, 20, 22.5, 25 and 50% acetonitrile/triethylamine (0.1%) solutions were collected and analyzed on a Fusion Orbitrap mass spectrometer.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eUbiquitylation Enrichment\u003c/h2\u003e \u003cp\u003eThe dried peptides were resuspended in PTMScan HS IAP Bind Buffer and placed on ice. PTMScan HS Magnetic Immunoaffinity Beads were washed four times with ice-cold 1X PBS. The dissolved peptides were combined with the washed magnetic beads and incubated on a tabletop mixer at 4\u0026deg;C for six hours. After incubation, magnetic beads with bound peptides were placed on a magnetic separator, and unmodified peptides were removed. The magnetic beads were washed four times with chilled HS IAP Wash Buffer, followed by two washes with chilled LC-MS Grade H\u003csub\u003e2\u003c/sub\u003eO. Ubiquitylated peptides were eluted twice by the addition of 0.15% TFA with gentle agitation for 10 minutes. Ubiquitylated peptides were placed into glass vials and dried prior to LC-MS analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eNano-LC/MS/MS analysis\u003c/h2\u003e \u003cp\u003eNanoliquid chromatography-nanospray tandem mass spectrometry (nano-LC‒MS/MS) for protein identification was performed on a Thermo Scientific orbitrap Fusion mass spectrometer equipped with an EASY-Spray\u0026trade; Sources operated in positive ion mode. The samples were separated on an easy spray nanocolumn (Pepmap\u0026trade; RSLC, C18 3 \u0026micro; 100A, 75 \u0026micro;m X250 mm Thermo Scientific) via a 2D RSLC HPLC system from Thermo Scientific. Mobile phase A was 0.1% formic acid in water, and acetonitrile (with 0.1% formic acid) was used as mobile phase B. The flow rate was set at 300 nL/min. For the TMT samples, a 3-hour gradient was used after the samples were desalted via a trap column. For ubiquitylated peptides, samples were directly loaded and separated on an easy spray nanocolumn bypassing the desalting column, and a 1-hour gradient was used for analysis.\u003c/p\u003e \u003cp\u003eMS/MS data were acquired with a spray voltage of 1.95 kV, and a capillary temperature of 305\u0026deg;C was used. The scan sequence of the mass spectrometer was based on the preview mode data-dependent TopSpeed\u0026trade; method. To achieve high mass accuracy MS determination, the full scan was performed in FT mode, and the resolution was set at 120,000 with internal mass calibration. For TMT-labeled samples, FT mode (resolution set at 50000) was used for MS2 data acquisition to ensure that mass tags that differ by only one N15 and C13 can be well resolved for accurate quantitation. For ubiquitinated samples, MSn was performed via HCD in ion trap (IT) mode to ensure the highest signal intensity of the MSn spectra. Three FAIMS compensation voltages (cv=-50, -65 and \u0026minus;\u0026thinsp;80v) were used for data acquisition. The AGC target ion number for the FT full scan was set at 4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e ions, the maximum ion injection time was set at 50 ms, and the microscan number was set at 1. The HCD collision energy was set at 32%. The AGC target ion number for the ion trap MSn scan was set at 3.0E4 ions, the maximum ion injection time was set at 35 ms, and the microscan number was set at 1. Dynamic exclusion is enabled with a repeat count of 1 within 60 s and a low mass width and high mass width of 10 ppm. Data analysis and quantitation were performed via MASCOT on Proteome Discoverer following workflows recommended by Thermo.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThe combined deletion of Scorpins causes the disappearance of GID8 and the functional inactivation of the CTLH complex\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWe previously generated RANBP9 shRNA-knockdown and complete knockout (KO) NSCLC cells \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. However, we did not assess the levels of RANBP10, which is reported to be expressed at lower levels than its paralog. For example, in HEK293T cells, the number of RANBP10 protein copies (7.6x10\u003csup\u003e4\u003c/sup\u003e) is estimated to be approximately one-third that of RANBP9 (2.3x10\u003csup\u003e5\u003c/sup\u003e) \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. To better characterize the role of both Scorpins in NSCLC in the context of the CTLH complex, we used CRISPR/Cas9 technology to generate RANBP10 knockout (KO) A549 and H460 cell lines. We also generated NSCLC cells in which both Scorpins were genetically inactivated (double-KO [DKO] cells) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). When tested for other CTLH members, DKO cells presented nearly complete ablation of GID8 and MAEA, whereas the levels of ARMC8 and WDR26 did not appear to consistently change among the two cell lines and different genotypes. On the other hand, the levels of MKLN1 were consistently elevated in DKO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The combined Scorpin deletion had similar effects on the levels of GID8 and MAEA in mouse embryonic fibroblasts (MEFs) (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e). These cells were derived from embryonic stem cell-generated mice deficient in RANBP9, RANBP10, or both Scorpins together (DKO), indicating that the disappearance of GID8 and MAEA is not dependent on cell type or species and is not caused by the use of CRISPR/Cas9 \u003csup\u003e42, 43\u003c/sup\u003e. MKLN1 levels were increased in DKO MEFs, which was consistent with observations in NSCLC cells, whereas WDR26 levels appeared to be unchanged in Scorpin-mutant MEFs (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRT‒PCR evaluation of GID8 and MAEA mRNA levels in both A549 and H460 DKO cells generally revealed a modest increase (less than 25%). The exception was GID8 mRNA, whose expression modestly but significantly decreased in H460 cells. These results indicated that changes in GID8 and MAEA protein levels were not due to changes in the corresponding transcripts (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B-C\u003c/b\u003e), which is in agreement with previous observations in cell lines \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOverall, both RANBP9 and RANBP10 can independently stabilize GID8 in mammalian cells, and only their stable combined deletion disrupts the formation of a functional CTLH complex via protein-mediated mechanisms.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRANBP9 or RANBP10 acute re-expression is sufficient to stabilize GID8 and MAEA and restore CTLH complex formation\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo further prove that both RANBP9 and RANBP10 can independently stabilize GID8 and enable the formation of a canonical CTLH complex, we generated A549 DKO cells in which either RANBP9 alone or RANBP10 alone can be re-expressed by a doxycycline (Doxy)-inducible system (\u003cb\u003eSupplementary Fig.\u0026nbsp;2A\u003c/b\u003e). The re-expression of RANBP9 or RANBP10 resulted in the clear reappearance of GID8 and MAEA, whereas the amount of MKLN1, which is also a substrate of the complex, decreased to levels similar to those of the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;2B\u003c/b\u003e) \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003ede novo\u003c/em\u003e expression of GFP had no appreciable effects on the Scorpins or other CTLH proteins. We concluded that in NSCLC cells, the expression of either one of the two Scorpins is sufficient to stabilize its assembly and restore the formation and function of the CTLH complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRANBP9 and RANBP10 cross-regulate each other\u0026rsquo;s expression\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWhen either RANBP9 shRNA-knockdown or CRISPR-KO NSCLC cells were generated, we previously reported that, in general, the expression of RANBP10 was greater than that in parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cb\u003eSupplementary Fig.\u0026nbsp;1C\u003c/b\u003e) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. This observation, together with the high similarity between the two proteins, led us to hypothesize that the two paralogs may functionally compensate for each other\u0026rsquo;s absence. To further investigate this phenomenon in a short period of time, we engineered A549 parental (Scorpin WT) cells bearing Doxy-inducible safe-harbor gene integration of either RANBP9 or RANBP10, similar to what we previously reported in A549 Scorpin DKO cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;2A\u003c/b\u003e). Time course experiments revealed that the protein expression of RANBP9 or RANBP10 was consistently upregulated from 6 to 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). When RANBP9 was induced, the amount of RANBP10 protein decreased accordingly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). The reverse was also true. When the expression of RANBP10 was induced, the expression of RANBP9 was proportionally downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). GID8, MAEA, and WDR26 trended toward increased expression with the induction of either RANBP9 or RANBP10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D; \u003cb\u003eSupplementary Fig.\u0026nbsp;3A\u003c/b\u003e). At the indicated times, Doxy treatment did not cause any appreciable change in Scorpin expression in the A549 parental line (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). The strong artificial increase in either the RANBP9 or RANBP10 transcript did not have a significant effect on the transcripts of their paralog or other CTLH complex members (\u003cb\u003eSupplementary Fig.\u0026nbsp;3C\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTogether with previous observations, these results demonstrate that the expression of the RANBP9 and RANBP10 proteins is cross regulated in NSCLC cells and that their dynamic changes reciprocally affect each other\u0026rsquo;s protein expression in the short term. Overall, CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e are subject to balanced expression at the protein level.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGID8 and RANBP9 are overexpressed, whereas RANBP10 is downregulated in NSCLC at both the RNA and protein levels\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe results above demonstrated that RANBP9 and RANBP10 acutely cross-regulate each other\u0026rsquo;s protein expression \u003cem\u003ein vitro\u003c/em\u003e, without being correlated with their transcript levels. Next, we proceeded to establish the relevance of these findings in NSCLC patients. We previously reported that the protein levels of RANBP9 are higher both in NSCLC cells and in patient samples than in their normal counterparts \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. However, we could not unequivocally determine how pervasive the overexpression of the RANBP9 protein was because of the small number of NSCLC samples we had available at the time \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. For this study, we acquired a collection of fifty (50) frozen NSCLC samples (T) with matched normal adjacent tissue (N), which included 16 adenocarcinoma (LUAD), 8 squamous carcinoma (LUSQ), 6 carcinoid, and other histotypes (OSU collection; \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e) samples. We measured the expression levels of the Scorpins and their binding partner GID8 via WB and RT‒PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e‒E\u003c/b\u003e; \u003cb\u003eSupplementary Fig.\u0026nbsp;4A\u003c/b\u003e; \u003cb\u003eSupplementary Materials\u003c/b\u003e). We found that the RANBP9 and GID8 proteins were significantly more abundant in T vs. matched N samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B; \u003cb\u003eSupplementary Fig.\u0026nbsp;4A\u003c/b\u003e). We also detected significant overexpression of GID8 and RANBP9 transcripts in T compared with N (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). On the other hand, we did not find a significant difference in the level of RANBP10 mRNA between the N and T stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Regrettably, the low affinity/specificity of the commercially available antibody did not allow reliable quantitation of the RANBP10 protein. To corroborate our findings concerning the overexpression of RANBP9 and GID8 in NSCLC, we analyzed the level of expression of the CTLH genes in the publicly available TCGA collection (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://crispr.mit.edu\" target=\"_blank\"\u003ewww.cbioportal.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.cbioportal.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) of lung cancer samples (T) with paired normal adjacent tissue (N) both squamous cell carcinoma (LUSQ) and adenocarcinoma (LUAD) samples. As shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;4B-C\u003c/b\u003e, RANBP9 and GID8 (a.k.a. C20orf11) were significantly higher in T than in N both in LUAD and LUSQ. Interestingly, RANBP10 mRNA levels were significantly lower in the T dataset than in the N dataset in both the LUAD and LUSQ datasets. Overall, the TCGA collection of NSCLC data revealed that RANBP9 and GID8 mRNAs were overexpressed, whereas RANBP10 transcript levels were decreased in both LUAD and LUSQ samples compared with those in T vs N samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, these results show that both the transcripts and proteins of GID8 and RANBP9 are upregulated in tumors of various histotypes in NSCLC patients. On the other hand, RANBP10 mRNA is expressed at low levels.\u003c/p\u003e \u003cp\u003eTo further investigate Scorpin expression in NSCLC, we sought to analyze the transcriptomic and proteomic data characterized by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) for lung adenocarcinoma (LUAD) and squamous lung cancer (LUSQ) recently published and available at kb.linkedomics.org \u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConsistent with our observations in the OSU collection of NSCLC samples analyzed by WB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B; \u003cb\u003eSupplementary Fig.\u0026nbsp;4A\u003c/b\u003e\u003cem\u003e)\u003c/em\u003e, the RANBP9 and GID8 proteins were significantly upregulated in both LUAD and LUSQ samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, D, E\u003cem\u003e)\u003c/em\u003e. On the other hand, the RANBP10 protein was significantly underexpressed compared with that in normal matched controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, F). The expression of mRNAs exhibited highly similar patterns (\u003cb\u003eSupplementary Fig.\u0026nbsp;5G-L\u003c/b\u003e). Although RANBP10 is expressed at lower levels on average, some tumors presented relatively high RANBP10 expression compared with normal controls. These latter cases appear to have correspondingly higher levels of GID8, as indicated by the significant positive correlation between GID8 and RANBP10 in both the LUAD and LUSQ datasets (\u003cb\u003eSupplementary Fig.\u0026nbsp;5A, B, D, E\u003c/b\u003e). We also determined that in both LUAD and LUSQ, the maximum values of either RANBP9 or RANBP10 were much more strongly correlated with GID8 protein levels than either of the two Scorpins alone were (\u003cb\u003eSupplementary Fig.\u0026nbsp;5C, F\u003c/b\u003e). The latter result is concordant with the stoichiometric relationship observed in our cell line experiments, suggesting that the predominant expression of one member suppresses the expression of its counterpart and that both RANBP9 and RANBP10 can stabilize the CTLH, protecting GID8 from degradation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, these data show that RANBP9 expression dominates RANBP10 expression at both the mRNA and protein levels, indicating that the chronic changes in CTLH protein levels are mediated by adjustments in transcript levels differently from those observed in cell lines in short-term experiments. Moreover, the expression of both RANBP9 and RANBP10 was positively correlated with the GID8 level.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRANBP9 and RANBP10 correlate with significantly different proteomes in NSCLC patient tumors\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eHaving established that RANBP9 and GID8 are upregulated while RANBP10 is downregulated or expressed at low levels in NSCLC patients, we next proceeded to assess their correlations with the global proteome in the CPTAC LUAD and LUSQ datasets. The regression analysis of the absolute expression of both Scorpins and their differential expression (RANBP9 minus RANBP10\u0026thinsp;=\u0026thinsp;delta [DRANBP] expression) revealed that RANBP9 and RANBP10 expression correlated negatively and positively with hundreds of other proteins both in LUAD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e‒C\u003c/b\u003e) and LUSQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e‒F\u003c/b\u003e; \u003cb\u003eSupplementary Material\u003c/b\u003e). As mentioned, both RANBP9 and RANBP10 were positively correlated with GID8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B, D, E; \u003cb\u003eSupplementary Fig.\u0026nbsp;6A-B\u003c/b\u003e) but negatively correlated with each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-F). In both LUAD and LUSQ, the list of proteins positively and negatively correlated with RANBP9 was significantly different from that associated with RANBP10. A selection of the top 200 proteins expressed in correlation with one of the three CTLH members in both types of tumors revealed that the overlapping proteins favor the comparisons RANBP10 LUAD vs RANBP10 LUSQ, RANBP9 LUAD vs RANBP9 LUSQ, and GID8 LUAD vs GID8 LUSQ, suggesting strongly concordant effects on tumor biology across distinct tumor histologies (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, I). RANBP9-associated proteins highly overlap with GID8-associated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, blue boxes), and relatively fewer proteins are unique to one group (\u003cb\u003eSupplementary Material\u003c/b\u003e). Conversely, only limited similarity was observed when comparing RANBP10 vs GID8, especially when comparing RANBP9 to RANBP10.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, these results indicate that RANBP9 and RANBP10 expression in NSCLC correlates with different proteins because they both positively correlate with GID8, thus suggesting a partially different functional role for the two paralogs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRANBP9 expression is associated with increased proliferation in NSCLC\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo gain biological insights into the proteomes potentially regulated by RANBP9 and RANBP10 in NSCLC, we performed two independent analyses to search for gene sets, pathways, and biological processes. First, we performed gene set enrichment analysis (GSEA) via the LinkedOmics website (kb.linkedomics.org) \u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, which queries associations with the publicly available WEB-basedGEne SeT AnaLysis Toolkit (WebGestalt: webgestalt.org) (\u003cb\u003eSupplementary Fig.\u0026nbsp;7A-H\u003c/b\u003e). Second, we used the list of proteins that were positively or negatively associated with RANBP9 or RANBP10 in the CPTAC LUSQ and LUAD collections (\u003cb\u003eSupplementary Material\u003c/b\u003e) to perform Metascape analyses (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org\u003c/span\u003e\u003cspan address=\"https://metascape.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (\u003cb\u003eSupplementary Fig.\u0026nbsp;7I‒P\u003c/b\u003e) \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBoth the WebGestalt and the Metascape results indicated that RANBP9 and RANBP10 have strong positive associations with gene sets related to all the different steps of RNA metabolism in both LUSQ and LUAD (\u003cb\u003eSupplementary Fig.\u0026nbsp;7A-P\u003c/b\u003e). The results also revealed that RANBP9 and RANBP10 were negatively correlated with processes related to different aspects of the immune response, both innate and adaptive, together with endocytosis, vesicle and membrane trafficking, and cell adhesion processes (\u003cb\u003eSupplementary Fig.\u0026nbsp;7I-P\u003c/b\u003e). However, both the GSEA and the Metascape analysis also revealed differences between RANBP9 and RANBP10. GSEA revealed that RANBP9 was positively associated with \u0026ldquo;DNA replication\u0026rdquo; in both LUSQ and LUAD, whereas RANBP10 was not (\u003cb\u003eSupplementary Fig.\u0026nbsp;7A-B\u003c/b\u003e). In the Metascape analysis, RANBP9 expression was strongly associated with terms related to cell proliferation, such as \u0026ldquo;cell cycle\u0026rdquo; (R-HSA-1640170), \u0026ldquo;DNA metabolic process\u0026rdquo; (GO:0051052), \u0026ldquo;mitotic cell cycle\u0026rdquo; (GO:0000278) or \u0026ldquo;mitotic cell cycle process\u0026rdquo; (GO:1903047) or \u0026ldquo;mitotic G2-G2M phases\u0026rdquo; (R-HSA-453274), \u0026ldquo;cell cycle checkpoints\u0026rdquo; (R-HSA-69620), \u0026ldquo;regulation of cell cycle process\u0026rdquo; (GO:0010564), \u0026ldquo;DNA replication\u0026rdquo; (GO:0006260 and WP466), \u0026ldquo;regulation of DNA replication\u0026rdquo; (GO:0007265), and \u0026ldquo;S-phase\u0026rdquo; (R-HSA-69242) (\u003cb\u003eSupplementary Fig.\u0026nbsp;7I-J\u003c/b\u003e). RANBP10 was associated with \u0026ldquo;regulation of cell cycle process\u0026rdquo; (GO:0010564) in LUAD and with \u0026ldquo;cell cycle\u0026rdquo; (R-HSA-1640170) in LUSQ, although the statistical significance of these latter associations was markedly lower.\u003c/p\u003e \u003cp\u003eCollectively, these results show that in NSCLC tumors, both the CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and the CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e configurations are potentially involved in the regulation of biological processes such as multiple steps of RNA metabolism, but they also have distinct preferential associations with other biological processes such as cell proliferation, which was found to be strongly associated with the increased protein ratio RANBP9/RANBP10.\u003c/p\u003e \u003cp\u003eTo corroborate the preferential association between cell proliferation and RANBP9 expression in comparison with RANBP10, we selected a recently published protein proliferation signature \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e and analyzed its correlation with the two Scorpins and GID8 in both the CPTAC LUAD and LUSQ datasets (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-F). Even if both RANBP9 and RANBP10 expression was positively correlated with GID8 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B, D, E; \u003cb\u003eSupplementary Fig.\u0026nbsp;6A-B\u003c/b\u003e), the results clearly revealed that RANBP9 and GID8 expression was positively correlated with the proliferation signature in both LUAD and LUSQ, whereas RANBP10 expression was not (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, these results indicate that the CTLH complexes formed by RANBP9 or RANBP10 are associated with a variety of fundamental biological processes and proteins, where a relatively high RANBP9/RANBP10 ratio is positively correlated with proliferation in NSCLC tumors.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCompared with RANBP9, the acute overexpression of RANBP10 causes different changes in the NSCLC proteome, downregulating several proliferation-associated proteins\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eNext, to gain mechanistic insight into the effects of RANBP9 overexpression versus RANBP10 overexpression, we aimed to establish whether the increase in RANBP9 or RANBP10 levels differentially affected iA549 cell proliferation. We previously reported that ablation of RANBP9 in NSCLC cells caused a modest but consistent reduction in cell proliferation \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In contrast, the downregulation of RANBP9 in HEK293 cells increased cell proliferation, and the silencing of RANBP10 was found to reduce glioblastoma cell proliferation \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. However, our A549 WT cell lines, where either RANBP9 (Scorpin WT A549 iBP9) or RANBP10 (Scorpin WT A549 iBP10) are induced without the constitutive ablation of endogenous genes, provide a tool that avoids artifacts due to \u003cem\u003ein vitro\u003c/em\u003e cell adaptation previously observed when manipulating CTLH proteins and better mimics human NSCLC tumors \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We treated Scorpin WT iBP9 and iBP10 cells with Doxy for 24 hours. Quadruplicate samples were collected and analyzed via tandem mass spectrometry via an isotopic labeling approach (\u003cb\u003eSupplementary Fig.\u0026nbsp;8A\u003c/b\u003e). For the CPTAC data collection, we considered the effects of RANBP9 induction, RANBP10 induction, and DRANBP separately (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe overexpression of the two Scorpins caused significant global proteome changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-C; \u003cb\u003eSupplementary Material\u003c/b\u003e). RANBP9 can affect the expression levels of other proteins both negatively and positively \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52 CR53\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. After the induction of RANBP9 in A549 iBP9 cells, 396 and 394 proteins correlated positively and negatively with RANBP9 expression (\u003csub\u003elog10\u003c/sub\u003e p\u0026le;1.3), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). When RANBP10 was induced in A549 iBP10 cells, 229 and 404 proteins correlated positively and negatively with RANBP10 levels, respectively (\u003csub\u003elog10\u003c/sub\u003e p\u0026le; 1.3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). In both induced cell lines, the amounts of GID8 and MAEA were positively correlated with either RANBP9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) or RANBP10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), which is in line with our previous results. When considering the expression of DRANBP, 288 proteins were found to be positively correlated with a higher DRANBP, and 293 proteins were positively correlated with a lower DRANBP \u003csub\u003e(log10\u003c/sub\u003e p\u0026le; 1.3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC; \u003cb\u003eSupplementary Material\u003c/b\u003e). These results are in line with the observations in the NSCLC CPTAC data, where the expression of the two paralogs correlated with only partially overlapping enriched proteomes, while both correlated positively with GID8 and other CTLH members (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-I).\u003c/p\u003e \u003cp\u003eProteins that were differentially expressed when RANBP9 was overexpressed were significantly similar to RANBP9-associated proteins in both LUSQ and LUAD (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). This similarity was in part due to an enrichment of proliferation-associated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-C, E-G), in agreement with the positive association of RANBP9 expression with proliferation observed in CPTAC patient tumors. However, our experiments also revealed that the overexpression of RANBP10 clearly downregulated the expression of proliferation-associated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB; \u003cb\u003eSupplementary Fig.\u0026nbsp;8B-C\u003c/b\u003e). RANBP9 loss of function or downregulation in NSCLC cells causes a modest but consistent reduction in cell proliferation \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Therefore, we investigated the effect of RANBP10 overexpression on iA549 cell proliferation. We again used iA549 WT iBP9 and iBP10 cells treated with or without doxycycline and monitored cell growth for three days. While the overexpression of RANBP9 did not significantly affect cell growth, the overexpression of RANBP10 caused a modest but significant decrease in the growth rate, which became evident after 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH). These results indicate that in iA549 cells, an increase in RANBP10 (a lower RANBP9/RANBP10 ratio) has a \u0026ldquo;braking effect\u0026rdquo; on cell proliferation.\u003c/p\u003e \u003cp\u003eCollectively, these results show that the artificial overexpression of the two Scorpins modulates distinct groups of proteins. Moreover, the overexpression of the CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e complex downregulates proliferation-associated proteins, leading to a measurable decrease in the growth rate. These observations are also consistent with the association of RANBP9 with an increased proliferative phenotype in CPTAC NSCLC tumors and decreased proliferation when RANBP9 is silenced or ablated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCompared with RANBP9, the acute overexpression of RANBP10 causes different changes in the A549 ubiquitylome, which includes proliferation-associated proteins\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eAll observations made thus far in NSCLC patients and cell lines have indicated that the CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e complexes coexist in a tightly regulated balance and that the functional effects of these two CTLH configurations are different, especially when considering cell proliferation. Next, we aimed to identify potential mechanistic candidates to guide future studies by assessing the effects of Scorpin manipulation on the A549 ubiquitylome. We repeated the Scorpin WT iA549 iBP9 and iBP10 cell induction and collected samples after proteasomal inhibition with MG132. Quadruplicate samples were collected and processed to enrich for ubiquitylated peptides with KGG remnants as proxies for ubiquitylation (\u003cb\u003eSupplementary Fig.\u0026nbsp;9A\u003c/b\u003e). We found that RANBP9 overexpression was positively correlated with 453 KGGs and negatively correlated with 436 KGGs \u003csub\u003e(log10\u003c/sub\u003e p\u0026le;1.3). On the other hand, the induction of RANBP10 was positively correlated with 1,765 KGGs and negatively correlated with 598 peptides with KGGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-C; \u003cb\u003eSupplementary Material\u003c/b\u003e). Considering the expression of DRANBP, 1559 genes were positively correlated with it, and 1611 genes were negatively correlated with it (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results clearly demonstrated that the upregulation of RANBP9 has significantly different effects on reshaping the ubiquitylome of iA549 cells than the increase in RANBP10 does.\u003c/p\u003e \u003cp\u003eWe found that proteins displaying more than one KGG peptide, including RANBP9 and RANBP10, were significantly enriched upon induction with the Scorpins themselves (\u003cb\u003eSupplementary Material\u003c/b\u003e). We constructed lists of ubiquitylated proteins associated either positively or negatively with RANBP9 or RANBP10 (\u003cb\u003eSupplementary Material\u003c/b\u003e) and performed a Metascape analysis to assess which gene sets, pathways, and biological processes were potentially perturbed upon manipulation of the Scorpin expression levels (\u003cb\u003eSupplementary Fig.\u0026nbsp;9B-E\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e\u0026ldquo;Metabolism of RNA\u0026rdquo; (R-HSA-8953854) was one of the most significantly enriched terms in all 4 different groups, further confirming that the two Scorpins participate together in the regulation of the transcriptome. The term \u0026ldquo;cell cycle\u0026rdquo; (R-HSA-164070) was among the top two enriched terms in the list compiled with ubiquitylated proteins associated either positively or negatively with RANBP10 (\u003cb\u003eSupplementary Fig.\u0026nbsp;9D-E\u003c/b\u003e). Importantly, the same \u0026ldquo;cell cycle\u0026rdquo; (R-HSA-164070) term was not significantly enriched in the Metascape analysis of ubiquitylated proteins positively or negatively associated with RANBP9 (\u003cb\u003eSupplementary Fig.\u0026nbsp;9B-C\u003c/b\u003e). However, other terms related to cell proliferation, such as \u0026ldquo;cell cycle, mitotic\u0026rdquo; (R-HSA-69278\u0026rdquo;) and \u0026ldquo;mitotic cell cycle process\u0026rdquo;, were significantly enriched with the proteins negatively associated with RANBP9.\u003c/p\u003e \u003cp\u003eOverall, these results indicate that a change in the balance between CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e at 24 hours results in hundreds of ubiquitylation changes in NSCLC cells, potentially fine-tuning a wide variety of key biological processes, including RNA processing and cell proliferation.\u003c/p\u003e \u003cp\u003eAnalysis of the ubiquitylome revealed that several proliferation-associated proteins had significantly different ubiquitylations when RANBP9 or RANBP10 was overexpressed in iA549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-C). To prioritize candidates most likely to be functionally relevant, we focused on the intersection of differentially ubiquitylated peptides whose total protein expression was also significantly altered by altered Scorpin expression. To broaden the list of putative candidates, we used a less stringent statistical cutoff for the total protein associations, requiring both a difference in Doxy induction with p\u0026thinsp;\u0026lt;\u0026thinsp;0.1 and a difference in the RANBP9\u0026thinsp;\u0026gt;\u0026thinsp;RANBP10 effect with P\u0026thinsp;\u0026lt;\u0026thinsp;0.1; for the ubiquitylation effect, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was used for Doxy vs the control in either RANBP9 or RANBP10 induction.\u003c/p\u003e \u003cp\u003eAs in our earlier results (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), a higher ratio of RANBP9\u0026thinsp;\u0026gt;\u0026thinsp;RANBP10 remained significantly enriched for proliferation-associated proteins with the use of relaxed statistical cutoffs (\u003cb\u003eSupplementary Fig.\u0026nbsp;9F; 27\u003c/b\u003e of 337; odds ratio 1.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Among the 337 RANBP9-associated proteins, 43 had at least one ubiquitylation site whose expression was increased by RANBP10 induction, and this subset presented the greatest enrichment of proliferation-associated proteins (8 of 43; odds ratio 4.9, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These differentially expressed proteins and their corresponding ubiquitylation sites putatively represent candidate substrates that may link CTLH E3 ubiquitin ligase activity with the regulation of cell growth and proliferation.\u003c/p\u003e \u003cp\u003eUltimately, we found that seven proliferation-associated proteins displayed significantly different specific ubiquitylation events that could explain the different levels of expression observed in the analysis of the proteome (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). This restricted list included the two members of the replisome MCM5 and MCM7, the calcium binding protein CACYBP1, which regulates replisome functions, the cohesin SMC1A, the core component of the RNA polymerase II POLR2B, the splicing factor SF3B3, and XAB2. Among these, CACYBP1, MCM5, MCM7, SMC1A, and SF3B3 were previously reported to be bona fide CTLH interactors in at least two different studies \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThese results demonstrate that the overexpression of RANBP9 or RANBP10 changes the total amount and ubiquitylation pattern of proteins critically involved in cell proliferation, such as members of the replisome and cohesins \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, with prioritized candidates that warrant further mechanistic validation in future studies.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite significant advances in patient survival, NSCLC remains the deadliest cancer in the United States \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. A better understanding of the pathogenesis of the disease is needed to design more efficacious biology-driven treatments \u003csup\u003e\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe present work illustrates the role of two variants of the CTLH complex, an E3 ligase that is emerging as a central node connecting cell signaling and metabolism, in NSCLC \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we show that RANBP9 and RANBP10, also called Scorpins \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, work in concert to modulate the ubiquitylation output of the CTLH complex in NSCLC. First, we demonstrated that the CTLH complex coexists in two different configurations, one based on the scaffold provided by RANBP9 (CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e) and the other built on RANBP10 (CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e). Both complexes are expressed in normal lung and NSCLC tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C, \u003cb\u003eSupplementary Fig.\u0026nbsp;1B-C\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-E, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F, \u003cb\u003eSupplementary Fig.\u0026nbsp;5A-L\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-F). In agreement with previous observations demonstrating the existence of both CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e during erythroid maturation, RANBP9 or RANBP10 were independently sufficient to protect their binding partner GID8 from proteolysis and stabilize the core on which the complex is formed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C, \u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;2A, B\u003c/b\u003e) \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e complexes acutely cross-regulate each other at the protein level. An artificial increase in the amount of RANBP9 caused a decrease in RANBP10, and \u003cem\u003evice versa\u003c/em\u003e, the forced increase in RANBP10 caused a proportional decrease in RANBP9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D; \u003cb\u003eSupplementary Fig.\u0026nbsp;3A, B\u003c/b\u003e). Notably, upon acute induction of the expression of RANBP9 or RANBP10, the transcripts of the uninduced paralog did not appreciably change within the short period we analyzed (\u003cb\u003eSupplementary Fig.\u0026nbsp;3C\u003c/b\u003e). Similarly, in single-KO NSCLC cells, the paralog transcript level did not significantly increase, whereas the protein level did (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B-C\u003c/b\u003e). In Scorpin double-KO (DKO) NSCLC cells, the disappearance of GID8 and MAEA, on the one hand, and the increase in MKLN1, on the other hand, are not in line with the levels of their relative transcripts (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B-C\u003c/b\u003e). It is not uncommon to find a poor correlation between the mRNA and protein levels of CTLH family members and proteins involved in proteostasis in general \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR66 CR67\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. However, albeit not in all tumors taken singularly, the overall increase in RANBP9 protein in NSCLC masses corresponded to an overall increase in the RANBP9 transcript, and the overall decrease in RANBP10 was also consistently observed at both the protein and mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F; \u003cb\u003eSupplementary Fig.\u0026nbsp;5G-L\u003c/b\u003e). Therefore, we can conclude that while acute changes in RANBP9 and RANBP10 cause changes in protein expression, long-term changes in Scorpin mRNA expression are involved in NSCLC tumorigenesis.\u003c/p\u003e \u003cp\u003eExisting evidence indicates that RANBP9 and RANBP10 originated from the duplication of the ancestral yeast Gid1 gene and that they can partially compensate for each other\u0026rsquo;s absence \u003csup\u003e22 69 70\u003c/sup\u003e. In contrast, there is evidence that these two proteins may have opposite functions \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Hence, the two paralogs should be considered partially antagonistic, similar to other proteins that originated from evolutionary duplications \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. In line with this concept, here, we show that the combined targeting of both Scorpins in NSCLC impairs the formation of functional CTLH complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C; \u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We also showed that, compared with RANBP10 induction, the controlled overexpression of RANBP9 in iA549 cells caused changes in the expression and ubiquitylation of a significantly different number of proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-D, \u003cb\u003eSupplementary Fig.\u0026nbsp;8A-C\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-C, \u003cb\u003eSupplementary Fig.\u0026nbsp;9B-F\u003c/b\u003e). The dynamics observed \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) were consistent with the observations in CPTAC patients, where RANBP9 was overexpressed, whereas RANBP10 was maintained at low levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-F). \u003cem\u003eIn vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e, the expression of either RANBP9 or RANBP10 was positively correlated with that of other members of the CTLH complex, but the two paralogs were negatively correlated with each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B). We identified proteins affected by the overexpression of RANBP9 and/or RANBP10 in both iA549 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B) and CPTAC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-F). Collectively, our data suggest that, in NSCLC, the increase in RANBP9, together with the decrease in RANBP10, works in concert to obtain proteome and ubiquitylome changes that potentially directly and indirectly modulate many other proteins (\u003cb\u003eSupplementary Fig.\u0026nbsp;7A-P\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe lists of proteins whose total amount or ubiquitylation was altered depending on the expression of the two Scorpins in CPTAC NSCLC patients and in iA549 cells indicate that CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e have the theoretical ability to regulate all aspects of cellular life (\u003cb\u003eSupplementary Fig.\u0026nbsp;7A-P\u003c/b\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-D; \u003cb\u003eSupplementary Fig.\u0026nbsp;9B-E\u003c/b\u003e) \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The iA549 proteome and ubiquitylome consistently showed a strong association of \u0026ldquo;RNA metabolism\u0026rdquo; with both RANBP9 and RANBP10 expression, which is in line with the CPTAC data. Both proteins appeared to be involved in the regulation of the transcriptome \u003cem\u003ein vitro\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;7A-P\u003c/b\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-C; \u003cb\u003eSupplementary Fig.\u0026nbsp;9B-E\u003c/b\u003e). This finding is in line with previous evidence indicating that RANBP9 is involved in RNA transcription and splicing \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. However, we also showed that RANBP10 can also modulate the RNAome and that the regulation exerted by Scorpins could be much more pervasive than previously thought (\u003cb\u003eSupplementary Fig.\u0026nbsp;7A-P\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe GO term \u0026ldquo;cell cycle\u0026rdquo; was also strongly associated with a higher BP9/BP10 ratio in the CPTAC LUAD and LUSQ proteomic data (\u003cb\u003eSupplementary Fig.\u0026nbsp;7I-P\u003c/b\u003e). We used an unbiased protein proliferation signature to corroborate this initial finding (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-F\u003cem\u003e)\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Given its elevated expression in NSCLC tumors, RANBP9 is expected to be protumorigenic and proproliferative compared with RANBP10. This assumption is consistent with the fact that RANBP9 enhances signaling through receptor tyrosine kinases such as MET (hepatocyte growth factor receptor), whereas RANBP10 ablates that effect and predominantly expresses RANBP9 in highly proliferative progenitor cells, in contrast with the predominance of RANBP10 in differentiated erythroid cells \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Therefore, we can hypothesize that increased expression of the CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e complex favors the stability of proteins that are associated with cell proliferation in NSCLC \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, since RANBP10 has been reported to promote glioblastoma cell growth and RANBP9 silencing can increase proliferation, we can speculate that these effects are likely cell type dependent.\u003c/p\u003e \u003cp\u003eIn iA549 cells, the preferential association of the proliferation-associated protein signature with increased RANBP9 expression was consistent with the increased BP9/BP10 ratio observed in CPTAC NSCLC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE-G\u003cem\u003e).\u003c/em\u003e However, we found that high levels of RANBP10 caused a significant decrease in the expression of selected proliferation proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-C; \u003cb\u003eSupplementary Fig.\u0026nbsp;8B-C\u003c/b\u003e\u003cem\u003e)\u003c/em\u003e. Moreover, Doxy-treated iBP10 cells exhibited decreased proliferation, which became significant at 48 h, whereas Doxy-treated iBP9 cells were indistinguishable from their noninduced controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH). These results showed that a decreased BP9/BP10 ratio decelerates cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH). We also found that the acute overexpression of RANBP9 or RANBP10 significantly changed the ubiquitylome landscape of A549 cells, which included specific ubiquitylations of proliferation-associated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-B). Therefore, while the decrease in proliferation observed upon RANBP10 overexpression could be explained by the concomitant decrease in RANBP9, the ubiquitylation and decrease in specific proliferation-associated proteins such as MCM5, MCM7, and SMC1A, for example, cannot be easily explained by the decrease in RANBP9.\u003c/p\u003e \u003cp\u003eThe specific RANBP9-associated lysine ubiquitylations that we observed are largely different from those found to be positively associated with RANBP9 expression in HEK293 cells \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. However, in that study, RANBP9 was stably silenced, the expression of RANBP10 was not investigated, and differences can be caused by technical and/or cell type-specific factors \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. An in-depth analysis of the observed lysine ubiquitylations suggests a model in which CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e are likely to act on proteins/targets that are CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e specific, CTLH\u003csup\u003e\u003cb\u003eBP\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e specific, or common between the two. Several ubiquitylations showed positive or negative associations only with RANBP9 or RANBP10 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-B; \u003cb\u003eSupplementary Material\u003c/b\u003e). Interestingly, we found that the ubiquitylation of one or more lysines in some proteins changed when RANBP9 or RANBP10 expression was induced. However, in some cases, the correlation was positive or negative for both Scorpins (concordant), but in other cases, the correlation was opposite (discordant), where the same ubiquitylated lysine was positively correlated with one Scorpin expression but negatively correlated with the other. We also identified cases in which the same protein presented two different ubiquitylated lysine residues, one correlating positively and the other with RANBP9 or RANBP10 negatively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-C; \u003cb\u003eSupplementary Material\u003c/b\u003e). Therefore, we can hypothesize that these proteins whose ubiquitylation changed in more than one residue likely represent proteins on which Scorpins converge to functionally regulate them either in the same or opposite functional direction (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). This model can explain how Scorpins have only partially overlapping functions, sometimes having a concordant final effect, and some other times an opposite outcome. This model is also in agreement with previously reported observations showing that the overexpression of RANBP9 increased the stabilization of several binding partners and not their degradation, as expected for an E3 ligase protein. We hypothesize that some proteins undergo degradation upon RANBP9 expression, whereas others are stabilized, and \u003cem\u003evice versa\u003c/em\u003e. A clear example is the proteins that we found to be differentially ubiquitylated on specific lysines that were also previously reported as both putative targets of the CTLH complex and part of the proliferation signature, such as members of the replisome (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). In this context, our findings indicate that RANBP9 and RANBP10 might finely regulate major complexes involved in DNA replication \u003csup\u003e\u003cspan additionalcitationids=\"CR76 CR77\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. These observations are supported by previous studies in which proteins involved in DNA replication were reported as putative interactors, including our recent study, which demonstrated that RanBP9 interacts with members of the replisome in normal lungs \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur study has several limitations. For example, we cannot conclude that the changes in ubiquitylation observed following altered expression of the CTLH complex are directly caused by changes in the abundance of proteins such as CACYBP, MCM7, and SMC1A. Moreover, we cannot completely exclude the possibility that the effects on protein abundance and ubiquitylation were indirect or that deubiquitylation could be involved \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. More than one-third of the modified iA549 ubiquitylome that we identified in this study upon Scorpin induction has been previously reported as a putative CTLH complex interactant (\u003cb\u003eSupplementary Material\u003c/b\u003e). Therefore, many proteins subject to changes in ubiquitylation are likely not direct targets. In this context, several proteins whose ubiquitylation changes upon RANBP9 or RANBP10 overexpression are E2 or E3 ligases themselves (\u003cb\u003eSupplementary Tables\u003c/b\u003e). These findings suggest that the CTLH complex may act indirectly through other ubiquitylation machineries. Evidence supporting this indirect model already exists in yeast, where it was shown that the GID complex can ubiquitylate rsp5 (Reverse Spt-phenotype 5), which is an E3 ligase of the NEDD4 family that in turn regulates vesicular trafficking \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. If confirmed in mammalian cells, this model would indicate that the CTLH complex has the ability to fine-tune all aspects of cellular life through protein-mediated mechanisms.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe two Scorpins should be considered as one functional unit that acts as a sophisticated rheostat to modulate the enzymatic output of the CTLH complex. Due to the substantial number of ubiquitylation events that changes when their expression changes, RANBP9 and RANBP10 significantly impact NSCLC pathogenesis, including tumor cell proliferation. Since their deletion disrupts the formation of a functional CTLH complex, they should be considered as potential targets for therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNSCLC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNon-Small Cell Lung Cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRANBP9\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRan Binding Protein 9\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRANBP10\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRan Binding Protein 10\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGID8\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlucose-induced degradation Deficient 8\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCTLH\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eC-Terminal to LisH\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCTLH\u003c/b\u003e\u003csup\u003e\u003cb\u003eBP9\u003c/b\u003e\u003c/sup\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCTLH complex built on RANBP9\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCTLH\u003c/b\u003e\u003csup\u003e\u003cb\u003eBP10\u003c/b\u003e\u003c/sup\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCTLH complex built on RANBP10\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSCORPIN\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSpry-COntaing Ran binding ProteIN\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eARMC8\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eArmadillo Repeat Containing protein 8\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGID4\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlucose-Induced degradation Deficient 4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMAEA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMacrophage Erythroblast Attacher E3 ligase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMKLN1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMuskelin 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRMND5A\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRequired in Meiotic Division 5 A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRMND5B\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRequired in Meiotic Division 5 B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eWDR26\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWD Repeat containing protein 26\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eYPEL5\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eYippee Like protein 5\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eKO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKnockout\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDKO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDouble KO\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMEFs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMouse Embryonic Fibroblasts\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDoxy\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edoxycycline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eLUAD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLung Adenocarcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eLUSQ\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLung Squamous carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor tissue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eN\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNormal adjacent tissue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eWB\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWestern blot\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCo-IP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCo-immunoprecipitation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCPTAC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClinical Proteomic Tumor Analysis Consortium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGSEA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Set Enrichment Analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eiA549 DKO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eScorpin DKO A549 Doxy-inducible controls\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eA549 DKO iBP9\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eScorpin DKO A549 with Doxy-inducible RANBP9\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eA549 DKO iBP10\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eScorpin DKO A549 with Doxy-inducible RANBP10\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eiA549 WT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eparental A549 Doxy-inducible controls\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eA549 WT iBP9\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eparental A549 with Doxy-inducible RANBP9\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eA549 WT iBP10\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eparental A549 with Doxy-inducible RANBP10\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot Applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eAll authors approved publication.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work is partially supported by the NIH R03CA259389 to V.C. and the Pelotonia Idea Award GR123713 \u0026ldquo;Establishing the role of CTLH proteins in NSCLC\u0026rdquo; to V.C.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDesigned the research study: A.O., Y.K., A.L., G.F., D.P., J.K., and V.C.; conducted the experiments: A.O., Y.K., L.R., A.T., S.H.S., L.Z., B.F., D.P., and V.C.; provided resources: L.T., R.V., D.P.C., V.C.; acquired the data: A.O., Y.K., L.Z.; analyzed the data: A.O., Y.K., L.Z., M.F., J.K., and V.C.; wrote and edited the manuscript: A.O., Y.K., A.T., L.T., J.A., R.V., D.P.C., A.A., A.L., G.F., M.F., D.P., J.K., and V.C.; project conception: V.C.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are extremely grateful to Laura Monovich of the Biospecimen Shared Resource (Director N. Single) of the Ohio State University and James Comprehensive Cancer Center (OSU-CCC) for the procurement of the 50 NSCLC patient samples through the Total Cancer Care Program. The authors would also like to thank the Flow Cytometry Shared Resource, the Genome Editing Shared Resource, the Genome Sequencing Shared Resource, and the Comparative Pathology and Digital Imaging Shared Resource of OSU-CCC, which were all supported by the P30 CA016058 grant to R. Pollock. This work was also supported by the Pelotonia Institute of Immuno-Oncology (PIIO). The content is solely the responsibility of the authors and does not necessarily represent the official views of the PIIO.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003e\u0026ldquo;Data is provided within the manuscript or supplementary information files\u0026rdquo;. Data is also available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDas S, Suresh B, Kim HH, Ramakrishna S. RanBPM: a potential therapeutic target for modulating diverse physiological disorders. Drug Discov Today (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuffman N, Palmieri D, Coppola V. The CTLH Complex in Cancer Cell Plasticity. \u003cem\u003eJ Oncol\u003c/em\u003e 2019, 4216750 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLampert F et al. The multi-subunit GID/CTLH E3 ubiquitin ligase promotes cell proliferation and targets the transcription factor Hbp1 for degradation. Elife 7 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalemi LM, Maitland MER, McTavish CJ, Schild-Poulter C. Cell signalling pathway regulation by RanBPM: molecular insights and disease implications. 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EMBO Rep. 2022;23:e53835.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lung cancer, non-small cell lung cancer, NSCLC, CTLH complex, GID complex, RANBP9, RANBPM, SCORPIN, ARMC8, GID4, GID8, TWA1, MAEA, MKLN1, RANBP10, RMND5A, RMND5B, WDR26, YPEL5","lastPublishedDoi":"10.21203/rs.3.rs-5707591/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5707591/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRANBP9 and RANBP10, also called Scorpins, are essential components of the C-terminal to LisH (CTLH) complex, an evolutionarily conserved poorly investigated multisubunit E3 ligase. Their role in non-small cell lung cancer (NSCLC) is unknown.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, first we used stable loss-of function and overexpression inducible cell lines to investigate the ability of either RANBP9 or RANBP10 to form their own functional CTLH complex. Then, we probed lysates from patient tumors and analyzed data from publicly available repositories to investigate the expression of RANBP9 and RANBP10. Finally, we used inducible cell lines in vitro to recapitulate the expression observed in patients and investigate the changes of the proteome and the ubiquitylome associated with either RANBP9 or RANBP10 in NSCLC.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHere, we show that the two Scorpins are both expressed in NSCLC cells and that either of them can independently support the formation of the CTLH complex. Short-term experiments revealed that the RANBP9 and RANBP10 proteins balance each other in terms of expression, and the acute overexpression of one or the other results in significant reshaping of the NSCLC cell proteome and ubiquitylome. A higher RANBP9/RANBP10 ratio is associated with greater proliferation in both NSCLC cell lines and patients. Acute increased expression of RANBP10 slows NSCLC cell proliferation and decreases the level of proliferation-associated proteins, including key players in DNA replication.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWe present evidence that the Scorpins act as partial antagonists and work together as one sophisticated rheostat to modulate the CTLH complex ubiquitylation output, which regulates cell proliferation and other key biological processes in NSCLC. These results suggest that the two Scorpins can be considered as targets for the treatment of NSCLC.\u003c/p\u003e","manuscriptTitle":"RANBP9 and RANBP10 cooperate in regulating non-small cell lung cancer proliferation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-03 08:44:05","doi":"10.21203/rs.3.rs-5707591/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-18T08:47:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-18T06:12:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86362429820578373514903819841077674214","date":"2025-01-07T00:02:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-03T10:08:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-03T07:24:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-03T07:23:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Experimental \u0026 Clinical Cancer Research","date":"2024-12-24T17:20:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d83e67f9-f746-40db-b9c0-4ad7df45ea07","owner":[],"postedDate":"February 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T15:58:52+00:00","versionOfRecord":{"articleIdentity":"rs-5707591","link":"https://doi.org/10.1186/s13046-025-03491-8","journal":{"identity":"journal-of-experimental-and-clinical-cancer-research","isVorOnly":false,"title":"Journal of Experimental \u0026 Clinical Cancer Research"},"publishedOn":"2025-08-29 15:56:55","publishedOnDateReadable":"August 29th, 2025"},"versionCreatedAt":"2025-02-03 08:44:05","video":"","vorDoi":"10.1186/s13046-025-03491-8","vorDoiUrl":"https://doi.org/10.1186/s13046-025-03491-8","workflowStages":[]},"version":"v1","identity":"rs-5707591","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5707591","identity":"rs-5707591","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}