the Interaction Proteome of Ribosomal 40S Components PNO1 and NOB1 Using TurboID Proximity Labeling Technology

the Interaction Proteome of Ribosomal 40S Components PNO1 and NOB1 Using TurboID Proximity Labeling Technology | 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 the Interaction Proteome of Ribosomal 40S Components PNO1 and NOB1 Using TurboID Proximity Labeling Technology Xingyuan Xu, Jiefu Zheng, Wenli Chen, Jian-You Liao, Shuang Zhu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4508442/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The ribosome assembly factors PNO1 and NOB1 play crucial roles in the maturation of the 40S ribosomal small subunit. TurboID is an efficient biotin ligase that can biotinylate proteins in proximity to the target protein and is widely used to study complex biological processes within cells.Here, we utilized this technology to investigate the complex interaction network of PNO1 and NOB1 within cells. Results Through immunofluorescence experiments, we found that PNO1 and NOB1 have different localizations within cells. By identifying the proximal proteins biotinylated by PNO1-TurboID and NOB1-TurboID, we discovered 871 proteins interacting with PNO1 and 1044 proteins interacting with NOB1, with 663 proteins overlapping. These results suggest that PNO1 and NOB1 are extensively involved in various biological processes within the cell. Furthermore, we found that PNO1 and NOB1 interact with translation-related proteins EIF4B and EIF4G2, indicating that they may participate in the mRNA translation process. Conclusion The interaction proteome results of PNO1 and NOB1 suggest that ribosome assembly factors are not only involved in ribosome biogenesis but also couple with multiple biological processes within the cell, such as mRNA translation. This provides a foundation for understanding the complex biological processes within the cell. PNO1 NOB1 TurboID Biotin labeling Translation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Ribosomes are molecular machines that translate genetic information from messenger RNA (mRNA) into proteins. They are composed of a 40S small subunit and a 60S large subunit assembled in a highly coordinated manner. Together, these subunits contain four ribosomal RNAs (rRNAs) and approximately 80 ribosomal proteins [ 1 , 2 ] . Ribosome biogenesis(RiBi)is a multi-step process initiated in the nucleolus with transcription, followed by export to the cytoplasm for final assembly, coordinated by over 200 trans-acting protein and RNA factors. This process is tightly controlled by multiple checkpoints and surveillance pathways [ 1 , 3 ] . Disruption of these checkpoints and pathways can lead to hyperactivation of ribosome biogenesis. Some studies [ 4 ] have shown that cancer cells contain a special class of ribosomes (cancer ribosomes) that promote oncogenic translation programs [ 5 ] and regulate cellular functions [ 6 ] . Mutations in ribosomal proteins, rRNA processing, and ribosome assembly factors lead to ribosomopathies, which are associated with an increased risk of malignancy [ 7 ] . Ribosome assembly factors dynamically interact with rRNA during its processing. These factors perform unique functions at different cellular locations, binding rRNA at specific times and sites for processing, and some factors dissociate from rRNA after processing is complete [ 8 ] . For example, in the nucleus, Dim1 [ 9 ] methylates two adenosines in the 3' region of 18S rRNA before separating from them prior to the final export of the mature ribosome to the cytoplasm. Assembly factors such as BYSL [ 10 ] , PNO1(Partner Of NOB1 Homolog) [ 11 ] , and RRP12 [ 12 ] remain bound during nuclear maturation and accompany the 40S precursor to the cytoplasm. Studies have shown that PNO1 and NOB1(NIN1 (RPN12) Binding Protein 1 Homolog) proteins bind together in the 3' region of the 18S rRNA precursor. NOB1 remains in an inactive state, inhibited by its partner PNO1. When PNO1 dissociates from the 18S rRNA, the endonuclease NOB1 is activated to cleave the 3' end of the 18S rRNA, marking the maturation of the 40S ribosomal small subunit [ 12 , 13 ] . Ribosome biogenesis is also considered a fundamental biological process closely related to tumor cell growth and proliferation, and it is one of the most energy-consuming processes [ 14 ] . PNO1 and NOB1 are not only key members of the ribosome assembly factors but also play a crucial role in the maturation of the 40S ribosomal small subunit. Whether they possess additional biological functions beyond their known roles in ribosomal RNA processing remains an area for further investigation. Therefore, we aim to study the interaction proteome of PNO1 and NOB1 in tumor cells and the molecular mechanisms they may regulate. This will enhance our understanding of ribosome assembly factors and provide new perspectives for targeted cancer therapy. Protein interaction networks form the basis of all signaling and regulatory processes within cells, participating in various cellular biological processes at different spatial and temporal levels [ 15 , 16 ] , such as cell cycle regulation [ 17 ] , protein synthesis and secretion, signal transduction, and metabolism. Therefore, studying protein interactions is crucial for understanding molecular regulatory networks. Enzyme-catalyzed proximity labeling [ 18 , 19 ] has become a new method for studying the spatial and interaction characteristics of proteins in living cells. TurboID [ 18 , 20 ] is a novel biotin ligase that converts biotin into a reactive intermediate that covalently labels proximal proteins with biotin. It has faster labeling kinetics and higher labeling yield than any other biotin ligase-related PL method. Its labeling time can be shortened to 30 minutes, and it maintains catalytic activity at lower temperatures. Additionally, TurboID can label proteins in live physiological environments without disrupting the cells [ 21 ] . Here, we constructed PNO1-TurboID and NOB1-TurboID systems to use TurboID technology to identify the interaction proteome of PNO1 and NOB1 within cells. This study aims to reveal their potential regulatory protein interaction networks and provide a theoretical basis for understanding the functions of ribosome assembly factors. Results Construction of the turboID systems To identify the interacting proteome of PNO1 and NOB1 at the molecular level, we constructed fusion expression plasmids for PNO1-TurboID and NOB1-TurboID. The TurboID sequence was fused to the C-terminal of the PNO1 and NOB1 coding sequences (CDS), connected by a flexible linker consisting of 15 amino acids (GGGGS)3, to maximize the identification of proteins interacting with PNO1 and NOB1. The construction of the overexpression plasmids for PNO1-TurboID and NOB1-TurboID is illustrated (Fig. 1A). These plasmids include a tag protein HA, the target gene (PNO1, NOB1) CDS sequence, the flexible linker sequence, the biotin-labeling enzyme TurboID sequence, and the tag protein V5. They also include two different selection markers, AmpR and Puro, where AmpR is used to screen positive clones in E.coli, and Puro is used to select cells transfected with and stably expressing PNO1 and NOB1. The workflow of the TurboID proximity labeling experiment is as follows (Fig. 1B-C). Using the gene PNO1 as an example, the constructed PNO1-TurboID plasmid was successfully transfected into 293T cells. After allowing the plasmid to express in the cells for 48 hours, the cells were incubated in an environment with an appropriate concentration of biotin for a suitable period to ensure complete biotin uptake by the cells. TurboID utilizes exogenously added biotin and intracellular ATP to convert biotin into biotin-AMP, which then biotinylates lysine residues of proximal proteins. Streptavidin magnetic beads were used to capture the biotinylated proteins, which were then subjected to protein affinity purification. The enriched proteins were eluted under denaturing conditions, followed by sample preparation for mass spectrometry analysis. The identification was performed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). PNO1 and NOB1 have different localizations in cells To determine the expression levels of the constructed TurboID overexpression plasmids in cells (Fig. 2A), we transfected 293T cells with PNO1-TurboID and NOB1-TurboID overexpression plasmids. Western Blot analysis using anti-PNO1 and anti-NOB1 antibodies revealed clear bands at molecular weights of 70 kDa and 77 kDa, respectively (with TurboID at 35 kDa, PNO1 at 35 kDa, and NOB1 at 42 kDa). This indicates that the TurboID fusion expression plasmids are stably expressed in cells. As essential members of the ribosomal small subunit 40S, PNO1 and NOB1 are crucial for the processing, assembly, and maturation of the 40S ribosomal subunit. It is known that PNO1 inhibits the cleavage activity of NOB1 on ribosomal RNA, but whether NOB1's functions outside the ribosome in the cell also depend on PNO1 remains unknown. To understand the functions and localization of PNO1 and NOB1 on the 40S pre-ribosomal subunit, we used the PDB database to analyze their spatial distribution in the Cryo-EM structure of the late-stage human 40S pre-ribosomal subunit. The results showed that PNO1 and NOB1 physically interact and are in close spatial proximity (Fig. 2B). These findings suggest that these two proteins may work together in ribosome processing. However, their roles in regulating other cellular functions beyond ribosome maturation are still largely unexplored, leading to the hypothesis that they might also jointly participate in various other biological processes within the cell. Due to the large size of the TurboID sequence, its fusion with target genes may affect the localization and biological function of the target genes in cells. To ensure that our constructed TurboID fusion expression plasmids are successful and do not affect the gene's localization and function within the cells, we transfected HeLa cells with GFP-PNO1 and GFP-NOB1 plasmids. Immunofluorescence experiments revealed that PNO1 is primarily localized in the nucleus and nucleolus, whereas NOB1 is mainly localized in the cytoplasm. Additionally, we transfected cells with HA-PNO1-TurboID and HA-NOB1-TurboID plasmids and used anti-HA antibodies to detect their localization. Immunofluorescence results demonstrated that the localization of the target genes fused with TurboID in cells matched the GFP localization (Fig. 2C-D), indicating that TurboID does not affect the function or proper localization of PNO1 and NOB1 in cells. This ensures the accuracy of subsequent mass spectrometry detection of proteins interacting with PNO1 and NOB1. Moreover, immunofluorescence results revealed that PNO1 and NOB1 have different localizations, with PNO1 in the nucleus and NOB1 in the cytoplasm, yet they exhibit physical interactions in spatial structure. This suggests that, besides participating in ribosome biogenesis, PNO1 and NOB1 may have additional functions. Therefore, we also utilized TurboID technology to explore the additional biological functions of these two proteins. Determination of biotin concentration and incubation time for the turboID system Based on the results shown above, to further confirm the interaction between PNO1 and NOB1, we performed co-immunoprecipitation experiments (Fig. 3A-B), which indicated that PNO1 and NOB1 interact with each other. Before conducting the formal biotin proximity labeling experiments, we first needed to determine the optimal incubation concentration and time for the TurboID system to biotinylate proximal proteins.By setting incubation times of 0, 15, 30, 60, 120, and 240 minutes, we found that, with a constant biotin concentration, the number of proteins labeled by TurboID increased with longer biotin incubation times. Compared to the control group, TurboID could label proximal proteins after 60 minutes of biotin incubation. To prevent false positives caused by excessive biotin labeling due to prolonged incubation, we chose a moderate incubation time of 60 minutes (Fig. 3C). Therefore, we selected 60 minutes as the optimal labeling time for subsequent experiments. By setting biotin incubation concentrations of 0, 50, 100, 150, 200, and 250 µM, we observed that the number of proteins labeled by TurboID did not increase with higher biotin concentrations. This may be because the TurboID system is limited, and excess exogenous biotin does not enhance TurboID activity. Compared to the control group, adding 50 µM of biotin allowed TurboID to label proximal proteins. To avoid non-specific binding caused by excess free biotin competing with streptavidin magnetic beads, which would reduce the specificity of the biotin-labeled proteins isolated by magnetic separation, we decided to use a biotin concentration of 50 µM for subsequent experiments (Fig. 3D).Through preliminary experiments, we confirmed the correct localization of PNO1-TurboID and NOB1-TurboID plasmids in cells, and determined the optimal biotin incubation concentration to be 50 µM and the optimal incubation time to be 60 minutes. Capture and identification of biotinylated proteins To demonstrate that the amount of endogenous biotin in cells is very low and that TurboID has low activity without the addition of a large amount of exogenous biotin, we set up a control group without added biotin. We performed Western blot analysis on the cell lysates. The results showed that, with equal sample loading, the control group without exogenous biotin had very few proteins labeled by TurboID, whereas the group with additional exogenous biotin had a large number of proximal proteins labeled by biotin (Fig. 4A).Using streptavidin magnetic beads, we enriched the biotinylated proteins and identified them through silver staining. The results indicated that the proximal proteins labeled by TurboID, PNO1-TurboID, and NOB1-TurboID in the presence of exogenous biotin were successfully enriched (Fig. 4B). We identified the enriched proximal proteins from these three groups using mass spectrometry, with the TurboID group serving as the control group. Each group was repeated three times. We processed the raw mass spectrometry data through database searches, initially screening for proteins with Unique Peptide ≥ 2 to obtain a preliminary protein set. Subsequently, we compared the overexpression groups to the control group (overexpression group/control group), conducting a secondary screening for proteins with Unique Peptide ≥ 2 to obtain the final protein set. These proteins were then subjected to enrichment analysis using KEGG, Reactome, and GO (Fig. 4C). Functional analysis of PNO1 and NOB1 protein interaction groups After identifying the proteins using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), we filtered the identified proteins based on criteria such as Unique Peptide, PSMs, protein abundance, and biotin modification to obtain a final protein set. We identified 2054 proteins interacting with PNO1 and 2363 proteins interacting with NOB1, with 1437 proteins common to both. KEGG enrichment analysis of proteins enriched by PNO1-TurboID and NOB1-TurboID revealed that the proteins were mainly involved in cell cycle, endocytosis, spliceosome, and ribosome functions (Fig. 5A-B). Reactome enrichment analysis showed that the proteins enriched by PNO1 and NOB1 were primarily involved in GTPase signaling, RNA metabolism, cell cycle, translation, and ribosomal RNA processing (Fig. 5C-D). Biological process analysis indicated that PNO1 and NOB1 were mainly involved in RNA metabolic processes, translation, and the assembly of protein-containing complexes. The enriched proteins were primarily localized in the nucleolus, microtubule cytoskeleton, and centrosome. The molecular functions of these proteins included adenosine nucleotide binding, mRNA binding, rRNA binding, and snRNA binding (Fig. 5E-F). Functional analysis of the proteins enriched by PNO1 and NOB1 revealed significant functional similarities between the two. Despite their different localizations, PNO1 and NOB1 shared many common regulatory functions, such as mRNA translation, which are crucial for cell growth. Protein interaction network of PNO1 and NOB1 The protein-protein interaction network is fundamental to understanding the intricate interactions and functional relationships between proteins within a cell. These networks illustrate how proteins interact physically and functionally, providing valuable insights into cellular processes and pathways. By mapping these interactions, researchers can elucidate the roles of proteins in various biological functions, such as signal transduction, metabolic pathways, and structural assembly.In this study, we constructed a protein-protein interaction network for the proteins interacting with both PNO1 and NOB1 using the CPDB database. The results revealed that PNO1 and NOB1 are primarily enriched in the pre-rRNA complex, mRNA translation complex, and IGF2BP1 complex(Fig. 6A). PNO1 and NOB1 may be involved in mRNA translation regulation mRNA translation is a crucial biochemical process within cells that converts genetic information from mRNA into proteins, which are the primary executors of cellular functions. The translation process itself is tightly regulated. By controlling the translation of specific mRNAs, cells can rapidly respond to endogenous and exogenous signals, enabling physiological changes and maintaining homeostasis. Through the biological functional analysis of mass spectrometry data from PNO1-TurboID and NOB1-TurboID, we surprisingly discovered that PNO1 and NOB1 share significant functional similarities and may be involved in the process of mRNA translation. Based on our mass spectrometry results, we identified translation-related proteins such as EIF4B and EIF4G2 (Fig. 7A-B). Analyzing the peptide segments of EIF4B and EIF4G2 through mass spectrometry database searches, we obtained the M/Z spectra for these proteins (Fig. 7C-D).To validate our mass spectrometry results, we performed co-immunoprecipitation experiments, which demonstrated that PNO1 and NOB1 interact with EIF4B and EIF4G2 (Fig. 7E-F). However, the mechanism by which PNO1 and NOB1 regulate the translation process remains unknown, and we look forward to further experimental investigations to explore this process. Discussion Ribosome assembly factors play a crucial role in the biosynthesis and function of ribosomes. In this study, we observed that ribosome assembly factors PNO1 and NOB1 have different localizations within cells through immunofluorescence experiments [ 1 ] . This piqued our interest, and to explore their biological functions within cells, we employed the TurboID proximity labeling technology to identify proteins interacting with PNO1 and NOB1 and to uncover the functional networks they are involved in. The identified proteins interacting with PNO1 and NOB1 were subjected to KEGG, Reactome, and GO gene annotation and functional analyses. The results indicated that these proteins were mainly enriched in functions related to the cell cycle, spliceosome, ribosome, RNA metabolic processes, translation, and assembly of protein-containing complexes. A total of 1437 proteins were found to interact with both PNO1 and NOB1, 617 proteins interacted exclusively with PNO1, and 926 proteins interacted exclusively with NOB1. We speculate that PNO1 and NOB1 might function together within cells.Moreover, our analysis of the PNO1 and NOB1 interaction protein network revealed that, in addition to their roles in ribosome processing and maturation, PNO1 and NOB1 are also involved in mRNA translation, mRNA splicing, RNA polymerase I transcription, and other processes critical for cell growth and proliferation. Ribosome assembly is a highly complex and tightly regulated process that requires the involvement of multiple assembly factors [ 22 ] . These assembly factors perform several crucial functions in ribosome biosynthesis, including rRNA modification and processing [ 3 ] , ribosomal protein binding, subunit folding and stabilization [ 23 ] , subunit transport, and quality control and error correction [ 24 ] . These functions are essential to ensure the correct assembly and function of ribosomes. Ribosome assembly factors not only play key roles in ribosome biosynthesis but also contribute to RNA metabolism [ 25 ] , protein translation regulation [ 26 ] , stress responses [ 27 ] , protein quality control, and cell differentiation [ 28 ] . These non-traditional roles further emphasize the importance and diversity of ribosome assembly factors in cellular biology. Therefore, investigating the protein interaction networks and biological functions of ribosome assembly factors within cells is of significant importance.TurboID is an advanced biotinylation technology used for efficient and specific labeling of protein-protein interactions in living cells [ 20 ] . Compared to traditional labeling methods, TurboID offers higher labeling efficiency, lower cytotoxicity, and higher specificity [ 29 ] . These advantages make TurboID a valuable tool for studying protein-protein interactions and have led to its widespread application [ 30 – 33 ] . In summary, our study reveals, through the use of TurboID technology, that ribosome assembly factors PNO1 and NOB1 are not only involved in ribosome processing but also participate in mRNA translation and other functions. Materials and Methods Cell Culture HEK293T and HeLa cells were purchased from the American Type Culture Collection (ATCC, Manassas, USA). All cells were grown in high-glucose DMEM supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin solution. The cells were cultured in a 5% CO2 incubator at 37°C. Plasmid Construction Human cDNA was used as a template for PCR amplification to obtain the PNO1 and NOB1 sequences. The TurboID sequence was sourced from the plasmid pLV3-CMV-EGFP-TurboID-NLS-3x FLAG-Puro purchased from Miaoling Bio. PNO1-TurboID and NOB1-TurboID sequences were amplified via fusion PCR. The amplified gene fragments and PCDH CMV plasmid were linearized using the restriction endonucleases XbaI and BamHI, and then ligated overnight at 16°C using T4 DNA ligase. The ligation products were transformed into Stbl3 competent cells and plated on AMP plates to select positive clones. Single colonies were sent to Guangzhou Aiji Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmids were named pCDH-CMV-HA-PNO1-Linker-V5-TurboID (abbreviated as PNO1-TurboID plasmid) and pCDH-CMV-HA-NOB1-Linker-V5-TurboID (abbreviated as NOB1-TurboID plasmid). All vectors constructed in this study were validated by Sanger sequencing before use. Antibody Anti-Biotin Antibody(#7075), Anti-HA-Tag Rabbit Antibody #3724(C29F4) were purchased from Cell Signaling Technology. Anti-PNO1 Polyclonal Antibody(# PA5-70579)were purchased from Invitrogen. Anti-NOB1 Monoclonal antibody(#66048-1-Ig),Anti-GAPDH Monoclonal antibody(#60004-1-Ig), Anti-Alpha Tubulin Monoclonal antibody(#11221-1-AP) were purchased from Proteintech. Anti-FLAG Monoclonal Antibody(#F1804)were purchased from Sigma. Western blot and Co-IP Briefly, HEK293T cells were successfully transfected with PKO187-SFB, SFB-PNO1, and SFB-NOB1 plasmids. After 48 hours of expression, cell proteins were collected and lysed in NETN buffer containing a protease inhibitor cocktail (Bimake, China) and benzonase nuclease (Merck Millipore) at 4°C for 30 minutes. The lysates were then centrifuged, and the supernatants were separated by SDS-PAGE and analyzed with specific antibodies.For Co-IP of exogenously tagged proteins, cell lysates were incubated with S-beads (Merck Millipore) at 4°C with rotation overnight. The next day, the beads were centrifuged and washed five times with NETN buffer. The precipitated proteins were then analyzed by Western blotting with the indicated antibodies. Immunofluorescence Assay HeLa cells transfected with the plasmids were cultured on glass-bottom dishes. The cells were then fixed with 4% paraformaldehyde at room temperature for 30 minutes. After fixation, the cells were incubated with 0.5% Triton X-100 for 5 minutes and then blocked with blocking solution (Beyotime) for 30 minutes. Primary antibody staining was performed overnight at 4°C, followed by secondary antibody incubation and DAPI staining. Images were acquired at room temperature using a Leica SP-8 STED 3X microscope. Biotin Labeling Time Screening HEK293T cells transfected with PNO1-TurboID and NOB1-TurboID were cultured in 6-well plates. When the cell density reached over 80%, the original medium was replaced with medium containing biotin (50 µmol/L). The cells were incubated and evaluated at six different time points (0 min, 15 min, 30 min, 1 h, 2 h, and 4 h). At each time point, the cells were collected, and the cell lysates were subjected to SDS-PAGE. Streptavidin-HRP was used as the antibody for detection. Preparation of Biotinylated Protein Samples Cells from each 100mm culture dish were collected into 1.5 mL EP tubes, and 1 mL of RIPA buffer was added to resuspend the cells. The cells were then lysed on ice for 15 minutes and centrifuged at 12,000 rpm for 10 minutes. The supernatant was transferred to a clean 1.5 mL EP tube.Streptavidin magnetic beads were washed with 1 mL of RIPA buffer five times until the liquid became clear. The prepared protein lysates were added to the washed beads, sealed with parafilm, and incubated overnight at 4°C on a rotator at 360 degrees.After overnight incubation, the supernatant was discarded, and the beads were washed with 1 mL of RIPA buffer for approximately 1.5 minutes. This process was repeated once. The beads were then washed once with 1 mL of KCl (1 mol/L), followed by three washes with Buffer 1 (prepared by mixing 67 mL of NaCl (3 mol/L), 1 mL of Tris-HCl (pH 7.4, 1 mol/L), and 32 mL of H2O in 100 mL of Buffer 1). The beads were washed once with Na2CO3 (0.1 mol/L) and once with 10% SDS for 2 minutes. Finally, after the last wash with RIPA buffer, one-third of the sample was mixed with 5X protein buffer and denatured for separation by SDS-PAGE and identification by silver staining. The remaining sample was washed twice with PBS, and after removing the supernatant, the beads were stored at -20°C. Data availability The mass spectrometry proteomics data generated in this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier IPX0008946000 (iProX- integrated Proteome resources). All data generated or analyzed during this study are included in this article and its Supplementary Information files. Source data are provided with this paper. Declarations Acknowledgements We thank Professor Kaishun Hu from Sun Yat-sen Memorial Hospital, Sun Yat-sen University, for providing the PKO187-SFB and PKO187-GFP plasmids and for his technical support. Mass spectrometry identification and analysis were conducted by the Bioinformatics and Omics Center at Sun Yat-sen Memorial Hospital, Sun Yat-sen University. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Funding This· study· was· supported by· grants from·the Natural Science Foundation of China (82273033, 82072924); Guangdong Science and Technology Department (2022B1515020100); Guangzhou Bureau of Science and Information Technology (202201020575). Author Contribution Xingyuan Xu wrote the main manuscript text, Xingyuan Xu,Jiefu Zheng and Wenli Chen jointly organized the images and mass spectrometry data. All authors reviewed the manuscript. Competing interests The authors confirm that there are no conflicts of interest. References AMEISMEIER M, ZEMP I, VAN DEN HEUVEL J et al. Structural basis for the final steps of human 40S ribosome maturation [J]. 2020, 587(7835): 683–7. KATHERINE E B, MARKUS T B J E. J. Uncovering the assembly pathway of human ribosomes and its emerging links to disease [J]. 2019, 38(13). ANTHONY K H, CéLIA P-C, MARIE-FRANçOISE OD et al. An overview of pre-ribosomal RNA processing in eukaryotes [J]. 2014, 6(2). ELHAMAMSY A, METGE B, ALSHEIKH H et al. Ribosome Biogenesis: A Central Player in Cancer Metastasis and Therapeutic Resistance [J]. 2022, 82(13): 2344–53. UNIACKE J, PERERA J, LACHANCE G et al. Cancer cells exploit eIF4E2-directed synthesis of hypoxia response proteins to drive tumor progression [J]. 2014, 74(5): 1379–89. BARNA M, PUSIC A, ZOLLO O et al. Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency [J]. 2008, 456(7224): 971–5. PENZO M, MONTANARO L, TRERé D et al. Ribosome Biogenesis-Cancer Connection [J]. 2019, 8(1). DöRNER K, RUGGERI C, ZEMP I et al. Ribosome biogenesis factors-from names to functions [J]. 2023, 42(7): e112699. WYLER E, ZIMMERMANN M. WIDMANN B, Tandem affinity purification combined with inducible shRNA expression as a tool to study the maturation of macromolecular assemblies [J]. 2011, 17(1): 189–200. CHAKER-MARGOT M, BARANDUN J, HUNZIKER M et al. Architecture of the yeast small subunit processome [J]. 2017, 355(6321). CHENG J, KELLNER N, BERNINGHAUSEN O et al. 3.2-Å-resolution structure of the 90S preribosome before A1 pre-rRNA cleavage [J]. 2017, 24(11): 954–64. JINGDONG MICHAELA. C, OTTO B, Visualizing late states of human 40S ribosomal subunit maturation [J]. 2018, 558(7709). ALLISON CL, KATRIN K J P N A S U S. A. Nob1 binds the single-stranded cleavage site D at the 3'-end of 18S rRNA with its PIN domain [J]. 2009, 106(34). EMMA T, SéBASTIEN F-C, ED H J J C. S. Eukaryotic ribosome biogenesis at a glance [J]. 2013, 126(0). MARTINO E, CHIARUGI S, MARGHERITI F et al. Mapping, Structure and Modulation of PPI [J]. 2021, 9(718405. SPEER S, ZHENG W, JIANG X et al. The intracellular environment affects protein-protein interactions [J]. 2021, 118(11). ZHOU L, ZHANG Q, DENG H et al. The SNHG1-Centered ceRNA Network Regulates Cell Cycle and Is a Potential Prognostic Biomarker for Hepatocellular Carcinoma [J]. 2022, 258(4): 265–76. KIM D, ROUX K J T I C B. Filling the Void: Proximity-Based Labeling of Proteins in Living Cells [J]. 2016, 26(11): 804–17. JOHNSON B, CHAFIN L. FARKAS D, MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics [J]. 2022, 21(7): 100256. BRANON T, BOSCH J, SANCHEZ A et al. Efficient proximity labeling in living cells and organisms with TurboID [J]. 2018, 36(9): 880–7. KANZLER C, DONOHUE M, DOWDLE M et al. TurboID functions as an efficient biotin ligase for BioID applications in Xenopus embryos [J]. 2022, 492(133–8. THOMSON E, FERREIRA-CERCA S, HURT E J J O C. S. Eukaryotic ribosome biogenesis at a glance [J]. 2013, 126(4815–21. DIETER K, ED H, JOCHEN B J B. B A. Driving ribosome assembly [J]. 2009, 1803(6). JESUS D L C, KATRIN K, JOHN L JR W J. A R B. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo [J]. 2015, 84(0). LIONEL T, CHRISTIANE Z, JEAN-LOUIS L et al. The complexity of human ribosome biogenesis revealed by systematic nucleolar screening of Pre-rRNA processing factors [J]. 2013, 51(4). KAREN J, CRISTIAN B DORIML et al. rRNA pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells [J]. 2011, 44(4). SéVERINE B, BELINDA J W, SASKIA H et al. The nucleolus under stress [J]. 2010, 40(2). JONATHAN R W, KERRI B M J. M C. How common are extraribosomal functions of ribosomal proteins? [J]. 2009, 34(1). KELVIN F C, TESS C B, NAMRATA D U et al. Proximity labeling in mammalian cells with TurboID and split-TurboID [J]. 2020, 15(12). ANDREA M, SHOU-LING X, TESS C B et al. Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID [J]. 2019, 8(0). YONGLIANG Z, YUANYUAN L, XINXIN Y et al. TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks [J]. 2020, 159). KAIXIN Z, YINYIN L, TENGBO H et al. Potential application of TurboID-based proximity labeling in studying the protein interaction network in plant response to abiotic stress [J]. 2022, 13(0). YANTING S, YUANYUAN G, JIEYU G et al. Study of FOXO1-interacting proteins using TurboID-based proximity labeling technology [J]. 2023, 24(1). Additional Declarations No competing interests reported. Supplementary Files PNO1TurboIDandNOB1TurboIDPCRPrimer.xlsx WBoriginalimage1.pdf WBoriginalimage2.pdf WBoriginalimage3.pdf Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4508442","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":311425281,"identity":"0403568e-90e0-426f-a5ff-2368f4814040","order_by":0,"name":"Xingyuan Xu","email":"","orcid":"","institution":"Guangdong Pharmaceutical University，","correspondingAuthor":false,"prefix":"","firstName":"Xingyuan","middleName":"","lastName":"Xu","suffix":""},{"id":311425282,"identity":"eb5fb236-c51d-4418-a724-170429322b2b","order_by":1,"name":"Jiefu Zheng","email":"","orcid":"","institution":"Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Jiefu","middleName":"","lastName":"Zheng","suffix":""},{"id":311425283,"identity":"36946e63-3704-40c4-90a9-ac14c9b34372","order_by":2,"name":"Wenli Chen","email":"","orcid":"","institution":"Guangdong Pharmaceutical University，","correspondingAuthor":false,"prefix":"","firstName":"Wenli","middleName":"","lastName":"Chen","suffix":""},{"id":311425284,"identity":"faa699de-cb6b-4dbe-a435-859d2ea0ee39","order_by":3,"name":"Jian-You Liao","email":"","orcid":"","institution":"Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Jian-You","middleName":"","lastName":"Liao","suffix":""},{"id":311425285,"identity":"df806078-46a5-4c60-b131-0189aa64c54e","order_by":4,"name":"Shuang Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACNvnDBx9+/GdT30+0Fj4JtmRjCbY0xpkNxGqRk+BRk+BhO8y44QDRDpPuYTaQ4GFmNj6evIHhR8U2IrTInD34oECCjc3szLMCxp4zt4nQwpCXbCBhwMNjdiPHgJmxjSgtOWYSPAkSEsYziNYiAdJywMDAQIJoLTzHko0lGxISJIB+OUiUX+Tbm4FR2fA/gb89eeODHxVEaEECCQYHSFIP1kKqjlEwCkbBKBghAACGRjkHEwMetQAAAABJRU5ErkJggg==","orcid":"","institution":"Guangdong Pharmaceutical University，","correspondingAuthor":true,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2024-05-31 10:58:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4508442/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4508442/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58575126,"identity":"04d7f389-7e00-4a4f-b569-36cdf34dd87e","added_by":"auto","created_at":"2024-06-18 11:57:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1803683,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of the turboID system\u003c/p\u003e\n\u003cp\u003e(A)The main components of the PNO1-TurboID and NOB1-TurboID plasmids are shown, including the CDS sequences of PNO1 and NOB1 proteins, the flexible linker sequence (GGGGS)3, the TurboID sequence, and HA and V5 tag sequences.(B)The PNO1-TurboID and NOB1-TurboID sequences were obtained by fusion PCR amplification and integrated into the PCDH CMV plasmid using restriction enzyme digestion and ligation. The plasmids were transfected into HEK293T cells. After successful expression of the plasmids in the cells, exogenous biotin was added to start biotin labeling of proximal proteins.(C)Taking PNO1 as an example, the PNO1-TurboID plasmid was successfully transfected into 293T cells. After 48 hours of expression, exogenous biotin was added and incubated at 37°C for an appropriate time. TurboID began biotin labeling lysine residues of proteins proximal to PNO1. Streptavidin magnetic beads were used to enrich the biotin-labeled proteins. After affinity purification to remove non-bound proteins, the enriched proteins were detected by mass spectrometry.\u003c/p\u003e","description":"","filename":"Figure1.ConstructionoftheTurboIDSystem.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4508442/v1/8755b7e16c69fa29b202a618.jpg"},{"id":58575647,"identity":"96370fb9-51dc-41a2-8f74-0fb57c306f54","added_by":"auto","created_at":"2024-06-18 12:05:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1391938,"visible":true,"origin":"","legend":"\u003cp\u003ePNO1 and NOB1 have different localizations in cells\u003c/p\u003e\n\u003cp\u003e(A)The PNO1-TurboID and NOB1-TurboID plasmids were transfected into HEK293T cells. Protein levels were detected by Western blot analysis using specific antibodies.(B)The distribution and interaction of PNO1 and NOB1 in the Cryo-EM structure of a late human pre-40S ribosomal subunit were analyzed using the PDB online database.(C)GFP-PNO1 and GFP-NOB1 plasmids were successfully transfected into HeLa cells. After 48 hours of expression, the localization of PNO1 and NOB1 proteins in the cells was detected by immunofluorescence.(D)Using the same experimental method, HA-PNO1-TurboID and HA-NOB1-TurboID plasmids were successfully transfected into HeLa cells. Immunofluorescence was used to observe the localization of the fusion proteins in the cells. The results showed that the fusion of PNO1 and NOB1 with TurboID did not affect the localization of the genes themselves.\u003c/p\u003e","description":"","filename":"Figure2.PNO1andNOB1HaveDifferentLocalizationsinCells.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4508442/v1/f0fa81e8987cf95643f8920c.jpg"},{"id":58575129,"identity":"175f75d7-9b29-4dc8-aed5-5b7cb7f6a199","added_by":"auto","created_at":"2024-06-18 11:57:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":995764,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of biotin concentration and incubation time for the turboID system\u003c/p\u003e\n\u003cp\u003e(A-B)HeLa cells were transfected with SFB-PNO1 and SFB-NOB1 plasmids for 48 hours. Cell lysates were collected and subjected to Co-IP using S beads, followed by Western blot analysis with specific antibodies. The results indicated that PNO1 and NOB1 interact with each other.(C)Determination of the optimal incubation time for TurboID biotin labeling of proximal proteins. The results showed that 60 minutes was sufficient for significant biotin labeling of proximal proteins by TurboID.(D)Determination of the optimal biotin concentration for TurboID biotin labeling of proximal proteins. The results showed that 50 µM biotin was sufficient for significant biotin labeling of proximal proteins by TurboID.\u003c/p\u003e","description":"","filename":"Figure3.ScreeningofBiotinConcentrationandIncubationTimefortheTurboIDSystem.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4508442/v1/09a2b8b040b3cb13fe4ca3c2.jpg"},{"id":58575127,"identity":"3823d14c-93c4-40a4-bbce-7fbe15fd21e1","added_by":"auto","created_at":"2024-06-18 11:57:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3030917,"visible":true,"origin":"","legend":"\u003cp\u003eCapture and identification of biotinylated proteins\u003c/p\u003e\n\u003cp\u003e(A)To exclude the interference of endogenous biotin, samples were divided into groups without exogenous biotin and with exogenous biotin.(B)Western blot and IP experiments were conducted on the Wild Type, TurboID, PNO1-TurboID, and NOB1-TurboID groups, with and without the addition of exogenous biotin. Anti-Biotin antibodies were used to detect biotinylated proteins in whole cell lysates and proteins enriched by streptavidin magnetic beads. The results showed a significant amount of proteins biotinylated by TurboID. Ponceau S staining of the PVDF membrane was used to ensure equal protein loading in the whole cell lysates.(C) Silver staining of proteins enriched by streptavidin magnetic beads demonstrated that the addition of exogenous biotin enables the TurboID system to rapidly biotinylate proteins proximal to the target gene.\u003c/p\u003e","description":"","filename":"Figure4.EnrichmentandIdentificationofBiotinylatedProteins.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4508442/v1/e6d08492bee4e4bfe07846b6.jpg"},{"id":58575130,"identity":"0dec9763-d8dc-4263-b1bd-3af934f79418","added_by":"auto","created_at":"2024-06-18 11:57:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3851247,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional analysis of PNO1 and NOB1 protein interaction groups\u003c/p\u003e\n\u003cp\u003e(A)KEGG pathway annotation of proteins interacting with PNO1. The proteins were mainly enriched in functions related to the cell cycle, endocytosis, spliceosome, and ribosome.(B)KEGG pathway annotation of proteins interacting with NOB1. The proteins were mainly enriched in functions related to the spliceosome, cell cycle, endocytosis, and ribosome.(C)Reactome pathway annotation of proteins interacting with PNO1. The proteins were mainly enriched in functions related to Rho GTPase signaling, RNA metabolism, cell cycle, translation, and rRNA processing. (D) Reactome pathway annotation of proteins interacting with NOB1. The proteins were mainly enriched in functions related to Rho GTPase signaling, RNA metabolism, cell cycle, translation, and rRNA processing. (E) GO analysis of proteins interacting with PNO1. Biological process results indicated that the proteins were mainly involved in RNA metabolic processing, translation, and cellular protein complex assembly. Cellular component results showed that the proteins were primarily enriched in the nucleolus, microtubule cytoskeleton, and centrosome. Molecular function results indicated that the proteins were mainly involved in adenosine nucleotide binding, pyrophosphatase activity, and mRNA binding.(F)GO analysis of proteins interacting with NOB1. Biological process results indicated that the proteins were mainly involved in RNA metabolic processing, translation, and peptide biosynthesis. Cellular component results showed that the proteins were primarily enriched in the microtubule cytoskeleton, nucleolus, and centrosome. Molecular function results indicated that the proteins were mainly involved in adenosine nucleotide binding, pyrophosphatase activity, and mRNA binding.\u003c/p\u003e","description":"","filename":"Figure5.FunctionalAnalysisofPNO1andNOB1ProteinInteractionGroups.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4508442/v1/44b96a2748e8f794f28d72c5.jpg"},{"id":58575649,"identity":"85dc68b5-50e4-4790-a3d0-a579660d6d4d","added_by":"auto","created_at":"2024-06-18 12:05:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1852619,"visible":true,"origin":"","legend":"\u003cp\u003eProtein interaction network of PNO1 and NOB1\u003c/p\u003e\n\u003cp\u003e(A) The protein sets interacting with PNO1 and NOB1 were overlapped, and the overlapping proteins were subjected to complex analysis using the DAVID database to construct a PPI network. The analysis revealed that PNO1 and NOB1 commonly interact with complexes such as the Nop56-associated pre-rRNA complex, the spliceosome complex, the translation complex, and the IGF2BP1 complex.\u003c/p\u003e","description":"","filename":"Figure6.ProteinInteractionNetworkofPNO1andNOB1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4508442/v1/7810dee18ff9d0505ae1f68e.jpg"},{"id":58575132,"identity":"5853baa6-72e1-4778-8a60-95e5d45f737d","added_by":"auto","created_at":"2024-06-18 11:57:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2492562,"visible":true,"origin":"","legend":"\u003cp\u003ePNO1 and NOB1 may be involved in mRNA translation regulation\u003c/p\u003e\n\u003cp\u003e(A)Intersection analysis of proteins interacting with PNO1 and NOB1 identified 1437 common interacting proteins. Among these, EIF4B and EIF4G2 were jointly identified by mass spectrometry.(B)In the mass spectrometry results, EIF4B and EIF4G2 showed high unique peptide counts, coverage, and PSM values. These two proteins are primarily involved in the regulation of mRNA translation.(C-D)The mass spectrometry search results for PNO1 and NOB1 indicated that EIF4B and EIF4G2 were biotinylated and had high protein abundance and specific peptides, suggesting potential interactions.(E) HeLa cells were transfected with SFB-PNO1 and SFB-NOB1 plasmids for 48 hours. Cell lysates were collected and subjected to Co-IP using S beads, followed by Western blot analysis with specific antibodies. The results showed that PNO1 and NOB1 interact with EIF4B and EIF4G2.\u003c/p\u003e","description":"","filename":"Figure7.PNO1andNOB1MayBeInvolvedinmRNATranslationRegulation.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4508442/v1/961ef2d2bb12e851bd09211c.jpg"},{"id":59241041,"identity":"ce356023-e5e7-439d-ad97-67e95ffc5d42","added_by":"auto","created_at":"2024-06-28 05:23:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13083375,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4508442/v1/774071fe-21f5-432a-af5e-f64b13cc8921.pdf"},{"id":58575648,"identity":"7a7d84dd-77f5-46f8-b36e-7f5d33e7df97","added_by":"auto","created_at":"2024-06-18 12:05:11","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":18611,"visible":true,"origin":"","legend":"","description":"","filename":"PNO1TurboIDandNOB1TurboIDPCRPrimer.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4508442/v1/767b463f138108c43f00c4fb.xlsx"},{"id":58575136,"identity":"490a1934-0c67-4076-833f-e94644e31ea3","added_by":"auto","created_at":"2024-06-18 11:57:12","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":3472582,"visible":true,"origin":"","legend":"","description":"","filename":"WBoriginalimage1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4508442/v1/6de76fcdd0aa98c8f1b64b94.pdf"},{"id":58575134,"identity":"944056ed-3a04-4ccd-8869-6f54d9b9329f","added_by":"auto","created_at":"2024-06-18 11:57:11","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":7497836,"visible":true,"origin":"","legend":"","description":"","filename":"WBoriginalimage2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4508442/v1/91966bf875ca90dca4a2b942.pdf"},{"id":58575135,"identity":"c9d24955-9c11-47de-9ae1-c167fcd7d299","added_by":"auto","created_at":"2024-06-18 11:57:12","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":3996169,"visible":true,"origin":"","legend":"","description":"","filename":"WBoriginalimage3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4508442/v1/19c77bdf683fadb69fa412f0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"the Interaction Proteome of Ribosomal 40S Components PNO1 and NOB1 Using TurboID Proximity Labeling Technology","fulltext":[{"header":"Background","content":"\u003cp\u003eRibosomes are molecular machines that translate genetic information from messenger RNA (mRNA) into proteins. They are composed of a 40S small subunit and a 60S large subunit assembled in a highly coordinated manner. Together, these subunits contain four ribosomal RNAs (rRNAs) and approximately 80 ribosomal proteins \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Ribosome biogenesis(RiBi)is a multi-step process initiated in the nucleolus with transcription, followed by export to the cytoplasm for final assembly, coordinated by over 200 trans-acting protein and RNA factors. This process is tightly controlled by multiple checkpoints and surveillance pathways \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Disruption of these checkpoints and pathways can lead to hyperactivation of ribosome biogenesis. Some studies\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e have shown that cancer cells contain a special class of ribosomes (cancer ribosomes) that promote oncogenic translation programs\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e and regulate cellular functions\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Mutations in ribosomal proteins, rRNA processing, and ribosome assembly factors lead to ribosomopathies, which are associated with an increased risk of malignancy\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRibosome assembly factors dynamically interact with rRNA during its processing. These factors perform unique functions at different cellular locations, binding rRNA at specific times and sites for processing, and some factors dissociate from rRNA after processing is complete\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. For example, in the nucleus, Dim1\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e methylates two adenosines in the 3' region of 18S rRNA before separating from them prior to the final export of the mature ribosome to the cytoplasm. Assembly factors such as BYSL\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, PNO1(Partner Of NOB1 Homolog)\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, and RRP12\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e remain bound during nuclear maturation and accompany the 40S precursor to the cytoplasm. Studies have shown that PNO1 and NOB1(NIN1 (RPN12) Binding Protein 1 Homolog) proteins bind together in the 3' region of the 18S rRNA precursor. NOB1 remains in an inactive state, inhibited by its partner PNO1. When PNO1 dissociates from the 18S rRNA, the endonuclease NOB1 is activated to cleave the 3' end of the 18S rRNA, marking the maturation of the 40S ribosomal small subunit\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRibosome biogenesis is also considered a fundamental biological process closely related to tumor cell growth and proliferation, and it is one of the most energy-consuming processes\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. PNO1 and NOB1 are not only key members of the ribosome assembly factors but also play a crucial role in the maturation of the 40S ribosomal small subunit. Whether they possess additional biological functions beyond their known roles in ribosomal RNA processing remains an area for further investigation. Therefore, we aim to study the interaction proteome of PNO1 and NOB1 in tumor cells and the molecular mechanisms they may regulate. This will enhance our understanding of ribosome assembly factors and provide new perspectives for targeted cancer therapy.\u003c/p\u003e \u003cp\u003eProtein interaction networks form the basis of all signaling and regulatory processes within cells, participating in various cellular biological processes at different spatial and temporal levels\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, such as cell cycle regulation\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, protein synthesis and secretion, signal transduction, and metabolism. Therefore, studying protein interactions is crucial for understanding molecular regulatory networks. Enzyme-catalyzed proximity labeling\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003ehas become a new method for studying the spatial and interaction characteristics of proteins in living cells. TurboID \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003eis a novel biotin ligase that converts biotin into a reactive intermediate that covalently labels proximal proteins with biotin. It has faster labeling kinetics and higher labeling yield than any other biotin ligase-related PL method. Its labeling time can be shortened to 30 minutes, and it maintains catalytic activity at lower temperatures. Additionally, TurboID can label proteins in live physiological environments without disrupting the cells\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Here, we constructed PNO1-TurboID and NOB1-TurboID systems to use TurboID technology to identify the interaction proteome of PNO1 and NOB1 within cells. This study aims to reveal their potential regulatory protein interaction networks and provide a theoretical basis for understanding the functions of ribosome assembly factors.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of the turboID systems\u003c/h2\u003e \u003cp\u003eTo identify the interacting proteome of PNO1 and NOB1 at the molecular level, we constructed fusion expression plasmids for PNO1-TurboID and NOB1-TurboID. The TurboID sequence was fused to the C-terminal of the PNO1 and NOB1 coding sequences (CDS), connected by a flexible linker consisting of 15 amino acids (GGGGS)3, to maximize the identification of proteins interacting with PNO1 and NOB1. The construction of the overexpression plasmids for PNO1-TurboID and NOB1-TurboID is illustrated (Fig.\u0026nbsp;1A). These plasmids include a tag protein HA, the target gene (PNO1, NOB1) CDS sequence, the flexible linker sequence, the biotin-labeling enzyme TurboID sequence, and the tag protein V5. They also include two different selection markers, AmpR and Puro, where AmpR is used to screen positive clones in E.coli, and Puro is used to select cells transfected with and stably expressing PNO1 and NOB1.\u003c/p\u003e \u003cp\u003eThe workflow of the TurboID proximity labeling experiment is as follows (Fig.\u0026nbsp;1B-C). Using the gene PNO1 as an example, the constructed PNO1-TurboID plasmid was successfully transfected into 293T cells. After allowing the plasmid to express in the cells for 48 hours, the cells were incubated in an environment with an appropriate concentration of biotin for a suitable period to ensure complete biotin uptake by the cells. TurboID utilizes exogenously added biotin and intracellular ATP to convert biotin into biotin-AMP, which then biotinylates lysine residues of proximal proteins. Streptavidin magnetic beads were used to capture the biotinylated proteins, which were then subjected to protein affinity purification. The enriched proteins were eluted under denaturing conditions, followed by sample preparation for mass spectrometry analysis. The identification was performed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePNO1 and NOB1 have different localizations in cells\u003c/h2\u003e \u003cp\u003eTo determine the expression levels of the constructed TurboID overexpression plasmids in cells (Fig.\u0026nbsp;2A), we transfected 293T cells with PNO1-TurboID and NOB1-TurboID overexpression plasmids. Western Blot analysis using anti-PNO1 and anti-NOB1 antibodies revealed clear bands at molecular weights of 70 kDa and 77 kDa, respectively (with TurboID at 35 kDa, PNO1 at 35 kDa, and NOB1 at 42 kDa). This indicates that the TurboID fusion expression plasmids are stably expressed in cells. As essential members of the ribosomal small subunit 40S, PNO1 and NOB1 are crucial for the processing, assembly, and maturation of the 40S ribosomal subunit. It is known that PNO1 inhibits the cleavage activity of NOB1 on ribosomal RNA, but whether NOB1's functions outside the ribosome in the cell also depend on PNO1 remains unknown. To understand the functions and localization of PNO1 and NOB1 on the 40S pre-ribosomal subunit, we used the PDB database to analyze their spatial distribution in the Cryo-EM structure of the late-stage human 40S pre-ribosomal subunit. The results showed that PNO1 and NOB1 physically interact and are in close spatial proximity (Fig.\u0026nbsp;2B). These findings suggest that these two proteins may work together in ribosome processing. However, their roles in regulating other cellular functions beyond ribosome maturation are still largely unexplored, leading to the hypothesis that they might also jointly participate in various other biological processes within the cell.\u003c/p\u003e \u003cp\u003eDue to the large size of the TurboID sequence, its fusion with target genes may affect the localization and biological function of the target genes in cells. To ensure that our constructed TurboID fusion expression plasmids are successful and do not affect the gene's localization and function within the cells, we transfected HeLa cells with GFP-PNO1 and GFP-NOB1 plasmids. Immunofluorescence experiments revealed that PNO1 is primarily localized in the nucleus and nucleolus, whereas NOB1 is mainly localized in the cytoplasm. Additionally, we transfected cells with HA-PNO1-TurboID and HA-NOB1-TurboID plasmids and used anti-HA antibodies to detect their localization. Immunofluorescence results demonstrated that the localization of the target genes fused with TurboID in cells matched the GFP localization (Fig.\u0026nbsp;2C-D), indicating that TurboID does not affect the function or proper localization of PNO1 and NOB1 in cells. This ensures the accuracy of subsequent mass spectrometry detection of proteins interacting with PNO1 and NOB1.\u003c/p\u003e \u003cp\u003eMoreover, immunofluorescence results revealed that PNO1 and NOB1 have different localizations, with PNO1 in the nucleus and NOB1 in the cytoplasm, yet they exhibit physical interactions in spatial structure. This suggests that, besides participating in ribosome biogenesis, PNO1 and NOB1 may have additional functions. Therefore, we also utilized TurboID technology to explore the additional biological functions of these two proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of biotin concentration and incubation time for the turboID system\u003c/h2\u003e \u003cp\u003eBased on the results shown above, to further confirm the interaction between PNO1 and NOB1, we performed co-immunoprecipitation experiments (Fig.\u0026nbsp;3A-B), which indicated that PNO1 and NOB1 interact with each other. Before conducting the formal biotin proximity labeling experiments, we first needed to determine the optimal incubation concentration and time for the TurboID system to biotinylate proximal proteins.By setting incubation times of 0, 15, 30, 60, 120, and 240 minutes, we found that, with a constant biotin concentration, the number of proteins labeled by TurboID increased with longer biotin incubation times. Compared to the control group, TurboID could label proximal proteins after 60 minutes of biotin incubation. To prevent false positives caused by excessive biotin labeling due to prolonged incubation, we chose a moderate incubation time of 60 minutes (Fig.\u0026nbsp;3C). Therefore, we selected 60 minutes as the optimal labeling time for subsequent experiments.\u003c/p\u003e \u003cp\u003eBy setting biotin incubation concentrations of 0, 50, 100, 150, 200, and 250 \u0026micro;M, we observed that the number of proteins labeled by TurboID did not increase with higher biotin concentrations. This may be because the TurboID system is limited, and excess exogenous biotin does not enhance TurboID activity. Compared to the control group, adding 50 \u0026micro;M of biotin allowed TurboID to label proximal proteins. To avoid non-specific binding caused by excess free biotin competing with streptavidin magnetic beads, which would reduce the specificity of the biotin-labeled proteins isolated by magnetic separation, we decided to use a biotin concentration of 50 \u0026micro;M for subsequent experiments (Fig.\u0026nbsp;3D).Through preliminary experiments, we confirmed the correct localization of PNO1-TurboID and NOB1-TurboID plasmids in cells, and determined the optimal biotin incubation concentration to be 50 \u0026micro;M and the optimal incubation time to be 60 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCapture and identification of biotinylated proteins\u003c/h2\u003e \u003cp\u003eTo demonstrate that the amount of endogenous biotin in cells is very low and that TurboID has low activity without the addition of a large amount of exogenous biotin, we set up a control group without added biotin. We performed Western blot analysis on the cell lysates. The results showed that, with equal sample loading, the control group without exogenous biotin had very few proteins labeled by TurboID, whereas the group with additional exogenous biotin had a large number of proximal proteins labeled by biotin (Fig.\u0026nbsp;4A).Using streptavidin magnetic beads, we enriched the biotinylated proteins and identified them through silver staining. The results indicated that the proximal proteins labeled by TurboID, PNO1-TurboID, and NOB1-TurboID in the presence of exogenous biotin were successfully enriched (Fig.\u0026nbsp;4B). We identified the enriched proximal proteins from these three groups using mass spectrometry, with the TurboID group serving as the control group. Each group was repeated three times. We processed the raw mass spectrometry data through database searches, initially screening for proteins with Unique Peptide\u0026thinsp;\u0026ge;\u0026thinsp;2 to obtain a preliminary protein set. Subsequently, we compared the overexpression groups to the control group (overexpression group/control group), conducting a secondary screening for proteins with Unique Peptide\u0026thinsp;\u0026ge;\u0026thinsp;2 to obtain the final protein set. These proteins were then subjected to enrichment analysis using KEGG, Reactome, and GO (Fig.\u0026nbsp;4C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFunctional analysis of PNO1 and NOB1 protein interaction groups\u003c/h2\u003e \u003cp\u003eAfter identifying the proteins using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), we filtered the identified proteins based on criteria such as Unique Peptide, PSMs, protein abundance, and biotin modification to obtain a final protein set. We identified 2054 proteins interacting with PNO1 and 2363 proteins interacting with NOB1, with 1437 proteins common to both. KEGG enrichment analysis of proteins enriched by PNO1-TurboID and NOB1-TurboID revealed that the proteins were mainly involved in cell cycle, endocytosis, spliceosome, and ribosome functions (Fig.\u0026nbsp;5A-B). Reactome enrichment analysis showed that the proteins enriched by PNO1 and NOB1 were primarily involved in GTPase signaling, RNA metabolism, cell cycle, translation, and ribosomal RNA processing (Fig.\u0026nbsp;5C-D). Biological process analysis indicated that PNO1 and NOB1 were mainly involved in RNA metabolic processes, translation, and the assembly of protein-containing complexes. The enriched proteins were primarily localized in the nucleolus, microtubule cytoskeleton, and centrosome. The molecular functions of these proteins included adenosine nucleotide binding, mRNA binding, rRNA binding, and snRNA binding (Fig.\u0026nbsp;5E-F).\u003c/p\u003e \u003cp\u003eFunctional analysis of the proteins enriched by PNO1 and NOB1 revealed significant functional similarities between the two. Despite their different localizations, PNO1 and NOB1 shared many common regulatory functions, such as mRNA translation, which are crucial for cell growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProtein interaction network of PNO1 and NOB1\u003c/h2\u003e \u003cp\u003eThe protein-protein interaction network is fundamental to understanding the intricate interactions and functional relationships between proteins within a cell. These networks illustrate how proteins interact physically and functionally, providing valuable insights into cellular processes and pathways. By mapping these interactions, researchers can elucidate the roles of proteins in various biological functions, such as signal transduction, metabolic pathways, and structural assembly.In this study, we constructed a protein-protein interaction network for the proteins interacting with both PNO1 and NOB1 using the CPDB database. The results revealed that PNO1 and NOB1 are primarily enriched in the pre-rRNA complex, mRNA translation complex, and IGF2BP1 complex(Fig.\u0026nbsp;6A).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePNO1 and NOB1 may be involved in mRNA translation regulation\u003c/h2\u003e \u003cp\u003emRNA translation is a crucial biochemical process within cells that converts genetic information from mRNA into proteins, which are the primary executors of cellular functions. The translation process itself is tightly regulated. By controlling the translation of specific mRNAs, cells can rapidly respond to endogenous and exogenous signals, enabling physiological changes and maintaining homeostasis. Through the biological functional analysis of mass spectrometry data from PNO1-TurboID and NOB1-TurboID, we surprisingly discovered that PNO1 and NOB1 share significant functional similarities and may be involved in the process of mRNA translation. Based on our mass spectrometry results, we identified translation-related proteins such as EIF4B and EIF4G2 (Fig.\u0026nbsp;7A-B). Analyzing the peptide segments of EIF4B and EIF4G2 through mass spectrometry database searches, we obtained the M/Z spectra for these proteins (Fig.\u0026nbsp;7C-D).To validate our mass spectrometry results, we performed co-immunoprecipitation experiments, which demonstrated that PNO1 and NOB1 interact with EIF4B and EIF4G2 (Fig.\u0026nbsp;7E-F). However, the mechanism by which PNO1 and NOB1 regulate the translation process remains unknown, and we look forward to further experimental investigations to explore this process.\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eRibosome assembly factors play a crucial role in the biosynthesis and function of ribosomes. In this study, we observed that ribosome assembly factors PNO1 and NOB1 have different localizations within cells through immunofluorescence experiments\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. This piqued our interest, and to explore their biological functions within cells, we employed the TurboID proximity labeling technology to identify proteins interacting with PNO1 and NOB1 and to uncover the functional networks they are involved in. The identified proteins interacting with PNO1 and NOB1 were subjected to KEGG, Reactome, and GO gene annotation and functional analyses. The results indicated that these proteins were mainly enriched in functions related to the cell cycle, spliceosome, ribosome, RNA metabolic processes, translation, and assembly of protein-containing complexes. A total of 1437 proteins were found to interact with both PNO1 and NOB1, 617 proteins interacted exclusively with PNO1, and 926 proteins interacted exclusively with NOB1. We speculate that PNO1 and NOB1 might function together within cells.Moreover, our analysis of the PNO1 and NOB1 interaction protein network revealed that, in addition to their roles in ribosome processing and maturation, PNO1 and NOB1 are also involved in mRNA translation, mRNA splicing, RNA polymerase I transcription, and other processes critical for cell growth and proliferation.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eRibosome assembly is a highly complex and tightly regulated process that requires the involvement of multiple assembly factors\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. These assembly factors perform several crucial functions in ribosome biosynthesis, including rRNA modification and processing\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, ribosomal protein binding, subunit folding and stabilization\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, subunit transport, and quality control and error correction\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. These functions are essential to ensure the correct assembly and function of ribosomes. Ribosome assembly factors not only play key roles in ribosome biosynthesis but also contribute to RNA metabolism\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, protein translation regulation\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, stress responses\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, protein quality control, and cell differentiation\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. These non-traditional roles further emphasize the importance and diversity of ribosome assembly factors in cellular biology. Therefore, investigating the protein interaction networks and biological functions of ribosome assembly factors within cells is of significant importance.TurboID is an advanced biotinylation technology used for efficient and specific labeling of protein-protein interactions in living cells\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Compared to traditional labeling methods, TurboID offers higher labeling efficiency, lower cytotoxicity, and higher specificity\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. These advantages make TurboID a valuable tool for studying protein-protein interactions and have led to its widespread application\u003csup\u003e[\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eIn summary, our study reveals, through the use of TurboID technology, that ribosome assembly factors PNO1 and NOB1 are not only involved in ribosome processing but also participate in mRNA translation and other functions.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eHEK293T and HeLa cells were purchased from the American Type Culture Collection (ATCC, Manassas, USA). All cells were grown in high-glucose DMEM supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin solution. The cells were cultured in a 5% CO2 incubator at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid Construction\u003c/h2\u003e \u003cp\u003eHuman cDNA was used as a template for PCR amplification to obtain the PNO1 and NOB1 sequences. The TurboID sequence was sourced from the plasmid pLV3-CMV-EGFP-TurboID-NLS-3x FLAG-Puro purchased from Miaoling Bio. PNO1-TurboID and NOB1-TurboID sequences were amplified via fusion PCR. The amplified gene fragments and PCDH CMV plasmid were linearized using the restriction endonucleases XbaI and BamHI, and then ligated overnight at 16\u0026deg;C using T4 DNA ligase. The ligation products were transformed into Stbl3 competent cells and plated on AMP plates to select positive clones. Single colonies were sent to Guangzhou Aiji Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmids were named pCDH-CMV-HA-PNO1-Linker-V5-TurboID (abbreviated as PNO1-TurboID plasmid) and pCDH-CMV-HA-NOB1-Linker-V5-TurboID (abbreviated as NOB1-TurboID plasmid). All vectors constructed in this study were validated by Sanger sequencing before use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAntibody\u003c/h2\u003e \u003cp\u003eAnti-Biotin Antibody(#7075), Anti-HA-Tag Rabbit Antibody #3724(C29F4) were purchased from Cell Signaling Technology.\u003c/p\u003e \u003cp\u003eAnti-PNO1 Polyclonal Antibody(# PA5-70579)were purchased from Invitrogen.\u003c/p\u003e \u003cp\u003eAnti-NOB1 Monoclonal antibody(#66048-1-Ig),Anti-GAPDH Monoclonal antibody(#60004-1-Ig), Anti-Alpha Tubulin Monoclonal antibody(#11221-1-AP) were purchased from Proteintech.\u003c/p\u003e \u003cp\u003eAnti-FLAG Monoclonal Antibody(#F1804)were purchased from Sigma.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot and Co-IP\u003c/h2\u003e \u003cp\u003eBriefly, HEK293T cells were successfully transfected with PKO187-SFB, SFB-PNO1, and SFB-NOB1 plasmids. After 48 hours of expression, cell proteins were collected and lysed in NETN buffer containing a protease inhibitor cocktail (Bimake, China) and benzonase nuclease (Merck Millipore) at 4\u0026deg;C for 30 minutes. The lysates were then centrifuged, and the supernatants were separated by SDS-PAGE and analyzed with specific antibodies.For Co-IP of exogenously tagged proteins, cell lysates were incubated with S-beads (Merck Millipore) at 4\u0026deg;C with rotation overnight. The next day, the beads were centrifuged and washed five times with NETN buffer. The precipitated proteins were then analyzed by Western blotting with the indicated antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence Assay\u003c/h2\u003e \u003cp\u003eHeLa cells transfected with the plasmids were cultured on glass-bottom dishes. The cells were then fixed with 4% paraformaldehyde at room temperature for 30 minutes. After fixation, the cells were incubated with 0.5% Triton X-100 for 5 minutes and then blocked with blocking solution (Beyotime) for 30 minutes. Primary antibody staining was performed overnight at 4\u0026deg;C, followed by secondary antibody incubation and DAPI staining. Images were acquired at room temperature using a Leica SP-8 STED 3X microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBiotin Labeling Time Screening\u003c/h2\u003e \u003cp\u003eHEK293T cells transfected with PNO1-TurboID and NOB1-TurboID were cultured in 6-well plates. When the cell density reached over 80%, the original medium was replaced with medium containing biotin (50 \u0026micro;mol/L). The cells were incubated and evaluated at six different time points (0 min, 15 min, 30 min, 1 h, 2 h, and 4 h). At each time point, the cells were collected, and the cell lysates were subjected to SDS-PAGE. Streptavidin-HRP was used as the antibody for detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Biotinylated Protein Samples\u003c/h2\u003e \u003cp\u003eCells from each 100mm culture dish were collected into 1.5 mL EP tubes, and 1 mL of RIPA buffer was added to resuspend the cells. The cells were then lysed on ice for 15 minutes and centrifuged at 12,000 rpm for 10 minutes. The supernatant was transferred to a clean 1.5 mL EP tube.Streptavidin magnetic beads were washed with 1 mL of RIPA buffer five times until the liquid became clear. The prepared protein lysates were added to the washed beads, sealed with parafilm, and incubated overnight at 4\u0026deg;C on a rotator at 360 degrees.After overnight incubation, the supernatant was discarded, and the beads were washed with 1 mL of RIPA buffer for approximately 1.5 minutes. This process was repeated once. The beads were then washed once with 1 mL of KCl (1 mol/L), followed by three washes with Buffer 1 (prepared by mixing 67 mL of NaCl (3 mol/L), 1 mL of Tris-HCl (pH 7.4, 1 mol/L), and 32 mL of H2O in 100 mL of Buffer 1). The beads were washed once with Na2CO3 (0.1 mol/L) and once with 10% SDS for 2 minutes. Finally, after the last wash with RIPA buffer, one-third of the sample was mixed with 5X protein buffer and denatured for separation by SDS-PAGE and identification by silver staining. The remaining sample was washed twice with PBS, and after removing the supernatant, the beads were stored at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe mass spectrometry proteomics data generated in this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier IPX0008946000 (iProX- integrated Proteome resources). All data generated or analyzed during this study are included in this article and its Supplementary Information files. Source data are provided with this paper.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank Professor Kaishun Hu from Sun Yat-sen Memorial Hospital, Sun Yat-sen University, for providing the PKO187-SFB and PKO187-GFP plasmids and for his technical support. Mass spectrometry identification and analysis were conducted by the Bioinformatics and Omics Center at Sun Yat-sen Memorial Hospital, Sun Yat-sen University.\u003c/p\u003e\n\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis\u0026middot; study\u0026middot; was\u0026middot; supported by\u0026middot; grants from\u0026middot;the Natural Science Foundation of China (82273033, 82072924); Guangdong Science and Technology Department (2022B1515020100); Guangzhou Bureau of Science and Information Technology (202201020575).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eXingyuan Xu wrote the main manuscript text, Xingyuan Xu,Jiefu Zheng\u0026nbsp;and Wenli Chen jointly organized the images and mass spectrometry data. All authors reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors confirm that there are no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAMEISMEIER M, ZEMP I, VAN DEN HEUVEL J et al. Structural basis for the final steps of human 40S ribosome maturation [J]. 2020, 587(7835): 683\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKATHERINE E B, MARKUS T B J E. J. Uncovering the assembly pathway of human ribosomes and its emerging links to disease [J]. 2019, 38(13).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eANTHONY K H, C\u0026eacute;LIA P-C, MARIE-FRAN\u0026ccedil;OISE OD et al. An overview of pre-ribosomal RNA processing in eukaryotes [J]. 2014, 6(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eELHAMAMSY A, METGE B, ALSHEIKH H et al. Ribosome Biogenesis: A Central Player in Cancer Metastasis and Therapeutic Resistance [J]. 2022, 82(13): 2344\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUNIACKE J, PERERA J, LACHANCE G et al. Cancer cells exploit eIF4E2-directed synthesis of hypoxia response proteins to drive tumor progression [J]. 2014, 74(5): 1379\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBARNA M, PUSIC A, ZOLLO O et al. Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency [J]. 2008, 456(7224): 971\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePENZO M, MONTANARO L, TRER\u0026eacute; D et al. Ribosome Biogenesis-Cancer Connection [J]. 2019, 8(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026ouml;RNER K, RUGGERI C, ZEMP I et al. Ribosome biogenesis factors-from names to functions [J]. 2023, 42(7): e112699.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWYLER E, ZIMMERMANN M. WIDMANN B, Tandem affinity purification combined with inducible shRNA expression as a tool to study the maturation of macromolecular assemblies [J]. 2011, 17(1): 189\u0026ndash;200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCHAKER-MARGOT M, BARANDUN J, HUNZIKER M et al. Architecture of the yeast small subunit processome [J]. 2017, 355(6321).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCHENG J, KELLNER N, BERNINGHAUSEN O et al. 3.2-\u0026Aring;-resolution structure of the 90S preribosome before A1 pre-rRNA cleavage [J]. 2017, 24(11): 954\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJINGDONG MICHAELA. C, OTTO B, Visualizing late states of human 40S ribosomal subunit maturation [J]. 2018, 558(7709).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eALLISON CL, KATRIN K J P N A S U S. A. Nob1 binds the single-stranded cleavage site D at the 3'-end of 18S rRNA with its PIN domain [J]. 2009, 106(34).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEMMA T, S\u0026eacute;BASTIEN F-C, ED H J J C. S. Eukaryotic ribosome biogenesis at a glance [J]. 2013, 126(0).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMARTINO E, CHIARUGI S, MARGHERITI F et al. Mapping, Structure and Modulation of PPI [J]. 2021, 9(718405.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSPEER S, ZHENG W, JIANG X et al. The intracellular environment affects protein-protein interactions [J]. 2021, 118(11).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZHOU L, ZHANG Q, DENG H et al. The SNHG1-Centered ceRNA Network Regulates Cell Cycle and Is a Potential Prognostic Biomarker for Hepatocellular Carcinoma [J]. 2022, 258(4): 265\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKIM D, ROUX K J T I C B. Filling the Void: Proximity-Based Labeling of Proteins in Living Cells [J]. 2016, 26(11): 804\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJOHNSON B, CHAFIN L. FARKAS D, MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics [J]. 2022, 21(7): 100256.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBRANON T, BOSCH J, SANCHEZ A et al. Efficient proximity labeling in living cells and organisms with TurboID [J]. 2018, 36(9): 880\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKANZLER C, DONOHUE M, DOWDLE M et al. TurboID functions as an efficient biotin ligase for BioID applications in Xenopus embryos [J]. 2022, 492(133\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTHOMSON E, FERREIRA-CERCA S, HURT E J J O C. S. Eukaryotic ribosome biogenesis at a glance [J]. 2013, 126(4815\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDIETER K, ED H, JOCHEN B J B. B A. Driving ribosome assembly [J]. 2009, 1803(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJESUS D L C, KATRIN K, JOHN L JR W J. A R B. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo [J]. 2015, 84(0).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLIONEL T, CHRISTIANE Z, JEAN-LOUIS L et al. The complexity of human ribosome biogenesis revealed by systematic nucleolar screening of Pre-rRNA processing factors [J]. 2013, 51(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKAREN J, CRISTIAN B DORIML et al. rRNA pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells [J]. 2011, 44(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS\u0026eacute;VERINE B, BELINDA J W, SASKIA H et al. The nucleolus under stress [J]. 2010, 40(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJONATHAN R W, KERRI B M J. M C. How common are extraribosomal functions of ribosomal proteins? [J]. 2009, 34(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKELVIN F C, TESS C B, NAMRATA D U et al. Proximity labeling in mammalian cells with TurboID and split-TurboID [J]. 2020, 15(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eANDREA M, SHOU-LING X, TESS C B et al. Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID [J]. 2019, 8(0).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYONGLIANG Z, YUANYUAN L, XINXIN Y et al. TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks [J]. 2020, 159).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKAIXIN Z, YINYIN L, TENGBO H et al. Potential application of TurboID-based proximity labeling in studying the protein interaction network in plant response to abiotic stress [J]. 2022, 13(0).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYANTING S, YUANYUAN G, JIEYU G et al. Study of FOXO1-interacting proteins using TurboID-based proximity labeling technology [J]. 2023, 24(1).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PNO1, NOB1, TurboID, Biotin labeling, Translation","lastPublishedDoi":"10.21203/rs.3.rs-4508442/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4508442/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe ribosome assembly factors PNO1 and NOB1 play crucial roles in the maturation of the 40S ribosomal small subunit. TurboID is an efficient biotin ligase that can biotinylate proteins in proximity to the target protein and is widely used to study complex biological processes within cells.Here, we utilized this technology to investigate the complex interaction network of PNO1 and NOB1 within cells.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThrough immunofluorescence experiments, we found that PNO1 and NOB1 have different localizations within cells. By identifying the proximal proteins biotinylated by PNO1-TurboID and NOB1-TurboID, we discovered 871 proteins interacting with PNO1 and 1044 proteins interacting with NOB1, with 663 proteins overlapping. These results suggest that PNO1 and NOB1 are extensively involved in various biological processes within the cell. Furthermore, we found that PNO1 and NOB1 interact with translation-related proteins EIF4B and EIF4G2, indicating that they may participate in the mRNA translation process.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe interaction proteome results of PNO1 and NOB1 suggest that ribosome assembly factors are not only involved in ribosome biogenesis but also couple with multiple biological processes within the cell, such as mRNA translation. This provides a foundation for understanding the complex biological processes within the cell.\u003c/p\u003e","manuscriptTitle":"the Interaction Proteome of Ribosomal 40S Components PNO1 and NOB1 Using TurboID Proximity Labeling Technology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-18 11:57:06","doi":"10.21203/rs.3.rs-4508442/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f8b81a93-bbea-4efd-93b7-803cb26582b1","owner":[],"postedDate":"June 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-28T04:59:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-18 11:57:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4508442","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4508442","identity":"rs-4508442","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}