Phosphorylation patterns of pre-ribosomal proteins associated with the RNA exosome

The RNA exosome is an essential and ubiquitous RNase with exonucleolytic activity, involved in ribosome biogenesis and RNA quality control in eukaryotes. It is present both in nucleus and cytoplasm, and interacts with specific cofactors in each cell compartment, which are essential for recruitment and activity control of the exosome. Posttranslational modifications are known to regulate enzyme activity and protein interaction , although their precise roles are individually specific. In this study, we investigated the phosphorylation status of proteins associated with the nuclear (Rrp6) and core (Rrp46) subunits of the RNA exosome in Saccharomyces cerevisiae. Using co -immunoprecipitation followed by phosphopeptide enrichment and high -resolution mass spectrometry, we identified 121 phosphorylation sites on proteins functionally related to rRNA processing. Differential phosphorylation patterns between Rrp6 and Rrp46 co- immunoprecipitations are consistent with distinct exosome assemblies and suggest potential regulatory roles for phosphorylation. The results shown here highlight the role of phosphorylation in the recruitment and control of the exosome in RNA processing and degradation, offering new insight s into the posttranscriptional control of gene expression. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 3

The RNA exosome complex is a ubiquitous RNase with 3’ -5’ exonucleolytic activity involved in processing and degradation of all classes of RNA in eukaryotes (Kilchert et al, 2016; Zinder & Lima, 2017). In budding yeast, the exosome is comprised of eleven different protein subunits, nine of which form the catalytically inactive core (Csl4, Rrp4, Rrp40 -43, Rrp45, Rrp46, Mtr3). The core associates with Rrp44/Dis3 in the cytoplasm, forming Exo -10, and in the nucleus, Exo-11 is composed of Exo -10+Rrp6. Additionally, the exo some binds to specific cofactors in each subcellular compartment (Schneider & Tollervey, 2013). The exosome was first identified in the yeast Saccharomyces cerevisiae and later detected in other eukaryotes and some archaea species (Allmang et al, 1999b; Hartung & Hopfner, 2009; Mitchell et al , 1997) . In the nucleus, the exosome pl ays an essential role in pre -rRNA processing and snoRNA and snRNA maturation (Allmang et al, 1999a). The exosome is recruited to the 90S SSU processome, the first pre - ribosomal particle to be formed cotranscriptionally , and is responsible for the degradation of the 5´ -ETS spacer sequence after its release from the 35S pre - rRNA (Cepeda et al, 2019; Lau et al, 2021). Later during ribosomal maturation, the exosome is recruited to the pre-60S particles to process the 7S intermediate and form the mature 5.8S rRNA (Allmang et al. , 1999a; Oliveira et al , 2002; Schuller et al, 2018; Thoms et al, 2015). The major interactors of the exosome in the nucleus are responsible for its recruitment to pre -ribosomal particles during maturation and include the RNA helicase Mtr4, which is also a subunit of the TRAMP complex, and ribosome assembly factors Nop53 and Utp18, which recruit the exosome through Mtr4 (Bagatelli et al, 2021; Cepeda et al., 2019; Houseley et al, 2006; LaCava et al, 2005; Thoms et al., 2015). In the cytoplasm, the exosome is recruited by the SKI complex to degrade its substrates (van Hoof et al, 2000; Zhang et al, 2019). Ski7 is a critical adaptor connecting the SKI complex (Ski2 -Ski3-Ski8) and the cytoplasmic exosome for mRNA degradation (Araki et al, 2001). Although the structure and function of the exosome has been extensively studied in recent years, a detailed understanding of how the control of its function and recruitment is achieved is still lacking. Posttranslational modifications are a preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 4 very important mechanism for controlling protein function, and protein phosphorylation is among the prevalent modifications. Phosphorylation of ribosomal proteins has long been known to influence translation (Bohlen et al, 2021; Cerezo et al, 2021; Nielsen et al, 1982), and has also been shown to affect ribosomal maturation and interaction between assembly factors (Ballesta et al, 1999; Filipek et al, 2024; Tomioka et al, 2018). Phosphorylation at specific sites on Dis3 (Ser809 and Tyr814) and Mrt4 (Thr1061 and Ser1067) from Schizosaccharomyces pombe has been shown to regulate exosome function (Telekawa et al, 2018). In this study, we investigated the role of phosphorylation in the regulation of the Saccharomyces cerevisiae exosome interaction with its cofactors. We selected Rrp6 and Rrp46 as representative components of the nuclear and core exosome complexes, respectively, to identify phosphorylation sites of proteins interacting with exosome -containing complexes that may be regulated during ribosome biogenesis, or cytoplasmic RNA processing/degradation processes.

Yeast strains and culture conditions Exosome subunits were affinity -purified from strains RRP46 -TAP::HIS3 and RRP6 -TAP::URA3 (Euroscarf) as described (Lourenco et al , 2013) . Maintenance and growth of yeast cultures were performed according to standard procedures (Sherman et al., 1986). They were initially grown at 30°C in minimal medium (YNB) supplemented with 2% glucose, in addition to the essential amino acids, according to their auxotrophic marks. Yeast wild type strain W303 was used as negative control for subsequent analysis. Cloning and site-directed mutagenesis For cloning of RRP4 and SKI7, the plasmids were linearized by inverse PCR and the inser ts were amplified from the yeast genomic DNA and recombinant plasmids were constructed with the InFusion HD (Clontech) recombination cloning system using competent Stellar (Clontech) cells. The site -directed mutagenesis was performed by overlap of PCR product and In -fusion DNA assembly kit, following the manufacturer’s instructions. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 5 Spot assay growth and growth curve Precultures of the conditional strain Δski7/yCP111-MET15::SKI7-TAP (Neto et al, 2025) and GAL1::13MYC-RRP4/yCP111-ADH1::RRP4-3HA, expressing the corresponding plasmid-borne wild type Ski7 or Rrp4, or mutants (Ski7-S90A and Rrp4-S152A) in log phase were diluted for OD 600nm=0.1 and then subjected to 10-fold serial dilutions, and spotted onto glucose -containing solid minimal medium methionine-free or methionine-containing (2 mM) and incubated at 30°C. Growth curve measurements were performed in microplates incubated over 12h of growth using a BioTek LogPhase 600 Microbiology Reader set to read OD600 every 60 min at 30°C in the case of Ski7, and 30°C and 37°C in the case of Rrp4. Coimmunoprecipitation of Rrp46, Rrp6 and W303 strains For the tandem affinity purification (TAP) method, these yeast strains were cultured in 6L YPD (1% yeast extract; 2% peptone; 2% glucose) medium. Biological triplicates were harvested at log phase (OD600 ~ 1) by centrifugation at 8000 rpm (F12-6×500 LEX Rotor, Sorvall R6 C Plus) for 30 min at 4ºC. The cell pellets were resuspended in cooled extraction buffer (60 mM Tris–HCl pH 8.0, 50 mM NaCl, 10 mM MgCl 2, 5% glycerol, 1 % PMSF, 1% triton, 1% EDTA, Pierce Protease Inhibitor Tablets, and Pierce Phosphatase Inhibitor Tablet) and flash - frozen in liquid N2. The coimmunoprecipitation assays were carried out as previously described (Puig et al , 2001) , with minor modifications. Briefly, the whole -cell lysates were obtained by cryogenic milling on a Ball Mill device (Retsch PM 100) and cleared by ultracentrifugation at 28,100 rpm (P50AT4 rotor, CP80NX Hitachi) for 60 min at 4°C. T he lysates were incubated for 2 h at 4°C with 200 μl IgG - Sepharose 6 Fast Flow (GE Healthcare) previously equilibrated with extraction buffer and centrifuged at 500 rpm at 4ºC for 2 min. The beads were washed twice with extraction buffer and twice with was h buffer (50 mM tris -HCl and 50 mM NaCl). Elution was performed in the same column by adding 1 ml of TEV cleavage buffer and 100 units of TEV protease. The beads are rotated overnight at 4ºC and the eluate was recovered by gravity flow. Eluates were lyophilized and stored at -80ºC until treatment for LC–MS/MS analysis. Both input and eluate fractions were separated for analysis by SDS-PAGE and western blot. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 6 SDS-PAGE and immunoblot Aliquots (~5%) of yeast coimmunoprecipitated proteins were resuspended in SDS sample buffer, resolved on 10% SDS-PAGE (Tris-glycine running buffer), transferred to nitrocellulose membranes (Ambion) and incubated with antibody against the CBP tag (Millipore). Secondary antibody conjugated to IRDye680RD (anti-rabbit IgG, LI -COR) was employed, and near -infrared Western blot detection was carried out using ChemiDoc MP Imaging System (BioRad). Peptide digestion and TiO2 phosphopeptide enrichment The lyophilized eluates were subjected to in -solution digestion for LC– MS/MS analysis. Briefly, proteins were resuspended in 50 mM NH 4HCO3, 8 M urea and reduced with DTT at a final concentration of 5 mM at 56°C for 25 min. Samples were then alkylated with 14 mM iodoacetamide for 30 min under protection from light. After dilution with 50 mM NH4HCO3, samples were digested for 16 h with sequencing -grade modified trypsin (Promega) at a 1:50 (E:S) ratio at 37°C. The proteolysis was stopped by adding TFA to a final concentration of 0.4% (v/v) and the resultant peptides were desal ted using SepPak tC18 cartridges (Waters). A High -Select TiO 2 kit (Thermo Fisher Scientific) was used to enrich phosphopeptides from tryptic peptides. The digested peptides were placed into TiO2 spin tips and the enrichment steps were performed according to the manufacturer’s instructions. After elution of the phosphopeptides, the eluate was vacuum-dried and stored at -80 ºC until mass spectrometry analysis. Mass spectrometry analysis and data acquisition Digested peptides were resuspended in 0.1% formic acid (FA) and separated on an in-house reversed-phase capillary emitter column (10 cm × 75 μm, filled with 5 μm particle diameter C18 Aqua resins-Phenomenex) coupled to a nano-HPLC (Thermo). Mobile phases consisted of 0.1% FA in water (buffer A) and 0.1% FA in acetonitrile (buffer B). Peptides were eluted at 300 nL min⁻¹ using a 60 min gradient (2–28% B for 45 min, 28–80% B for 13 min, and a return to 5% B in 2 min) . The eluted peptides were analyzed by an LTQ -Orbitrap Velos (Thermo Scientific) (source voltage of 1.9 kV, capil lary temperature of 200°C). The mass spectrometer was operated in DDA mode with dynamic exclusion preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 7 enabled (exclusion duration of 45 seconds), MS1 resolution of 30,000, and MS2 normalized collision energy of 30. For each cycle, one full MS1 scan range of 200–2000 m/z was followed by ten MS2 scans (for the most intense ions) using an isolation window size of 1.0 m/z and collision-induced dissociation (CID). The 10 most intense precursor ions were selected for fragmentation by CID in DDA. The .raw data from the LTQ Velo s-Orbitrap were analyzed with the MaxQuant (version 2.6.7.0) software using the UniProt database for Saccharomyces cerevisiae (strain ATCC 204508 / S288c), setting tolerance for the MS of 20 ppm and the MS/MS of 0.5 Da; carbamidomethylation of cystein e as a fixed modification; and phosphorylation at STY residues as variable modifications; and 1% False Discovery Rate (FDR). The match between runs option was used to increase the number of trusted IDs. The normalized Intensity values of the Phospho (STY)S ites.txt output and the Intensity columns for each biological replicate were used for downstream analyses . Identifier for the r aw mass spectrometry data: PXD073587. Label-free phosphoproteomics data analysis Initial data analysis was performed using Perseus software (version 2.1.3.0, Max Planck Institute of Biochemistry). The affinity purification assays using dif ferent baits were analyzed together. The MaxQuant output data was initially filtered removing contaminants, hits to the reverse database, and phosphoproteins with localization probability ≥ 0.70 . The intensities were log2 - transformed and the biological triplicates were grouped by categorical annotation in the two conditions assessed, Rrp46 and Rrp6. Furthermore, phosphopeptides were filtered to include only those detected in at least two of the three biological replicates or present uniquely in one condition (presence/absence filtering for exclusive identifications). For differential phosphorylation analysis, intensity values log ₂- transformed were subjected to Student ’s t -test. Only phosphopeptides with statistically significant differences (FDR-adjusted p-value (Benjamini–Hochberg) ≤ 0.05) were considered for downstream analysis. Additionally, phosphopeptides identified in control W303 samples were excluded to reduce false -positive identifications. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 8

To investigate the possible regulatory role of phosphorylation in the recruitment of the exosome to pre -ribosomal complexes, we conducted a label- free ph osphoproteomic analysis using Saccharomyces cerevisiae strains expressing TAP -tagged versions of the nuclear (Rrp6) o r core (cytoplasmic/nuclear - Rrp46) subunits of the RNA exosome. These subunits interact with distinct protein partners and ribonucleoprotein complexes during ribosome biogenesis, or cytoplasmic RNA degradation, and our goal was to identify phosphorylation events that may modulate these interactions. Protein co- immunoprecipitation experiments were therefore performed using total extracts of yeast cells expressing either Rrp6 -TAP or Rrp46 -TAP. Eluted proteins from biological triplicates were then digested with trypsin and resulting peptides were subjected to phosphopeptide enrichment and identification by mass spectrometry. The protein baits were detected both by mass spec and immunoblots (Fig. 1A). Extracts from wild-type W303 strain, processed in parallel, were used and proteins/phosphosites detected in control runs were excluded from downstream analyses to reduce background binders. The similarity between replicates was compared by a principal component analysis (PCA), which show two clusters, one for each sample, Rrp46 or Rrp6, with good representation (Fig. 1B). preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 9 A total of 756 phos phorylated peptides were identified (Suppl. Table 1), corresponding to phosphosites present on proteins co-purifying with the exosome subunits. Gene ontology analysis of proteins identified in these affinity -purified fractions showed prevalence of rRNA processing factors, which was expected due to the essenti al role played by the exosome in the ribosome maturation pathway (Fig. 2). Among the rRNA processing factors recovered, those involved in 5.8S rRNA maturation were the most abundant (Fig. 2), which reinforces the validation of our results, since the exosom e is directly involved in mature 5.8S formation. From the initial peptides recovered, we selected a total of 121 phosphorylated residues that correspond to proteins functionally related to RNA processing for further analyses. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 10 We investigated the distribution and intensity of phosphorylation events across the two experimental conditions. A heatmap based on phosphosite intensities for rRNA processing -associated proteins revealed both shared phosphorylation profiles and condition-associated patterns (Fig. 3). preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 11 Utp20, Nop4, Sqs1, and Nog2, among other ribosome maturation factors, were uniquely detected in Rrp46 samples, whereas other assembly factors, including Enp2, Pwp2, Kri1 and Utp22 were phosphorylated in both samples (Fig. 3), suggesting a he terogeneity of nuclear exosome complexes. Notably, the phosphosites on the N-terminal unstructured portion of Utp20 detected here have not been previously reported. The selective enrichment of phosphorylated peptides in Rrp46 samples suggests that the asso ciation of the exosome at different stages of ribosomal maturation may be mediated by additional assembly preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 12 factors, other than Utp18 (SSU processome) and Nop53 (pre-60S) via Mtr4-Rrp6 direct interaction (Schuch et al, 2014). A smaller set of phosphosites was uniquely detected in Rrp6 samples. Among those, Sas10 (residues S336 and S339) and nuclear pore -associated export factor Nup2 (Y15) were consistently phosphoryl ated only in the Rrp6 - associated fractions (Fig. 3). Sas10 is functionally linked to the SSU processome, and has been shown to bind Rrp6-Rrp47 heterodimer (Mitchell, 2010), confirming the interaction of the exosome with factors involved in early rRNA processing steps in the nucleolus (Cepeda et al. , 2019) . Further demonstrating the consistency of our results, proteins with multiple phosphorylation sites showed particular phosphorylation states, depending on the residue and the complex they were purified with. Utp20, for example, had its four phosphorylated serine residues purified exclusively with Rrp46, while Sas10 -S336 and S339 we re phosphorylated exclusively in Rrp6 samples . Sas10-S477 and Mpp10, on the other hand, a protein of the same complex as Sas10, were phosphorylated in both samples (Fig. 3; Table 1). These differential phosphorylation states might indicate regulation of protein function, affecting interactions throughout ribosomal maturation. Several phosphosites exhibited comparable phosphorylation levels in both Rrp6 and Rrp46 complexes, suggesting that these modifications play constitutive regulatory roles irrespective of the context of the ribosomal maturation stage with which the exosome is associated. These shared sites were found in central assembly factors such as Mpp10, Utp22, Utp14, Rrp5, Kri1, preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 13 Enp2, Bud21, Bud22, Esf1, and Pwp2 (Fig. 3). These proteins are essential for 90S pre-ribosome formation, and the conserved presence of their phosphorylated forms in both conditions indicates that their regulation is fundamental and that the Exo11 complex remains bound to pre-ribosomal particles at different maturation stages. Strengthening this hypothesis, Pwp1, which is also phosphorylated in both conditions, is an example of a protein identified here that participates in pre- 60S maturation (Fig. 3). In addition to the phosphorylated peptides c o-purified with the exosome subunits, phosphosites of three different exosome subunits were identified, Csl4- S94, Rrp6 -S709, and Rrp4 -S152, S237 (Fig. 3; Table 1). Interestingly, these residues were phosphorylated in both samples, purified with Rrp6 and Rr p46, with the exception of Rrp4 -S237, which was co-purified exclusively with Rrp46, suggesting that the phosphorylation of this residue is involved in cytoplasmic function of the exosome, either for activity control, or recruitment of the complex by its co factors. Importantly, this residue is exposed on the exosome complex structure (Makino & Conti, 2013) (Fig. 4; Suppl. Fig. 1), indicating its suitability for being modified. Csl4 -S94 and Rrp4 -S152 phosphorylation have already been described (Synowsky et al , 2006) , confirming the high reproducibility of our purification method. Csl4-S94 is present in a predicted disordered domain of this protein. Rrp4-S152, on the other hand, is imbedded in its S1 RNA binding domain and is accessible for post-translational modifications (Fig. 4; Suppl. Fig. 1 ), and is conserved in the human ortholog EXOSC2 (Ramos et al, 2006; Synowsky et al., 2006) . The importance of Ser152 phosphorylation for Rrp4 function was assessed by substituting Ala for this Ser (Rrp4-S152A) and analyzing growth of yeast cells expressing this mutant. For this analysis, t he endogenous promoter of RRP4 was replaced by GAL1 promoter, which is activated in the presence of galactose, and inhibited in glucose medium , and this strain ( GAL1::Myc-RRP4) was transformed with a plasmid expressing either wild type Rrp4, or the mutant Rrp4-S152A, fused to the HA tag and under control of the constitutive promoter ADH1. Growth was then analyzed in glucose medium, condition in which only the plasmid-encoded Rrp4 -HA proteins are expressed . Despite the evolutionary conservation of this residue, however, its change to the non-phophorylatable Ala does not affect growth (Fig. 5), suggesting that phosphorylation at position 152 does not interfere with the RNA binding activity of Rrp4. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 14 Rrp6-S709 is present in the lasso domain, involved in RNA binding and stimulation of exosome activity, and is inse rted in the canonical nuclear localization signal (NLS) of Rrp6 (697 -723) (Gonzales-Zubiate et al , 2017; Wasmuth & Lima, 2017) , potentially affecting both RNA bindin g and Rrp6 interaction with karyopherins. Phosphosites S42, S119, S150 of the exosome cofactor Mpp6 (Schilders et al, 2005; Wasmuth et al, 2017) were found in bo th samples (Table 1). Mpp6 has already been identified as a phosphorylated protein in high-throughput analyses (Leutert et al, 2023), validating our results. A minimal portion of Mpp6 comprising amino acid positions 90 through 118 was crystallized in complex with the exosome and shown to bind directly Rrp40 (Wasmuth et al., 2017). Due to its proximity to the Mpp6 portion binding the exosome, it is possible, therefore, that phosphorylation of S119 might influence the Mpp6 association with preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 15 the exosome, possibly positively, since this phosphosite was recovered with both baits. Mtr4 has been shown to participate in pre -rRNA maturation by directly interacting and recruiting the exosome for processing (de la Cruz et al, 1998; Lingaraju et al, 2019). Although the phosphosite T34 was detected here, and also previously (Leutert et al. , 2023) , Mtr4 N -terminal 75 amino acids have been shown not to be necessary for the interaction with the exosome (Lingaraju et al., 2019). T34 phosphorylation may therefore not be involved in exosome activity control. Mtr4 is a subunit of the TRAMP complex, of which Air2 and Trf4 are also part (LaCava et al. , 2005) . We identified the phosphosite S49 of Air2, in a predicted low complexity region of the protein that controls Mtr4 RNA binding activity (Falk et al, 2014). S49 does not directly interact with Mtr4 (distances > 20 Å), but the intrinsically disordered region that contains S49, is enriched in charged residues that promote its high flexibility, suggesting that phosphorylation of S49 might reorient it toward Mtr4. This could stabilize th e Air2-Mtr4 interaction and modulate TRAMP complex activity during rRNA processing (Suppl. Fig. 2). Interestingly, although the TRAMP complex localizes to the nucleus, Air2 -S49 was only found phosphorylated in the Rrp46 sample, confirming that by using this bait, we isolate both, cytoplasmic and nuclear exosome complexes. These results also further imply a heterogeneity of nuclear exosome complexes, which might have distinct functional roles. Phosphorylated Ski7, a known cytoplasmic exosome adaptor (Araki et al., 2001), was exclusively detected in Rrp46 samples (Fig. 3; Table 1), validating the use of this core subunit as bait for identifying exosome interactors. Phosphosite preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 16 S90 is present in the middle of the N -terminal portion of Ski7, between the SKI complex-interacting and the exosome-interacting regions of Ski7, whereas S307 is inserted in its GTPase domain (Fig. 6; Suppl Fig. 3), which suggests that the phosphorylation of these residues might affect Ski7 function (Keidel et al, 2023; Kowalinski et al, 2016). Based on the observation that overexpression of Ski7 traps th e exosome in the cytoplasm, therefore affecting ribosome synthesis (Neto et al., 2025), to confirm the hypothesis that S90 phosphorylation might have a functional relevance, the genes of wild type Ski7 and the mutant Ski7-S90A were cloned in a plasmid, under control of MET25 promoter, and transformed into Δski7 strain to test the effects of their overexpression on cell growth. The results show that while Ski7 overexpression strongly inhibits growth, the non-phosphorylatable Ski7-S90A does not impair growth (Fig. 7), growing very similarly to the condition preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 17 when Ski7 is expressed at low levels (+Met; Fig. 7 ). These results c onfirm the importance of Ski7 phosphorylation for the regulation of its function. SSU processome subunit Utp14 phosphosites were also recovered in our samples, with the phosphoresidues S423, S424, and S488 identified both in Rrp6 and Rrp46 eluates (Table 1). These residues are present in Utp14 portion involved in the recruitment of the DEAH-box helicase Dhr1 (Buzovetsky & Klinge, 2025) and, therefore, the phosphorylation of these Ser residues might have functional relevance. Interestingly, we also identified phospho S738 exclusively in Rrp6 samples. This residue is located in the Utp14 region responsible for Dhr1 activation, which occurs right after the release of Rrp6 from the SSU processome (Buzovetsky & Klinge, 2025) . Importantly, all the Utp14 phosphosites have preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 18 previously been identified in global yeast proteomics analyses (Leutert et al. , 2023), validating our results. To investigate the evolutionary relevance of the phosphorylation sites identified here, we assessed the conservation of phosphorylated residues across orthologous proteins in human and mouse. The sequence alignments identified conserved phosphorylation sit es in corresponding positions of the orthologous sequences. The majority of phosphosites (approximately 58%) were not conserved in either human or mouse orthologs, but a subset of residues (12.4%) was conserved in both mammalian species, including those of Air2 (S49), Ctr9 (S1015, 1017), Enp2 (S550, S555), Nop13 (S2), Pxr1 (S230), Rps21B (S68), Rps6B (S232, S233), Rps7A (T117, T119), Rrp3 (S43, S45, S47), and Rrp4 (S152) (Table 2). The observed conservation of these phosphoresidues suggests potential functional importance across eukaryotes. To evaluate the functional importance of the identified phosphosites, we performed a broad evolutionary analysis, focusing on ribosomal proteins and ribosome assembly factors. We aligned the phosphopeptides identified for these proteins, with their corresponding sequences from 16 additional organisms and observed that many of these conserved peptides also contain conserved phosphoresidues. We then analysed the relative proportions of serine, threonine and tyrosine (STY) residues, as well as aspartic acid and glutamic acid (DE), relative to the total conserved peptides (Fig.8). preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 19 The Saccharomycetaceae clade has a strong conservation of STY - containing peptides (Fig. 8A, blue bars). As expected, the degree of conservation decreases as more evolutionar ily distant the organisms are from S. cerevisiae, as indicated by an increase in residues unrelated to phosphorylation (grey bars). In more distant organisms, the conservation of phosphorylatable “STY” residues decreases to approximately 25%. Interestingly, this reduction is accompanied by an increas e in phosphomimetic (negatively charged) amino acids (D or E) , represented by orange bars . Although these organisms hav e lost their phosphorylatable residues at these positions, the ir replacement by phosphomimetic residues suggests that the negative charge at these sites is important for protein structure or function (Fig. 8A). The identified S. cerevisiae phosphoresidues of exosome subunits an d cofactors, and of ribosome assembly factors were aligned with sequences from 16 additional organ isms and grouped by complex to analyze the evolutionary conservation of t he phosphorylation. The conservation profile mir rors th e one presented above, showing the phosphorylatable S , T, Y residues conserved positions in Saccharomycetaceae, while D, E substitute these residues in more distant organisms, preserving the negative charge (Fig. 8B). Interestingly, several residues are conserved from yeast to higher eukaryotes. This conservation underscores the importance of these phosphorylation sites for the structure / function of the RNA exosome complex and ribosome assembly factors, highlighting th e role of phosphorylation as a regulatory mechanism in RNA metabolism. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 20 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 21

The RNA exosome is involved in processing and degradation of all classes of RNA, both in nucleus and cytoplasm (Chlebowski et al, 2013; Januszyk & Lima, 2014; Lykke-Andersen et al, 2009; Schneider & Tollervey, 2013). In each cell compartment, the exosome interacts with and is regulated by a specific set of cofactors and interactors (Zinder & Lima, 2017), and these protein interactions may be affected by phosphorylation. In this study, we present a phosphoproteomic investigation of proteins associated with the Saccharomyces cerevisiae exosome by using subunits Rrp6 and Rrp46 as baits, and show evidence that phosphorylation may contribute to the structural and regulatory dynamics of the exosome in association with distinct pre -ribosomal intermediate particles and cytoplasmic cofactors. The majority of proteins interacting with the exosome participate in RNA processing pathway and our data show that these factors exhibit a large number of phosphorylated residues, with a predominance of serine phosphorylation, consistent with global eukaryotic phosphoproteome patterns (Ochoa et al, 2020). Phosphorylation was detected both within structural domains and in intrinsically disordered regions (IDRs), the latter being known for facilitating dynamic protein interactions. Our findings are in agreement wi th previous studies demonstrating that the recruitment of the exosome to pre -ribosomes is mediated by cofactors (Schneider & Tollervey, 2013; Sloan et al, 2012) and show their phosphorylation status in distinct complexes, which may influence those interactions. Although most of the phosphosites isolated here have already been identified in global analyses, which further validates our data, we detected phosphoresidues being co -purified with the exosome as part of pre -ribosomal particles, showing the maturation stage in which their phosphorylation is functionally relevant . In addition, we also detected phosphosites that had not been previously described. The evidence that the exosome is associated with pre-ribosomes is given by the number of proteins identified that participate in various pre-rRNA processing steps. We identified proteins associated with the pre-ribosomal particle 90S (e.g. Utp14, Mpp10), pre -60S (e.g. Nop4, Tif6), proteins involved in nuclea r transport of ribosomal particles (e.g. Mtr2, Srm1), subunits of snoRNP particles (e.g. Nop58), ribosomal proteins (e.g. Rps7, Rps14) and nuclear pore complex components (e.g. Nup2, Nup159), which give evidence preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 22 of the participation of the exosome in multiple steps of ribosomal maturation. The distinct phosphosites identified in the two samples further strengthen this conclusion. Based on these differences, we also hypothesize that the exosome complexes present in the nucleus may not be homogeneous, but rat her exist as Exo11 (containing Rrp6) or Exo10 (without Rrp6). This conclusion could also be inferred by previous co-immunoprecipitation of the exosome with Rrp43, in which Rrp6 was not co-purified (Lourenco et al., 2013), suggesting a loose association of this subunit with the exosome core. In addition to ribosome factors, we identified the phosphorylated exosome subunits Csl4, Rrp4 and Rrp6. Interestingly, Rrp4 -S237 phosphosite was only identified in Rrp4 6 samples, strengthening the hypothesis of these phosphorylation events being important for function, in this case, probably in the cytoplasm, or as part of the nuclear Exo10 . Furthermore, the cytoplasmic exosome cofactor Ski7 was identified exclusively in Rrp46 samples, validating the purification method. The importance of Ski7-S90 phosphorylation was confirmed here by changing this residue to alanine, which abolishes the negative effect of Ski7 overexpression on cell growth. Interestingly, S90 is part of helix H3 in the SKI complex-interacting portion of Ski7, in close proximity to the exosome- interacting region of Ski7 (positions 116-225; Fig. 6) (Kowalinski et al., 2016; Liu et al, 2016). It is possible, therefore, that phosphorylation of S90 might influence Ski7 interaction with the SKI complex, and also affect its interaction with the exosome, since Ski7 overexpression has been shown to trap the exosome in the cytoplasm (Neto et al., 2025). Our results corroborate the hypothesis that S90 phosphorylation is important for function , and might modulat e cytoplasmic RNA degradation pathways. The analysis of the phosphorylated residues across seventeen organisms ranging from yeast to humans show their evolutionary conservation, further strengthening the validity of our results. A lso confirming the impor tance of the phosphorylation sites identified here, posttranslational modifications of S. pombe exosome subunits Dis3 and Rrp6, and cofactor Mtr4, have been sho wn to be essential for normal cell growth and to fin e-tune exosome activity regulation (Telekawa et al., 2018). In conclusion, here we have identified ribosome assembly factors that are phosphorylated, modification that may affect their affinity for binding the preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 23 intermediate pre-ribosomal particles, and show evidence that the exosome may not be a homogeneous complex in the nucleus, but rather be present as Exo11 and Exo10 + Rrp6 in this cell compartment, and its subunits may also be subject to phosphorylation for functional regulation. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 30, 2026. ; https://doi.org/10.64898/2026.01.29.702549doi: bioRxiv preprint 24

We are grateful to all members of the Oliveira laboratory for help, reagents, advice and discussion. Funding This work was supported by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP - 20/00901-1 to C.C.O.). F.A.A., V.G.N., R.B.J., M.R.A.B and B.R.S.Q were supported by FAPESP fellowships ( 2021/14620-7, 22/00071-4, 2025/06224-5, 2021/14137-4, 2023/06344-5, respectively). LPPC L.P.C. was supported by a CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) fellowship.

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