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RNA splicing entails the coordinated interaction of more than 150 proteins in the spliceosome, one of the most complex of the cell's molecular machines. We previously discovered that the RNA-binding motif protein 17 (RBM17), a component of the spliceosome, is essential for survival and cell maintenance. Here, we find that it interacts with the spli...
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... also evaluated the mutual dependence among RBM17, U2SURP, and CHERP protein levels by measuring the abundance of U2SURP and CHERP proteins in response to a gradient of RBM17 protein obtained by treating HEK293T cells with different concentrations of siRBM17 ( Figures S4A and S4B). Levels of U2SURP and CHERP directly correlate to the amount of RBM17 at siRBM17 concentrations of 10 nM and lower ( Figure S4B), doses at which we could establish a clear and detectable RBM17 protein gradient. ...
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... also evaluated the mutual dependence among RBM17, U2SURP, and CHERP protein levels by measuring the abundance of U2SURP and CHERP proteins in response to a gradient of RBM17 protein obtained by treating HEK293T cells with different concentrations of siRBM17 ( Figures S4A and S4B). Levels of U2SURP and CHERP directly correlate to the amount of RBM17 at siRBM17 concentrations of 10 nM and lower ( Figure S4B), doses at which we could establish a clear and detectable RBM17 protein gradient. ...
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... even though reduced RBM17 protein levels do not seem to destabilize all the proteins that we validated as interactors (Figures 2A and 2B), we decided to explore the possibility that RBM17 reduction could affect the stability of the entire U2 snRNP spliceosomal complex. We measured the levels of seven additional factors that the Spliceosome database (Cvitkovic and Jurica, 2013) defines as either core or associated with the 17S-U2 snRNP complex ( Figures 4A and 4B); five of these were not detected in our IP-MS, and the remaining two, DDX42 and DDX46, did not validate by IP-WB as RBM17 interactors. This experiment showed that decreased levels of RBM17 in HEK293T cells slightly reduce the levels of these factors, but the effect size is smaller than what we observed for U2SURP and CHERP, with only U2AF1 showing about 30% reduction ( Figures 4A and 4B). ...
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... measured the levels of seven additional factors that the Spliceosome database (Cvitkovic and Jurica, 2013) defines as either core or associated with the 17S-U2 snRNP complex ( Figures 4A and 4B); five of these were not detected in our IP-MS, and the remaining two, DDX42 and DDX46, did not validate by IP-WB as RBM17 interactors. This experiment showed that decreased levels of RBM17 in HEK293T cells slightly reduce the levels of these factors, but the effect size is smaller than what we observed for U2SURP and CHERP, with only U2AF1 showing about 30% reduction ( Figures 4A and 4B). ...
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Citations
... For example, in S. cerevisiae, Cmg1, Ntr1, Pxr1 and Sqs1 bind to helicase Prp43, and Spp2 associates with Prp2 helicase [21,22,[25][26][27][28]. In human cells, the G-patch proteins CMTR1, GPATCH2, NKRF, PINX1, RBM5, RBM17, TFIP11 and ZGPAT interact with DHX15 helicase and GPKOW interact with DHX16 helicase [29][30][31][32]. In S. pombe, the G-patch proteins Ntr1 and Gpl1 were found to interact with Prp43 and Gih35 helicases, respectively [33][34][35]. ...
Pre-mRNA splicing plays a key role in the regulation of gene expression. Recent discoveries suggest that defects in pre-mRNA splicing, resulting from the dysfunction of certain splicing factors, can impact the expression of genes crucial for genome surveillance mechanisms, including those involved in cellular response to DNA damage. In this study, we analyzed how cells with a non-functional spliceosome-associated Gpl1–Gih35–Wdr83 complex respond to DNA damage. Additionally, we investigated the role of this complex in regulating the splicing of factors involved in DNA damage repair. Our findings reveal that the deletion of any component within the Gpl1–Gih35–Wdr83 complex leads to a significant accumulation of unspliced pre-mRNAs of DNA repair factors. Consequently, mutant cells lacking this complex exhibit increased sensitivity to DNA-damaging agents. These results highlight the importance of the Gpl1–Gih35–Wdr83 complex in regulating the expression of DNA repair factors, thereby protecting the stability of the genome following DNA damage.
... PPIA protein complex identification following 3XF-PPIA immunoprecipitation. Immunopurified samples were analysed by MS-based proteomics, as previously described 65 . Minor modifications from the previously cited protocol are listed here. ...
... Separated peptides were directly electro-sprayed into the mass spectrometer, controlled by Xcalibur software (Thermo Scientific) in data-dependent acquisition mode, selecting fragmentation spectra of the top 25 and 35 strongest ions for first samples and second samples, respectively. MS/MS spectra were searched against the target-decoy human RefSeq database (released January 2019, containing 73,637 entries) with the software interface and search parameters previously described 65 . Variable modification of methionine oxidation and lysine acetylation was allowed. ...
... Following sample lysis and overnight trypsin digestion, reconstituted peptidic fractions were loaded onto a nano-HPLC 1000 system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific), with identical acquisition settings as previously described 67 . The trap and capillary HPLC columns have been described previously 65 . The search of resultant MS/MS spectra against the target-decoy mouse RefSeq database (released June 2015, containing 58,549 entries) was carried out with the Proteome Discoverer 2.1 interface (Thermo Fisher) with the Mascot 2.4 algorithm (Matrix Science). ...
Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid–liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing.
... Previous research has shown this protein to regulate the expression and splicing of RNA-processing proteins. 33 Moreover, this RBM17 knockdown and control contrast reveals differential expression of several protein factors that facilitate APA. 34 These differential core APA factors include NUDT21, CSTF3, FIP1L1, PPP1CB, CPSF3, PPP1CA, PAPOLA, CSTF2, PABPN1, RBBP6, and PCF11 ( Figure 5A). ...
... We used CrypSplice [8,50] to infer novel cryptic splice sites. CrypSplice evade full-length isoform and ASM quantifications and is based only on junction counts. ...
MATR3 is an RNA-binding protein implicated in neurodegenerative and neurodevelopmental diseases. However, little is known regarding the role of MATR3 in cryptic splicing within the context of functional genes and how disease-associated variants impact this function. We show that loss of MATR3 leads to cryptic exon inclusion in many transcripts. We reveal that ALS-linked S85C pathogenic variant reduces MATR3 solubility but does not impair RNA binding. In parallel, we report a novel neurodevelopmental disease-associated M548T variant, located in the RRM2 domain, which reduces protein solubility and impairs RNA binding and cryptic splicing repression functions of MATR3. Altogether, our research identifies cryptic events within functional genes and demonstrates how disease-associated variants impact MATR3 cryptic splicing repression function.
... One identified protein that depends on this modification is the RNA-binding motif protein 17 (RBM17) (Lim et al., 2008). RBM17, a member of spliceosome, is crucial for mRNA splicing, and it was shown to be important for Purkinje cell maintenance (De Maio et al., 2018;Tan et al., 2016). In this study, the expanded polyglutamine tract increased the binding of ataxin-1 to RBM17, in mutant ataxin-1-expressing HEK293 cells and a SCA1 knock-in mouse. ...
Polyglutamine spinocerebellar ataxias (PolyQ SCAs) represent a group of monogenetic diseases in which the expanded polyglutamine repeats give rise to a mutated protein. The abnormally expanded polyglutamine protein produces aggregates and toxic species, causing neuronal dysfunction and neuronal death. The main symptoms of these disorders include progressive ataxia, motor dysfunction, oculomotor impairment, and swallowing problems. Nowadays, the current treatments are restricted to symptomatic alleviation, and no existing therapeutic strategies can reduce or stop the disease progression. Even though the origin of these disorders has been associated with polyglutamine‐induced toxicity, RNA toxicity has recently gained relevance in polyQ SCAs molecular pathogenesis. Therefore, the research's focus on RNA metabolism has been increasing, especially on RNA‐binding proteins (RBPs). The present review summarizes RNA metabolism, exposing the different processes and the main RBPs involved. We also explore the mechanisms by which RBPs are dysregulated in PolyQ SCAs. Finally, possible therapies targeting the RNA metabolism are presented as strategies to reverse neuropathological anomalies and mitigate physical symptoms.
... Whereas the number of genes affected in our study is fewer compared to another recent study that has used siRNA-mediated knockdown of CHERP in similar cell types, there was a significant overlap of differentially expressed genes. 40 We surmise that this difference in gene number is probably due to a different knockdown approach. ...
... 51,52 Recent research has identified CHERP as part of a stable complex with another two splicing factors, U2SURP and RBM17. 40 This complex and its associated alternative splicing events are implicated in colorectal tumorigenesis development. 22 The combination of our recent findings with previous data provides strong evidence implicating the CHERP/U2SURP/RBM17 complex in splicing events of numerous transcription factors driving tumorigenesis. ...
During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modifications on the C-terminal domain (CTD) of the largest subunit, generating an information-rich PTM landscape that transcriptional regulators bind. The phosphorylation of Ser5 and Ser2 of CTD heptad occurs spatiotemporally with the transcriptional stages, recruiting different transcriptional regulators to Pol II. To delineate the protein interactomes at different transcriptional stages, we reconstructed phosphorylation patterns of the CTD at Ser5 and Ser2 in vitro. Our results showed that distinct protein interactomes are recruited to RNA polymerase II at different stages of transcription by the phosphorylation of Ser2 and Ser5 of the CTD heptads. In particular, we characterized calcium homeostasis endoplasmic reticulum protein (CHERP) as a regulator bound by phospho-Ser2 heptad. Pol II association with CHERP recruits an accessory splicing complex whose loss results in broad changes in alternative splicing events. Our results shed light on the PTM-coded recruitment process that coordinates transcription.
... Interestingly, a significant number (40%) of the identified RBM5 interactors also constitute known HTT interactors (Fig. 6d) 79,80 , with the shared interactors between RBM5 and HTT highly enriched for proteins involved in mRNA metabolic processes, neuron specific processes and Huntington's disease ( Supplementary Fig. 8e). This was specific to RBM5, as a much lower overlap was observed between proteins interacting with HTT and the interactomes of three RBPs unrelated to this study ( Supplementary Fig. 8f) [81][82][83] . ...
RNA-binding proteins (RBPs) are key players regulating RNA processing and are associated with disorders ranging from cancer to neurodegeneration. Here, we present a proteomics workflow for large-scale identification of RBPs and their RNA-binding regions in the mammalian brain identifying 526 RBPs. Analysing brain tissue from males of the Huntington’s disease (HD) R6/2 mouse model uncovered differential RNA-binding of the alternative splicing regulator RBM5. Combining several omics workflows, we show that RBM5 binds differentially to transcripts enriched in pathways of neurodegeneration in R6/2 brain tissue. We further find these transcripts to undergo changes in splicing and demonstrate that RBM5 directly regulates these changes in human neurons derived from embryonic stem cells. Finally, we reveal that RBM5 interacts differently with several known huntingtin interactors and components of huntingtin aggregates. Collectively, we demonstrate the applicability of our method for capturing RNA interactor dynamics in the contexts of tissue and disease.
... We benchmarked PolyAMiner-Bulk against several of the most common bulk RNA-seq-based APA methods to examine the APA dynamics in a bulk RNA-seq dataset of immortalized human embryonic kidney (HEK293) cells with and without siRNA-mediated knockdown of RNA-binding motif protein 17 (RBM17) (GEO: GSE107648). Previous research has shown this protein to regulate the expression and splicing of RNA-processing proteins (36). Moreover, this RBM17 knockdown and control contrast reveals differential expression of several protein factors that facilitate alternative polyadenylation (37). ...
More than half of human genes exercise alternative polyadenylation (APA) and generate mRNA transcripts with varying 3' untranslated regions (UTR). However, current computational approaches for identifying C/PASs and quantifying 3'UTR length changes from bulk RNA-seq data fail to unravel actual tissue- and disease-specific APA dynamics. Here, we developed a next-generation bioinformatics algorithm and application, PolyAMiner-Bulk, that utilizes an attention-based machine learning architecture and an improved vector projection-based engine to assess differential APA dynamics accurately. When applied to earlier studies, PolyAMiner-Bulk accurately identified more than twice the number of APA changes in an RBM17 knockdown bulk RNA-seq benchmarking dataset compared to current generation tools. Moreover, on a separate dataset, PolyAMiner-Bulk revealed novel APA dynamics and pathways in scleroderma pathology and identified a novel APA gene that was independently identified as being involved in scleroderma pathogenesis in a separate study. Lastly, as a proof-of-principle, we used PolyAMiner-Bulk to analyze bulk RNA-seq data of post-mortem prefrontal cortexes from the ROSMAP data consortium and unraveled novel APA dynamics in Alzheimer's Disease. Our method, PolyAMiner-Bulk, creates a paradigm for future alternative polyadenylation analysis from bulk RNA-seq data.
... ; https://doi.org/10.1101/2022.11.14.516533 doi: bioRxiv preprint Sharma et al., 2008). RBM17 also interacts with DHX15 and contributes to alternative splicing (De Maio et al., 2018). We observed minimal overlap between introns affected by RBM17 and DHX15, but substantial overlap and correlation between introns repressed by DHX15 and SUGP1 (Fig. 4D), suggesting that SUGP1 is the primary G-patch partner of DHX15 for splicing QC. ...
Pre-mRNA splicing is surveilled at different stages by quality control (QC) mechanisms. The leukemia-associated DExH-box family helicase hDHX15/scPrp43, is known to disassemble spliceosomes after splicing. Here, using rapid protein depletion and analysis of nascent and mature RNA to enrich for direct effects, we identified a widespread splicing QC function for DHX15 in human cells, consistent with recent in vitro studies. We found that suboptimal introns with weak splice sites, multiple branch points, and cryptic introns are repressed by DHX15, suggesting a general role in promoting splicing fidelity. We identified SUGP1 as a G-patch factor that activates the splicing QC function of DHX15. This interaction is dependent on both the ATPase activity of DHX15 and the ULM domain of SUGP1. Together, our results support a model in which DHX15 plays a major role in splicing QC when recruited and activated by SUGP1.
... (c) Differences of the chemical shift values between the S1BRp-bound form of SURP1 and the chimeric form of SURP1 in the chimera. The values were obtained from [ 1 H, 15 N]-HSQC spectrum of labeled SURP1 in the presence of S1BRp and from that of the labeled chimera ( Figure S4b). The absolute values of the chemical shift difference between the two forms Δδ( 15 N + 1 H N ) are shown. ...
... In the chemical shift perturbation and biochemical experiments, 15 N-and 13 C-labeled and non-labeled SF3A1 SURP1 were prepared as previously described. 8 Briefly, DNA encoding SURP1 (res. ...
... 295-335), a 41-residue synthesized peptide, was dissolved in 20 mM sodium phosphate buffer (pH 7.0) containing 1 mM d-DTT to prepare a 2 mM stock solution. 2D [ 1 H, 15 N]-HSQC spectra of SURP1 at a concentration of 180 μM were recorded in the absence of S1BRp, whereas those at a concentration of 900 μM were recorded in the presence of S1BRp at the same concentration. The spectra for the sequence-specific assignments of the resonances are listed in Table S4. ...
SURP domains are exclusively found in splicing‐related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1–α2–310–α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α‐helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16‐residue α‐helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio‐layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 μM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2.
