The U1-70K and SRSF1 interaction is modulated by phosphorylation during the early stages of spliceosome assembly.

In eukaryotes, pre-mRNA splicing is vital for RNA processing and orchestrated by the spliceosome, whose assembly starts with the interaction between U1-70K and SR proteins. Despite the significance of the U1-70K/SR interaction, the dynamic nature of the complex and the challenges in obtaining soluble U1-70K have impeded a comprehensive understanding of the interaction at the structural level for decades. We overcome the U1-70K solubility issues, enabling us to characterize the interaction between U1-70K and SRSF1, a representative SR protein. We unveil specific interactions: phosphorylated SRSF1 RS with U1-70K BAD1, and SRSF1 RRM1 with U1-70K RRM. The RS/BAD1 interaction plays a dominant role, whereas the interaction between the RRM domains further enhances the stability of the U1-70K/SRSF1 complex. The RRM interaction involves the C-terminal extension of U1-70K RRM and the conserved acid patches on SRSF1 RRM1 that is involved in SRSF1 phase separation. Our circular dichroism spectra reveal that BAD1 adapts an α-helical conformation and RS is intrinsically disordered. Intriguingly, BAD1 undergoes a conformation switch from α-helix to ß-strand and random coil upon RS binding. In addition to the regulatory mechanism via SRSF1 phosphorylation, the U1-70K/SRSF1 interaction is also regulated by U1-70K BAD1 phosphorylation. We find that U1-70K phosphorylation inhibits the U1-70K and SRSF1 interaction. Our structural findings are validated through in vitro splicing assays and in-cell saturated domain scanning using the CRISPR method, providing new insights into the intricate regulatory mechanisms of pre-mRNA splicing.

Unearthing a novel function of SRSF1 in binding and unfolding of RNA G-quadruplexes.

SRSF1 governs splicing of over 1500 mRNA transcripts. SRSF1 contains two RNA-recognition motifs (RRMs) and a C-terminal Arg/Ser-rich region (RS). It has been thought that SRSF1 RRMs exclusively recognize single-stranded exonic splicing enhancers, while RS lacks RNA-binding specificity. With our success in solving the insolubility problem of SRSF1, we can explore the unknown RNA-binding landscape of SRSF1. We find that SRSF1 RS prefers purine over pyrimidine. Moreover, SRSF1 binds to the G-quadruplex (GQ) from the ARPC2 mRNA, with both RRMs and RS being crucial. Our binding assays show that the traditional RNA-binding sites on the RRM tandem and the Arg in RS are responsible for GQ binding. Interestingly, our FRET and circular dichroism data reveal that SRSF1 unfolds the ARPC2 GQ, with RS leading unfolding and RRMs aiding. Our saturation transfer difference NMR results discover that Arg residues in SRSF1 RS interact with the guanine base but not other nucleobases, underscoring the uniqueness of the Arg/guanine interaction. Our luciferase assays confirm that SRSF1 can alleviate the inhibitory effect of GQ on gene expression in the cell. Given the prevalence of RNA GQ and SR proteins, our findings unveil unexplored SR protein functions with broad implications in RNA splicing and translation.

Discovery of a pre-mRNA structural scaffold as a contributor to the mammalian splicing code.

The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of 3D structural scaffold of AdML-a model pre-mRNA substrate-guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5' and 3' splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a 3D structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable 3D structural scaffolds to the splicing code across the mammalian transcriptome.

The RNA-binding profile of the splicing factor SRSF6 in immortalized human pancreatic ß-cells.

In pancreatic ß-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes ß-cell demise through splicing dysregulation of central genes for ß-cells function and survival, but how RNAs are targeted by SRSF6 remains poorly understood. Here, we define the SRSF6 binding landscape in the human pancreatic ß-cell line EndoC-ßH1 by integrating individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) under basal conditions with RNA sequencing after SRSF6 knockdown. We detect thousands of SRSF6 bindings sites in coding sequences. Motif analyses suggest that SRSF6 specifically recognizes a purine-rich consensus motif consisting of GAA triplets and that the number of contiguous GAA triplets correlates with increasing binding site strength. The SRSF6 positioning determines the splicing fate. In line with its role in ß-cell function, we identify SRSF6 binding sites on regulated exons in several diabetes susceptibility genes. In a proof-of-principle, the splicing of the susceptibility gene LMO7 is modulated by antisense oligonucleotides. Our present study unveils the splicing regulatory landscape of SRSF6 in immortalized human pancreatic ß-cells.

Analysis of the acrolein-modified sites of apolipoprotein B-100 in LDL.

We have reported that acrolein-conjugated low-density lipoprotein (Acro-LDL) uptake by scavenger receptor class A type 1 (SR-A1) can mediate macrophage foam cell formation. The purpose of this study was to determine which amino acid residues of apoB protein in LDL are conjugated with acrolein. Acro-apoB was prepared by incubation of LDL with acrolein (10 to 60 µM) at 37 °C for 7 days. Identification of acrolein-conjugated amino acid residues in apoB was performed using LC-MS/MS. The levels of acrolein-conjugated amino acid residues of apoB as well as crosslinking apoB increased in proportion to acrolein concentration. The level of LDL uptake by macrophages was parallel with the acrolein-conjugated monomer apoB. Acrolein-conjugated amino acid residues in apoB were C212, K327, K742, K949, K1087, H1923, K2634, K3237 and K3846. The NH2-teriminal four amino acid residues (C212, K327, K742 and K949) were located at the scavenger receptor SR-A1 recognition site, suggesting that these four acrolein-conjugated amino acids are involved in the rapid uptake of Acro-LDL by macrophages. It is proposed that the rapid uptake of LDL by macrophages is dependent on acrolein conjugation of four amino acids residues at the scavenger receptor recognition site of apoB in LDL.

AMP-activated protein kinase regulates ß-catenin protein synthesis by phosphorylating serine/arginine-rich splicing factor 9.

ß-catenin is a multi-functional protein with a central role in regulating embryonic development and tissue homeostasis. The abnormal accumulation of ß-catenin, due to disrupted ß-catenin degradation or unregulated ß-catenin synthesis, causes the development of cancer. A recent study showed that the overexpression of proto-oncogene serine/arginine-rich splicing factor 9 (SRSF9) promotes ß-catenin accumulation via binding ß-catenin mRNA and enhancing its translation in a manner that is dependent on the mechanistic target of rapamycin (mTOR). However, the regulation of the interaction between SRSF9 and mRNA of ß-catenin remains unclear. Here, we show that AMP-activated protein kinase (AMPK) phosphorylates SRSF9 at the RNA-interacting SWQDLKD motif that plays a major role in determining substrate specificity. The phosphorylation by AMPK inhibits the binding of SRSF9 to ß-catenin mRNA and suppresses ß-catenin protein synthesis caused by SRSF9 overexpression without changing the ß-catenin mRNA levels. Our findings suggest that AMPK activators are potential therapeutic targets for SRSF9-derived overproduction of ß-catenin in cancer cells.

Identification and subcellular localization of splicing factor arginine/serine-rich 10 in the microsporidian Nosema bombycis.

Splicing factors are important components of RNA editing in eukaryotic organisms and can produce many functional and coding genes, which is an indispensable step for the correct expression of corresponding proteins. In this study, we identified splicing factor arginine/serine-rich 10 protein in the microsporidian Nosema bombycis and named it NbSRSF10. The NbSRSF10 gene contains a complete ORF of 1449 bp in length that encodes a 482-amino acid polypeptide. The isoelectric point (pI) of the protein encoded by NbSRSF10 gene was 4.94. NbSRSF10 has a molecular weight of 54.6 kD and has no signal peptide. NbSRSF10 is comprised of arginine (11.41%), glutamic acid (11.41%) and serine (9.54%) among the total amino acids, and 7 α-helix, 7 ß-sheet and 15 random coils in secondary structure, and contains 71 phosphorylation sites, 22 N-glycosylation sites and 20 O-glycosylation sites. The three-dimensional structure of NbSRSF10 is similar to that of transformer-2 beta of Homo sapiens (hTra2-ß). Indirect immunofluorescence showed that the NbSRSF10 is localized in the cytoplasm of the dormant microsporidian spore and is transferred to the nuclei when N. bombycis develops into the proliferative and sporogonic phase. qPCR revealed that the relative expression of NbSRSF10 increased in the meronts stage and was found at a relatively low level in the sporogonic phase of development of N. bombycis, and was up-regulated again during infection in the host cell and early proliferative phase of second life cycle. These results suggested that the NbSRSF10 may participate in the whole life cycle and play an important role in transcription regulation of N. bombycis.

HnRNPA1 interacts with G-quadruplex in the TRA2B promoter and stimulates its transcription in human colon cancer cells.

The human TRA2B gene consists of 10 exons and 9 introns and produces 5 splice isoforms (TRA2ß1 to TRA2ß5). TRA2B exon 2 encodes multiple premature termination codons. TRA2ß1 lacks exon 2 and is translated into a functional transformer 2ß (Tra2ß) protein, whereas TRA2ß4 contains 10 exons and works as a functional RNA. Overexpressed Tra2ß and ectopic expression of TRA2ß4 may be oncogenic. We found that heterogeneous nuclear ribonucleoprotein (hnRNP)A1 and hnRNPU interacted with TRA2ß4 exon 2. Minigene assays revealed that hnRNPA1 facilitated inclusion of exon 2, whereas hnRNPU promoted its skipping. However, knockdown of hnRNPA1 or hnRNPU reduced both TRA2ß1 and TRA2ß4 levels, and overexpression of these hnRNPs increased levels of both isoforms, suggesting that hnRNPA1 and hnRNPU mainly regulate the transcription of TRA2B. In fact, hnRNPA1 and hnRNPU positively regulated the promoter activity of TRA2B. Circular dichroism analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated the presence of G-quadruplex (G4) formation in the promoter of TRA2B. Formation of G4 suppressed TRA2B transcription, whereas hnRNPA1, but not hnRNPU, interacted with the G4 to facilitate transcription. Our results suggest that hnRNPA1 may modulate TRA2B transcription through its regulation of G4 formation in its promoter in colon cancer cells.

Modeling RNA-Binding Protein Specificity In Vivo by Precisely Registering Protein-RNA Crosslink Sites.

RNA-binding proteins (RBPs) regulate post-transcriptional gene expression by recognizing short and degenerate sequence motifs in their target transcripts, but precisely defining their binding specificity remains challenging. Crosslinking and immunoprecipitation (CLIP) allows for mapping of the exact protein-RNA crosslink sites, which frequently reside at specific positions in RBP motifs at single-nucleotide resolution. Here, we have developed a computational method, named mCross, to jointly model RBP binding specificity while precisely registering the crosslinking position in motif sites. We applied mCross to 112 RBPs using ENCODE eCLIP data and validated the reliability of the discovered motifs by genome-wide analysis of allelic binding sites. Our analyses revealed that the prototypical SR protein SRSF1 recognizes clusters of GGA half-sites in addition to its canonical GGAGGA motif. Therefore, SRSF1 regulates splicing of a much larger repertoire of transcripts than previously appreciated, including HNRNPD and HNRNPDL, which are involved in multivalent protein assemblies and phase separation.

B52 promotes alternative splicing of Dscam in Chinese mitten crab, Eriocheir sinensis.

B52 is a member of the classical serine/arginine (SR)-rich proteins, which are phylogenetically conserved and play significant roles in mRNA maturation, including alternative splicing. In the present study, the docking site, selector sequences and locus control region of the Chinese mitten crab (Eriocheir sinensis) Down syndrome cell adhesion molecule (EsDscam) were identified. Alternative splicing of Dscam is essential to generate different isoforms. We also isolated and characterised the B52 gene from E. sinensis (EsB52). The 876 bp open reading frame of EsB52 encodes a 291 amino acid residue polypeptide, and EsB52 has two RNA recognition motifs (RRMs) at the N-terminus and an arginine/serine-rich domain at the C-terminus. Each RRM contains two degenerate short submotifs, RNP-1 and RNP2. Analysis of tissue distribution revealed that EsB52 mRNA expression was widespread in all tested tissues, and especially high in brain and hemocytes. In hemocytes, EsB52 was upregulated significantly after stimulation with pathogen-associated molecular patterns and bacteria. Furthermore, EsB52 RNAi decreased the number of Ig7 inclusion in mRNA rather than Ig2 or Ig3. Taken together, these findings suggest that EsB52 acts as an alternative splicing activator of EsDscam.

Distinct mechanisms govern the phosphorylation of different SR protein splicing factors.

Serine-arginine (SR) proteins are essential splicing factors containing a canonical RNA recognition motif (RRM), sometimes followed by a pseudo-RRM, and a C-terminal arginine/serine-rich (RS) domain that undergoes multisite phosphorylation. Phosphorylation regulates the localization and activity of SR proteins, and thus may provide insight into their differential biological roles. The phosphorylation mechanism of the prototypic SRSF1 by serine-arginine protein kinase 1 (SRPK1) has been well-studied, but little is known about the phosphorylation of other SR protein members. In the present study, interaction and kinetic assays unveiled how SRSF1 and the single RRM-containing SRSF3 are phosphorylated by SRPK2, another member of the SRPK family. We showed that a conserved SRPK-specific substrate-docking groove in SRPK2 impacts the binding and phosphorylation of both SR proteins, and the localization of SRSF3. We identified a nonconserved residue within the groove that affects the kinase processivity. We demonstrated that, in contrast to SRSF1, for which SRPK-mediated phosphorylation is confined to the N-terminal region of the RS domain, SRSF3 phosphorylation sites are spread throughout its entire RS domain in vitro Despite this, SRSF3 appears to be hypophosphorylated in cells at steady state. Our results suggest that the absence of a pseudo-RRM renders the single RRM-containing SRSF3 more susceptible to dephosphorylation by phosphatase. These findings suggest that the single RRM- and two RRM-containing SR proteins represent two subclasses of phosphoproteins in which phosphorylation statuses are maintained by unique mechanisms, and pose new directions to explore the distinct roles of SR proteins in vivo.

Molecular interactions connecting the function of the serine-arginine-rich protein SRSF1 to protein phosphatase 1.

Splicing generates many mRNA strands from a single precursor mRNA, expanding the proteome and enhancing intracellular diversity. Both initial assembly and activation of the spliceosome require an essential family of splicing factors called serine-arginine (SR) proteins. Protein phosphatase 1 (PP1) regulates the SR proteins by controlling phosphorylation of a C-terminal arginine-serine-rich (RS) domain. These modifications are vital for the subcellular localization and mRNA splicing function of the SR protein. Although PP1 has been shown to dephosphorylate the prototype SR protein splicing factor 1 (SRSF1), the molecular nature of this interaction is not understood. Here, using NMR spectroscopy, we identified two electrostatic residues in helix α2 and a hydrophobic residue in helix α1 in the RNA recognition motif 1 (RRM1) of SRSF1 that constitute a binding surface for PP1. Substitution of these residues dissociated SRSF1 from PP1 and enhanced phosphatase activity, reducing phosphorylation in the RS domain. These effects lead to shifts in alternative splicing patterns that parallel increases in SRSF1 diffusion from speckles to the nucleoplasm brought on by regiospecific decreases in RS domain phosphorylation. Overall, these findings establish a molecular and biological connection between PP1-targeted amino acids in an RRM with the phosphorylation state and mRNA-processing function of an SR protein.

Dynamic association of human mRNP proteins with mitochondrial tRNAs in the cytosol.

Cytoplasmic localization, stability, and translation of mRNAs are controlled by their dynamic association of numerous mRNA-binding (mRNP) proteins, including cold shock domain (CSD)-containing proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), and serine/arginine-rich (SR) proteins. Here, we demonstrate that the most abundant human mRNP protein, the CSD-containing Y-box-binding protein 1 (YBX1), the closely related YBX3 protein, and other mRNP proteins, such as SRSF1, SRSF2, SRSF3, hnRNP A1, and H, specifically and efficiently interact with overlapping sets of mitochondrial tRNAs (mt tRNAs). In vitro reconstitution and in vivo binding experiments show that YBX1 recognizes the D- and/or T-stem-loop regions of mt tRNAs through relying on the RNA-binding capacity of its CSD. Cell fractionation and in vivo RNA-protein cross-linking experiments demonstrate that YBX1 and YBX3 interact with mt tRNAs in the cytosol outside of mitochondria. Cell fractionation and fluorescence in situ hybridization experiments provide evidence that mitochondrial autophagy promotes the release of mt tRNAs from the mitochondria into the cytoplasm. Association of mRNP proteins with mt tRNAs is highly dynamic; it is rapidly increased upon transcription inhibition and decreased during apoptosis. Although the cytoplasmic function of mt tRNAs remains elusive, their dynamic interactions with key mRNA-binding proteins may influence cytoplasmic mRNA stability and/or translation.

Identification of Novel Functional Variants of SIN3A and SRSF1 among Somatic Variants in Acute Myeloid Leukemia Patients.

The advent of massively parallel sequencing, also called next-generation sequencing (NGS), has dramatically influenced cancer genomics by accelerating the identification of novel molecular alterations. Using a whole genome sequencing (WGS) approach, we identified somatic coding and noncoding variants that may contribute to leukemogenesis in 11 adult Korean acute myeloid leukemia (AML) patients, with serial tumor samples (primary and relapse) available for 5 of them; somatic variants were identified in 187 AML-related genes, including both novel (SIN3A, C10orf53, PTPRR, and RERGL) and well-known (NPM1, RUNX1, and CEPBA) AML-related genes. Notably, SIN3A expression shows prognostic value in AML. A newly designed method, referred to as "hot-zone" analysis, detected two putative functional noncoding variants that can alter transcription factor binding affinity near PPP1R10 and SRSF1. Moreover, the functional importance of the SRSF1 noncoding variant was further investigated by luciferase assays, which showed that the variant is critical for the regulation of gene expression leading to leukemogenesis. We expect that further functional investigation of these coding and noncoding variants will contribute to a more in-depth understanding of the underlying molecular mechanisms of AML and the development of targeted anti-cancer drugs.

Mechanism of Nonsense-Mediated mRNA Decay Stimulation by Splicing Factor SRSF1.

The splicing factor SRSF1 promotes nonsense-mediated mRNA decay (NMD), a quality control mechanism that degrades mRNAs with premature termination codons (PTCs). Here we show that transcript-bound SRSF1 increases the binding of NMD factor UPF1 to mRNAs while in, or associated with, the nucleus, bypassing UPF2 recruitment and promoting NMD. SRSF1 promotes NMD when positioned downstream of a PTC, which resembles the mode of action of exon junction complex (EJC) and NMD factors. Moreover, splicing and/or EJC deposition increase the effect of SRSF1 on NMD. Lastly, SRSF1 enhances NMD of PTC-containing endogenous transcripts that result from various events. Our findings reveal an alternative mechanism for UPF1 recruitment, uncovering an additional connection between splicing and NMD. SRSF1's role in the mRNA's journey from splicing to decay has broad implications for gene expression regulation and genetic diseases.

SRp55 Regulates a Splicing Network That Controls Human Pancreatic ß-Cell Function and Survival.

Progressive failure of insulin-producing ß-cells is the central event leading to diabetes, but the signaling networks controlling ß-cell fate remain poorly understood. Here we show that SRp55, a splicing factor regulated by the diabetes susceptibility gene GLIS3, has a major role in maintaining the function and survival of human ß-cells. RNA sequencing analysis revealed that SRp55 regulates the splicing of genes involved in cell survival and death, insulin secretion, and c-Jun N-terminal kinase (JNK) signaling. In particular, SRp55-mediated splicing changes modulate the function of the proapoptotic proteins BIM and BAX, JNK signaling, and endoplasmic reticulum stress, explaining why SRp55 depletion triggers ß-cell apoptosis. Furthermore, SRp55 depletion inhibits ß-cell mitochondrial function, explaining the observed decrease in insulin release. These data unveil a novel layer of regulation of human ß-cell function and survival, namely alternative splicing modulated by key splicing regulators such as SRp55, that may cross talk with candidate genes for diabetes.

Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing.

Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3-dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3-dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3-dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3ß phosphorylated these proteins in vitro RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing.

Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function.

RBM25 is a global splicing factor promoting inclusion of alternatively spliced exons and is itself regulated by lysine mono-methylation.

In eukaryotes, precursor mRNA (pre-mRNA) splicing removes non-coding intron sequences to produce mature mRNA. This removal is controlled in part by RNA-binding proteins that regulate alternative splicing decisions through interactions with the splicing machinery. RNA binding motif protein 25 (RBM25) is a putative splicing factor strongly conserved across eukaryotic lineages. However, the role of RBM25 in global splicing regulation and its cellular functions are unknown. Here we show that RBM25 is required for the viability of multiple human cell lines, suggesting that it could play a key role in pre-mRNA splicing. Indeed, transcriptome-wide analysis of splicing events demonstrated that RBM25 regulates a large fraction of alternatively spliced exons throughout the human genome. Moreover, proteomic analysis indicated that RBM25 interacts with components of the early spliceosome and regulators of alternative splicing. Previously, we identified an RBM25 species that is mono-methylated at lysine 77 (RBM25K77me1), and here we used quantitative mass spectrometry to show that RBM25K77me1 is abundant in multiple human cell lines. We also identified a region of RBM25 spanning Lys-77 that binds with high affinity to serine- and arginine-rich splicing factor 2 (SRSF2), a crucial protein in exon definition, but only when Lys-77 is unmethylated. Together, our findings uncover a pivotal role for RBM25 as an essential regulator of alternative splicing and reveal a new potential mechanism for regulation of pre-mRNA splicing by lysine methylation of a splicing factor.

Comparative analyses of the thermodynamic RNA binding signatures of different types of RNA recognition motifs.

RNA recognition motifs (RRMs) are structurally versatile domains important in regulation of alternative splicing. Structural mechanisms of sequence-specific recognition of single-stranded RNAs (ssRNAs) by RRMs are well understood. The thermodynamic strategies are however unclear. Therefore, we utilized microcalorimetry and semi-empirical analyses to comparatively analyze the cognate ssRNA binding thermodynamics of four different RRM domains, each with a different RNA binding mode. The different binding modes are: canonical binding to the ß-sheet surface; canonical binding with involvement of N- and C-termini; binding to conserved loops; and binding to an α-helix. Our results identify enthalpy as the sole and general force driving association at physiological temperatures. Also, networks of weak interactions are a general feature regulating stability of the different RRM-ssRNA complexes. In agreement, non-polyelectrolyte effects contributed between ∼75 and 90% of the overall free energy of binding in the considered complexes. The various RNA binding modes also displayed enormous heat capacity differences, that upon dissection revealed large differential changes in hydration, conformations and dynamics upon binding RNA. Altogether, different modes employed by RRMs to bind cognate ssRNAs utilize various thermodynamics strategies during the association process.