Serine/arginine-rich Proteins Research Articles

Transcription and splicing variations of SR genes accompany with genome-wide accumulation of long-introns in pine

Most of mRNAs in Eukaryote were matured after the removal of introns in their pre-mRNA transcripts. Serine/arginine-rich (SR) proteins are a group of splicing regulators regulating the splicing processes globally. Expressions of SR proteins themselves were extensively regulated, at both transcription and splicing levels, under different environmental conditions, specially heat stress conditions. The pine genome is characterized by super-long and easily methylated introns in a large number of genes that derived from the extensive accumulation of transposons (TEs). Here, we identified and analyzed the phylogenetic characteristics of 24 SR proteins and their encoding genes from the pine genome. Then we explored transcription and pre-mRNA splicing expression patterns of SR genes in P. massoniana seedlings under normal and heat stress temperature conditions. Our results showed that the transcription patterns of SR genes in pine exhibited significant changes compared to other plant species, and these changes were not strictly correlated with the intron length and DNA methylation intensity of the SR genes. Interestingly, none of the long introns of SR genes underwent alternative splicing (AS) in our experiment. Furthermore, the intensity of AS regulation may be related to the potential DNA methylation intensity of SR genes. Taken together, this study explores for the first time the characteristics of significant variations in the transcription and splicing patterns of SR proteins in a plant species with an over-accumulation of super-long introns.

SCR106 splicing factor modulates abiotic stress responses by maintaining RNA splicing in rice.

Plants employ sophisticated molecular machinery to fine-tune their responses to growth, developmental, and stress cues. Gene expression influences plant cellular responses through regulatory processes such as transcription and splicing. Pre-mRNA is alternatively spliced to increase the genome coding potential and further regulate expression. Serine/arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize splicing cis-elements and regulate both constitutive and alternative splicing. Several studies have reported SR protein genes in the rice genome, subdivided into six subfamilies based on their domain structures. Here, we identified a new splicing factor in rice with an RNA recognition motif (RRM) and SR-dipeptides, which is related to the SR proteins, subfamily SC. OsSCR106 regulates pre-mRNA splicing under abiotic stress conditions. It localizes to the nuclear speckles, a major site for pre-mRNA splicing in the cell. The loss-of-function scr106 mutant is hypersensitive to salt, abscisic acid, and low-temperature stress, and harbors a developmental abnormality indicated by the shorter length of the shoot and root. The hypersensitivity to stress phenotype was rescued by complementation using OsSCR106 fused behind its endogenous promoter. Global gene expression and genome-wide splicing analysis in wild-type and scr106 seedlings revealed that OsSCR106 regulates its targets, presumably through regulating the alternative 3'-splice site. Under salt stress conditions, we identified multiple splice isoforms regulated by OsSCR106. Collectively, our results suggest that OsSCR106 is an important splicing factor that plays a crucial role in accurate pre-mRNA splicing and regulates abiotic stress responses in plants.

Identification of two short peptide motifs from serine/arginine-rich protein ribonucleic acid recognition motif-1 domain acting as splicing regulators.

Serine/arginine-rich (SR) proteins regulate pre-mRNA splicing. However, structurally similar proteins often behave differently in splicing regulation and the underlying mechanisms are largely unknown. Here, using SMN1/2 minigenes we extensively analyzed four SR proteins, SRSF1/5/6/9. In this study, the effects of these proteins on SMN1/2 exon 7 splicing when tethered at either intron 6 or 7 were evaluated using an MS2-tethering assay. Deletion analysis in four SR proteins and co-overexpression analysis were performed. Splicing outcomes varied among all four SR proteins, SRSF1 and SRSF5 function the same at the two sites, acting as repressor and stimulator, respectively; while SRSF6 and SRSF9 promote exon 7 inclusion at only one site. Further, the key domains of each SR proteins were investigated, which identified a potent inhibitory nonapeptide in the C-terminus of SRSF1/9 ribonucleic acid recognition motif-1 (RRM1) and a potent stimulatory heptapeptide at the N-terminus of SRSF5/6 RRM1. The insight of the four SR proteins and their domains in affecting SMN gene splicing brings a new perspective on the modes of action of SR proteins; and the functional peptides obtained here offers new ideas for developing splice switching-related therapies.

Posttranslational splicing modifications as a key mechanism in cytarabine resistance in acute myeloid leukemia

Despite the approval of several drugs for AML, cytarabine is still widely used as a therapeutic approach. However, 85% of patients show resistance and only 10% overcome the disease. Using RNA-seq and phosphoproteomics, we show that RNA splicing and serine-arginine-rich (SR) proteins phosphorylation were altered during cytarabine resistance. Moreover, phosphorylation of SR proteins at diagnosis were significantly lower in responder than non-responder patients, pointing to their utility to predict response. These changes correlated with altered transcriptomic profiles of SR protein target genes. Notably, splicing inhibitors were therapeutically effective in treating sensitive and resistant AML cells as monotherapy or combination with other approved drugs. H3B-8800 and venetoclax combination showed the best efficacy in vitro, demonstrating synergistic effects in patient samples and no toxicity in healthy hematopoietic progenitors. Our results establish that RNA splicing inhibition, alone or combined with venetoclax, could be useful for the treatment of newly diagnosed or relapsed/refractory AML.

The functions of a 5' tRNA-Ala-derived fragment in gene expression.

Transfer RNA (tRNA) can produce smaller RNA fragments called tRNA-derived fragments (tRFs). tRFs play critical roles in multiple cellular programs, although the functional mechanisms of tRFs remain largely unknown in plants. In this study, we examined the phenotype associated with 5' tRF-Ala (tRF-Ala, produced from tRNA-Ala) overexpression and knockdown lines (tDR-Ala-OE and tDR-Ala-kd, respectively) and the mechanisms by which tRF-Ala affects mRNA levels in Arabidopsis (Arabidopsis thaliana). We investigated the candidate proteins associated with tRF-Ala by quantitative proteomics and confirmed the direct interaction between tRF-Ala and the splicing factor SERINE-ARGININE RICH PROTEIN 34 (SR34). A transcriptome sequencing analysis showed that 318 genes among all the genes (786) with substantial alternative splicing (AS) variance in tDR-Ala-OE lines are targets of SR34. tRF-Ala diminished the binding affinity between SR34 and its targets by direct competition for interaction with SR34. These findings reveal the critical roles of tRF-Ala in regulating mRNA levels and splicing.

Repeat RNA structure and protein interactions influence RAN translation and neurodegenerative phenotypes in models of FTD and ALS

AbstractBackgroundThe most common genetic cause of Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) is an intronic GGGGCC hexanucleotide repeat expansion in C9orf72 (C9FTD/ALS). Transcribed GGGGCC repeat RNAs sequester RNA‐binding proteins (RBPs) into nuclear and cytoplasmic foci, which inhibits their normal functions. GGGGCC repeat RNAs also support translation of toxic repeat peptides through a process known as repeat associated non‐AUG (RAN) translation. Here we sought to understand how repeat RNA secondary structure and RBP engagements that occur during RNA transcription, processing, and trafficking influence GGGGCC RAN translation and repeat elicited toxicity, with a goal of identifying novel therapeutic targets.MethodWe developed a cellular RNA‐capture methodology coupled to mass spectrometry to identify GGGGCC repeat RNA‐interacting proteins within specific cellular compartments. In parallel, we used chemical approaches to probe GGGGCC repeat RNA secondary structures inside cells and RAN translation assays to determine whether repeat RNA secondary structures and/or interacting RBPs influence RAN translation efficiency. We employed Drosophila and neuronal models of repeat expansions to evaluate potential RBPs as disease modifiers.ResultOur RNA structural studies revealed that GGGGCC repeat RNAs form primarily hairpin structures, rather than G‐quadruplex structures, in cells. Folding repat RNAs into such hairpins supports RAN translation whereas folding repeat RNAs into g‐quadruplex structures strongly inhibits their translation. We identified multiple RBPs enriching in the nuclear fraction of GGGGCC RNA‐interactome, including hnRNPH1 and SR (serine/arginine‐rich domain) proteins, which are known components of the nuclear foci found in C9FTD/ALS patient cells. Our comparative analysis found that lowering SR protein expression or modifying its phosphorylation modified toxicity in Drosophila models of multiple repeat expansion disorders. Currently we are investigating how repeat RNA structures influence RBP interactions and whether structure dependent RBP interactions selectively modulate RAN translation.ConclusionWe present a comprehensive approach to evaluate the roles of repeat RNA secondary structures and critical RBPs in RNA toxicity underlying dementia‐associated repeat expansion disorders.

Plant serine/arginine-rich proteins: versatile players in RNA processing.

Serine/arginine-rich (SR) proteins participate in RNA processing by interacting with precursor mRNAs or other splicing factors to maintain plant growth and stress responses. Alternative splicing is an important mechanism involved in mRNA processing and regulation of gene expression at the posttranscriptional level, which is the main reason for the diversity of genes and proteins. The process of alternative splicing requires the participation of many specific splicing factors. The SR protein family is a splicing factor in eukaryotes. The vast majority of SR proteins' existence is an essential survival factor. Through its RS domain and other unique domains, SR proteins can interact with specific sequences of precursor mRNA or other splicing factors and cooperate to complete the correct selection of splicing sites or promote the formation of spliceosomes. They play essential roles in the composition and alternative splicing of precursor mRNAs, providing pivotal functions to maintain growth and stress responses in animals and plants. Although SR proteins have been identified in plants for three decades, their evolutionary trajectory, molecular function, and regulatory network remain largely unknown compared to their animal counterparts. This article reviews the current understanding of this gene family in eukaryotes and proposes potential key research priorities for future functional studies.

Cassava MeRS40 is required for the regulation of plant salt tolerance

Soil salinity affects the expression of serine/arginine-rich (SR) genes and isoforms by alternative splicing, which in turn regulates the adaptation of plants to stress. We previously identified the cassava spliceosomal component 35 like (SCL) and SR subfamilies, belonging to the SR protein family, which are extensively involved in responses to abiotic stresses. However, the post-transcriptional regulatory mechanism of cassava arginine/serine-rich (RS) subfamily in response to salt stress remains to be explored. In the current study, we identified 37 genes of the RS subfamily from 11 plant species and systematically investigated the transcript levels of the RS40 and RS31 genes under diverse abiotic stress conditions. Subsequently, an analysis of the conserved protein domains revealed that plant RS subfamily genes were likely to preserve their conserved molecular functions and played critical functional roles in responses to abiotic stresses. Importantly, we found that overexpression of MeRS40 in Arabidopsis enhanced salt tolerance by maintaining reactive oxygen species homeostasis and up-regulating the salt-responsive genes. However, overexpression of MeRS40 gene in cassava reduced salt tolerance due to the depression of its endogenous gene expression by negative autoregulation of its own pre-mRNA. Moreover, the MeRS40 protein interacted with MeU1-70Ks (MeU1-70Ka and MeU1-70Kb) in vivo and in vitro, respectively. Therefore, our ?ndings highlight the critical role of cassava SR proteins in responses to salt stress in plants.

SRSF2 in Sertoli cells is essential for testicular development and spermatogenesis in mice.

Sertoli cells are essential for testis development and normal spermatogenesis by providing support and nutrients. Pre-messenger RNA (pre-mRNA) processing is the basic mechanism required for gene expression, and members of the serine/arginine-rich protein (SR) family are key components of the machines that perform these basic processing events. Serine/arginine-rich splicing factor 2 (SRSF2) is an important member of the SR family; however, the physiological functions of SRSF2 in Sertoli cells are still unclear. Here, we found that SRSF2 was localized in the nuclei of Sertoli and germ cells in male mice at all stages by breeding Amh-Cre mice obtained with Srsf2-specific knockout in Sertoli cells to define the function of SRSF2 in Sertoli cells. The experimental results showed that specific deletion of SRSF2 impaired fetal Sertoli cell proliferation and induced abnormal apoptosis and severe DNA damage in seminiferous tubules, resulting in severe testicular dysplasia, seminiferous tubule atrophy, and almost no normal seminiferous tubules at postnatal day 14. Eventually, these changes resulted in failure to produce normal sperm and infertility. Further RNA-seq results showed that many key genes related to proliferation and apoptosis were downregulated; Racgap1 mRNA undergoes exon skipping. Thus, SRSF2-dependent Sertoli cells are essential for testicular development and male reproduction.

Arabidopsis SRPKII family proteins regulate flowering via phosphorylation of SR proteins and effects on gene expression and alternative splicing.

Alternative splicing of pre-mRNAs is crucial for plant growth and development. Serine/arginine-rich (SR) proteins are a conserved family of RNA binding proteins that are critical for both constitutive and alternative splicing. However, how phosphorylation of SR proteins regulates gene transcription and alternative splicing during plant development is poorly understood. We found that the Arabidopsis thaliana L. SR protein-specific kinase II family proteins (SRPKIIs) play an important role in plant development, including flowering. SRPKIIs regulate the phosphorylation status of a subset of specific SR proteins, including SR45 and SC35, which subsequently mediates their subcellular localization. A phospho-dead SR45 mutant inhibits the assembly of the ASAP (apoptosis-and splicing-associated protein) complex and thereby upregulates the expression of FLC (FLOWERING LOCUS C) via epigenetic modification. The splicing efficiency of FLC introns was significantly increased in the shoot apex of the srpkii mutant. Transcriptomic analysis revealed that SRPKIIs regulate the alternative splicing of approximately four hundred genes, which largely overlap with those regulated by SR45 and SC35-SCL family proteins. In summary, we found that Arabidopsis SRPKIIs specifically affect the phosphorylation status of a subset SR proteins and regulate the expression and alternative splicing of FLC to control flowering time.

Investigation of the regulatory effects of synthesized antisense oligonucleotides on androgen receptor (AR) exon 3 splicing in prostate cancer cells

The Androgen Receptor (AR) gene plays a key role in castration-resistant prostate cancer (CRPC). Controlling the progression of CRPC by inhibiting AR gene expression is one of the core directions for prostate cancer (Pca) drug development. A 23-amino acids retention, named exon 3a, into the DNA binding domain of the splice variant AR23 has been shown to prevent AR from entering the nucleus and restore the sensitivity of cancer cells to related therapies. In this study, we conducted a preliminary investigation of the splicing modulation of the AR gene in order to develop a splice-switching therapy for Pca by promoting exon 3a inclusion. Using mutagenesis-coupled RT-PCR with AR minigene and over-expression of certain splicing factors, we found that serine/arginine-rich (SR) proteins are key factors facilitating the recognition of the 3′ splice site of exon 3a (L-3′ SS), while the deletion or blocking of the polypyrimidine tract (PPT) region of the original 3′ splice site of exon 3 (S-3′ SS) could strongly enhance exon 3a splicing without affecting the function of any SR protein. Furthermore, we designed a series of antisense oligonucleotides (ASOs) to screen drug candidates, and ASOs targeting S-3′ SS and its PPT region or the exonic region of exon 3 turned out to be most effective in rescuing exon 3a splicing. A dose–response test indicated ASO12 as the lead candidate drug significantly promoting the inclusion of exon 3a to more than 85%. MTT assay confirmed that the cell proliferation was significantly inhibited after ASO treatment. Our results provide the first glance to AR splicing regulation. With several promising therapeutic ASO candidates obtained here, further development of ASO drugs to treat CRPC is strongly encouraged.

SGC-CLK-1: A chemical probe for the Cdc2-like kinases CLK1, CLK2, and CLK4

Small molecule modulators are important tools to study both basic biology and the complex signaling of protein kinases. The cdc2-like kinases (CLK) are a family of four kinases that have garnered recent interest for their involvement in a diverse set of diseases such as neurodegeneration, autoimmunity, and many cancers. Targeted medicinal chemistry around a CLK inhibitor hit identified through screening of a kinase inhibitor set against a large panel of kinases allowed us to identify a potent and selective inhibitor of CLK1, 2, and 4. Here, we present the synthesis, selectivity, and preliminary biological characterization of this compound – SGC-CLK-1 (CAF-170). We further show CLK2 has the highest binding affinity, and high CLK2 expression correlates with a lower IC50 in a screen of multiple cancer cell lines. Finally, we show that SGC-CLK-1 not only reduces serine arginine-rich (SR) protein phosphorylation but also alters SR protein and CLK2 subcellular localization in a reversible way. Therefore, we anticipate that this compound will be a valuable tool for increasing our understanding of CLKs and their targets, SR proteins, at the level of phosphorylation and subcellular localization.

Serine and arginine rich splicing factor 1: a potential target for neuroprotection and other diseases.

Alternative splicing is the process of producing variably spliced mRNAs by choosing distinct combinations of splice sites within a messenger RNA precursor. This splicing enables mRNA from a single gene to synthesize different proteins, which have different cellular properties and functions and yet arise from the same single gene. A family of splicing factors, Serine-arginine rich proteins, are needed to initiate the assembly and activation of the spliceosome. Serine and arginine rich splicing factor 1, part of the arginine/serine-rich splicing factor protein family, can either activate or inhibit the splicing of mRNAs, depending on the phosphorylation status of the protein and its interaction partners. Considering that serine and arginine rich splicing factor 1 is either an activator or an inhibitor, this protein has been studied widely to identify its various roles in different diseases. Research has found that serine and arginine rich splicing factor 1 is a key target for neuroprotection, showing its promising potential use in therapeutics for neurodegenerative disorders. Furthermore, serine and arginine rich splicing factor 1 might be used to regulate cancer development and autoimmune diseases. In this review, we highlight how serine and arginine rich splicing factor 1 has been studied concerning neuroprotection. In addition, we draw attention to how serine and arginine rich splicing factor 1 is being studied in cancer and immunological disorders, as well as how serine and arginine rich splicing factor 1 acts outside the central or peripheral nervous system.

The rice blast fungus SR protein 1 regulates alternative splicing with unique mechanisms.

Serine/arginine-rich (SR) proteins are well known as splicing factors in humans, model animals and plants. However, they are largely unknown in regulating pre-mRNA splicing of filamentous fungi. Here we report that the SR protein MoSrp1 enhances and suppresses alternative splicing in a model fungal plant pathogen Magnaporthe oryzae. Deletion of MoSRP1 caused multiple defects, including reduced virulence and thousands of aberrant alternative splicing events in mycelia, most of which were suppressed or enhanced intron splicing. A GUAG consensus bound by MoSrp1 was identified in more than 94% of the intron or/and proximate exons having the aberrant splicing. The dual functions of regulating alternative splicing of MoSrp1 were exemplified in enhancing and suppressing the consensus-mediated efficient splicing of the introns in MoATF1 and MoMTP1, respectively, which both were important for mycelial growth, conidiation, and virulence. Interestingly, MoSrp1 had a conserved sumoylation site that was essential to nuclear localization and enhancing GUAG binding. Further, we showed that MoSrp1 interacted with a splicing factor and two components of the exon-joining complex via its N-terminal RNA recognition domain, which was required to regulate mycelial growth, development and virulence. In contrast, the C-terminus was important only for virulence and stress responses but not for mycelial growth and development. In addition, only orthologues from Pezizomycotina species could completely rescue defects of the deletion mutants. This study reveals that the fungal conserved SR protein Srp1 regulates alternative splicing in a unique manner.

SR Splicing Factors Promote Cancer via Multiple Regulatory Mechanisms.

Substantial emerging evidence supports that dysregulated RNA metabolism is associated with tumor initiation and development. Serine/Arginine-Rich proteins (SR) are a number of ultraconserved and structurally related proteins that contain a characteristic RS domain rich in arginine and serine residues. SR proteins perform a critical role in spliceosome assembling and conformational transformation, contributing to precise alternative RNA splicing. Moreover, SR proteins have been reported to participate in multiple other RNA-processing-related mechanisms than RNA splicing, such as genome stability, RNA export, and translation. The dysregulation of SR proteins has been reported to contribute to tumorigenesis through multiple mechanisms. Here we reviewed the different biological roles of SR proteins and strategies for functional rectification of SR proteins that may serve as potential therapeutic approaches for cancer.

An IGF-1R-mTORC1-SRPK2 signaling Axis contributes to FASN regulation in breast cancer

BackgroundFatty acid synthase (FASN) expression is associated with a more aggressive breast cancer phenotype and is regulated downstream of receptor tyrosine kinase (RTK) signaling pathways. Recently, post transcriptional regulation of lipogenic transcripts have been demonstrated as being mediated downstream of serine-arginine rich protein kinase 2 (SRPK2), which acts to phosphorylate serine-arginine rich splicing factors (SRSFs), resulting in RNA binding and various RNA regulatory processes. Though post-transcriptional regulation of FASN has been studied previously, the upstream mediators of these pathways have not been elucidated.MethodsWestern blotting and RT-qPCR were utilized to demonstrate alterations in FASN and mRNA expression upon modulation of the IGF-1-mTORC1-SRPK2 pathway by small molecule inhibitors or RNAi mediated silencing. RNA stability was accessed by using the transcriptional inhibitor actinomycin-D followed by RT-qPCR. Further, we employed RNA-immunoprecipitation to demonstrate the direct binding of SRSF-1 to FASN transcripts.ResultsIn the current study, we demonstrated an IGF-1 induced increase in FASN mRNA and protein expression that was attenuated by mTORC1 inhibition. This mTORC1 inhibition also resulted in decreases in total and nuclear p-SRPK2 in response to IGF-1 exposure. Upon SRPK2 knockdown and inhibition, we observed a decrease in FASN protein and mRNA stability, respectively, in response to IGF-1 exposure that was specific to triple negative and HER2+ breast cancer cell lines. As we explored further, IGF-1 exposure resulted in an altered localization of eGFP expressed SRSF-1, pEGFP-SRSF-1 that was rescued upon both SRPK2 knockdown and mTORC1 inhibition. Further, we observed an increase binding of SRSF-1 to FASN RNA upon IGF-1 exposure, which was abrogated by SRPK2 knockdown.ConclusionThese current findings establish a potential IGF-1-mTORC1-SRPK2-FASN axis in breast cancer, which could be a potential therapeutic target for cancers that overexpress FASN and components of the IGF-1R pathway.

Insights into sweet potato SR proteins: from evolution to species-specific expression and alternative splicing.

SR proteins from sweet potato have conserved functional domains and similar gene structures as that of Arabidopsis and rice in general. However, expression patterns and alternative splicing regulations of SR genes from different species have changed under stresses. Novel alternative splicing regulations were found in sweet potato SR genes. Serine/arginine-rich (SR) proteins play important roles in plant development and stress response by regulating the pre-mRNA splicing process. However, SR proteins have not been identified so far from an important crop sweet potato. Through bioinformatics analysis, our study identified 24 SR proteins from sweet potato, with comprehensively analyzing of protein characteristics, gene structure, chromosome localization, and cis-acting elements in promotors. Salt, heat, and mimic drought stresses triggered extensive but different expressional regulations on sweet potato SR genes. Interestingly, heat stress caused the most active disturbances in both gene transcription and pre-mRNA alternative splicing (AS). Tissue and species-specific transcriptional and pre-mRNA AS regulations in response to stresses were found in sweet potato, in comparison with Arabidopsis and rice. Moreover, novel patterns of pre-mRNA alternative splicing were found in SR proteins from sweet potato. Our study provided an insight into similarities and differences of SR proteins in different plant species from gene sequences to gene structures and stress responses, indicating SR proteins may regulate their downstream genes differently between different species and tissues by varied transcriptional and pre-mRNA AS regulations.

Regulatory Network of Serine/Arginine-Rich (SR) Proteins: The Molecular Mechanism and Physiological Function in Plants.

Serine/arginine-rich (SR) proteins are a type of splicing factor. They play significant roles in constitutive and alternative pre-mRNA splicing, and are involved in post-splicing activities, such as mRNA nuclear export, nonsense-mediated mRNA decay, mRNA translation, and miRNA biogenesis. In plants, SR proteins function under a complex regulatory network by protein–protein and RNA–protein interactions between SR proteins, other splicing factors, other proteins, or even RNAs. The regulatory networks of SR proteins are complex—they are regulated by the SR proteins themselves, they are phosphorylated and dephosphorylated through interactions with kinase, and they participate in signal transduction pathways, whereby signaling cascades can link the splicing machinery to the exterior environment. In a complex network, SR proteins are involved in plant growth and development, signal transduction, responses to abiotic and biotic stresses, and metabolism. Here, I review the current status of research on plant SR proteins, construct a model of SR proteins function, and ask many questions about SR proteins in plants.

Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly.

We recently reported that serine–arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65 and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.

Expansion and functional analysis of the SR-related protein family across the domains of life.

Serine/arginine-rich (SR) proteins comprise a family of proteins that is predominantly found in eukaryotes and plays a prominent role in RNA splicing. A characteristic feature of SR proteins is the presence of an S/R-rich low-complexity domain (RS domain), often in conjunction with spatially distinct RNA recognition motifs (RRMs). To date, 52 human proteins have been classified as SR or SR-related proteins. Here, using an unbiased series of composition criteria together with enrichment for known RNA binding activity, we identified >100 putative SR-related proteins in the human proteome. This method recovers known SR and SR-related proteins with high sensitivity (∼94%), yet identifies a number of additional proteins with many of the hallmark features of true SR-related proteins. Newly identified SR-related proteins display slightly different amino acid compositions yet similar levels of post-translational modification, suggesting that these new SR-related candidates are regulated in vivo and functionally important. Furthermore, candidate SR-related proteins with known RNA-binding activity (but not currently recognized as SR-related proteins) are nevertheless strongly associated with a variety of functions related to mRNA splicing and nuclear speckles. Finally, we applied our SR search method to all available reference proteomes, and provide maps of RS domains and Pfam annotations for all putative SR-related proteins as a resource. Together, these results expand the set of SR-related proteins in humans, and identify the most common functions associated with SR-related proteins across all domains of life.