Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, sugIN that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain ofSF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for spicin.. SR proteins are a family of pre-mRNA splicing factors characterized by the presence of RNA binding motifs and the SR domain, which consists largely of serine/arginine repeats (1). They are essential splicing factors, playing critical roles in the initiation of spliceosome assembly on pre-mRNA substrates (2-6). SR proteins are functionally similar when assayed for splicing in vitro (7,8). However, recent studies have shown that each member displays a distinct spectrum of substrate specificity toward pre-mRNAs (5, 9-11). SR proteins can also alter alternative splice site selection in vitro by promoting the use of proximal 5' and 3' splice sites in a concentration-dependent manner (8,10,12,13 (21) a cell-cycle-regulated kinase specific for SR proteins, SR protein kinase 1 (SRPK1), and ahigh level ofthis kinase can inhibit splicing. Thus, these studies show that dephosphorylation is important for splicing. However, whether or not phosphorylation of SR proteins and other splicing factors containing a similar SR domain is essential for splicing and the possibility that phosphorylation and dephosphorylation of these proteins occur during different stages of splicing remain to be addressed.In this paper, we report the purification and characterization of SRPK1. We show that the phosphoepitope on SR proteins specifically recognized by mAb 104 can be restored by SRPK1 to a bacterially produced SR protein, SF2/ASF, suggesting that SRPK1 is responsible for phosphorylation of SR proteins in vivo. Finally, we provide evidence that phosphorylation of SR proteins by SRPK1 is also important for splicing because highly conservative changes in the SR domain of SF2/ASF, which were previously shown (23) to abolish its function in splicing, also affected the recognition and phosphorylation of this splicing factor by SRPK1. Further investigation of phosphorylation regul...

The molecular factors involved in the development of Post-Traumatic Stress Disorder (PTSD) remain poorly understood. Previous transcriptomic studies investigating the mechanisms of PTSD apply targeted approaches to identify individual genes under a cross-sectional framework lack a holistic view of the behaviours and properties of these genes at the system-level. Here we sought to apply an unsupervised gene-network based approach to a prospective experimental design using whole-transcriptome RNA-Seq gene expression from peripheral blood leukocytes of U.S. Marines (N=188), obtained both pre- and post-deployment to conflict zones. We identified discrete groups of co-regulated genes (i.e., co-expression modules) and tested them for association to PTSD. We identified one module at both pre- and post-deployment containing putative causal signatures for PTSD development displaying an over-expression of genes enriched for functions of innate-immune response and interferon signalling (Type-I and Type-II). Importantly, these results were replicated in a second non-overlapping independent dataset of U.S. Marines (N=96), further outlining the role of innate immune and interferon signalling genes within co-expression modules to explain at least part of the causal pathophysiology for PTSD development. A second module, consequential of trauma exposure, contained PTSD resiliency signatures and an over-expression of genes involved in hemostasis and wound responsiveness suggesting that chronic levels of stress impair proper wound healing during/after exposure to the battlefield while highlighting the role of the hemostatic system as a clinical indicator of chronic-based stress. These findings provide novel insights for early preventative measures and advanced PTSD detection, which may lead to interventions that delay or perhaps abrogate the development of PTSD.

We report striking differences in the substrate specificities of two human SR proteins, SF2/ASF and SC35, in constitutive splicing. ␤-Globin pre-mRNA (exons 1 and 2) is spliced indiscriminately with either SR protein.Human immunodeficiency virus tat pre-mRNA (exons 2 and 3) and immunoglobulin -chain (IgM) pre-mRNA (exons C3 and C4) are preferentially spliced with SF2/ASF and SC35, respectively. Using in vitro splicing with mutated or chimeric derivatives of the tat and IgM pre-mRNAs, we defined specific combinations of segments in the downstream exons, which mediate either positive or negative effects to confer SR protein specificity. A series of recombinant chimeric proteins consisting of domains of SF2/ASF and SC35 in various combinations was used to localize trans-acting domains responsible for substrate specificity. The RS domains of SF2/ASF and SC35 can be exchanged without effect on substrate specificity. The RNA recognition motifs (RRMs) of SF2/ASF are active only in the context of a two-RRM structure, and RRM2 has a dominant role in substrate specificity. In contrast, the single RRM of SC35 can function alone, but its substrate specificity can be influenced by the presence of an additional RRM. The RRMs behave as modules that, when present in different combinations, can have positive, neutral, or negative effects on splicing, depending upon the specific substrate. We conclude that SR protein-specific recognition of specific positive and negative pre-mRNA exonic elements via one or more RRMs is a crucial determinant of the substrate specificity of SR proteins in constitutive splicing.Pre-mRNA splicing is an essential step in the expression of eukaryotic genes (see review chapters in references 17 and 20). Introns are excised with a high degree of precision in two successive transesterification reactions, despite the enormous variability in intron number, size, and sequence in higher eukaryotic genes. The critical sequences involved in the transesterification reactions, at the 5Ј splice site, the branch site, and the 3Ј splice site, are relatively short and only weakly conserved. Many silent or cryptic splice site signals are present in both exons and introns, but they are normally ignored in the presence of the authentic signals. On the other hand, a number of pre-mRNAs show flexibility in the choice of alternative splice sites, often in response to tissue-specific, physiologically, or developmentally regulated states. Alternative splicing is a common strategy for the regulation of cellular and viral gene expression.Pre-mRNA splicing takes place within a large complex, the spliceosome, which includes the small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4/U6, and U5 and a large number of non-snRNP splicing factors. Biochemical characterization of the spliceosome, together with genetic studies in budding yeast, predicts that over 50 proteins are essential for constitutive splicing. Considerable effort has been devoted to dissecting the cis elements and trans-acting factors involved in the complex...

Competing Interests B.M.N. is on the Scientific Advisory Board at Deep Genomics and Camp4 Therapeutics Corporation, and on the Biogen Genomics Advisory Panel. M.F. is an employee of Verily Life Sciences. Accession codes Data included in this manuscript have been deposited in the database of Genotypes and Phenotypes (dbGaP) under accession number phs001196.v1. Data collection and analysis were not performed blind to the conditions of the experiments. Data availability Data included in this manuscript have been deposited in the database of Genotypes and Phenotypes (dbGaP) under accession number phs001196.v1. Data collection and analysis were not performed blind to the conditions of the experiments.

Pre-mRNA splicing requires a large number of RNA-binding proteins that have one or more RNArecognition motifs (RRMs). Among these is the SR protein family, whose members are essential for splicing and are able to commit pre-mRNAs to the splicing pathway with overlapping but distinct substrate specificity. Some SR proteins, such as SC35, contain an N-terminal RRM and a C-terminal arginine͞serine-rich (RS) domain, whereas others, such as SF2͞ASF, also contain a second, atypical RRM. Although both the RRMs and the RS domain of SR proteins are required for constitutive splicing, it is unclear which domain(s) defines their substrate specificity, and whether two RRMs in a given SR protein function independently or act coordinately. Using domain swaps between SC35 and SF2͞ASF and a functional commitment assay, we demonstrate that individual domains are functional modules, RS domains are interchangeable, and substrate specificity is defined by the RRMs. The atypical RRM of SF2͞ASF does not appear to function alone in splicing, but can either activate or suppress the splicing specificity of an N-terminal RRM. Therefore, multiple RRMs in SR proteins act coordinately to achieve a unique spectrum of pre-mRNA substrate specificity.Pre-mRNA splicing is a critical step in the posttranscriptional regulation of gene expression and requires both small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP factors. These factors assemble on pre-mRNA into a large complex known as a spliceosome, in which splicing takes place (for review, see ref. 1). A family of arginine͞serine-rich non-snRNP splicing factors called SR proteins mediate various steps of spliceosomal assembly (for reviews, see refs. 2 and 3). In constitutive splicing, binding of SR proteins is sufficient to commit pre-mRNA to the splicing pathway (4), probably by facilitating U1 snRNP binding to a functional 5Ј splice site (5), stabilizing complex assembly at the 3Ј splice site (6), and bridging complexes assembled at the 5Ј and 3Ј splice sites (7-9). Additionally, SR proteins can modulate splice site selection, which is consistent with their involvement in early steps of splice site recognition (for review, see ref. 10).SR proteins are essential splicing factors, but they have overlapping functions, at least in vitro, because all SR proteins can complement the splicing-deficient S100 cytoplasmic extract (11, 12). However, individual SR proteins clearly perform distinct activities in splicing different pre-mRNA substrates, and bind to different RNA sequence elements (for reviews, see refs. 2 and 3). It has been shown that the SR protein B52͞ SRp55 is essential for Drosophila development, although splicing of a number of transcripts examined in mutant larvae was not affected, indicating that these transcripts are not dependent on B52͞SRp55 (13, 14). More recently, another SR protein, SF2͞ASF, was analyzed by targeted gene disruption in a chicken B-cell line and shown to be essential for cell viability, indicating that this SR protein also has at least one n...