Summary SR proteins have been studied extensively as a family of RNA binding proteins that participate in both constitutive and regulated pre-mRNA splicing in mammalian cells. However, SR proteins were first discovered as factors that interact with transcriptionally active chromatin. Recent studies have now uncovered properties that connect these once apparently disparate functions, showing that a subset of SR proteins seem to bind directly to the histone 3 tail, play an active role in transcriptional elongation, and co-localize with genes that are engaged in specific intra- and inter-chromosome interactions for coordinated regulation of gene expression in the nucleus. These transcription-related activities are also coupled with a further expansion of putative functions of specific SR protein family members in RNA metabolism downstream of mRNA splicing, from RNA export to stability control to translation. These findings therefore highlight the broader roles of SR proteins in vertical integration of gene expression and provide mechanistic insights into their contributions to genome stability and proper cell cycle progression in higher eukaryotic cells.

It has been widely accepted that the early spliceosome assembly begins with U1 small nuclear ribonucleoprotein (U1 snRNP) binding to the 5′ splice site (5′SS), which is assisted by the Ser/Arg (SR)-rich proteins in mammalian cells. In this process, the RS domain of SR proteins is thought to directly interact with the RS motif of U1-70K, which is subject to regulation by RS domain phosphorylation. Here we report that the early spliceosome assembly event is mediated by the RNA recognition domains (RRM) of serine/arginine-rich splicing factor 1 (SRSF1), which bridges the RRM of U1-70K to pre-mRNA by using the surface opposite to the RNA binding site. Specific mutation in the RRM of SRSF1 that disrupted the RRM-RRM interaction also inhibits the formation of spliceosomal E complex and splicing. We further demonstrate that the hypo-phosphorylated RS domain of SRSF1 interacts with its own RRM, thus competing with U1-70K binding, whereas the hyper-phosphorylated RS domain permits the formation of a ternary complex containing ESE, an SR protein, and U1 snRNP. Therefore, phosphorylation of the RS domain in SRSF1 appears to induce a key molecular switch from intra-to intermolecular interactions, suggesting a plausible mechanism for the documented requirement for the phosphorylation/dephosphorylation cycle during pre-mRNA splicing.RNA splicing | spliceosome complex | exonic splicing enhancer | protein phosphorylation P re-mRNA splicing is essential for gene expression by precise removal of intervening sequences known as introns. Because splice site sequences are often insufficient to direct faithful recognition of authentic splice sites, such a lack of sequence stringency imposes a great challenge for the splicing machinery to assemble on functional sites while avoiding numerous cryptic splice sites in the pre-mRNA (1).Regulatory elements, such as exonic splicing enhancer (ESE) sequences, provide a key strategy to compensate for sequence variations on authentic splice sites. ESE typically consists of highly degenerate 6-8 nucleotide motifs (2, 3) that acts as positive regulators for splice site selection, and many of them are specifically recognized by SR proteins (3). ESE-bound SR proteins are involved in the recruitment of snRNPs, although the precise mechanisms of these recruitment events are only vaguely understood (4-7). This process also plays a crucial role in splicing regulation with the sequence elements, such as exonic and intronic silencer sequences (ESS and ISS, respectively) act as negative regulators by recruiting splicing repressors, such as heterogeneous nuclear RNPs (hnRNPs) (8, 9). The balance between these opposing functional elements determines the overall splicing strength in alternative splicing.In addition to their well known activities in the regulation of both constitutive and alternative splicing, SR proteins also participate in postsplicing activities, such as mRNA nuclear export, nonsense-mediated mRNA decay, and mRNA translation (10, 11). SR proteins are characterized by having RNA recognition m...

Phosphorylation is essential for the SR family of splicing factors/regulators to function in constitutive and regulated pre-mRNA splicing; yet both hypo-and hyperphosphorylation of SR proteins are known to inhibit splicing, indicating that SR protein phosphorylation must be tightly regulated in the cell. However, little is known how SR protein phosphorylation might be regulated during development or in response to specific signaling events. Here, we report that SRPK1, a ubiquitously expressed SR protein-specific kinase, directly binds to the cochaperones Hsp40/DNAjc8 and Aha1, which mediate dynamic interactions of the kinase with the major molecular chaperones Hsp70 and Hsp90 in mammalian cells. Inhibition of the Hsp90 ATPase activity induces dissociation of SRPK1 from the chaperone complexes, which can also be triggered by a stress signal (osmotic shock), resulting in translocation of the kinase from the cytoplasm to the nucleus, differential phosphorylation of SR proteins, and alteration of splice site selection. These findings connect the SRPK to the molecular chaperone system that has been implicated in numerous signal transduction pathways and provide mechanistic insights into complex regulation of SR protein phosphorylation and alternative splicing in response to developmental cues and cellular signaling.[Keywords: SR protein-specific kinase; molecular chaperones; stress signaling; SR protein phosphorylation alternative splicing] Supplemental material is available at http://www.genesdev.org.

Reversible phosphorylation of the SR family of splicing factors plays an important role in pre-mRNA processing in the nucleus. Interestingly, the SRPK family of kinases specific for SR proteins is localized in the cytoplasm, which is critical for nuclear import of SR proteins in a phosphorylation-dependent manner. Here, we report molecular dissection of the mechanism involved in partitioning SRPKs in the cytoplasm. Common among all SRPKs, the bipartite kinase catalytic core is separated by a unique spacer sequence. The spacers in mammalian SRPK1 and SRPK2 share little sequence homology, but they function interchangeably in restricting the kinases in the cytoplasm. Removal of the spacer in SRPK1 had little effect on the kinase activity, but it caused a quantitative translocation of the kinase to the nucleus and consequently induced aggregation of splicing factors in the nucleus. Rather than carrying a nuclear export signal as suggested previously, we found multiple redundant signals in the spacer that act together to anchor the kinase in the cytoplasm. Interestingly, a cell cycle signal induced nuclear translocation of the kinase at the G2/M boundary. These findings suggest that SRPKs may play an important role in linking signaling to RNA metabolism in higher eukaryotic cells.

Conformational change in human IKK2 permits dimers to form higher-order oligomers that support interaction between kinase domains and promote activation through trans auto-phosphorylation.