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 RRMcontaining SRSF3 are phosphorylated by SRPK2, another member of the SRPK family. We showed that a conserved SRPKspecific 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 RRMcontaining 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.

Mutations of splice sites, auxiliary splicing elements and the splicing machinery cause a wide range of genetic disease. Here we report that many of the complex effects of splicing mutations can be predicted from background splicing information, with emphasis on BRCA1, BRCA2 and DMD. Background splicing arises from very low level splicing between rarely used background splice sites and from low-level exon skipping between intron splice sites. We show how this information can be downloaded from the Snaptron database of spliced RNA, which we then compared with databases of human splice site mutations. We report that inactivating mutations of intron splice sites typically caused the non-mutated partner splice site to splice to a known background splice site in over 90% of cases and to the strongest background splice site in the large majority of cases. Consequently, background splicing information can usefully predict the effects of splice site mutations, which include cryptic splice activation and single or multiple exon skipping. In addition, de novo splice sites and splice sites involved in pseudoexon formation, recursive splicing and aberrant splicing in cancer show a 90% match to background splice sites, so establishing that the enhancement of background splicing causes a wide range of splicing aberrations. We also discuss how background splicing information can identify cryptic splice sites that might be usefully targeted by antisense oligonucleotides (ASOs) and how it might indicate possible multiple exon skipping side effects of ASOs designed to induce single exon skipping.