SR Proteins Induce Alternative Exon Skipping through Their Activities on the Flanking Constitutive Exons

Abstract

SR proteins are well known to promote exon inclusion in regulated splicing through exonic splicing enhancers. SR proteins have also been reported to cause exon skipping, but little is known about the mechanism. We previously characterized SRSF1 (SF2/ASF)-dependent exon skipping of the CaMKII␦ gene during heart remodeling. By using mouse embryo fibroblasts derived from conditional SR protein knockout mice, we now show that SR protein-induced exon skipping depends on their prevalent actions on a flanking constitutive exon and requires collaboration of more than one SR protein. These findings, coupled with other established rules for SR proteins, provide a theoretical framework to understand the complex effect of SR protein-regulated splicing in mammalian cells. We further demonstrate that heart-specific CaMKII␦ splicing can be reconstituted in fibroblasts by downregulating SR proteins and upregulating a RBFOX protein and that SR protein overexpression impairs regulated CaMKII␦ splicing and neuronal differentiation in P19 cells, illustrating that SR protein-dependent exon skipping may constitute a key strategy for synergism with other splicing regulators in establishing tissue-specific alternative splicing critical for cell differentiation programs.The splicing machinery is largely conserved in eukaryotic cells. However, compared to budding yeast, where critical splicing signals are nearly invariant, higher eukaryotic cells rely on auxiliary factors to help define functional splice sites that are only loosely conserved. Most genes in higher eukaryotic cells also undergo alternative splicing, which is subject to regulation by a variety of RNA binding proteins (2). SR proteins are unique to higher eukaryotes and are among the best-characterized RNA binding proteins involved in both constitutive and regulated splicing (29,31,48). Intensive biochemical analysis in the past 2 decades has established that the RNA recognition motifs (RRMs) of SR proteins are responsible for sequence-specific binding to the pre-mRNA, whereas the RS domain appears to mediate both protein-protein and protein-RNA interactions during the splicing reaction (17, 39).Individual SR proteins exhibit distinct RNA binding specificities for various exonic splicing enhancers (ESEs), a second code in higher eukaryotic genomes that is critical for defining functional splice sites. In many cases, multiple SR proteins bind to several ESEs within the same exon, which is thought to provide redundant functions to ensure constitutive splicing against variation of SR proteins in different cell types and tissues. However, it has become abundantly clear that individual SR proteins are not functionally redundant in vivo (1, 4, 30). Because exons are short whereas introns are highly variable in length, functional splice sites in most mammalian genes are initially recognized by the exon definition mechanism, in which ESE-bound SR proteins promote U2AF recognition of the 3Ј splice site and U1 binding to the downstream 5Ј splice site across the exon (16). Initial exon...

Isolated pseudo–RNA-recognition motifs of SR proteins can regulate splicing using a noncanonical mode of RNA recognition

“…Interestingly, our data obtained with the SRSF1 pseudo-RRM alone fit perfectly with the above-mentioned mode of action of SR proteins in splicing regulation (22). Indeed, the binding of SRSF1 pseudo-RRM to the alternative exon of IGF1 itself activates its inclusion (Fig.…”

“…Their binding to intronic sequences was reported primarily to promote exon skipping (58), but the effect of their interaction with exonic sequences seems to be more complex. It was proposed that the binding of SR proteins to alternative exons promotes their inclusion, whereas their interaction with a flanking exon causes skipping of the internal alternative exon (22,59). Here, we found that the isolated pseudo-RRM of SRSF1 is sufficient to modulate splicing of nearly half of the tested SRSF1 target genes (Fig.…”

“…In the inhibitor model SR proteins activate splicing by preventing the binding of splicing repressors, such as hnRNP A1, around the regulated splice site (4). In some cases, SR proteins have been shown not to enhance splicing but rather to promote exon skipping (19)(20)(21)(22)(23)(24)(25)(26). However, their mode of action in these cases is still unclear.…”

SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export

“…6D), in line with observed redundancies in RNA binding. Because inclusion of alternative exons is often facilitated by SR protein binding (Han et al 2011;Erkelenz et al 2013), we tested for binding of cognate SR proteins within those exons (Fig. 6E).…”

Exonic Mutations in the SLC12A3 Gene Cause Exon Skipping and Premature Termination in Gitelman Syndrome

“…The ESEfinder software was programmed to predict exon skipping by scoring the sequences matched with the putative ESEs, as previously reported. 23,24 The ESEs effectively contribute to the exons with the authentic splice sites displaying reduced homology with the consensus sequences. We therefore assessed the homology of all of the splice sites in the SLC12A3 gene to identify the vulnerable exons depending on the ESEs, as described in a previous article, using the Berkeley Drosophila Genome Project splicing predictor program (http://www.fruitfly.org/ seq_tools/splice.html).…”