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

Abstract

Serine/arginine (SR) proteins, one of the major families of alternativesplicing regulators in Eukarya, have two types of RNA-recognition motifs (RRMs): a canonical RRM and a pseudo-RRM. Although pseudo-RRMs are crucial for activity of SR proteins, their mode of action was unknown. By solving the structure of the human SRSF1 pseudo-RRM bound to RNA, we discovered a very unusual and sequence-specific RNA-binding mode that is centered on one α-helix and does not involve the β-sheet surface, which typically mediates RNA binding by RRMs. Remarkably, this mode of binding is conserved in all pseudo-RRMs tested. Furthermore, the isolated pseudo-RRM is sufficient to regulate splicing of about half of the SRSF1 target genes tested, and the bound α-helix is a pivotal element for this function. Our results strongly suggest that SR proteins with a pseudo-RRM frequently regulate splicing by competing with, rather than recruiting, spliceosome components, using solely this unusual RRM.NMR | protein-RNA complex | splicing factor S erine/arginine (SR) proteins are highly conserved in Eukarya and are key regulators of gene expression. These proteins are required for pre-mRNA splicing, regulate alternative splicing events, and play crucial roles in genomic stability, mRNA transcription, nuclear export, nonsense-mediated mRNA decay, and translation (1-5). Several studies have revealed links between these proteins and diseases (6), making the proteins potential therapeutic targets (7). For the last two decades, SR proteins have been studied intensively as regulators of alternative splicing, a mechanism used to modulate the expression of more than 95% of human genes (8, 9). Importantly, it is estimated that 15-50% of human disease-causing mutations affect splicing (10). Alternative splicing consists of the alternative selection of splice sites present within pre-mRNA, leading to different versions of mature mRNAs from a single gene (11). As a result, in some cases, proteins with opposite functions can be generated. In the context of cancer, alternative splicing can generate pro-or antiapoptotic isoforms (12). As an example, alternatively spliced variants of FAS/CD95 can be generated by inclusion or skipping of exon 6, depending on competition between antagonistic splicing factors (13-16). In the absence of Fas exon 6, the apoptotic receptor lacks the transmembrane domain and becomes soluble and antiapoptotic (17).Each SR protein has a modular structure with one or two RNA-recognition motifs (RRMs) at its N-terminal part, followed by a C-terminal arginine-serine-rich (RS) domain containing multiple RS dipeptide repeats. In many cases, SR proteins have been shown to interact with purine-rich exonic splicing enhancers (ESEs) and to promote inclusion of the targeted exon in mRNAs (18). Two main models have been proposed to explain the mode of action of ESE-bound SR proteins in splicing regulation. In the recruitment model SR proteins recruit U1-70K and/or U2AF35 to 5′ and 3′ splice sites, respectively, to promote spliceosome assembly (4)....

SRSF1 RNA Recognition Motifs Are Strong Inhibitors of HIV-1 Replication

“…Expression of a mutant clone carrying only RRM2 decreased transactivation with efficiency similar to the wild-type protein (10-fold), whereas RRM1 reduced transactivation by Ͻ50%. These data are consistent with our previous observations suggesting an inhibition mechanism solely dependent on the RNA binding specificity of SRSF1 for a sequence overlapping the Tat binding site onto TAR and with findings suggesting that the RNA binding activity of SRSF1 is mostly dependent on RRM2 (28).…”

“…The RNA-binding specificity of SRSF1 appears to be mostly dependent on RRM2, since it interacts with the target RNA with efficiency similar to the one for RRM1 and RRM2 combined (28). Our data indicate that inhibition of viral replication differs greatly upon the expression of RRM2 and RRM1ϩ2 (ϳ200-fold inhibition versus Ͼ2,000-fold inhibition); thus, factors other than the RNA-binding specificity of these subdomains may account for their antiviral activity.…”

“…Given the heterogeneity of the sequences recognized by SRSF1 and the different roles played by the single domains in determining its RNA-binding specificity (28,33), we expected substantial differences in the total and relative amounts of viral mRNAs generated by divergent viruses upon expression of each SRSF1 mutant. Nevertheless, the synthesis and processing of viral mRNAs was severely disrupted in all of the molecular clones tested, and the production of all three viral subtypes analyzed was strongly inhibited by SRSF1 and its single RBD.…”

“…On the contrary, its pervasive role in mRNA splicing regulation has been widely studied, and it is crucial for cell survival, development, and replication. To determine whether the deletion clones were differently affecting the splicing of cellular genes, we selected a panel of 15 genes, whose splicing is regulated by SRSF1 (28,34), and we measured the inclusion of specific exons. Only 4 of the genes analyzed were similarly regulated by the wild type and the deletion mutants (Fig.…”

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

“…5a). The unusual orientation of the C-terminal loop and α-helix of Snu17p within the RNA-binding site suggests that the RES-RNA interaction is potentially another example of a noncanonical RRM-RNA recognition mode [38][39][40][49][50][51][52][53] . The increase in RNA affinity when c Pml1p is bound to c Snu17p and the C-terminal α-helix of Snu17p is formed (Supplementary Fig.…”