No abstract

Summary Splicing factor SRSF1 is upregulated in human breast tumors, and its overexpression promotes transformation of mammary cells. Using RNA-seq, we identified SRSF1-regulated alternative splicing (AS) targets in organotypic three-dimensional MCF-10A cell cultures that mimic a context relevant to breast cancer. We identified and validated hundreds of endogenous SRSF1-regulated AS events. De-novo discovery of the SRSF1 binding motif reconciled discrepancies in previous motif analyses. Using a Bayesian model, we determined positional effects of SRSF1 binding on cassette exons: binding close to the 5′ splice site generally promoted exon inclusion, whereas binding near the 3′ splice site promoted either exon skipping or inclusion. Finally, we identified SRSF1-regulated AS events deregulated in human tumors; overexpressing one such isoform, exon-9-included CASC4, increased acinar size and proliferation, and decreased apoptosis, partially recapitulating SRSF1's oncogenic effects. Thus, we uncovered SRSF1 positive and negative regulatory mechanisms, and oncogenic AS events that represent potential targets for therapeutics development.

Highlights d Structures of FUS ZnF bound to GGU and FUS RRM bound to an RNA stem loop were solved d The RGG motif destabilizes the RNA structure, suggesting a role in remodeling RNA d The RGG motif increases RNA binding without forming an ordered structure d RNA binding by the ZnF and RRM domains contribute to FUSmediated splicing functions

Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures.

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)....