The RNA Binding Domain (RBD) of Serine-Arginine rich Splicing Factor 1 (SRSF1) is the most investigated RBD of SR proteins. It contains an N-terminal region and two tandem RNA Recognition Motifs (RRMs), called RRM1 and RRM2, connected by a linker. Exonic Splicing Enhancer (ESE) selection by RBDs is crucial to alternative splicing because it regulates pre-mRNA splicing patterns depending on the binding affinities of ESEs for the RBDs. Because this binding mechanism is still poorly characterized, we study how each part of the SRSF1 RBD facilitates RNA binding.  According to our binding assay data, the absence of either of the RRMs severely weakens RNA binding, and RRM2 governs the RNA binding of the RBD. Although we detect a few new RRM1 RNA binding sites of the RBD, the isolated and tandem forms of each RRM bind its cognate RNA motif similarly. Our NMR data identifies the linker as highly flexible, and our binding data further suggest that basic linker residues slightly mediate RNA binding by forming transient interactions with RNA. The linker flexibility changes the binding affinity of the RBD for RNA sequences bearing the RRM1-cognate motif located either upstream or downstream of the RRM2-cognate motif. This affinity change caused by such a mode of RNA binding further varies when the length of the RNA spacer between the two RRM-cognate motifs is changed from 0 to 10 nucleotides. These findings suggest that the presence of long RNA spacers and a downstream RRM1-cognate motif disfavors RNA binding of the RBD. By employing NMR, we discover that RRM1 and the linker are subject to more conformational changes than the RRM2 domain, upon swapping RRM cognate motifs. To elaborate on these data, we simulate ensemble structures of the RNA-bound RBD that illustrate how the RBD interacts with RNA upon swapping RRM recognition motifs and varying the RNA spacer length. These structures divulge the formation of loops in the RNA spacer and more extended conformations of the protein linker when the RRM1 cognate motif is located downstream of the RRM2 cognate motif. These structural changes lead to an entropy loss that varies the RNA binding affinity of the RBD. Therefore, ESEs recognized by the RBD could be longer than their typical length of 6-8 nucleotides. Next, we should study how the relative positions of the RRM cognate motifs and the RNA spacer in between them modulate alternative splicing patterns caused by SR proteins containing tandem RRMs including SRSF1.

Inter-domain flexibility of human SRSF1 allows variable spacer length in cognate RNA’s bipartite motifs

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