It has been widely accepted that the early spliceosome assembly begins with U1 small nuclear ribonucleoprotein (U1 snRNP) binding to the 5′ splice site (5′SS), which is assisted by the Ser/Arg (SR)-rich proteins in mammalian cells. In this process, the RS domain of SR proteins is thought to directly interact with the RS motif of U1-70K, which is subject to regulation by RS domain phosphorylation. Here we report that the early spliceosome assembly event is mediated by the RNA recognition domains (RRM) of serine/arginine-rich splicing factor 1 (SRSF1), which bridges the RRM of U1-70K to pre-mRNA by using the surface opposite to the RNA binding site. Specific mutation in the RRM of SRSF1 that disrupted the RRM-RRM interaction also inhibits the formation of spliceosomal E complex and splicing. We further demonstrate that the hypo-phosphorylated RS domain of SRSF1 interacts with its own RRM, thus competing with U1-70K binding, whereas the hyper-phosphorylated RS domain permits the formation of a ternary complex containing ESE, an SR protein, and U1 snRNP. Therefore, phosphorylation of the RS domain in SRSF1 appears to induce a key molecular switch from intra-to intermolecular interactions, suggesting a plausible mechanism for the documented requirement for the phosphorylation/dephosphorylation cycle during pre-mRNA splicing.RNA splicing | spliceosome complex | exonic splicing enhancer | protein phosphorylation P re-mRNA splicing is essential for gene expression by precise removal of intervening sequences known as introns. Because splice site sequences are often insufficient to direct faithful recognition of authentic splice sites, such a lack of sequence stringency imposes a great challenge for the splicing machinery to assemble on functional sites while avoiding numerous cryptic splice sites in the pre-mRNA (1).Regulatory elements, such as exonic splicing enhancer (ESE) sequences, provide a key strategy to compensate for sequence variations on authentic splice sites. ESE typically consists of highly degenerate 6-8 nucleotide motifs (2, 3) that acts as positive regulators for splice site selection, and many of them are specifically recognized by SR proteins (3). ESE-bound SR proteins are involved in the recruitment of snRNPs, although the precise mechanisms of these recruitment events are only vaguely understood (4-7). This process also plays a crucial role in splicing regulation with the sequence elements, such as exonic and intronic silencer sequences (ESS and ISS, respectively) act as negative regulators by recruiting splicing repressors, such as heterogeneous nuclear RNPs (hnRNPs) (8, 9). The balance between these opposing functional elements determines the overall splicing strength in alternative splicing.In addition to their well known activities in the regulation of both constitutive and alternative splicing, SR proteins also participate in postsplicing activities, such as mRNA nuclear export, nonsense-mediated mRNA decay, and mRNA translation (10, 11). SR proteins are characterized by having RNA recognition m...

SR proteins promote spliceosome formation by recognizing exonic splicing enhancers (ESEs) during pre-mRNA splicing. Each SR protein binds diverse ESEs using strategies that are yet to be elucidated. Here, we show that the RNA-binding domain (RBD) of SRSF1 optimally binds to decameric purine rich ESE sequences although locations of purines are not stringently specified. The presence of uracils either within or outside of the recognition site is detrimental for binding with SRSF1. The entire RBD, comprised of two RRMs and a glycine-rich linker, is essential for ESE binding. Mutation within each segment reduced or nearly abolished binding, suggesting that these segments mediate cooperative binding. The linker plays a decisive role in organizing ESE binding. The flanking basic regions of the linker appear to communicate with each other in bringing the two RRMs close together to form the complex with RNA. Our study thus suggests semi-conservative adaptable interaction between ESE and SRSF1, and such binding mode is not only essential for the recognition of plethora of physiological ESE sequences but may also be essential for the interaction with various factors during the spliceosome assembly.

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