Mechanisms of modulation of pre-mRNA 3D structural scaffolds for splicing substrate definition

Coordination of different serine-arginine-rich (SR) proteins - a class of critical splicing activators - facilitates recognition of the highly degenerate cognate splice signal sequences against the background sequences. Yet, the mechanistic details of their actions remain unclear. Here we show that cooperative binding of SR proteins to exonic and intronic motifs remodels the pre-mRNA 3D structural scaffold. The scaffold generated by pre-mRNA-specific combinations of different SR proteins in an appropriate stoichiometry is recognized by U1 snRNP. A large excess of U1 snRNP particles displaces the majority of the bound SR protein molecules from the remodeled pre-mRNA. A higher than optimal stoichiometry of SR proteins occludes the binding sites on the pre-mRNA, raising the U1 snRNP levels required for SR protein displacement and potentially impeding spliceosome assembly. This novel step is important for distinguishing the substrate and the non-substrate by U2AF65 - the primary 3' splice site-recognizing factor. Overall, this work elucidates early regulatory steps of mammalian splicing substrate definition by SR proteins.