Proceedings of the National Academy of Sciences | 2021
CLK1 reorganizes the splicing factor U1-70K for early spliceosomal protein assembly
Abstract
Significance Proteome size is greatly enhanced through alternative mRNA splicing, a process catalyzed by the macromolecular spliceosome. The initial step in spliceosome assembly involves the binding of U1 snRNP to the 5′ splice site in mRNA. SR proteins ensure the fidelity of this step by simultaneously binding to mRNA and U1-70K, a protein component of U1 snRNP. Although phosphorylation of U1-70K is required for coupling to the SR protein, the enzyme that catalyzes this modification and the activating mechanisms are unknown. Here, we show that the protein kinase CLK1 phosphorylates a specific serine in the splicing factor U1-70K, reorganizing its structure and relieving a repressor contact for integration into U1 snRNP and stable binding to the SR protein. Early spliceosome assembly requires phosphorylation of U1-70K, a constituent of the U1 small nuclear ribonucleoprotein (snRNP), but it is unclear which sites are phosphorylated, and by what enzyme, and how such modification regulates function. By profiling the proteome, we found that the Cdc2-like kinase 1 (CLK1) phosphorylates Ser-226 in the C terminus of U1-70K. This releases U1-70K from subnuclear granules facilitating interaction with U1 snRNP and the serine-arginine (SR) protein SRSF1, critical steps in establishing the 5′ splice site. CLK1 breaks contacts between the C terminus and the RNA recognition motif (RRM) in U1-70K releasing the RRM to bind SRSF1. This reorganization also permits stable interactions between U1-70K and several proteins associated with U1 snRNP. Nuclear induction of the SR protein kinase 1 (SRPK1) facilitates CLK1 dissociation from U1-70K, recycling the kinase for catalysis. These studies demonstrate that CLK1 plays a vital, signal-dependent role in early spliceosomal protein assembly by contouring U1-70K for protein–protein multitasking.