Phosphorylation of SF3B1 by CDK11 1 orchestrates spliceosome activation via SNIP1 dependent RES complex recruitment

Splicing Factor 3b Subunit 1 (SF3B1), a core component of the spliceosome, undergoes dynamic phosphorylation and dephosphorylation during the splicing cycle to regulate pre-mRNA splicing. Twenty-eight threonine/proline repeats are phosphorylated by CDK11 during spliceosome activation and remain phosphorylated in the catalytically active spliceosomes. The function of phosphorylated SF3B1 (P-SF3B1), and the identity of spliceosomes stalled by CDK11 inhibition remain unclear. Using quantitative proteomics of chromatin-associated spliceosomes, we identify a previously uncharacterized intermediate complex BOTS964, arrested by CDK11 inhibitor OTS964, that incorporates the nineteen-related (NTR) but not nineteen (NTC) complex. iCLIP-seq revealed that P-SF3B1 engages with the U6 snRNA internal stem-loop (ISL), suggesting a potential role in stabilizing the RNA catalytic core. We further demonstrate that P-SF3B1 is recognized by forkhead-associated (FHA) domain of SNIP1, which promotes recruitment of retention and splicing (RES) complex during spliceosome activation. Acute SNIP1 depletion disrupts RES incorporation, causes widespread splicing defects, and promotes hyperphosphorylation of SF3B1 by CDK11. Mutations in SNIP1 FHA domain, including the neurodevelopmental disorder associated E366G variant, impair P-SF3B1 binding, pre-mRNA splicing, and cell viability. Together, these findings uncover the phosphorylation-dependent CDK11/P-SF3B1/SNIP1 signaling axis that is critical for pre-mRNA splicing and cellular proliferation and provide a mechanistic insight into its dysregulation in disease.