CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA–protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes 1 , 2 . Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex 1 – 5 . Splicing factor 3B subunit 1 (SF3B1) protein—a subunit of the U2 small nuclear ribonucleoprotein 6 —is phosphorylated during spliceosome activation 7 – 10 , but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the B act complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex B act and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.