Structural basis of the regulation by CDK11 kinase of early spliceosome activation and evidence for its proofreading by DHX15 helicase.

Abstract

Formation of the activated human spliceosome (B^act) involves major structural rearrangements, leading to the catalytically active U2/U6 RNA core. This process involves at least two intermediates, pre-B^act-1 and pre-B^act-2, and is regulated by CDK11-mediated phosphorylation of the U2 snRNP protein SF3B1. However, the mechanisms of this essential step are poorly understood. Here we present the cryo-EM structure of a spliceosome stalled - by the CDK11 inhibitor OTS964 - in a previously undescribed early-activated state, termed pre-B^act-OTS, shortly after dissociation of U4 snRNP. In pre-B^act-OTS, the U2-SF3B6 protein is retained in a C-terminal region of the super-helical U2-SF3B1 HEAT domain (SF3B1^HEAT) that clamps the U2/branch-site helix. In contrast, in pre-B^act-1, SF3B6 is repositioned to SF3B1's N-terminal HEAT repeats, thereby preventing a steric clash of SF3B6 with PRP8 during the pre-B^act-OTS-to-pre-B^act-1 transition. We infer that the CDK11-mediated phosphorylation of SF3B1 drives the relocation of SF3B6, gating progression to B^act formation. In pre-B^act-OTS, we also located the RNA helicase DHX15 at the N-terminal region of SF3B1^HEAT, assisted by the SR140/SPF45/CHERP/SUGP1 protein complex. These results suggest the involvement of DHX15 in kinase-mediated proofreading of the early-activated spliceosome, by competing with CDK11's phosphorylation of SF3B1, and thus with relocation of SF3B6 at SF3B1^HEAT.