RESUMEN
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 complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, 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 Bact 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 Bact 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.
Asunto(s)
Quinasas Ciclina-Dependientes , Fosfoproteínas , Precursores del ARN , Empalme del ARN , Ribonucleoproteína Nuclear Pequeña U2 , Empalmosomas , Cromatina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Activación Enzimática/efectos de los fármacos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación , Quinolonas/farmacología , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN/efectos de los fármacos , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Empalmosomas/efectos de los fármacos , Empalmosomas/metabolismo , Treonina/metabolismoRESUMEN
More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex.
Asunto(s)
ADN/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Unión Competitiva/efectos de los fármacos , ADN/química , ADN/genética , Polarización de Fluorescencia , Humanos , Cinética , Unión Proteica/efectos de los fármacos , Complejo Shelterina , Cloruro de Sodio/farmacología , Espectrometría de Fluorescencia , Electricidad Estática , Resonancia por Plasmón de Superficie , Telómero/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/genética , Proteína 2 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
Replication-dependent histones (RDH) are required for packaging of newly synthetized DNA into nucleosomes during the S phase when their expression is highly upregulated. However, the mechanisms of this upregulation in metazoan cells remain poorly understood. Using iCLIP and ChIP-seq, we found that human cyclin-dependent kinase 11 (CDK11) associates with RNA and chromatin of RDH genes primarily in the S phase. Moreover, its amino-terminal region binds FLASH, an RDH-specific 3'-end processing factor, which keeps the kinase on the chromatin. CDK11 phosphorylates serine 2 (Ser2) of the carboxy-terminal domain of RNA polymerase II (RNAPII), which is initiated when RNAPII reaches the middle of RDH genes and is required for further RNAPII elongation and 3'-end processing. CDK11 depletion leads to decreased number of cells in S phase, likely owing to the function of CDK11 in RDH gene expression. Thus, the reliance of RDH expression on CDK11 could explain why CDK11 is essential for the growth of many cancers.