RESUMO
Accurate regulation of mRNA termination is required for correct gene expression. Here, we describe a role for SCAF4 and SCAF8 as anti-terminators, suppressing the use of early, alternative polyadenylation (polyA) sites. The SCAF4/8 proteins bind the hyper-phosphorylated RNAPII C-terminal repeat domain (CTD) phosphorylated on both Ser2 and Ser5 and are detected at early, alternative polyA sites. Concomitant knockout of human SCAF4 and SCAF8 results in altered polyA selection and subsequent early termination, leading to expression of truncated mRNAs and proteins lacking functional domains and is cell lethal. While SCAF4 and SCAF8 work redundantly to suppress early mRNA termination, they also have independent, non-essential functions. SCAF8 is an RNAPII elongation factor, whereas SCAF4 is required for correct termination at canonical, distal transcription termination sites in the presence of SCAF8. Together, SCAF4 and SCAF8 coordinate the transition between elongation and termination, ensuring correct polyA site selection and RNAPII transcriptional termination in human cells.
Assuntos
RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Elongação da Transcrição Genética , Terminação da Transcrição Genética , Células HEK293 , Humanos , Poli A/genética , Poli A/metabolismo , Domínios Proteicos , RNA Polimerase II/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina/genéticaRESUMO
CTDK-I is a yeast kinase complex that phosphorylates the C-terminal repeat domain (CTD) of RNA polymerase II (Pol II) to promote transcription elongation. CTDK-I contains the cyclin-dependent kinase Ctk1 (homologous to human CDK9/CDK12), the cyclin Ctk2 (human cyclin K), and the yeast-specific subunit Ctk3, which is required for CTDK-I stability and activity. Here we predict that Ctk3 consists of a N-terminal CTD-interacting domain (CID) and a C-terminal three-helix bundle domain. We determine the X-ray crystal structure of the N-terminal domain of the Ctk3 homologue Lsg1 from the fission yeast Schizosaccharomyces pombe at 2.0 Å resolution. The structure reveals eight helices arranged into a right-handed superhelical fold that resembles the CID domain present in transcription termination factors Pcf11, Nrd1, and Rtt103. Ctk3 however shows different surface properties and no binding to CTD peptides. Together with the known structure of Ctk1 and Ctk2 homologues, our results lead to a molecular framework for analyzing the structure and function of the CTDK-I complex.