RESUMEN
Facilitates chromatin transcription (FACT) is a histone chaperone that supports transcription through chromatin in vitro, but its functional roles in vivo remain unclear. Here, we analyze the in vivo functions of FACT with the use of multi-omics analysis after rapid FACT depletion from human cells. We show that FACT depletion destabilizes chromatin and leads to transcriptional defects, including defective promoter-proximal pausing and elongation, and increased premature termination of RNA polymerase II. Unexpectedly, our analysis revealed that promoter-proximal pausing depends not only on the negative elongation factor (NELF) but also on the +1 nucleosome, which is maintained by FACT.
Asunto(s)
Cromatina , Proteínas del Grupo de Alta Movilidad , Nucleosomas , Regiones Promotoras Genéticas , ARN Polimerasa II , Transcripción Genética , Factores de Elongación Transcripcional , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Humanos , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Cromatina/metabolismo , Cromatina/genética , Nucleosomas/metabolismo , Nucleosomas/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Células HeLa , Ensamble y Desensamble de Cromatina , Células HEK293 , Elongación de la Transcripción Genética , Terminación de la Transcripción GenéticaRESUMEN
Transcription by RNA polymerase II (RNA Pol II) relies on the elongation factors PAF1 complex (PAF), RTF1, and SPT6. Here, we use rapid factor depletion and multi-omics analysis to investigate how these elongation factors influence RNA Pol II elongation activity in human cells. Whereas depletion of PAF subunits PAF1 and CTR9 has little effect on cellular RNA synthesis, depletion of RTF1 or SPT6 strongly compromises RNA Pol II activity, albeit in fundamentally different ways. RTF1 depletion decreases RNA Pol II velocity, whereas SPT6 depletion impairs RNA Pol II progression through nucleosomes. These results show that distinct elongation factors stimulate either RNA Pol II velocity or RNA Pol II progression through chromatin in vivo. Further analysis provides evidence for two distinct barriers to early elongation: the promoter-proximal pause site and the +1 nucleosome. It emerges that the first barrier enables loading of elongation factors that are required to overcome the second and subsequent barriers to transcription.
Asunto(s)
ARN Polimerasa II/metabolismo , ARN/biosíntesis , Factores de Transcripción/metabolismo , Humanos , Células K562 , Nucleosomas/genética , Nucleosomas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Polimerasa II/genética , Factores de Transcripción/genéticaRESUMEN
The stability of mRNA is one of the major determinants of gene expression. Although a wealth of sequence elements regulating mRNA stability has been described, their quantitative contributions to half-life are unknown. Here, we built a quantitative model for Saccharomyces cerevisiae based on functional mRNA sequence features that explains 59% of the half-life variation between genes and predicts half-life at a median relative error of 30%. The model revealed a new destabilizing 3' UTR motif, ATATTC, which we functionally validated. Codon usage proves to be the major determinant of mRNA stability. Nonetheless, single-nucleotide variations have the largest effect when occurring on 3' UTR motifs or upstream AUGs. Analyzing mRNA half-life data of 34 knockout strains showed that the effect of codon usage not only requires functional decapping and deadenylation, but also the 5'-to-3' exonuclease Xrn1, the nonsense-mediated decay genes, but not no-go decay. Altogether, this study quantitatively delineates the contributions of mRNA sequence features on stability in yeast, reveals their functional dependencies on degradation pathways, and allows accurate prediction of half-life from mRNA sequence.
Asunto(s)
Estabilidad del ARN/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regiones no Traducidas 3'/genética , Secuencia de Bases , Codón/genética , Codón/metabolismo , Técnicas de Inactivación de Genes , Genes Fúngicos , Semivida , Modelos Biológicos , Degradación de ARNm Mediada por Codón sin Sentido/genética , Iniciación de la Cadena Peptídica Traduccional , Elementos Reguladores de la Transcripción , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
At the end of protein-coding genes, RNA polymerase (Pol) II undergoes a concerted transition that involves 3'-processing of the pre-mRNA and transcription termination. Here, we present a genome-wide analysis of the 3'-transition in budding yeast. We find that the 3'-transition globally requires the Pol II elongation factor Spt5 and factors involved in the recognition of the polyadenylation (pA) site and in endonucleolytic RNA cleavage. Pol II release from DNA occurs in a narrow termination window downstream of the pA site and requires the "torpedo" exonuclease Rat1 (XRN2 in human). The Rat1-interacting factor Rai1 contributes to RNA degradation downstream of the pA site. Defects in the 3'-transition can result in increased transcription at downstream genes.
