RESUMEN
Prefoldin is a heterohexameric complex conserved from archaea to humans that plays a cochaperone role during the co-translational folding of actin and tubulin monomers. Additional functions of prefoldin have been described, including a positive contribution to transcription elongation and chromatin dynamics in yeast. Here we show that prefoldin perturbations provoked transcriptional alterations across the human genome. Severe pre-mRNA splicing defects were also detected, particularly after serum stimulation. We found impairment of co-transcriptional splicing during transcription elongation, which explains why the induction of long genes with a high number of introns was affected the most. We detected genome-wide prefoldin binding to transcribed genes and found that it correlated with the negative impact of prefoldin depletion on gene expression. Lack of prefoldin caused global decrease in Ser2 and Ser5 phosphorylation of the RNA polymerase II carboxy-terminal domain. It also reduced the recruitment of the CTD kinase CDK9 to transcribed genes, and the association of splicing factors PRP19 and U2AF65 to chromatin, which is known to depend on CTD phosphorylation. Altogether the reported results indicate that human prefoldin is able to act locally on the genome to modulate gene expression by influencing phosphorylation of elongating RNA polymerase II, and thereby regulating co-transcriptional splicing.
Asunto(s)
Chaperonas Moleculares/fisiología , Empalme del ARN , ARN Mensajero/metabolismo , Transcripción Genética , Línea Celular , Humanos , Intrones , ARN Polimerasa II/metabolismo , Precursores del ARN/metabolismo , Factores de Empalme de ARN/metabolismo , Proteínas Represoras/fisiología , TranscriptomaRESUMEN
Ribosome assembly requires the concerted expression of hundreds of genes, which are transcribed by all three nuclear RNA polymerases. Transcription elongation involves dynamic interactions between RNA polymerases and chromatin. We performed a synthetic lethal screening in Saccharomyces cerevisiae with a conditional allele of SPT6, which encodes one of the factors that facilitates this process. Some of these synthetic mutants corresponded to factors that facilitate pre-rRNA processing and ribosome biogenesis. We found that the in vivo depletion of one of these factors, Arb1, activated transcription elongation in the set of genes involved directly in ribosome assembly. Under these depletion conditions, Spt6 was physically targeted to the up-regulated genes, where it helped maintain their chromatin integrity and the synthesis of properly stable mRNAs. The mRNA profiles of a large set of ribosome biogenesis mutants confirmed the existence of a feedback regulatory network among ribosome assembly genes. The transcriptional response in this network depended on both the specific malfunction and the role of the regulated gene. In accordance with our screening, Spt6 positively contributed to the optimal operation of this global network. On the whole, this work uncovers a feedback control of ribosome biogenesis by fine-tuning transcription elongation in ribosome assembly factor-coding genes.
Asunto(s)
Redes Reguladoras de Genes , Chaperonas de Histonas/genética , Biogénesis de Organelos , Ribosomas/genética , Proteínas de Saccharomyces cerevisiae/genética , Elongación de la Transcripción Genética , Factores de Elongación Transcripcional/genética , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/genética , Retroalimentación Fisiológica , Chaperonas de Histonas/metabolismo , Mutación , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Mutaciones Letales Sintéticas , Factores de Elongación Transcripcional/metabolismo , TranscriptomaRESUMEN
Cell cycle regulation is a very accurate process that ensures cell viability and the genomic integrity of daughter cells. A fundamental part of this regulation consists in the arrest of the cycle at particular points to ensure the completion of a previous event, to repair cellular damage, or to avoid progression in potentially risky situations. In this work, we demonstrate that a reduction in nucleotide levels or the depletion of RNA polymerase I or III subunits generates a cell cycle delay at the G1/S transition in Saccharomyces cerevisiae. This delay is concomitant with an imbalance between ribosomal RNAs and proteins which, among others, provokes an accumulation of free ribosomal protein L5. Consistently with a direct impact of free L5 on the G1/S transition, rrs1 mutants, which weaken the assembly of L5 and L11 on pre-60S ribosomal particles, enhance both the G1/S delay and the accumulation of free ribosomal protein L5. We propose the existence of a surveillance mechanism that couples the balanced production of yeast ribosomal components and cell cycle progression through the accumulation of free ribosomal proteins. This regulatory pathway resembles the p53-dependent nucleolar-stress checkpoint response described in human cells, which indicates that this is a general control strategy extended throughout eukaryotes.
Asunto(s)
Fase G1/fisiología , Proteínas Ribosómicas/biosíntesis , Ribosomas/metabolismo , Fase S/fisiología , Saccharomyces cerevisiae/metabolismo , Humanos , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3) in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA-damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/fisiología , Fase G1/fisiología , Proteínas del Grupo de Alta Movilidad/fisiología , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Factores de Elongación Transcripcional/fisiología , Northern Blotting , Inmunoprecipitación de Cromatina , Ciclinas/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Proteínas del Grupo de Alta Movilidad/metabolismo , Fosforilación , Regiones Promotoras Genéticas , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
Eukaryotic life is possible due to the multitude of complex and precise phenomena that take place in the cell. Essential processes like gene transcription, mRNA translation, cell growth, and proliferation, or membrane traffic, among many others, are strictly regulated to ensure functional success. Such systems or vital processes do not work and adjusts independently of each other. It is required to ensure coordination among them which requires communication, or crosstalk, between their different elements through the establishment of complex regulatory networks. Distortion of this coordination affects, not only the specific processes involved, but also the whole cell fate. However, the connection between some systems and cell fate, is not yet very well understood and opens lots of interesting questions. In this review, we focus on the coordination between the function of the three nuclear RNA polymerases and cell cycle progression. Although we mainly focus on the model organism Saccharomyces cerevisiae, different aspects and similarities in higher eukaryotes are also addressed. We will first focus on how the different phases of the cell cycle affect the RNA polymerases activity and then how RNA polymerases status impacts on cell cycle. A good example of how RNA polymerases functions impact on cell cycle is the ribosome biogenesis process, which needs the coordinated and balanced production of mRNAs and rRNAs synthesized by the three eukaryotic RNA polymerases. Distortions of this balance generates ribosome biogenesis alterations that can impact cell cycle progression. We also pay attention to those cases where specific cell cycle defects generate in response to repressed synthesis of ribosomal proteins or RNA polymerases assembly defects.
RESUMEN
RNA polymerase II backtracking is a well-known phenomenon, but its involvement in gene regulation is yet to be addressed. Structural studies into the backtracked complex, new reactivation mechanisms and genome-wide approaches are shedding some light on this interesting aspect of gene transcription. In this review, we briefly summarise these new findings, comment about some results recently obtained in our laboratory, and propose a new model for the influence of the chromatin context on RNA polymerase II backtracking.