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ABSTRACT: Termination of RNA polymerase II (RNAPII) transcription of protein-coding genes occurs downstream of cleavage/polyadenylation sites. According to the "torpedo" model, the 5'-3' exonuclease Rat1p/Xrn2p attacks the newly formed 5' end of the cleaved pre-mRNA, causing the still transcribing RNAPII to terminate. Here we demonstrate a similar role of S. cerevisiae Rat1p within the gene body. We find that the transcription processivity defect imposed on RNAPII by the rpb1-N488D mutation is corrected upon Rat1p inactivation. Importantly, Rat1p-dependent transcription termination occurs upstream the polyadenylation site. Genetic and biochemical evidence demonstrate that mRNA capping is defective in rpb1-N488D cells, which leads to increased levels of Rat1p all along the gene locus. Consistently, Rat1p-dependent RNAPII termination is also observed in the capping-deficient ceg1-63 strain. Our data suggest that Rat1p serves to terminate RNAPII molecules engaged in the production of uncapped RNA, regardless of their position on the gene locus.
Molecular cell 02/2010; 37(4):580-7. · 14.61 Impact Factor
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ABSTRACT: Transcription elongation by RNA polymerase II was often considered an invariant non-regulated process. However, genome-wide studies have shown that transcriptional pausing during elongation is a frequent phenomenon in tightly-regulated metazoan genes. Using a combination of ChIP-on-chip and genomic run-on approaches, we found that the proportion of transcriptionally active RNA polymerase II (active versus total) present throughout the yeast genome is characteristic of some functional gene classes, like those related to ribosomes and mitochondria. This proportion also responds to regulatory stimuli mediated by protein kinase A and, in relation to cytosolic ribosomal-protein genes, it is mediated by the silencing domain of Rap1. We found that this inactive form of RNA polymerase II, which accumulates along the full length of ribosomal protein genes, is phosphorylated in the Ser5 residue of the CTD, but is hypophosphorylated in Ser2. Using the same experimental approach, we show that the in vivo-depletion of FACT, a chromatin-related elongation factor, also produces a regulon-specific effect on the expression of the yeast genome. This work demonstrates that the regulation of transcription elongation is a widespread, gene class-dependent phenomenon that also affects housekeeping genes.
PLoS Genetics 09/2009; 5(8):e1000614. · 8.69 Impact Factor
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ABSTRACT: For efficient transcription, RNA PolII must overcome the presence of nucleosomes. The p38-related MAPK Hog1 is an important regulator of transcription upon osmostress in yeast and thereby it is involved in initiation and elongation. However, the role of this protein kinase in elongation has remained unclear. Here, we show that during stress there is a dramatic change in the nucleosome organization of stress-responsive loci that depends on Hog1 and the RSC chromatin remodelling complex. Upon stress, the MAPK Hog1 physically interacts with RSC to direct its association with the ORF of osmo-responsive genes. In RSC mutants, PolII accumulates on stress promoters but not in coding regions. RSC mutants also display reduced stress gene expression and enhanced sensitivity to osmostress. Cell survival under acute osmostress might thus depend on a burst of transcription that in turn could occur only with efficient nucleosome eviction. Our results suggest that the selective targeting of the RSC complex by Hog1 provides the necessary mechanistic basis for this event.
The EMBO Journal 02/2009; 28(4):326-36. · 9.20 Impact Factor
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PLoS Genetics 02/2009; 5(1). · 8.69 Impact Factor
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ABSTRACT: Rebound of HIV viremia after interruption of anti-retroviral therapy is due to the small population of CD4+ T cells that remain latently infected. HIV-1 transcription is the main process controlling post-integration latency. Regulation of HIV-1 transcription takes place at both initiation and elongation levels. Pausing of RNA polymerase II at the 5' end of HIV-1 transcribed region (5'HIV-TR), which is immediately downstream of the transcription start site, plays an important role in the regulation of viral expression. The activation of HIV-1 transcription correlates with the rearrangement of a positioned nucleosome located at this region. These two facts suggest that the 5'HIV-TR contributes to inhibit basal transcription of those HIV-1 proviruses that remain latently inactive. However, little is known about the cell elements mediating the repressive role of the 5'HIV-TR. We performed a genetic analysis of this phenomenon in Saccharomyces cerevisiae after reconstructing a minimal HIV-1 transcriptional system in this yeast. Unexpectedly, we found that the critical role played by the 5'HIV-TR in maintaining low levels of basal transcription in yeast is mediated by FACT, Spt6, and Chd1, proteins so far associated with chromatin assembly and disassembly during ongoing transcription. We confirmed that this group of factors plays a role in HIV-1 postintegration latency in human cells by depleting the corresponding human orthologs with shRNAs, both in HIV latently infected cell populations and in particular single-integration clones, including a latent clone with a provirus integrated in a highly transcribed gene. Our results indicate that chromatin reassembly factors participate in the establishment of the equilibrium between activation and repression of HIV-1 when it integrates into the human genome, and they open the possibility of considering these factors as therapeutic targets of HIV-1 latency.
PLoS Genetics 02/2009; 5(1):e1000339. · 8.69 Impact Factor
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ABSTRACT: The FACT complex stimulates transcription elongation on nucleosomal templates. In vivo experiments also involve FACT in the reassembly of nucleosomes traversed by RNA polymerase II. Since several features of chromatin organization vary throughout the genome, we wondered whether FACT is equally required for all genes. We show in this study that the in vivo depletion of Spt16, one of the subunits of Saccharomyces cerevisiae FACT, strongly affects transcription of three genes, GAL1, PHO5, and Kluyveromyces lactis LAC4, which exhibit positioned nucleosomes at their transcribed regions. In contrast, showing a random nucleosome structure, YAT1 and Escherichia coli lacZ are only mildly influenced by Spt16 depletion. We also show that the effect of Spt16 depletion on GAL1 expression is suppressed by a histone mutation and that the insertion of a GAL1 fragment, which allows the positioning of two nucleosomes, at the 5' end of YAT1 makes the resulting transcription unit sensitive to Spt16 depletion. These results indicate that FACT requirement for transcription depends on the chromatin organization of the 5' end of the transcribed region.
Molecular and Cellular Biology 01/2007; 26(23):8710-21. · 5.53 Impact Factor
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