RESUMEN
Rpb5 is a subunit shared by the three eukaryotic RNA polymerases although its role in transcription remains unclear. It has been proposed that it makes contact with the promoter DNA and to participate in the coordination of the opening/closing of the RNA polymerase II DNA cleft. Here, we report the specific role of Rpb5 in the function of the yeast RNA polymerase II. The rpb5-P151T mutation specifically impairs transcription elongation by RNA polymerase II but does not influence the functions of RNA polymerases I or III. The comparison of RNA polymerase II ChIP and run-on signals indicates a higher tendency to backtrack by this mutant, in agreement with its lower elongation rate and its genetic interactions with dst1Δ mutant. This phenotype is particularly striking shortly after transcription initiation and is linked to differences in the phosphorylation state of the RNA polymerase II and reduced recruitment of Spt5 to transcribe chromatin, thus influencing its anti-backtracking activity. All together, our results reveal an important role of Rpb5 in the transition from initiation to elongation mediated by the RNA polymerase II, by modulating the Spt5 association, and the backtracking activity of the enzyme.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética/genética , Factores de Elongación Transcripcional/genética , Cromatina/genética , Unión Proteica , Saccharomyces cerevisiae/genéticaRESUMEN
Bud27, the yeast orthologue of human URI/RMP, is a member of the prefoldin-like family of ATP-independent molecular chaperones. It has recently been shown to mediate the assembly of the three RNA polymerases in an Rpb5-dependent manner. In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation. We show that Bud27 associates with RNA pol II phosphorylated forms (CTD-Ser5P and CTD-Ser2P), and that its absence affects RNA pol II occupancy of transcribed genes. We also reveal that Bud27 associates in vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II. Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Factores de Iniciación de Péptidos/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Elongación de la Transcripción Genética , Cromatina/metabolismo , Mutación , Factores de Iniciación de Péptidos/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The unconventional prefoldin URI/RMP, in humans, and its orthologue in yeast, Bud27, have been proposed to participate in the biogenesis of the RNA polymerases. However, this role of Bud27 has not been confirmed and is poorly elucidated. Our data help clarify the mechanisms governing biogenesis of the three eukaryotic RNA pols. We show evidence that Bud27 is the first example of a protein that participates in the biogenesis of the three eukaryotic RNA polymerases and the first example of a protein modulating their assembly instead of their nuclear transport. In addition we demonstrate that the role of Bud27 in RNA pols biogenesis depends on Rpb5. In fact, lack of BUD27 affects growth and leads to a substantial accumulation of the three RNA polymerases in the cytoplasm, defects offset by the overexpression of RPB5. Supporting this, our data demonstrate that the lack of Bud27 affects the correct assembly of Rpb5 and Rpb6 to the three RNA polymerases, suggesting that this process occurs in the cytoplasm and is a required step prior to nuclear import. Also, our data support the view that Rpb5 and Rpb6 assemble somewhat later than the rest of the complexes. Furthermore, Bud27 Rpb5-binding but not PFD-binding domain is necessary for RNA polymerases biogenesis. In agreement, we also demonstrate genetic interactions between BUD27, RPB5, and RPB6. Bud27 shuttles between the nucleus and the cytoplasm in an Xpo1-independent manner, and also independently of microtubule polarization and possibly independently of its association with the RNA pols. Our data also suggest that the role of Bud27 in RNA pols biogenesis is independent of the chaperone prefoldin (PFD) complex and of Iwr1. Finally, the role of URI seems to be conserved in humans, suggesting conserved mechanisms in RNA pols biogenesis.
