Subunits Common to RNA Polymerases

RNA polymerases are heteromultimeric complexes responsible of RNA synthesis. In yeast, as in the other eukaryotes, these complexes contain five common subunits (Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12) that must have similar functions in the three RNA polymerases. However, some of these proteins have been shown to also have specific roles. In the last few decades, substantial progress has been made to understand the role of these common subunits in transcription, but their participation in the activity of each enzyme remains unclear. This review gives a comprehensive overview of current knowledge on the five common subunits of eukaryotic RNA pol, placing attention not only on their common roles in the activity of the RNA pols but also on describing specific roles for some of the complexes

Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration

mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5ʹ exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3ʹ was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.This work has been supported by grants from the Ministerio de Economía, Industria y Competitividad – Agencia Estatal de Investigación, and European Union funds (FEDER) [BFU2016-77728-C3-1-P to S. C.],[BFU2016-77728-C3-3-P to J.E.P-O & P.A], [BFU2016-77728-C3-2-P to F.N.] and RED2018‐102467‐T to J.E.P-O, F.N. and S.C.; by FPI grants from the Spanish Government to V.B and A.C-B; by the Regional Andalusian Government [BIO271 and US-1256285 to S. C.], [BIO258 and FEDER-UJA 1260360 to F.N.] and from the Regional Valencian Government [AICO/2019/088 to J.E.P-O]. Funding for open access charge: [BFU2016-77728-C3-1-P]

Rpb4/7 heterodimer regulates RNAPII phosphorylation

Póster presentado a la EMBO Conferences: Gene Transcription in Yeast: From regulatory networks to mechanisms, celebrada en Sant Feliu de Guixols (España) del 14 al 19 de junio de 2014.The 12-subunit RNA polymerase II enzyme in eukaryotic cells is essential for transcription of protein-coding genes. The Rpb4 and Rpb7 subunits bind to each other forming a complex in archaebacteria and in eukaryotic cells, and display some unique features that distinguish them from the remaining subunits. In S. cerevisiae, the Rpb4/7 heterodimer can dissociate from the rest of the holoenzyme. These subunits are important for transcription initiation, elongation and, in particular, Rpb4 contributes to contranscriptional recruitment of 3’-end processing factors. However, recent evidences suggests that they are also involved in DNA repair, mRNA export and decay, as well as translation. Additionally, several facts suggest that Rpb4/7 may play a role on RNAPII phosphorylation: 1) The close proximity of the Rpb4/7 heterodimer to the CTD of the Rpb1. 2) The in vitro concerted interaction of Rpb4 and the CTD phosphatase, Fcp1, in S. pombe, suggesting that Rpb4 may play an important role in the assembly of Fcp1 into the RNAPII complex and thus, also in dephosphorylation. 3) The Rpb4-Fcp1 interaction is also conserved in Drosophila; and 4) in Saccharomyces cerevisiae, Rpb7 genetically interacts with the propyl isomerase Ess1, also involved in CTD phosphorylation regulation. Here, we present genetic and biochemical data showing that effectively Rpb4/7 subcomplex is important for RNAPII dephosphorylation in S. cerevisiae, regulating several CTD modifying enzymes.This work was funded by the MINECO (BFU2009-07179/BMC).Peer Reviewe

The Association of Rpb4 with RNA Polymerase II Depends on CTD Ser5P Phosphatase Rtr1 and Influences mRNA Decay in <i>Saccharomyces cerevisiae</i>

Rtr1 is an RNA polymerase II (RNA pol II) CTD-phosphatase that influences gene expression during the transition from transcription initiation to elongation and during transcription termination. Rtr1 interacts with the RNA pol II and this interaction depends on the phosphorylation state of the CTD of Rpb1, which may influence dissociation of the heterodimer Rpb4/7 during transcription. In addition, Rtr1 was proposed as an RNA pol II import factor in RNA pol II biogenesis and participates in mRNA decay by autoregulating the turnover of its own mRNA. Our work shows that Rtr1 acts in RNA pol II assembly by mediating the Rpb4/7 association with the rest of the enzyme. RTR1 deletion alters RNA pol II assembly and increases the amount of RNA pol II associated with the chromatin that lacks Rpb4, decreasing Rpb4-mRNA imprinting and, consequently, increasing mRNA stability. Thus, Rtr1 interplays RNA pol II biogenesis and mRNA decay regulation. Our data also indicate that Rtr1 mediates mRNA decay regulation more broadly than previously proposed by cooperating with Rpb4. Interestingly, our data include new layers in the mechanisms of gene regulation and in the crosstalk between mRNA synthesis and decay by demonstrating how the association of Rpb4/7 to the RNA pol II influences mRNA decay

The conserved foot domain of RNA pol II associates with proteins involved in transcriptional initiation and/or early elongation

RNA polymerase (pol) II establishes many protein-protein interactions with transcriptional regulators to coordinate different steps of transcription. Although some of these interactions have been well described, little is known about the existence of RNA pol II regions involved in contact with transcriptional regulators. We hypothesize that conserved regions on the surface of RNA pol II contact transcriptional regulators. We identified such an RNA pol II conserved region that includes the majority of the >foot> domain and identified interactions of this region with Mvp1, a protein required for sorting proteins to the vacuole, and Spo14, a phospholipase D. Deletion of MVP1 and SPO14 affects the transcription of their target genes and increases phosphorylation of Ser5 in the carboxyterminal domain (CTD). Genetic, phenotypic, and functional analyses point to a role for these proteins in transcriptional initiation and/ or early elongation, consistent with their genetic interactions with CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme. © 2011 by the Genetics Society of America.This work was supported by grants of Spanish Ministry of Education and Science, Ministry of Sciencie and Innovation and FEDER (BFU2007-67575-C03-03/BMC, BFU2010-21975-C03-02 Spain) and Junta de Andalucía (BIO258, P08-CVI-03508) to F.N, BFU2007-67575-C03-01/BMC to J.E.P-O., and BFU 2009-07179 to O.C.. A.I. G-G. was recipient of predoctoral fellowships from Universidad de Jaén and currently from Ministry of Education and Culture. A.G. was supported by a fellowship from the Junta de Castilla y León.Peer Reviewe

Prefoldin-like Bud27 influences the transcription of ribosomal components and ribosome biogenesis in Saccharomyces cerevisiae

Understanding the functional connection that occurs for the three nuclear RNA polymerases to synthesize ribosome components during the ribosome biogenesis process has been the focal point of extensive research. To preserve correct homeostasis on the production of ribosomal components, cells might require the existence of proteins that target a common subunit of these RNA polymerases to impact their respective activities. This work describes how the yeast prefoldin-like Bud27 protein, which physically interacts with the Rpb5 common subunit of the three RNA polymerases, is able to modulate the transcription mediated by the RNA polymerase I, likely by influencing transcription elongation, the transcription of the RNA polymerase III, and the processing of ribosomal RNA. Bud27 also regulates both RNA polymerase II-dependent transcription of ribosomal proteins and ribosome biogenesis regulon genes, likely by occupying their DNA ORFs, and the processing of the corresponding mRNAs. With RNA polymerase II, this association occurs in a transcription rate-dependent manner. Our data also indicate that Bud27 inactivation alters the phosphorylation kinetics of ribosomal protein S6, a readout of TORC1 activity. We conclude that Bud27 impacts the homeostasis of the ribosome biogenesis process by regulating the activity of the three RNA polymerases and, in this way, the synthesis of ribosomal components. This quite likely occurs through a functional connection of Bud27 with the TOR signaling pathway

Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration.

mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5' exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3' was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation