The association of the RSC remodeler complex with chromatin is influenced by the prefoldin-like Bud27 and determines nucleosome positioning and polyadenylation sites usage in Saccharomyces cerevisiae.

The tripartite interaction between the chromatin remodeler complex RSC, RNA polymerase subunit Rpb5 and prefoldin-like Bud27 is necessary for proper RNA pol II elongation. Indeed lack of Bud27 alters this association and affects transcription elongation. This work investigates the consequences of lack of Bud27 on the chromatin association of RSC and RNA pol II, and on nucleosome positioning. Our results demonstrate that RSC binds chromatin in gene bodies and lack of Bud27 alters this association, mainly around polyA sites. This alteration impacts chromatin organization and leads to the accumulation of RNA pol II molecules around polyA sites, likely due to pausing or arrest. Our data suggest that RSC is necessary to maintain chromatin organization around those sites, and any alteration of this organization results in the widespread use of alternative polyA sites. Finally, we also find a similar molecular phenotype that occurs upon TOR inhibition with rapamycin, which suggests that alternative polyadenylation observed upon TOR inhibition is likely Bud27-dependent.

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

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.

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.

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.

A Yeast Chromatin-enriched Fractions Purification Approach, yChEFs, from Saccharomyces cerevisiae.

We have adapted a previous procedure and improved an approach that we named yChEFs (yeast Chromatin Enriched Fractions) for purifying chromatin fractions. This methodology allows the easy, reproducible and scalable recovery of proteins associated with chromatin. By using yChEFs, we bypass subcellular fractionation requirements involved when using zymolyase to obtain the spheroplast, which is employed in many other procedures. Employing small amount of culture cells and small volumes of solutions during the yChEFs procedure is very useful to allow many samples to be handled at the same time, and also reduces costs and efforts. The purified proteins associated with chromatin fractions obtained by yChEFs can be analyzed by Western blot (Figure 1) or combined with mass spectrometry for proteomic analyses.

The mRNA degradation factor Xrn1 regulates transcription elongation in parallel to Ccr4.

Co-transcriptional imprinting of mRNA by Rpb4 and Rpb7 subunits of RNA polymerase II (RNAPII) and by the Ccr4-Not complex conditions its post-transcriptional fate. In turn, mRNA degradation factors like Xrn1 are able to influence RNAPII-dependent transcription, making a feedback loop that contributes to mRNA homeostasis. In this work, we have used repressible yeast GAL genes to perform accurate measurements of transcription and mRNA degradation in a set of mutants. This genetic analysis uncovered a link from mRNA decay to transcription elongation. We combined this experimental approach with computational multi-agent modelling and tested different possibilities of Xrn1 and Ccr4 action in gene transcription. This double strategy brought us to conclude that both Xrn1-decaysome and Ccr4-Not regulate RNAPII elongation, and that they do it in parallel. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII. We found that xrn1Δ and ccr4Δ exhibited very different patterns of TFIIS versus RNAPII occupancy, which confirmed their distinct role in controlling transcription elongation. We also found that the relative influence of Xrn1 and Ccr4 is different in the genes encoding ribosomal proteins as compared to the rest of the genome.

The Yeast Prefoldin Bud27.

Bud27 and its human orthologue URI (unconventional prefoldin RPB5-interactor) are members of the prefoldin (PFD) family of ATP-independent molecular chaperones binding the Rpb5 subunit to all three nuclear eukaryotic RNA polymerases (RNA pols). Bud27/URI are considered to function as a scaffold protein able to assemble additional members of the prefoldin (PDF) family in both human and yeast. Bud27 and URI are not subunits of the canonical PFD/GimC complex and not only the composition but also other functions independent of the PFD/GimC complex have been described for Bud27 and URI. Bud27 interacts only with Pfd6 but no other components of the R2TP/PFDL. Furthermore previously reported interaction between Bud27 and Pfd2 was not later confirmed. These results point to major differences in the prefoldin-like complex composition between yeast and other organisms, suggesting also important differences in functions. Furthermore, this assumption could be extended to the R2TP/PFDL complex, which has been shown to differ between different organisms and has not been identified in yeast. This casts doubt on whether Bud27 cooperation with prefoldin and other components of the R2TP/PFDL modules are required for its action. This could be extended to URI and point to a role of Bud27/URI in cell functions more relevant than this previously proposed as co-prefoldin.

The yeast prefoldin-like URI-orthologue Bud27 associates with the RSC nucleosome remodeler and modulates transcription.

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.