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.

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.

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.I N eukaryotes as in archaea, bacteria, chloroplasts, some mitochondria, and nucleocytoplasmic DNA viruses, transcription is ensured by heteromultimeric DNA-dependent RNA polymerases (Thuriaux and Sentenac 1992;Vassylyev et al. 2002;Werner and Weinzierl 2002;Iyer et al. 2006). RNA polymerase II (RNA pol II) produces all mRNAs and many noncoding RNAs. Although it transcribes most of the nuclear genome, it contributes ,10% of the total RNA present in growing cells (Hahn 2004). To transcribe a gene, RNA pol II requires the action of general transcription factors, coregulators, specific transcription activators, and repressors. In fact, the RNA pol II transcription machinery is the most complex of those associated with the three RNA polymerases, with a total of nearly 60 polypeptides (Hahn 2004).Knowledge of both the architecture making up this complex and the function of its different parts is essential to understand their role in the different transcription steps (Cramer 2006;Zaros et al. 2007;Venters and Pugh 2009). Structural data gathered over the last few years on Saccharomyces cerevisiae RNA pol II have provided a detailed map of the physical interactions between the different subunits, establishing regions that are important for transcription (Cramer et al. 2001;Bushnell et al. 2002;Armache et al. 2003;Meyer et al. 2009). Notably, recent work has contributed to the understanding of how RNA pol II amino acid regions or subunits are involved in the contact with transcriptional regulators such as TFIIS, TFIIB, TFIIE, TFIIF, or Mediator, among others, although the data are sometimes imprecise or controversial (Guglielmi et al. 2004;Chadick and Asturias 2005;Chen et al. 2007;Meyer et al. 2009;Kostrewa et al. 2009).A major question that remains unexplored is the identification of domains of RNA pol II that could be involved in the interaction with elements of the transcriptional machinery and that could participate in coordinating with them. The...

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