The prefoldin bud27 mediates the assembly of the eukaryotic RNA polymerases in an rpb5-dependent manner

15 páginasThe 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.This work was supported by grants from the Spanish Ministry of Education and Science, Ministry of Science and Innovation, and FEDER (BFU2010-21975-C03-02 Spain; http://www.idi.mineco.gob.es/portal/site/MICINN/) to FN and SR-N (BFU2011-23418; http://www.idi.mineco.gob.es/portal/site/MICINN/) and from Junta de Andalucı´a (BIO258, P10-CVI6521, P08-CVI-03508; http://www.juntadeandalucia.es/organismos/economiainnovacioncienciayempleo.html) to FN. AIG-G and VG-M are recipients of predoctoral fellowships from MEC, and FH-T of postdoctoral fellowships from Junta de Andalucı´a. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe