The Prefoldin Bud27 Mediates the Assembly of the Eukaryotic RNA Polymerases in an Rpb5-Dependent Manner

Abstract

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.

Correct Assembly of RNA Polymerase II Depends on the Foot Domain and Is Required for Multiple Steps of Transcription in Saccharomyces cerevisiae

“…Recent observations in yeast and humans are consistent with RNA pol assembly in the cytoplasm as a prerequisite for its nuclear import (23,(30)(31)(32). Thus, our results do not eliminate the possibility that RNA pol II foot mutations affect the correct assembly of Rpb1 with the rest of the complex and that, thus, nonassociated Rpb1 could be degraded in the cytoplasm.…”

“…Thus, our results do not eliminate the possibility that RNA pol II foot mutations affect the correct assembly of Rpb1 with the rest of the complex and that, thus, nonassociated Rpb1 could be degraded in the cytoplasm. To help elucidate this possibility, we tested the localization and accumulation of Rpb1 upon deletion of BUD27, a prefoldin whose inactivation alters the assembly of RNA pol II, leading to its cytoplasmic accumulation (23). In immunocytochemistry experiments, clear Rpb1 cytoplasmic accumulation was found in a bud27⌬ mutant strain, although nuclear staining was also detected, indicating that RNA pol II was partially mislocalized (Fig.…”

Subunits Common to RNA Polymerases

“…In S. cerevisiae, assembly of the RNA pols occurs in the cytoplasm prior their entry to the nucleus, and Rpb6 and Rpb5 assemble in a process dependent on the prefoldin-like Bud27 [42]. Similarly, cytoplasmic RNA pol I assembly has been previously proposed in human [44].…”

“…In accordance with the role of Rpb6 in RNA pols assembly, the lack of Bud27 alters the correct cytoplasmic assembly of Rpb5 and Rpb6 to the three RNA polymerases, leading to a more instable RNA pol II [42]. Intriguingly, of the five shared subunits, both Rpb6 and Rpb5 have two paralogues in Trypanosome brucei, T. cruzi and Leishmania major [17].…”

“…Owing to RPB5-URI interaction, RPB5 could participate in regulating the androgen receptor in human cells [41]. This interaction also extends to S. cerevisiae and the correct association between Rpb5 and the URI orthologue, Bud27, is essential for the correct cytoplasmic assembly of the three RNA pols before their entry to the nucleus [42]. Notably, in mammals, RPB5 forms a complex with UXT, WDR92/Monad, PDRG1, URI, PFDN2 and PFDN6, which is thought to adopt a prefoldin-like structure and cooperates with the cochaperone R2TP complex to assembly of RNA pol II [43][44][45].…”