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.
Highlights
Eukaryotic RNA polymerases are a family of multimeric enzymes, RNA pol I, II, and III, responsible for the specific synthesis of different RNAs
Only the physical interaction between Bud27 and Rpb5 in yeast has been further demonstrated and no clear data concerning the association of Bud27 with the other two RNA polymerases have been reported
To gain insights into the association of Bud27 with the three RNA pols and to identify proteins that associate with Bud27 in yeast, we used the BUD27 gene TAP-tagged at its 39 end in TAP purifications
Summary
Eukaryotic RNA polymerases are a family of multimeric enzymes, RNA pol I, II, and III, responsible for the specific synthesis of different RNAs. RNA pol I is specialized in the synthesis of the pre-rRNA precursor of the three largest rRNA and typically account for about 75% of the entire transcription output in fast-growing yeast cells. RNA pol III transcribes mostly tRNAs and 5S rRNA, together with several short non-translated RNAs, while transcription corresponds to about 15% of the total RNA. RNA pol II, the enzyme that produces all mRNAs and many noncoding ones, transcribes most of the nuclear genome but contributes to less than 10% of total RNA in growing cells. Despite intensive studies concerning the structure and the transcriptional regulation of the three RNA polymerases [4,5], little is known about the mechanisms governing their assembly and their nuclear import
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