RRC ID 78071
Author McNamar R, Abu-Adas Z, Rothblum K, Knutson BA, Rothblum LI.
Title Conditional depletion of the RNA polymerase I subunit PAF53 reveals that it is essential for mitosis and enables identification of functional domains.
Journal J Biol Chem
Abstract Our knowledge of the mechanism of rDNA transcription has benefited from the combined application of genetic and biochemical techniques in yeast. Nomura's laboratory (Nogi, Y., Vu, L., and Nomura, M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7026-7030 and Nogi, Y., Yano, R., and Nomura, M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3962-3966) developed a system in yeast to identify genes essential for ribosome biogenesis. Such systems have allowed investigators to determine whether a gene was essential and to determine its function in rDNA transcription. However, there are significant differences in both the structures and components of the transcription apparatus and the patterns of regulation between mammals and yeast. Thus, there are significant deficits in our understanding of mammalian rDNA transcription. We have developed a system combining CRISPR/Cas9 and an auxin-inducible degron that enables combining a "genetics-like"approach with biochemistry to study mammalian rDNA transcription. We now show that the mammalian orthologue of yeast RPA49, PAF53, is required for rDNA transcription and mitotic growth. We have studied the domains of the protein required for activity. We have found that the C-terminal, DNA-binding domain (tandem-winged helix), the heterodimerization, and the linker domain were essential. Analysis of the linker identified a putative helix-turn-helix (HTH) DNA-binding domain. This HTH constitutes a second DNA-binding domain within PAF53. The HTH of the yeast and mammalian orthologues is essential for function. In summary, we show that an auxin-dependent degron system can be used to rapidly deplete nucleolar proteins in mammalian cells, that PAF53 is necessary for rDNA transcription and cell growth, and that all three PAF53 domains are necessary for its function.
Volume 294(52)
Pages 19907-19922
Published 2019-12-27
DOI 10.1074/jbc.RA119.009902
PII S0021-9258(20)30014-4
PMID 31727736
PMC PMC6937585
MeSH Amino Acid Sequence Animals CRISPR-Cas Systems / genetics DNA, Ribosomal / metabolism Dimerization Helix-Turn-Helix Motifs Indoleacetic Acids / metabolism Mice Mitosis* Protein Subunits / chemistry Protein Subunits / genetics Protein Subunits / metabolism RNA Polymerase I / chemistry RNA Polymerase I / genetics RNA Polymerase I / metabolism* RNA, Guide, CRISPR-Cas Systems / metabolism S Phase Cell Cycle Checkpoints Saccharomyces cerevisiae / growth & development Saccharomyces cerevisiae / metabolism* Saccharomyces cerevisiae Proteins / chemistry Saccharomyces cerevisiae Proteins / metabolism Transcription, Genetic
Yeast BY27858