Rothblum, L. | Search bioRxiv

bioRxiv2021

Targeted knockdown of the PAF49 component of the PAF53/PAF49 heterodimer causes the degradation of PAF53.

There are significant differences in the components of the ribosomal DNA transcription apparatuses of yeast and mammals. Moreover, the patterns of regulation between mammals and yeast are also different. To overcome, deficits in our understanding of mammalian rDNA transcription, we have developed a system to introduce an inducible degron into the endogenous genes of mammalian cells. This allows us to combine a knock out the endogenous gene product and replace it with mutant proteins in order to study their function in ribosomal DNA transcription. Using this system, we show that the knockout of PAF49, the mammalian ortholog of yeast A34, results in the relatively rapid degradation of PAF53, the ortholog of yeast A49. Interestingly, the steady-state levels of the core subunits of RNA polymerase I are unaffected.

molecular biology↗

bioRxiv2019

Inducible degron-dependent depletion of the RNA polymerase I associated factor PAF53 demonstrates it is essential for cell growth and allows for the analysis of functional domains

Our knowledge of the mechanism of rDNA transcription has benefitted from the combined application of genetic techniques in yeast, and progress on the biochemistry of the various components of yeast rDNA transcription. Nomuras laboratory derived a system in yeast for screening for mutants essential for ribosome biogenesis. Such systems have allowed investigators to not only determine if a gene was essential, but to analyze domains of the proteins for different functions in rDNA transcription in vivo. However, because there are significant differences in both the structures and components of the transcription apparatus and the patterns of regulation between mammals and yeast, there are significant deficits in our understanding of mammalian rDNA transcription. We have developed a system combining CRISPR/Cas9 and an inducible degron that allows us to combine a \"genetics-like\" approach to studying mammalian rDNA transcription with biochemistry. Using this system, we show that the mammalian homologue of yeast A49, PAF53, is required for rDNA transcription and mitotic growth. Further, we have been able to study the domains of the protein required for activity. We have found that while the C-terminal, DNA-binding domain (tWH) was necessary for complete function, the heterodimerization and linker domains were also essential. Analysis of the linker identified a putative DNA-binding domain. We have confirmed that the helix-turn-helix (HTH) of the linker constitutes a second DNA-binding domain within PAF53 and that the HTH is essential for PAF53 function.

molecular biology↗