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

Abstract

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. Nomura’s 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.

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

“…We targeted PAF53 for degradation as described previously (51) and determined the level of PAF49. As shown in Figure 1A, the knockdown of PAF53 for 24 hours is not associated with the depletion of PAF49 (compare lanes 1 and 2).…”

“…However, as discussed above, neither of the yeast homologs of PAF53 or PAF49 were found to be essential for rDNA transcription. Additional evidence in support of this model came from CRISPR/Cas9 studies that demonstrated that both subunits were essential for mammalian cell proliferation (30) and our own that PAF53 was essential (51,53). However, these studies did not shed light upon the mechanism(s) that regulate the steady-state levels of the proteins.…”

“…Interestingly, the dimerization domain of yA49 is not required for the protein to function in transcription. On the other hand, the dimerization domain of its mammalian homolog, PAF53, is required for rDNA transcription and cell proliferation (51). If PAF53 could bind to Pol I independent of dimerization, then the dimerization domain would not be essential for transcription like its yeast homolog.…”

“…post transfection. The established cell line expressing NLS-TIR1 was described previously, as was the cell line expressing both NLS-TIR1 and AIDtagged PAF53 (51). The AID tag was introduced into the 5' coding region of the PAF49 gene using CRISPR/Cas9 and microhomology-mediated end-joining (MMEJ) repair (56).…”

“…The analyses presented were carried out after three hours of treatment with 500 μM IAA (+) or water (−). Cells were harvested andwestern blots for PAF53 and PAF49 were carried out as described(26,51) .…”