PAF49: An RNA Polymerase I subunit essential for rDNA transcription and stabilization of PAF53.

The application of genetic and biochemical techniques in yeast has informed our knowledge of transcription in mammalian cells. 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 the nature of the transcription factors essential for transcription by Pol I in yeast and mammalian cells, and yeast RNA polymerase I contains 14 subunits while mammalian polymerase contains 13 subunits. We previously reported the adaptation of the auxin-dependent degron that enabled a combination of a "genetics-like" approach and biochemistry to study mammalian rDNA transcription. Using this system, we studied the mammalian orthologue of yeast RPA34.5, PAF49, and found that it is essential for rDNA transcription and cell division. The auxin-induced degradation of PAF49 induced nucleolar stress and the accumulation of P53. Interestingly, the auxin-induced degradation of AID-tagged PAF49 led to the degradation of its binding partner, PAF53, but not vice versa. A similar pattern of co-dependent expression was also found when we studied the non-essential, yeast orthologues. An analysis of the domains of PAF49 that are essential for rDNA transcription demonstrated a requirement for both the dimerization domain and an "arm" of PAF49 that interacts with PolR1B. Further, we demonstrate this interaction can be disrupted to inhibit Pol I transcription in normal and cancer cells which leads to the arrest of normal cells and cancer cell death. In summary, we have shown that both PAF53 and PAF49 are necessary for rDNA transcription and cell growth.

Conditional depletion of the RNA polymerase I subunit PAF53 reveals that it is essential for mitosis and enables identification of functional domains.

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.

Dynamics of the RNA polymerase I TFIIF/TFIIE-like subcomplex: a mini-review.

RNA polymerase I (Pol I) is the most specialized eukaryotic Pol. It is only responsible for the synthesis of pre-ribosomal RNA (rRNA), the precursor of 18S, 5.8S and 28S rRNA, the most abundant cellular RNA types. Aberrant Pol I transcription is observed in a wide variety of cancers and its down-regulation is associated with several genetic disorders. The regulation and mechanism of Pol I transcription is increasing in clarity given the numerous high-resolution Pol I structures that have helped bridge seminal genetic and biochemical findings in the field. Here, we review the multifunctional roles of an important TFIIF- and TFIIE-like subcomplex composed of the Pol I subunits A34.5 and A49 in yeast, and PAF49 and PAF53 in mammals. Recent analyses have revealed a dynamic interplay between this subcomplex at nearly every step of the Pol I transcription cycle in addition to new roles in chromatin traversal and the existence of a new helix-turn-helix (HTH) within the A49/PAF53 linker domain that expands its dynamic functions during the Pol I transcription process.

The Mammalian and Yeast A49 and A34 Heterodimers: Homologous but Not the Same.

Ribosomal RNA synthesis is the rate-limiting step in ribosome biogenesis. In eukaryotes, RNA polymerase I (Pol I) is responsible for transcribing the ribosomal DNA genes that reside in the nucleolus. Aberrations in Pol I activity have been linked to the development of multiple cancers and other genetic diseases. Therefore, it is key that we understand the mechanisms of Pol I transcription. Recent studies have demonstrated that there are many differences between Pol I transcription in yeast and mammals. Our goal is to highlight the similarities and differences between the polymerase-associated factors (PAFs) in yeast and mammalian cells. We focus on the PAF heterodimer A49/34 in yeast and PAF53/49 in mammals. Recent studies have demonstrated that while the structures between the yeast and mammalian orthologs are very similar, they may function differently during Pol I transcription, and their patterns of regulation are different.