Recovery of RNA Polymerase III Transcription from the Glycerol-repressed State

Abstract

Background: Maf1 is a global repressor of RNA polymerase (pol) III transcription whose function is phospho-regulated by nutrient and stress signaling pathways. Results: We tested the hypothesis that CK2 phosphorylation of Maf1 is required for derepression of pol III transcription. Conclusion:The hypothesis is not supported. Significance: CK2 regulation of pol III transcription is likely to involve targets other than Maf1.

Regulation of pol III transcription by nutrient and stress signaling pathways

“…The results of several unbiased or Maf1-targeted phosphoproteomic studies [33,35–38] combined with in vitro phosphorylation assays and functional studies of phosphosite and kinase/signaling mutants [31–33,39] have found that the biologically important sites in yeast Maf1 are seven serine residues (amino acids 90, 101, 177–179, 209 and 210), six of which correspond to consensus PKA/Sch9 sites (RR/KXS/T)[40]. Phosphorylation of these sites changes the localization of Maf1 in the cell and alters the interaction of Maf1 with the polymerase [29–33,41,42]. Mutagenesis studies indicate that no single phosphosite is critical, their contributions are cumulative and that residues 209 and 210 account for most of the phosphorylation on Maf1 [32].…”

“…Mutagenesis studies indicate that no single phosphosite is critical, their contributions are cumulative and that residues 209 and 210 account for most of the phosphorylation on Maf1 [32]. Indeed, recent experiments with an internally-deleted, functional MAF1 allele ( MAF1-id ) that is missing five of the seven phosphosites (noted above) suggest that phosphorylation of serines 209 and 210 is sufficient for the normal regulated localization of this protein [42]. Despite the fact that Maf1 is phosphorylated on many sites, the protein usually separates into just two species on high resolving one-dimensional SDS gels [29,30,32].…”

“…Thus, Maf1 mobility in these gels does not correlate with the total level of phosphorylation of the protein. Immunoprecipitation and western blotting with a phospho-PKA substrate specific antibody [31,32,43] or the use of Phostag acrylamide gels are able to report the extent of Maf1 phosphorylation [42]. For human Maf1, a combination of mass spectrometry, Phostag acrylamide gel analysis and mutagenesis identified serines 60, 68 and 75 as the major regulated phosphosites [24].…”

“…The phospho-regulation of serine 75 has been independently examined in two other studies [23,34]. It is noteworthy that although the sequence in this region of human Maf1 is not conserved in the yeast protein, the sites are positionally similar to serines 209/210 in yeast Maf1 relative to the phylogenetically conserved domain B [42] and appear to be functionally equivalent [23,24,34]. …”

Emerging Roles for Maf1 beyond the Regulation of RNA Polymerase III Activity

“…Caesin Kinase 2 (CK2) stimulates RNA pol III transcription via phosphorylation of TFIIIB (CK2 forms a stable complex with TFIIIB) and has also been reported to directly phosphorylate Maf1 in yeast and mammalian cell culture [35]. There are contradicting reports however on whether CK2 phosphorylation of Maf1 is required for derepression of RNA pol III transcription in yeast[35, 36] and a role for CK2 has yet to be investigated in other species. Taken together, phosphorylation clearly plays an important regulatory role in Maf1 function, but the details underlying this control need further refinement (Fig.…”