Maf1, a New Player in the Regulation of Human RNA Polymerase III Transcription

BACKGROUND: Human RNA polymerase III (pol III) transcription is regulated by several factors, including the tumor suppressors P53 and Rb, and the proto-oncogene c-Myc. In yeast, which lacks these proteins, a central regulator of pol III transcription, called Maf1, has been described. Maf1 is required for repression of pol III transcription in response to several signal transduction pathways and is broadly conserved in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We show that human endogenous Maf1 can be co-immunoprecipitated with pol III and associates in vitro with two pol III subunits, the largest subunit RPC1 and the α-like subunit RPAC2. Maf1 represses pol III transcription in vitro and in vivo and is required for maximal pol III repression after exposure to MMS or rapamycin, treatments that both lead to Maf1 dephosphorylation. CONCLUSIONS/SIGNIFICANCE: These data suggest that Maf1 is a major regulator of pol III transcription in human cells

Regulations of RNA Polymerase III transcription by Maf1 in mammalian cells

RNA polymerase (pol) III produces essential components of the biosynthetic machinery; therefore, its output is tightly coupled with the rate of cell growth and proliferation. In <i>Saccharomyces cerevisiae</i>, Maf1 is an essential mediator of pol III repression in response to starvation. We demonstrate that a Maf1 ortholog is also used to restrain pol III activity in mouse and human cells. Mammalian Maf1 represses pol III transcription <i>in vitro</i> and in transfected fibroblasts. Furthermore, genetic deletion of Maf1 elevates pol III transcript expression, thus confirming the role of endogenous Maf1 as an inhibitor of mammalian pol III output. Maf1 is detected at chromosomal pol III templates in rodent and human cells. It interacts with pol III as well as its associated initiation factor TFIIIB and is phosphorylated in a serum-sensitive manner <i>in vivo</i>. These aspects of Maf1 function have been conserved between yeast and mammals and are therefore likely to be of fundamental importance in controlling pol III transcriptional activit

Casein kinase II-mediated phosphorylation of general repressor Maf1 triggers RNA polymerase III activation

Maf1 protein is a global negative regulator of RNA polymerase (Pol) III transcription conserved from yeast to man. We report that phosphorylation of Maf1 by casein kinase II (CK2), a highly evolutionarily conserved eukaryotic kinase, is required for efficient Pol III transcription. Both recombinant human and yeast CK2 were able to phosphorylate purified human or yeast Maf1, indicating that Maf1 can be a direct substrate of CK2. Upon transfer of Saccharomyces cerevisiae from repressive to favorable growth conditions, CK2 activity is required for the release of Maf1 from Pol III bound to a tRNA gene and for subsequent activation of tRNA transcription. In a yeast strain lacking Maf1, CK2 inhibition showed no effect on tRNA synthesis, confirming that CK2 activates Pol III via Maf1. Additionally, CK2 was found to associate with tRNA genes, and this association is enhanced in absence of Maf1, especially under repressive conditions. These results corroborate the previously reported TFIIIB–CK2 interaction and indicate an important role of CK2-mediated Maf1 phosphorylation in triggering Pol III activation

Protein kinase A regulates RNA polymerase III transcription through the nuclear localization of Maf1

Maf1 is an essential and specific mediator of transcriptional repression in the RNA polymerase (pol) III system. Maf1-dependent repression occurs in response to a wide range of conditions, suggesting that the protein itself is targeted by the major nutritional and stress-signaling pathways. We show that Maf1 is a substrate for cAMP-dependent PKA in vitro and is differentially phosphorylated on PKA sites in vivo under normal versus repressing conditions. PKA activity negatively regulates Maf1 function because strains with unregulated high PKA activity block repression of pol III transcription in vivo, and strains lacking all PKA activity are hyperrepressible. Nuclear accumulation of Maf1 is required for transcriptional repression and is regulated by two nuclear localization sequences in the protein. An analysis of PKA phosphosite mutants shows that the localization of Maf1 is affected via the N-terminal nuclear localization sequence. In particular, mutations that prevent phosphorylation at PKA consensus sites promote nuclear accumulation of Maf1 without inducing repression. These results indicate that negative regulation of Maf1 by PKA is achieved by inhibiting its nuclear import and suggest that a PKA-independent activation step is required for nuclear Maf1 to function in the repression of pol III transcription. Finally, we report a previously undescribed phenotype for Maf1 in tRNA gene-mediated silencing of nearby RNA pol II transcription