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 genemediated silencing of nearby RNA pol II transcription.nuclear import ͉ phosphorylation ͉ tRNA biosynthesis T he action of all three nuclear RNA polymerases (pols) in the synthesis of rRNAs, ribosomal protein mRNAs, and tRNAs is coordinately regulated to control ribosome biogenesis and cell growth in response to nutrients and many other conditions (1). In Saccharomyces cerevisiae, Maf1 has been identified as an absolute and specific effector of repression in the pol III system (2). The diversity of conditions that signal repression, combined with the essential role of Maf1 in this process, suggests that the Maf1 protein is targeted by multiple signaling pathways.Genomewide localization of the pol III transcription apparatus has shown that nutrient deprivation and entry into stationary phase causes a significant decrease in polymerase occupancy on pol III genes (3, 4). This change is Maf1-dependent and is presumably a consequence of the direct interaction of Maf1 with the polymerase (5, 6). Consistent with this view, an in vitro system that recapitulates Maf1-dependent repression identified two steps that are inhibited as follows: polymerase recruitment to existing TFIIIB-DNA complexes and de novo assembly of the initiation factor TFIIIB onto DNA (5). In the latter step, Maf1 is thought to target the activity of TFIIIB via a direct interaction with one of its subunits, Brf1 (2, 5). However, the mechanism by which Maf1 inhibits TFIIIB-DNA assembly and transcription is not yet known.Maf1 is a phylogenetically conserved and structurally novel protein that lacks homology to any motifs of known function (6). However, three conserved doma...

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1 −/− mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1 −/− mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD + , and is associated with obesity resistance. Consistent with this, NAD + levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.

Sir2 is a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase involved in gene silencing and longevity. Cellular stresses affect Sir2 activity, but the mechanisms of Sir2 regulation are debated. Nicotinamide has been proposed as a physiological regulator that inhibits Sir2 deacetylase activity by chemical reversal of a covalent reaction intermediate. We demonstrate a chemical strategy to activate Sir2-dependent transcriptional silencing and present evidence that the endogenous level of nicotinamide limits Sir2 activity in wild-type (wt) yeast cells. Nicotinamide inhibition of Sir2 is antagonized in vitro by isonicotinamide, which causes an increase in Sir2 deacetylation activity. Isonicotinamide also substantially increases transcriptional silencing at Sir2-regulated loci in wt strains and in strains lacking key NAD+ salvage pathway enzymes (PNC1 and NPT1). Thus, a nicotinamide antagonist is a Sir2 agonist in vitro and in vivo.

In Saccharomyces cerevisiae, Maf1 is essential for mediating the repression of transcription by RNA polymerase (pol) III in response to diverse cellular conditions. These conditions activate distinct signaling pathways that converge at or above Maf1. Thus, Maf1-dependent repression is thought to involve a common set of downstream inhibitory effects on the pol III machinery. Here we provide support for this view and define two steps in Maf1-dependent transcriptional repression. We show that chlorpromazine (CPZ)-induced repression of pol III transcription is achieved by inhibiting de novo assembly of transcription factor (TF) IIIB onto DNA as well as the recruitment of pol III to preassembled TFIIIB⅐DNA complexes. Additionally Brf1 was identified as a target of repression in extracts of CPZ-treated cells. Maf1-Brf1 and Maf1-pol III interactions were implicated in the inhibition of TFIIIB⅐DNA complex assembly and polymerase recruitment by recombinant Maf1. Co-immunoprecipitation experiments confirmed these interactions in yeast extracts and demonstrated that Maf1 does not differentially sequester Brf1 or pol III under repressing conditions. The results suggest that Maf1 functions by a non-stoichiometric mechanism to repress pol III transcription.Transcription of the large rRNAs by RNA polymerase (pol) 1 I and of 5 S rRNA and tRNAs by pol III is tightly co-regulated under essentially all conditions (1-3). This coordinate regulation is biologically important as it is conserved in all eukaryotes where transcription by pols I and III has been examined. The principal evolutionary imperatives that are thought to underlie this conserved regulation are the common function of rRNAs and tRNA in protein synthesis and the high energetic cost of their synthesis, which accounts for about 80% of nuclear gene transcription in actively growing cells (1, 3). The levels of pol I and pol III transcription are critical determinants of cell growth rate, and the deregulation of this transcription is a hallmark of cell transformation and tumorigenesis (4, 5). In addition, for single cell eukaryotes whose biological niche exposes them to periods when nutrients are in short supply and/or harsh environmental conditions, the ability to rapidly shut off the synthesis of rRNAs and tRNA is thought to be of vital importance for achieving metabolic economy (6) and hence is likely to impact cell survival.In higher eukaryotes, p53 and Rb and its relatives p107 and p130 play an important role in controlling pol I and pol III transcription and in coordinating the production of new protein synthetic capacity with cell proliferation (2, 5). These repressors function by binding directly to components of the pol I and pol III transcription machinery and thereby prevent proteinprotein interactions required for transcription. Specific components of this machinery are also substrates for phosphorylation by various kinases including extracellular signal-regulated kinase, casein kinase II, and cyclin-dependent kinases, which can either activate or repress tran...

Maf1 is a conserved repressor of transcription that functions at the downstream end of multiple nutrient and stress signaling pathways. How these different signaling pathways converge on Maf1 is not known. Previous work in yeast indicates that protein kinase A (PKA) regulates RNA polymerase (pol) III transcription, in part, by phosphorylating multiple sites in Maf1. Here we present additional evidence for this view and show that a parallel nutrient and stress-sensing pathway involving Sch9, an homologous kinase to metazoan S6 kinase, targets Maf1 at a subset of PKA sites. Using ATP analog-sensitive alleles of PKA and Sch9, we find that these two kinases account for the bulk of the phosphorylation on consensus PKA sites in Maf1. Deletion of Sch9 reduces RNA pol III transcription in a Maf1-dependent manner, yet the cells remain susceptible to further repression by rapamycin and other treatments. Because the rapamycin-sensitive kinase activity of the TORC1 complex is necessary for Sch9 function in vivo and in vitro, our results show that transcriptional regulation of RNA pol III and the coordinate control of ribosomal protein genes can be achieved by Sch9-dependent and -independent branches of the target of rapamycin (TOR) signaling pathway.Maf1 is essential for repressing transcription by RNA polymerase (pol) 2 III in yeast, and this function is conserved in mammals (1-4). In addition, human Maf1 is known to directly repress RNA pol II transcription of certain protein coding genes such as the TATA box-binding protein, TBP, and to indirectly repress transcription by RNA pol I (3). Repression by Maf1 occurs in response to a wide variety of nutritional and stress conditions, but the nature of the signaling pathways mediating these responses and how they converge on Maf1 is largely unknown (5).In budding yeast, Maf1 is phosphorylated on consensus PKA sites under optimal growth conditions and is rapidly dephosphorylated under starvation or stress conditions that repress transcription (5, 6). Dephosphorylation of these PKA sites correlates with the relocation of Maf1 from the cytoplasm to the nucleus and is thought to regulate its inhibitory interaction with RNA pol III (5-9). Nuclear import of Maf1 is directed by two redundant nuclear localization sequences (NtNLS and CtNLS) that are differentially sensitive to PKA site phosphorylation. Specifically, the NtNLS is inhibited by phosphorylation as acidic substitutions at the PKA sites, which are normally fully functional, can prevent nuclear import and repression when the CtNLS is disabled (6). The localization of Maf1 in the cytoplasm serves to fine-tune its regulation but is not essential for preventing repression at inappropriate times. The accumulation of Maf1 in the nucleus, either by mutation of the protein or by deletion of the exportin of the protein, Msn5, does not cause repression in the absence of additional changes triggered by cellular signaling pathways (6, 10).Numerous studies have implicated the RAS/PKA pathway and the rapamycin-sensitive TOR pathway as po...