Signaling to and from the RNA Polymerase III Transcription and Processing Machinery.

RNA polymerase (Pol) III has a specialized role in transcribing the most abundant RNAs in eukaryotic cells, transfer RNAs (tRNAs), along with other ubiquitous small noncoding RNAs, many of which have functions related to the ribosome and protein synthesis. The high energetic cost of producing these RNAs and their central role in protein synthesis underlie the robust regulation of Pol III transcription in response to nutrients and stress by growth regulatory pathways. Downstream of Pol III, signaling impacts posttranscriptional processes affecting tRNA function in translation and tRNA cleavage into smaller fragments that are increasingly attributed with novel cellular activities. In this review, we consider how nutrients and stress control Pol III transcription via its factors and its negative regulator, Maf1. We highlight recent work showing that the composition of the tRNA population and the function of individual tRNAs is dynamically controlled and that unrestrained Pol III transcription can reprogram central metabolic pathways.

Defective myelination in an RNA polymerase III mutant leukodystrophic mouse.

RNA polymerase (Pol) III synthesizes abundant short noncoding RNAs that have essential functions in protein synthesis, secretion, and other processes. Despite the ubiquitous functions of these RNAs, mutations in Pol III subunits cause Pol III-related leukodystrophy, an early-onset neurodegenerative disease. The basis of this neural sensitivity and the mechanisms of disease pathogenesis are unknown. Here we show that mice expressing pathogenic mutations in the largest Pol III subunit, Polr3a, specifically in Olig2-expressing cells, have impaired growth and developmental delay, deficits in cognitive, sensory, and fine sensorimotor function, and hypomyelination in multiple regions of the cerebrum and spinal cord. These phenotypes reflect a subset of clinical features seen in patients. In contrast, the gross motor defects and cerebellar hypomyelination that are common features of severely affected patients are absent in the mice, suggesting a relatively mild form of the disease in this conditional model. Our results show that disease pathogenesis in the mice involves defects that reduce both the number of mature myelinating oligodendrocytes and the ability of these cells to produce a myelin sheath of normal thickness. The findings suggest unique sensitivities of oligodendrogenesis and myelination to perturbations of Pol III transcription.

Loss of the RNA polymerase III repressor MAF1 confers obesity resistance.

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.

Conserved Herpesvirus Kinase ORF36 Activates B2 Retrotransposons during Murine Gammaherpesvirus Infection.

Short interspersed nuclear elements (SINEs) are RNA polymerase III (RNAPIII)-transcribed, retrotransposable noncoding RNA (ncRNA) elements ubiquitously spread throughout mammalian genomes. While normally silenced in healthy somatic tissue, SINEs can be induced during infection with DNA viruses, including the model murine gammaherpesvirus 68 (MHV68). Here, we explored the mechanisms underlying MHV68 activation of SINE ncRNAs. We demonstrate that lytic MHV68 infection of B cells, macrophages, and fibroblasts leads to robust activation of the B2 family of SINEs in a cell-autonomous manner. B2 ncRNA induction requires neither host innate immune signaling factors nor involvement of the RNAPIII master regulator Maf1. However, we identified MHV68 ORF36, the conserved herpesviral kinase, as playing a key role in B2 induction during lytic infection. SINE activation is linked to ORF36 kinase activity and can also be induced by inhibition of histone deacetylases 1 and 2 (HCAC 1/2), which is one of the known ORF36 functions. Collectively, our data suggest that ORF36-mediated changes in chromatin modification contribute to B2 activation during MHV68 infection and that this activity is conserved in other herpesviral protein kinase homologs.IMPORTANCE Viral infection dramatically changes the levels of many types of RNA in a cell. In particular, certain oncogenic viruses activate expression of repetitive genes called retrotransposons, which are normally silenced due to their ability to copy and spread throughout the genome. Here, we established that infection with the gammaherpesvirus MHV68 leads to a dramatic induction of a class of noncoding retrotransposons called B2 SINEs in multiple cell types. We then explored how MHV68 activates B2 SINEs, revealing a role for the conserved herpesviral protein kinase ORF36. Both ORF36 kinase-dependent and kinase-independent functions contribute to B2 induction, perhaps through ORF36 targeting of proteins involved in controlling the accessibility of chromatin surrounding SINE loci. Understanding the features underlying induction of these elements following MHV68 infection should provide insight into core elements of SINE regulation, as well as disregulation of SINE elements associated with disease.

Metabolic programming a lean phenotype by deregulation of RNA polymerase III.

As a master negative regulator of RNA polymerase (Pol) III, Maf1 modulates transcription in response to nutrients and stress to balance the production of highly abundant tRNAs, 5S rRNA, and other small noncoding RNAs with cell growth and maintenance. This regulation of Pol III transcription is important for energetic economy as mice lacking Maf1 are lean and resist weight gain on normal and high fat diets. The lean phenotype of Maf1 knockout (KO) mice is attributed in part to metabolic inefficiencies which increase the demand for cellular energy and elevate catabolic processes, including autophagy/lipophagy and lipolysis. A futile RNA cycle involving increased synthesis and turnover of Pol III transcripts has been proposed as an important driver of these changes. Here, using targeted metabolomics, we find changes in the liver of fed and fasted Maf1 KO mice consistent with the function of mammalian Maf1 as a chronic Pol III repressor. Differences in long-chain acylcarnitine levels suggest that energy demand is higher in the fed state of Maf1 KO mice versus the fasted state. Quantitative metabolite profiling supports increased activity in the TCA cycle, the pentose phosphate pathway, and the urea cycle and reveals changes in nucleotide levels and the creatine system. Metabolite profiling also confirms key predictions of the futile RNA cycle hypothesis by identifying changes in many metabolites involved in nucleotide synthesis and turnover. Thus, constitutively high levels of Pol III transcription in Maf1 KO mice reprogram central metabolic pathways and waste metabolic energy through a futile RNA cycle.

Diurnal regulation of RNA polymerase III transcription is under the control of both the feeding-fasting response and the circadian clock.

RNA polymerase III (Pol III) synthesizes short noncoding RNAs, many of which are essential for translation. Accordingly, Pol III activity is tightly regulated with cell growth and proliferation by factors such as MYC, RB1, TRP53, and MAF1. MAF1 is a repressor of Pol III transcription whose activity is controlled by phosphorylation; in particular, it is inactivated through phosphorylation by the TORC1 kinase complex, a sensor of nutrient availability. Pol III regulation is thus sensitive to environmental cues, yet a diurnal profile of Pol III transcription activity is so far lacking. Here, we first use gene expression arrays to measure mRNA accumulation during the diurnal cycle in the livers of (1) wild-type mice, (2) arrhythmic Arntl knockout mice, (3) mice fed at regular intervals during both night and day, and (4) mice lacking the Maf1 gene, and so provide a comprehensive view of the changes in cyclic mRNA accumulation occurring in these different systems. We then show that Pol III occupancy of its target genes rises before the onset of the night, stays high during the night, when mice normally ingest food and when translation is known to be increased, and decreases in daytime. Whereas higher Pol III occupancy during the night reflects a MAF1-dependent response to feeding, the rise of Pol III occupancy before the onset of the night reflects a circadian clock-dependent response. Thus, Pol III transcription during the diurnal cycle is regulated both in response to nutrients and by the circadian clock, which allows anticipatory Pol III transcription.

TOR signaling regulates ribosome and tRNA synthesis via LAMMER/Clk and GSK-3 family kinases.

Target of rapamycin (TOR)-dependent signaling and the control of cell growth is deregulated in many cancers. However, the signaling molecules downstream of TOR that coordinately regulate the synthesis of ribosomes and tRNAs are not well defined. Here, we show in yeast that conserved kinases of the LAMMER/Cdc-like and GSK-3 families function downstream of TOR complex 1 to repress ribosome and tRNA synthesis in response to nutrient limitation and other types of cellular stress. As a part of this response, we found that the LAMMER kinase Kns1 is differentially expressed and hyperphosphorylated and accumulates in the nucleus after rapamycin treatment, whereupon it primes the phosphorylation of the RNA polymerase III subunit Rpc53 by a specific GSK-3 family member, Mck1. In cooperation with another polymerase subunit, Rpc11, this phosphorylation of Rpc53 modifies the function of the enzyme and together with dephosphorylation of the Maf1 repressor inhibits the growth-promoting activity of RNA polymerase III transcription.

Participatory approaches, local stakeholders and cultural relevance facilitate an impactful community-based project in Uganda.

Sanitation is a major global challenge that is often addressed at national and international levels, while community opinions and beliefs are neglected. To promote water, sanitation and hygiene (WASH) we organized a cross-cultural knowledge exchange workshop to assess participatory methods for engaging local stakeholders. The workshop included 22 participants from all sectors of society. Practical solutions to sanitation challenges were identified and later shared with a local community. Qualitative and quantitative analyses were used to assess impact and showed participatory methods were highly valued to encourage information sharing among widely varied stakeholders, and that video was a particularly successful approach when engaging with local communities. An 8-month follow-up survey of village members revealed excellent information recall, positive behaviour changes and a desire for future visits. Our evidence suggests that community-based participation helped identify solutions to WASH issues affecting rural communities in resource-poor settings. Engaging in a multicultural knowledge-share was particularly valuable as it enabled participants to recognize they have common challenges and allowed them to share low-cost solutions from their different communities. Our use of video was widely viewed as an ideal means of circulating findings, as it communicated information to people with a wide variety of community roles and to all age groups. Its relevance was increased by adopting a culturally appropriate context by involving local communities in workshop activities. We recommend that research in low- and middle-income countries should be mindful of the environmental context in which WASH is implemented, and encourage acceptance by engaging with communities through the use of varied participatory methods.

Surface Pond Energy Absorption Across Four Himalayan Glaciers Accounts for 1/8 of Total Catchment Ice Loss.

Glaciers in High Mountain Asia, many of which exhibit surface debris, contain the largest volume of ice outside of the polar regions. Many contain supraglacial pond networks that enhance melt rates locally, but no large-scale assessment of their impact on melt rates exists. Here we use surface energy balance modeling forced using locally measured meteorological data and monthly satellite-derived pond distributions to estimate the total melt enhancement for the four main glaciers within the 400-km2 Langtang catchment, Nepal, for a 6-month period in 2014. Ponds account for 0.20 ± 0.03 m/year of surface melt, representing a local melt enhancement of a factor of 14 ± 3 compared with the debris-covered area, and equivalent to 12.5 ± 2.0% of total catchment ice loss. Given the prevalence of supraglacial ponds across the region, our results suggest that effective incorporation of melt enhancement by ponds is essential for accurate predictions of future mass balance change in the region.

Quantifying ChIP-seq data: a spiking method providing an internal reference for sample-to-sample normalization.

Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments are widely used to determine, within entire genomes, the occupancy sites of any protein of interest, including, for example, transcription factors, RNA polymerases, or histones with or without various modifications. In addition to allowing the determination of occupancy sites within one cell type and under one condition, this method allows, in principle, the establishment and comparison of occupancy maps in various cell types, tissues, and conditions. Such comparisons require, however, that samples be normalized. Widely used normalization methods that include a quantile normalization step perform well when factor occupancy varies at a subset of sites, but may miss uniform genome-wide increases or decreases in site occupancy. We describe a spike adjustment procedure (SAP) that, unlike commonly used normalization methods intervening at the analysis stage, entails an experimental step prior to immunoprecipitation. A constant, low amount from a single batch of chromatin of a foreign genome is added to the experimental chromatin. This "spike" chromatin then serves as an internal control to which the experimental signals can be adjusted. We show that the method improves similarity between replicates and reveals biological differences including global and largely uniform changes.

Differential phosphorylation of a regulatory subunit of protein kinase CK2 by target of rapamycin complex 1 signaling and the Cdc-like kinase Kns1.

Transcriptional regulation of ribosome and tRNA synthesis plays a central role in determining protein synthetic capacity and is tightly controlled in response to nutrient availability and cellular stress. In Saccharomyces cerevisiae, the regulation of ribosome and tRNA synthesis was recently shown to involve the Cdc-like kinase Kns1 and the GSK-3 kinase Mck1. In this study, we explored additional roles for these conserved kinases in processes connected to the target of rapamycin complex 1 (TORC1). We conducted a synthetic chemical-genetic screen in a kns1Δ mck1Δ strain and identified many novel rapamycin-hypersensitive genes. Gene ontology analysis showed enrichment for TORC1-regulated processes (vesicle-mediated transport, autophagy, and regulation of cell size) and identified new connections to protein complexes including the protein kinase CK2. CK2 is considered to be a constitutively active kinase and in budding yeast, the holoenzyme comprises two regulatory subunits, Ckb1 and Ckb2, and two catalytic subunits, Cka1 and Cka2. We show that Ckb1 is differentially phosphorylated in vivo and that Kns1 mediates this phosphorylation when nutrients are limiting and under all tested stress conditions. We determined that the phosphorylation of Ckb1 does not detectably affect the stability of the CK2 holoenzyme but correlates with the reduced occupancy of Ckb1 on tRNA genes after rapamycin treatment. Thus, the differential occupancy of tRNA genes by CK2 is likely to modulate its activation of RNA polymerase III transcription. Our data suggest that TORC1, via its effector kinase Kns1, may regulate the association of CK2 with some of its substrates by phosphorylating Ckb1.

Isotopic Ratio Outlier Analysis of the S. cerevisiae Metabolome Using Accurate Mass Gas Chromatography/Time-of-Flight Mass Spectrometry: A New Method for Discovery.

Isotopic ratio outlier analysis (IROA) is a (13)C metabolomics profiling method that eliminates sample to sample variance, discriminates against noise and artifacts, and improves identification of compounds, previously done with accurate mass liquid chromatography/mass spectrometry (LC/MS). This is the first report using IROA technology in combination with accurate mass gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS), here used to examine the S. cerevisiae metabolome. S. cerevisiae was grown in YNB media, containing randomized 95% (13)C, or 5%(13)C glucose as the single carbon source, in order that the isotopomer pattern of all metabolites would mirror the labeled glucose. When these IROA experiments are combined, the abundance of the heavy isotopologues in the 5%(13)C extracts, or light isotopologues in the 95%(13)C extracts, follows the binomial distribution, showing mirrored peak pairs for the molecular ion. The mass difference between the (12)C monoisotopic and the (13)C monoisotopic equals the number of carbons in the molecules. The IROA-GC/MS protocol developed, using both chemical and electron ionization, extends the information acquired from the isotopic peak patterns for formulas generation. The process that can be formulated as an algorithm, in which the number of carbons, as well as the number of methoximations and silylations are used as search constraints. In electron impact (EI/IROA) spectra, the artifactual peaks are identified and easily removed, which has the potential to generate "clean" EI libraries. The combination of chemical ionization (CI) IROA and EI/IROA affords a metabolite identification procedure that enables the identification of coeluting metabolites, and allowed us to characterize 126 metabolites in the current study.

SCS3 and YFT2 link transcription of phospholipid biosynthetic genes to ER stress and the UPR.

The ability to store nutrients in lipid droplets (LDs) is an ancient function that provides the primary source of metabolic energy during periods of nutrient insufficiency and between meals. The Fat storage-Inducing Transmembrane (FIT) proteins are conserved ER-resident proteins that facilitate fat storage by partitioning energy-rich triglycerides into LDs. FIT2, the ancient ortholog of the FIT gene family first identified in mammals has two homologs in Saccharomyces cerevisiae (SCS3 and YFT2) and other fungi of the Saccharomycotina lineage. Despite the coevolution of these genes for more than 170 million years and their divergence from higher eukaryotes, SCS3, YFT2, and the human FIT2 gene retain some common functions: expression of the yeast genes in a human embryonic kidney cell line promotes LD formation, and expression of human FIT2 in yeast rescues the inositol auxotrophy and chemical and genetic phenotypes of strains lacking SCS3. To better understand the function of SCS3 and YFT2, we investigated the chemical sensitivities of strains deleted for either or both genes and identified synthetic genetic interactions against the viable yeast gene-deletion collection. We show that SCS3 and YFT2 have shared and unique functions that connect major biosynthetic processes critical for cell growth. These include lipid metabolism, vesicular trafficking, transcription of phospholipid biosynthetic genes, and protein synthesis. The genetic data indicate that optimal strain fitness requires a balance between phospholipid synthesis and protein synthesis and that deletion of SCS3 and YFT2 impacts a regulatory mechanism that coordinates these processes. Part of this mechanism involves a role for SCS3 in communicating changes in the ER (e.g. due to low inositol) to Opi1-regulated transcription of phospholipid biosynthetic genes. We conclude that SCS3 and YFT2 are required for normal ER membrane biosynthesis in response to perturbations in lipid metabolism and ER stress.

Regulation of pol III transcription by nutrient and stress signaling pathways.

Transcription by RNA polymerase III (pol III) is responsible for ~15% of total cellular transcription through the generation of small structured RNAs such as tRNA and 5S RNA. The coordinate synthesis of these molecules with ribosomal protein mRNAs and rRNA couples the production of ribosomes and their tRNA substrates and balances protein synthetic capacity with the growth requirements of the cell. Ribosome biogenesis in general and pol III transcription in particular is known to be regulated by nutrient availability, cell stress and cell cycle stage and is perturbed in pathological states. High throughput proteomic studies have catalogued modifications to pol III subunits, assembly, initiation and accessory factors but most of these modifications have yet to be linked to functional consequences. Here we review our current understanding of the major points of regulation in the pol III transcription apparatus, the targets of regulation and the signaling pathways known to regulate their function. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Molecular basis of neurodegeneration in a mouse model of Polr3 -related disease.

Pathogenic variants in subunits of RNA polymerase (Pol) III cause a spectrum of Polr3 -related neurodegenerative diseases including 4H leukodystrophy. Disease onset occurs from infancy to early adulthood and is associated with a variable range and severity of neurological and non-neurological features. The molecular basis of Polr3 -related disease pathogenesis is unknown. We developed a postnatal whole-body mouse model expressing pathogenic Polr3a mutations to examine the molecular mechanisms by which reduced Pol III transcription results primarily in central nervous system phenotypes. Polr3a mutant mice exhibit behavioral deficits, cerebral pathology and exocrine pancreatic atrophy. Transcriptome and immunohistochemistry analyses of cerebra during disease progression show a reduction in most Pol III transcripts, induction of innate immune and integrated stress responses and cell type-specific gene expression changes reflecting neuron and oligodendrocyte loss and microglial activation. Earlier in the disease when integrated stress and innate immune responses are minimally induced, mature tRNA sequencing revealed a global reduction in tRNA levels and an altered tRNA profile but no changes in other Pol III transcripts. Thus, changes in the size and/or composition of the tRNA pool have a causal role in disease initiation. Our findings reveal different tissue- and brain region-specific sensitivities to a defect in Pol III transcription.

Recovery of RNA polymerase III transcription from the glycerol-repressed state: revisiting the role of protein kinase CK2 in Maf1 phosphoregulation.

Maf1 is a conserved regulator of RNA polymerase (pol) III transcription and is required for transcriptional repression under diverse stress conditions. In yeast, Maf1 function is negatively regulated at seven phosphosites by the overlapping action of protein kinase A (PKA) and the TORC1-regulated kinase Sch9. Under stress conditions, Maf1 is dephosphorylated at these sites leading to its nuclear accumulation, increased association with pol III genes and direct physical interactions with the polymerase which ultimately inhibit transcription. These changes are reversed upon return to optimal growth conditions. Transcription in this system is also regulated by protein kinase CK2. CK2 stimulates pol III transcription in yeast and human cells via phosphorylation of the initiation factor TFIIIB. Recently it was proposed that CK2 phosphorylation of Maf1 is required for reactivation of pol III transcription following growth on glycerol. We have examined this hypothesis using two Maf1 mutants (Maf1-id S388A and Maf1-ck2(0)) which lack all of the CK2 phosphosites implicated in the response. Both mutant proteins are phosphoregulated, function normally during repression and transcription is fully restored to the wild-type level upon transfer from glycerol to glucose. Additionally, phos-tag gel analysis of Maf1 7SA, a functional mutant that cannot be phosphorylated by PKA/Sch9, did not reveal any evidence for differential phosphorylation of Maf1 during carbon source switching. Together, these data do not support the proposed requirement for CK2 phosphorylation of Maf1 during derepression of pol III transcription.

Contrasting effects of whole-body and hepatocyte-specific deletion of the RNA polymerase III repressor Maf1 in the mouse.

MAF1 is a nutrient-sensitive, TORC1-regulated repressor of RNA polymerase III (Pol III). MAF1 downregulation leads to increased lipogenesis in Drosophila melanogaster, Caenorhabditis elegans, and mice. However, Maf1 -/- mice are lean as increased lipogenesis is counterbalanced by futile pre-tRNA synthesis and degradation, resulting in increased energy expenditure. We compared Chow-fed Maf1 -/- mice with Chow- or High Fat (HF)-fed Maf1 hep-/- mice that lack MAF1 specifically in hepatocytes. Unlike Maf1 -/- mice, Maf1 hep-/- mice become heavier and fattier than control mice with old age and much earlier under a HF diet. Liver ChIPseq, RNAseq and proteomics analyses indicate increased Pol III occupancy at Pol III genes, very few differences in mRNA accumulation, and protein accumulation changes consistent with increased lipogenesis. Futile pre-tRNA synthesis and degradation in the liver, as likely occurs in Maf1 hep-/- mice, thus seems insufficient to counteract increased lipogenesis. Indeed, RNAseq and metabolite profiling indicate that liver phenotypes of Maf1 -/- mice are strongly influenced by systemic inter-organ communication. Among common changes in the three phenotypically distinct cohorts, Angiogenin downregulation is likely linked to increased Pol III occupancy of tRNA genes in the Angiogenin promoter.

Mechanism of down-regulation of RNA polymerase III-transcribed non-coding RNA genes in macrophages by Leishmania.

The parasitic protozoan Leishmania invades mammalian macrophages to establish infection. We reported previously that Leishmania manipulates the expression of several non-coding RNA genes (e.g. Alu RNA, B1 RNA, and signal recognition particle RNA) in macrophages to favor the establishment of their infection in the phagolysosomes of these cells (Ueda, Y., and Chaudhuri, G. (2000) J. Biol. Chem. 275, 19428-19432; Misra, S., Tripathi, M. K., and Chaudhuri, G. (2005) J. Biol. Chem. 280, 29364-29373). We report here the mechanism of this down-regulation. We found that the non-coding RNA (ncRNA) genes that are repressed by Leishmania infection in macrophages contain a "B-box" in their promoters and thus require the polymerase III transcription factor TFIIIC for their expression. We also found that Leishmania promastigotes through their surface protease (leishmanolysin or gp63) activate the thrombin receptor PAR1 in the macrophages. This activation of PAR1 raised the cytosolic concentration of Ca(2+) into the micromolar range, thereby activating the Ca(2+)-dependent protease µ-calpain. µ-Calpain then degraded TFIIIC110 to inhibit the expression of the selected ncRNA genes. Avirulent stocks of Leishmania not expressing surface gp63 failed to down-regulate ncRNAs in the exposed macrophages. Inhibition of PAR1 or calpain 1 in macrophages made them resistant to Leishmania infection. These data suggest that macrophage PAR1 and calpain 1 are potential drug targets against leishmaniasis.

Integration of nutritional and stress signaling pathways by Maf1.

Repression of RNA polymerase III transcription under many conditions requires Maf1, a yeast protein with close sequence homologs in all eukaryotes. Three recent studies have identified key aspects of Maf1 regulation, which suggest that Maf1 integrates the responses from convergent nutritional and stress signaling pathways. These new findings indicate that the opposing actions of protein kinase A and protein phosphatase 2A alter the phosphorylation state of Maf1 and thereby regulate its localization and repressing activity.