Robyn D. Moir

Repression of Ribosome and tRNA Synthesis in Secretion-Defective Cells Is Signaled by a Novel Branch of the Cell Integrity Pathway

ABSTRACT The transcription of ribosomal DNA, ribosomal protein (RP) genes, and 5S and tRNA genes by RNA polymerases (Pols) I, II, and III, respectively, is rapidly and coordinately repressed upon interruption of the secretory pathway in Saccharomyces cerevisiae. We find that repression of ribosome and tRNA synthesis in secretion-defective cells involves activation of the cell integrity pathway. Transcriptional repression requires the upstream components of this pathway, including the Wsc family of putative plasma membrane sensors and protein kinase C (PKC), but not the downstream Bck1–Mkk1/2–Slt2 mitogen-activated protein kinase cascade. These findings reveal a novel PKC effector pathway that controls more than 85% of nuclear transcription. It is proposed that the coordination of ribosome and tRNA synthesis with cell growth may be achieved, in part, by monitoring the turgor pressure of the cell.

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.

Two Steps in Maf1-dependent Repression of Transcription by RNA Polymerase III

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 TFIIIB·DNA 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 TFIIIB·DNA 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.

Regulation of RNA Polymerase III Transcription Involves SCH9-dependent and SCH9-independent Branches of the Target of Rapamycin (TOR) Pathway

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.

Nhp6, an HMG1 Protein, Functions in SNR6 Transcription by RNA Polymerase III in S. cerevisiae

Nhp6A and Nhp6B are HMG1-like proteins required for the growth of S. cerevisiae at elevated temperatures. We show that the conditional lethality of an nhp6 strain results from defective transcription of SNR6 (U6 snRNA) by RNA polymerase III. Overexpression of U6 snRNA or Brf1, a limiting component of TFIIIB, and an activating mutation (PCF1-1) in TFIIIC were each found to suppress the nhp6 growth defect. Additionally, U6 snRNA levels, which are reduced over 10-fold in nhp6 cells at 37 degrees C, were restored by Brf1 overexpression and by PCF1-1. Nhp6A protein specifically enhanced TFIIIC-dependent, but not TATA box-dependent, SNR6 transcription in vitro by facilitating TFIIIC binding to the SNR6 promoter. Thus, Nhp6 has a direct role in transcription complex assembly at SNR6.

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.

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.

A tetratricopeptide repeat mutation in yeast transcription factor IIIC131 (TFIIIC131) facilitates recruitment of TFIIB-related factor TFIIIB70.

Transcription factor IIIC (TFIIIC) plays an important role in assembling the initiation factor TFIIIB on genes transcribed by RNA polymerase III (Pol III). In Saccharomyces cerevisiae, assembly of the TFIIIB complex by promoter-bound TFIIIC is thought to be initiated by its tetratricopeptide repeat (TPR)-containing subunit, TFIIIC131, which interacts directly with the TFIIB-related factor, TFIIIB70/Brf1. In this work, we have identified 10 dominant mutations in TFIIIC131 that increase Pol III gene transcription. All of these mutations are found within a discrete 53-amino-acid region of the protein encompassing TPR2. Biochemical studies of one of the mutations (PCF1-2) show that the increase in transcription is due to an increase in the recruitment of TFIIIB70 to TFIIC-DNA. The PCF1-2 mutation does not affect the affinity of TFIIIC for DNA, and the differential in both transcription and TFIIIB complex assembly is observed at saturating levels of TFIIIB70. This indicates that mutant and wild-type TFIIIC-DNA complexes have the same affinity for TFIIIB70 and suggests that the increased recruitment of this factor is achieved by a nonequilibrium binding mechanism. A novel mechanism of activation in which the TPR mutations facilitate a conformational change in TFIIIC that is required for TFIIIB70 binding is proposed. The implications of this model for the regulation of processes involving TPR proteins are discussed.

Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells

The endoplasmic reticulum (ER) unfolded protein response (UPR) is correlated with changes in unfolded secretory levels. A novel fluorescence biosensor now reports changes in the unfolded protein burden. This reporter reveals a form of ER stress—inositol withdrawal—that stimulates the UPR without changes in unfolded protein levels.

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.