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1.
Annu Rev Biochem ; 87: 75-100, 2018 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-29328783

RESUMEN

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.


Asunto(s)
ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Animales , Humanos , Modelos Biológicos , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Fosforilación , Conformación Proteica , ARN Polimerasa III/química , Procesamiento Postranscripcional del ARN , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Estrés Fisiológico , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética
2.
Proc Natl Acad Sci U S A ; 118(40)2021 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-34583988

RESUMEN

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.


Asunto(s)
Enfermedades Desmielinizantes/fisiopatología , Mutación , ARN Polimerasa III/genética , Animales , Enfermedades Desmielinizantes/genética , Crecimiento , Humanos , Masculino , Ratones , Ratones Mutantes
3.
Genes Dev ; 29(9): 934-47, 2015 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-25934505

RESUMEN

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.


Asunto(s)
Proteínas Represoras/genética , Animales , Autofagia/genética , Ingestión de Alimentos/genética , Metabolismo Energético/genética , Metabolismo de los Lípidos/genética , Longevidad/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/genética , Obesidad/genética , ARN de Transferencia/metabolismo , Espermidina/metabolismo
4.
Proc Natl Acad Sci U S A ; 115(48): 12182-12187, 2018 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-30429315

RESUMEN

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.


Asunto(s)
Peso Corporal , Metaboloma , ARN Polimerasa III/metabolismo , Animales , Regulación de la Expresión Génica , Hígado/metabolismo , Redes y Vías Metabólicas , Ratones , Ratones Noqueados , Fenotipo , ARN Polimerasa III/genética , ARN Ribosómico 5S/genética , ARN Ribosómico 5S/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo
5.
Mol Cell ; 45(6): 836-43, 2012 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-22364741

RESUMEN

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.


Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN de Transferencia/biosíntesis , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Ribosomas/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Sirolimus/farmacología , Factores de Transcripción/genética
6.
J Biol Chem ; 290(11): 7221-33, 2015 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-25631054

RESUMEN

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.


Asunto(s)
Quinasa de la Caseína II/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Antifúngicos/farmacología , Quinasa de la Caseína II/genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Polimerasa III/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sirolimus/farmacología , Estrés Fisiológico , Activación Transcripcional
7.
PLoS Genet ; 8(8): e1002890, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22927826

RESUMEN

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.


Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiología , Animales , Estrés del Retículo Endoplásmico , Eliminación de Gen , Redes Reguladoras de Genes , Humanos , Inositol/metabolismo , Metabolismo de los Lípidos , Fosfolípidos/metabolismo , Saccharomyces cerevisiae/genética , Respuesta de Proteína Desplegada
8.
Biochim Biophys Acta ; 1829(3-4): 361-75, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23165150

RESUMEN

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.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación de la Expresión Génica , Transducción de Señal , Estrés Fisiológico , Transcripción Genética , Animales , Alimentos , Humanos , Proteínas Represoras/metabolismo , Factores de Transcripción TFIII/metabolismo , Levaduras/genética , Levaduras/metabolismo
9.
bioRxiv ; 2024 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-38168294

RESUMEN

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.

10.
J Biol Chem ; 287(36): 30833-41, 2012 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-22810236

RESUMEN

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.


Asunto(s)
Crioprotectores/farmacología , Glicerol/farmacología , ARN Polimerasa III/metabolismo , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/enzimología , Transcripción Genética/efectos de los fármacos , Sustitución de Aminoácidos , Quinasa de la Caseína II , Humanos , Mutación Missense , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Polimerasa III/genética , Proteínas Represoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Transcripción Genética/fisiología
12.
Trends Biochem Sci ; 32(2): 51-3, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17174096

RESUMEN

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.


Asunto(s)
Estrés Oxidativo , ARN Polimerasa III/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Señales de Localización Nuclear , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 2 , ARN Polimerasa III/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Transcripción Genética
13.
PLoS Genet ; 4(7): e1000112, 2008 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-18604275

RESUMEN

Transcriptional repression of ribosomal components and tRNAs is coordinately regulated in response to a wide variety of environmental stresses. Part of this response involves the convergence of different nutritional and stress signaling pathways on Maf1, a protein that is essential for repressing transcription by RNA polymerase (pol) III in Saccharomyces cerevisiae. Here we identify the functions buffering yeast cells that are unable to down-regulate transcription by RNA pol III. MAF1 genetic interactions identified in screens of non-essential gene-deletions and conditionally expressed essential genes reveal a highly interconnected network of 64 genes involved in ribosome biogenesis, RNA pol II transcription, tRNA modification, ubiquitin-dependent proteolysis and other processes. A survey of non-essential MAF1 synthetic sick/lethal (SSL) genes identified six gene-deletions that are defective in transcriptional repression of ribosomal protein (RP) genes following rapamycin treatment. This subset of MAF1 SSL genes included MED20 which encodes a head module subunit of the RNA pol II Mediator complex. Genetic interactions between MAF1 and subunits in each structural module of Mediator were investigated to examine the functional relationship between these transcriptional regulators. Gene expression profiling identified a prominent and highly selective role for Med20 in the repression of RP gene transcription under multiple conditions. In addition, attenuated repression of RP genes by rapamycin was observed in a strain deleted for the Mediator tail module subunit Med16. The data suggest that Mediator and Maf1 function in parallel pathways to negatively regulate RP mRNA and tRNA synthesis.


Asunto(s)
Proteínas Represoras/genética , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Transcripción Genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Complejo Mediador , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/biosíntesis , Proteínas Represoras/metabolismo , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirolimus/farmacología , Factores de Transcripción/metabolismo
14.
Gene ; 768: 145259, 2021 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-33148458

RESUMEN

Mutations in RNA polymerase III (Pol III) cause hypomeylinating leukodystrophy (HLD) and neurodegeneration in humans. POLR3A and POLR3B, the two largest Pol III subunits, together form the catalytic center and carry the majority of disease alleles. Disease-causing mutations include invariant and highly conserved residues that are predicted to negatively affect Pol III activity and decrease transcriptional output. A subset of HLD missense mutations in POLR3A cluster in the pore region that provides nucleotide access to the Pol III active site. These mutations were engineered at the corresponding positions in the Saccharomyces cerevisiae homolog, Rpc160, to evaluate their functional deficits. None of the mutations caused a growth or transcription phenotype in yeast. Each mutation was combined with a frequently occurring pore mutation, POLR3A G672E, which was also wild-type for growth and transcription. The double mutants showed a spectrum of phenotypes from wild-type to lethal, with only the least fit combinations showing an effect on Pol III transcription. In one slow-growing temperature-sensitive mutant the steady-state level of tRNAs was unaffected, however global tRNA synthesis was compromised, as was the synthesis of RPR1 and SNR52 RNAs. Affinity-purified mutant Pol III was broadly defective in both factor-independent and factor-dependent transcription in vitro across genes that represent the yeast Pol III transcriptome. Thus, the robustness of yeast Rpc160 to single Pol III leukodystrophy mutations in the pore domain can be overcome by a second mutation in the domain.


Asunto(s)
Proteínas de Unión al ADN/biosíntesis , ARN Polimerasa III/genética , ARN de Transferencia/biosíntesis , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/genética , Proteínas de Unión al ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , Humanos , Enfermedad de Pelizaeus-Merzbacher/genética , ARN Polimerasa III/metabolismo , ARN de Transferencia/genética , Transcripción Genética/genética
15.
Nat Struct Mol Biol ; 27(3): 229-232, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32066962

RESUMEN

Maf1 is a conserved inhibitor of RNA polymerase III (Pol III) that influences phenotypes ranging from metabolic efficiency to lifespan. Here, we present a 3.3-Å-resolution cryo-EM structure of yeast Maf1 bound to Pol III, establishing that Maf1 sequesters Pol III elements involved in transcription initiation and binds the mobile C34 winged helix 2 domain, sealing off the active site. The Maf1 binding site overlaps with that of TFIIIB in the preinitiation complex.


Asunto(s)
ARN Polimerasa III/química , Proteínas Represoras/química , Proteínas de Saccharomyces cerevisiae/química , Factor de Transcripción TFIIIB/química , Factores de Transcripción/química , Transcripción Genética , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
16.
Sci Rep ; 10(1): 11956, 2020 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-32686713

RESUMEN

Maf1-/- mice are lean, obesity-resistant and metabolically inefficient. Their increased energy expenditure is thought to be driven by a futile RNA cycle that reprograms metabolism to meet an increased demand for nucleotides stemming from the deregulation of RNA polymerase (pol) III transcription. Metabolic changes consistent with this model have been reported in both fasted and refed mice, however the impact of the fasting-refeeding-cycle on pol III function has not been examined. Here we show that changes in pol III occupancy in the liver of fasted versus refed wild-type mice are largely confined to low and intermediate occupancy genes; high occupancy genes are unchanged. However, in Maf1-/- mice, pol III occupancy of the vast majority of active loci in liver and the levels of specific precursor tRNAs in this tissue and other organs are higher than wild-type in both fasted and refed conditions. Thus, MAF1 functions as a chronic repressor of active pol III loci and can modulate transcription under different conditions. Our findings support the futile RNA cycle hypothesis, elaborate the mechanism of pol III repression by MAF1 and demonstrate a modest effect of MAF1 on global translation via reduced mRNA levels and translation efficiencies for several ribosomal proteins.


Asunto(s)
Regulación de la Expresión Génica , ARN Polimerasa III/genética , Proteínas Represoras/metabolismo , Animales , Secuenciación de Inmunoprecipitación de Cromatina , Biología Computacional/métodos , Ontología de Genes , Estudio de Asociación del Genoma Completo , Hígado/metabolismo , Ratones , Unión Proteica , Precursores del ARN , ARN de Transferencia/genética , Proteínas Represoras/genética , Transcriptoma
17.
Mol Cell Biol ; 26(16): 5946-56, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16880507

RESUMEN

The binding of Brf1 to the tetratricopeptide repeat (TPR)-containing transcription factor IIIC (TFIIIC) subunit (Tfc4) represents a rate-limiting step in the ordered assembly of the RNA polymerase III initiation factor TFIIIB. Tfc4 contains multiple binding sites for Brf1 within its amino terminus and adjacent TPR arrays, but the access of Brf1 to these sites is limited by autoinhibition. Moreover, the Brf1 binding sites in Tfc4 overlap with sites important for the subsequent recruitment of another TFIIIB subunit, Bdp1, implying that repositioning of Brf1 is required after its initial interaction with Tfc4. As a starting point for dissecting the steps in TFIIIC-directed assembly of TFIIIB, we conducted yeast two-hybrid screens of Brf1 peptide libraries against different TPR-containing Tfc4 fragments. Short, biochemically active peptides were identified in three distinct regions of Brf1. Two peptides defined conserved but distal regions of Brf1 that participate in stable binding of Brf1 to TFIIIC-DNA. Remarkably, a third peptide that binds specifically to TPR6-9 of Tfc4 was found to promote the formation of both TFIIIC-DNA and Brf1-TFIIIC-DNA complexes and to reduce the mobility of these complexes in native gels. The data are consistent with this peptide causing a conformational change in TFIIIC that overcomes Tfc4 autoinhibition of Brf1 binding and suggest a structural model for the Brf1-Tfc4 interaction.


Asunto(s)
Péptidos/metabolismo , Subunidades de Proteína/metabolismo , Factor de Transcripción TFIIIB/metabolismo , Factores de Transcripción TFIII/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , ADN de Hongos/metabolismo , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIIIB/química , Factores de Transcripción TFIII/química , Técnicas del Sistema de Dos Híbridos
19.
Mol Brain ; 12(1): 59, 2019 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-31221184

RESUMEN

Recessive mutations in the ubiquitously expressed POLR3A and POLR3B genes are the most common cause of POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), a rare childhood-onset disorder characterized by deficient cerebral myelin formation and cerebellar atrophy. POLR3A and POLR3B encode the two catalytic subunits of RNA Polymerase III (Pol III), which synthesizes numerous small non-coding RNAs. We recently reported that mice homozygous for the Polr3a mutation c.2015G > A (p.Gly672Glu) have no neurological abnormalities and thus do not recapitulate the human POLR3-HLD phenotype. To determine if other POLR3-HLD mutations can cause a leukodystrophy phenotype in mouse, we characterized mice carrying the Polr3b mutation c.308G > A (p.Arg103His). Surprisingly, homozygosity for this mutation was embryonically lethal with only wild-type and heterozygous animals detected at embryonic day 9.5. Using proteomics in a human cell line, we found that the POLR3B R103H mutation severely impairs assembly of the Pol III complex. We next generated Polr3aG672E/G672E/Polr3b+/R103Hdouble mutant mice but observed that this additional mutation was insufficient to elicit a neurological or transcriptional phenotype. Taken together with our previous study on Polr3a G672E mice, our results indicate that missense mutations in Polr3a and Polr3b can variably impair mouse development and Pol III function. Developing a proper model of POLR3-HLD is crucial to gain insights into the pathophysiological mechanisms involved in this devastating neurodegenerative disease.


Asunto(s)
Pérdida del Embrión/enzimología , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Mutación/genética , ARN Polimerasa III/genética , Animales , Secuencia de Bases , Pérdida del Embrión/genética , Regulación Enzimológica de la Expresión Génica , Técnicas de Sustitución del Gen , Células HEK293 , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/fisiopatología , Homocigoto , Humanos , Ratones Endogámicos C57BL , Ratones Mutantes , Actividad Motora , Vaina de Mielina/metabolismo , ARN Polimerasa III/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
20.
Metabolites ; 8(1)2018 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-29346327

RESUMEN

Identifying non-annotated peaks may have a significant impact on the understanding of biological systems. In silico methodologies have focused on ESI LC/MS/MS for identifying non-annotated MS peaks. In this study, we employed in silico methodology to develop an Isotopic Ratio Outlier Analysis (IROA) workflow using enhanced mass spectrometric data acquired with the ultra-high resolution GC-Orbitrap/MS to determine the identity of non-annotated metabolites. The higher resolution of the GC-Orbitrap/MS, together with its wide dynamic range, resulted in more IROA peak pairs detected, and increased reliability of chemical formulae generation (CFG). IROA uses two different 13C-enriched carbon sources (randomized 95% 12C and 95% 13C) to produce mirror image isotopologue pairs, whose mass difference reveals the carbon chain length (n), which aids in the identification of endogenous metabolites. Accurate m/z, n, and derivatization information are obtained from our GC/MS workflow for unknown metabolite identification, and aids in silico methodologies for identifying isomeric and non-annotated metabolites. We were able to mine more mass spectral information using the same Saccharomyces cerevisiae growth protocol (Qiu et al. Anal. Chem 2016) with the ultra-high resolution GC-Orbitrap/MS, using 10% ammonia in methane as the CI reagent gas. We identified 244 IROA peaks pairs, which significantly increased IROA detection capability compared with our previous report (126 IROA peak pairs using a GC-TOF/MS machine). For 55 selected metabolites identified from matched IROA CI and EI spectra, using the GC-Orbitrap/MS vs. GC-TOF/MS, the average mass deviation for GC-Orbitrap/MS was 1.48 ppm, however, the average mass deviation was 32.2 ppm for the GC-TOF/MS machine. In summary, the higher resolution and wider dynamic range of the GC-Orbitrap/MS enabled more accurate CFG, and the coupling of accurate mass GC/MS IROA methodology with in silico fragmentation has great potential in unknown metabolite identification, with applications for characterizing model organism networks.

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