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1.
Curr Issues Mol Biol ; 44(7): 3001-3017, 2022 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-35877431

RESUMO

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that load amino acids to their cognate tRNA molecules. The expression of certain ARSs and tRNAs has been shown to be deregulated in cancer, presumably to accommodate elevated protein synthesis requirements. In this work, the expression of cytoplasmic ARSs and tRNAs in ten TCGA cancers has been systematically examined. ARSs were found to be mostly upregulated in tumours and their upregulation often correlated with worse patient survival. tRNAs were found to be either upregulated or downregulated in tumours and their expression sometimes correlated to worse survival outcomes. However, although the expression of most ARSs and tRNAs was deregulated in tumours when compared to healthy adjacent tissues, only in a few cases, and independently, did it correlate to patient survival. These data point to the general uncoupling of concomitant ARS and tRNA expression deregulation and patient survival, highlighting the different ARS and tRNA requirements in cancers.

2.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35042798

RESUMO

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.


Assuntos
Instabilidade Cromossômica/genética , DNA Helicases/metabolismo , Enzimas Multifuncionais/metabolismo , RNA Helicases/metabolismo , Ataxias Espinocerebelares/congênito , Animais , Apraxias/genética , Ataxia/genética , Linhagem Celular , Ataxia Cerebelar/genética , DNA Helicases/genética , Reparo do DNA/genética , Perfilação da Expressão Gênica/métodos , Instabilidade Genômica/genética , Genômica/métodos , Humanos , Camundongos , Células-Tronco Embrionárias Murinas , Enzimas Multifuncionais/genética , Mutação/genética , Doenças Neurodegenerativas/genética , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , RNA Helicases/genética , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/fisiopatologia , Transcriptoma/genética
3.
Mol Cell Biol ; 40(1)2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31658995

RESUMO

Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, a drug that is widely used for immunosuppression in organ transplantation and autoimmune diseases, as well as anticancer chemotherapy. It inhibits IMP dehydrogenase, a rate-limiting enzyme in de novo synthesis of guanidine nucleotides. MPA treatment interferes with transcription elongation, resulting in a drastic reduction of pre-rRNA and pre-tRNA synthesis, the disruption of the nucleolus, and consequently cell cycle arrest. Here, we investigated the mechanism whereby MPA inhibits RNA polymerase III (Pol III) activity, in both yeast and mammalian cells. We show that MPA rapidly inhibits Pol III by depleting GTP. Although MPA treatment can activate p53, this is not required for Pol III transcriptional inhibition. The Pol III repressor MAF1 is also not responsible for inhibiting Pol III in response to MPA treatment. We show that upon MPA treatment, the levels of selected Pol III subunits decrease, but this is secondary to transcriptional inhibition. Chromatin immunoprecipitation (ChIP) experiments show that Pol III does not fully dissociate from tRNA genes in yeast treated with MPA, even though there is a sharp decrease in the levels of newly transcribed tRNAs. We propose that in yeast, GTP depletion may lead to Pol III stalling.


Assuntos
Inibidores Enzimáticos/farmacologia , Imunossupressores/farmacologia , Ácido Micofenólico/farmacologia , RNA Polimerase III/antagonistas & inibidores , RNA de Transferência/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Camundongos , Células RAW 264.7 , RNA Polimerase III/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Cell ; 168(5): 843-855.e13, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28215706

RESUMO

The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.


Assuntos
Processamento Alternativo/efeitos da radiação , DNA Helicases/genética , RNA não Traduzido/genética , Transcrição Gênica , Raios Ultravioleta , Linhagem Celular , Éxons , Humanos , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elongação da Transcrição Genética/efeitos da radiação , Iniciação da Transcrição Genética/efeitos da radiação
5.
Genes Dev ; 30(4): 408-20, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26883360

RESUMO

Genome instability is a recurring feature of tumorigenesis. Mutation in MLL2, encoding a histone methyltransferase, is a driver in numerous different cancer types, but the mechanism is unclear. Here, we present evidence that MLL2 mutation results in genome instability. Mouse cells in which MLL2 gene deletion can be induced display elevated levels of sister chromatid exchange, gross chromosomal aberrations, 53BP1 foci, and micronuclei. Human MLL2 knockout cells are characterized by genome instability as well. Interestingly, MLL2 interacts with RNA polymerase II (RNAPII) and RECQL5, and, although MLL2 mutated cells have normal overall H3K4me levels in genes, nucleosomes in the immediate vicinity of RNAPII are hypomethylated. Importantly, MLL2 mutated cells display signs of substantial transcription stress, and the most affected genes overlap with early replicating fragile sites, show elevated levels of γH2AX, and suffer frequent mutation. The requirement for MLL2 in the maintenance of genome stability in genes helps explain its widespread role in cancer and points to transcription stress as a strong driver in tumorigenesis.


Assuntos
Instabilidade Genômica/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Transcrição Gênica/genética , Animais , Linhagem Celular , Dano ao DNA/genética , Histona-Lisina N-Metiltransferase , Humanos , Camundongos , Mutação , RNA Polimerase II/metabolismo , RecQ Helicases/metabolismo
6.
Cell ; 157(5): 1037-49, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24836610

RESUMO

RECQL5 is the sole member of the RECQ family of helicases associated with RNA polymerase II (RNAPII). We now show that RECQL5 is a general elongation factor that is important for preserving genome stability during transcription. Depletion or overexpression of RECQL5 results in corresponding shifts in the genome-wide RNAPII density profile. Elongation is particularly affected, with RECQL5 depletion causing a striking increase in the average rate, concurrent with increased stalling, pausing, arrest, and/or backtracking (transcription stress). RECQL5 therefore controls the movement of RNAPII across genes. Loss of RECQL5 also results in the loss or gain of genomic regions, with the breakpoints of lost regions located in genes and common fragile sites. The chromosomal breakpoints overlap with areas of elevated transcription stress, suggesting that RECQL5 suppresses such stress and its detrimental effects, and thereby prevents genome instability in the transcribed region of genes.


Assuntos
Instabilidade Genômica , RecQ Helicases/metabolismo , Elongação da Transcrição Genética , Transcrição Gênica , Genoma Humano , Células HEK293 , Humanos , RNA Polimerase II/metabolismo
8.
Proc Natl Acad Sci U S A ; 107(26): 11823-8, 2010 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-20543138

RESUMO

Synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III is regulated by the mTOR pathway in mammalian cells. The mTOR kinase localizes to tRNA and 5S rRNA genes, providing an opportunity for direct control. Its presence at these sites can be explained by interaction with TFIIIC, a DNA-binding factor that recognizes the promoters of these genes. TFIIIC contains a TOR signaling motif that facilitates its association with mTOR. Maf1, a repressor that binds and inhibits pol III, is phosphorylated in a mTOR-dependent manner both in vitro and in vivo at serine 75, a site that contributes to its function as a transcriptional inhibitor. Proximity ligation assays confirm the interaction of mTOR with Maf1 and TFIIIC in nuclei. In contrast to Maf1 regulation in yeast, no evidence is found for nuclear export of Maf1 in response to mTOR signaling in HeLa cells. We conclude that mTOR associates with TFIIIC, is recruited to pol III-transcribed genes, and relieves their repression by Maf1.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição TFIII/metabolismo , Sequência de Bases , Sítios de Ligação , Imunoprecipitação da Cromatina , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Fosforilação , RNA Polimerase III/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Serina/química , Serina-Treonina Quinases TOR
9.
Nucleic Acids Res ; 38(4): 1228-39, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19965767

RESUMO

Dr1 (also known as NC2beta) was identified as a repressor of RNA polymerase (pol) II transcription. It was subsequently shown to inhibit pol III transcription when expressed at high levels in vitro or in yeast cells. However, endogenous Dr1 was not detected at pol III-transcribed genes in growing yeast. In contrast, we demonstrate that endogenous Dr1 is present at pol III templates in human cells, as is its dimerization partner DRAP1 (also called NC2alpha). Expression of tRNA by pol III is selectively enhanced by RNAi-mediated depletion of endogenous human Dr1, but we found no evidence that DRAP1 influences pol III output in vivo. A stable association was detected between endogenous Dr1 and the pol III-specific transcription factor Brf1. This interaction may recruit Dr1 to pol III templates in vivo, as crosslinking to these sites increases following Brf1 induction. On the basis of these data, we conclude that the physiological functions of human Dr1 include regulation of pol III transcription.


Assuntos
Regulação da Expressão Gênica , Fosfoproteínas/metabolismo , RNA de Transferência/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Humanos , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Interferência de RNA , RNA Polimerase III/metabolismo , RNA de Transferência/biossíntese , Proteínas Repressoras/análise , Fator de Transcrição TFIIIB/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição Gênica
10.
Biochem J ; 419(3): 655-60, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19193195

RESUMO

Interaction of SM (Sec1/Munc18) proteins with their cognate syntaxins represents an important regulatory mechanism of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor)-mediated membrane fusion. Understanding the conserved mechanisms by which SM proteins function in this process has proved challenging, largely due to an apparent lack of conservation of binding mechanisms between different SM-syntaxin pairs. In the present study, we have identified a hitherto uncharacterized mode of binding between syntaxin 4 and Munc18c that is independent of the binding mode shown previously to utilize the N-terminal peptide of syntaxin 4. Our data demonstrate that syntaxin 4 and Munc18c interact via two distinct modes of binding, analogous to those employed by syntaxin 1a-Munc18a and syntaxin 16-Vps45p (vacuolar protein sorting 45). These data support the notion that all syntaxin/SM proteins bind using conserved mechanisms, and pave the way for the formulation of unifying hypotheses of SM protein function.


Assuntos
Proteínas Munc18/metabolismo , Proteínas Qa-SNARE/metabolismo , Animais , Proteínas Mutantes/metabolismo , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo
11.
J Immunol ; 180(11): 7471-9, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18490747

RESUMO

Structural polymorphisms (L263P, M313V, and S331P) in the third intracellular loop of the murine histamine receptor H(1) (H(1)R) are candidates for Bphs, a shared autoimmune disease locus in experimental allergic encephalomyelitis and experimental allergic orchitis. The P-V-P haplotype is associated with increased disease susceptibility (H(1)R(S)) whereas the L-M-S haplotype is associated with less severe disease (H(1)R(R)). In this study, we show that selective re-expression of the H(1)R(S) allele in T cells fully complements experimental allergic encephalomyelitis susceptibility and the production of disease-associated cytokines while selective re-expression of the H(1)R(R) allele does not. Mechanistically, we show that the two H(1)R alleles exhibit differential cell surface expression and altered intracellular trafficking, with the H(1)R(R) allele being retained within the endoplasmic reticulum. Moreover, we show that all three residues (L-M-S) comprising the H(1)R(R) haplotype are required for altered expression. These data are the first to demonstrate that structural polymorphisms influencing cell surface expression of a G protein-coupled receptor in T cells regulates immune functions and autoimmune disease susceptibility.


Assuntos
Doenças Autoimunes/genética , Encefalomielite Autoimune Experimental/genética , Orquite/genética , Receptores Histamínicos H1/genética , Alelos , Animais , Doenças Autoimunes/imunologia , Linhagem Celular , Membrana Celular/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Retículo Endoplasmático/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Predisposição Genética para Doença , Haplótipos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Orquite/imunologia , Polimorfismo Genético , Transporte Proteico , Receptores Histamínicos H1/deficiência , Receptores Histamínicos H1/imunologia , Receptores Histamínicos H1/metabolismo , Linfócitos T/metabolismo
12.
J Mol Biol ; 378(3): 481-91, 2008 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-18377933

RESUMO

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 Saccharomyces cerevisiae, 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 in vitro 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 in vivo. 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 activity.


Assuntos
RNA Polimerase III/metabolismo , Proteínas Repressoras/fisiologia , Transcrição Gênica , Animais , Células-Tronco Embrionárias/metabolismo , Células HeLa , Humanos , Camundongos , Fosforilação , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transfecção
13.
Nucleic Acids Res ; 35(9): 3046-52, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17439968

RESUMO

The tumour suppressor protein ARF provides a defence mechanism against hyperproliferative stresses that can result from the aberrant activation of oncogenes. Accordingly, ARF is silenced or deleted in many human cancers. Activation of ARF can arrest growth and cell cycle progression, or trigger apoptosis. A principle mediator of these effects is p53, which ARF stabilizes by binding and inhibiting MDM2. However, ARF has additional targets and remains able to block growth in the absence of p53, albeit less efficiently. For example, ARF can suppress rRNA production in a p53-independent manner. We have found that the synthesis of tRNA by RNA polymerase III is also inhibited in response to ARF. However, in contrast to its effects on rRNA synthesis, ARF is unable to inhibit tRNA gene transcription when p53 is ablated. These results add to the growing list of cellular changes that can be triggered by ARF induction.


Assuntos
RNA Polimerase III/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Linhagem Celular , Fator de Transcrição E2F1/metabolismo , Humanos , Transcrição Gênica , Proteína Supressora de Tumor p53/fisiologia
14.
Biochem Soc Symp ; (73): 141-54, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16626295

RESUMO

The proto-oncogene product c-Myc can induce cell growth and proliferation. It regulates a large number of RNA polymerase II-transcribed genes, many of which encode ribosomal proteins, translation factors and other components of the biosynthetic apparatus. We have found that c-Myc can also activate transcription by RNA polymerases I and III, thereby stimulating production of rRNA and tRNA. As such, c-Myc may possess the unprecedented capacity to induce expression of all ribosomal components. This may explain its potent ability to drive cell growth, which depends on the accumulation of ribosomes. The activation of RNA polymerase II transcription by c-Myc is often inefficient, but its induction of rRNA and tRNA genes can be very strong in comparison. We will describe what is known about the mechanisms used by c-Myc to activate transcription by RNA polymerases I and II.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ativação Transcricional , Animais , RNA Polimerases Dirigidas por DNA/genética , Humanos , Camundongos , Modelos Biológicos , Proto-Oncogene Mas , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , RNA Ribossômico/genética , RNA de Transferência/genética
15.
Proc Natl Acad Sci U S A ; 102(51): 18350-5, 2005 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-16344466

RESUMO

The human La autoantigen can bind to nascent RNA transcripts and has also been postulated to act as an RNA polymerase III (pol III) transcription initiation and termination factor. Here, we show by chromatin immunoprecipitation (ChIP) that La is associated with pol III-transcribed genes in vivo. In contrast, the Ro autoantigen, which can also bind pol III transcripts, is not found at these genes. The putative pol III transcription factors NF1 and TFIIA are also not detected at class III genes. Binding of La remains when transcription is repressed at mitosis and does not correlate with the presence of polymerase at the gene. However, gene occupancy depends on the phosphorylation status of La, with the less prevalent, unphosphorylated form being found selectively on pol III templates.


Assuntos
Autoantígenos/metabolismo , RNA Polimerase III/metabolismo , Ribonucleoproteínas/metabolismo , Transcrição Gênica , Imunoprecipitação da Cromatina , Inativação Gênica , Células HeLa , Humanos , Neurofibromina 1/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Moldes Genéticos , Fator de Transcrição TFIIA/metabolismo , Antígeno SS-B
16.
Mol Cell Biol ; 25(9): 3763-73, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15831480

RESUMO

We have previously identified poly(A)-binding protein 1 (PABP1) as a ligand for paxillin and shown that the paxillin-PABP1 complex undergoes nucleocytoplasmic shuttling. By targeting the paxillin-binding subdomain sequences in PABP1, we have generated mutants of PABP1 that do not bind to cellular paxillin. Here we report that paxillin association is necessary for efficient nuclear export of PABP1 and that RNA interference of paxillin drives the nuclear accumulation of PABP1. Furthermore, ablation of paxillin-PABP1 association impeded a number of indices of cell motility including spreading on fibronectin, cell migration on two-dimensional matrices, and transmigration in Boyden chambers. These data indicate that PABP1 must associate with paxillin in order to be efficiently transported from the nucleus to the cytoplasm and that this event is necessary for cells to remodel their focal adhesions during cell migration.


Assuntos
Movimento Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Adesões Focais/metabolismo , Fosfoproteínas/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , Citoplasma/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Fibroblastos/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Mutação/genética , Paxilina , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Proteína I de Ligação a Poli(A)/análise , Proteína I de Ligação a Poli(A)/genética , Estrutura Terciária de Proteína , Interferência de RNA , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Transfecção
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