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1.
Emerg Microbes Infect ; 9(1): 1418-1428, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32529952

RESUMO

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Interferons/antagonistas & inibidores , Interferons/metabolismo , Metiltransferases/metabolismo , Pneumonia Viral/metabolismo , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/genética , Linhagem Celular , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Endorribonucleases/genética , Exorribonucleases/genética , Interações Hospedeiro-Patógeno , Humanos , Interferons/genética , Metiltransferases/genética , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/virologia , RNA Helicases/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
2.
Cells ; 9(5)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32443810

RESUMO

The current coronavirus disease-2019 (COVID-19) pandemic is due to the novel coronavirus SARS-CoV-2. The scientific community has mounted a strong response by accelerating research and innovation, and has quickly set the foundation for understanding the molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key stage of the SARS-CoV-2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of the structural and functional data on the key actors of the replicatory machinery of SARS-CoV-2, to fill the gaps in the currently available structural data, which is mainly obtained through homology modeling. Moreover, learning from similar viruses, we collect data from the literature to reconstruct the pattern of interactions among the protein actors of the SARS-CoV-2 RNA polymerase machinery. Here, an important role is played by co-factors such as Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors that hold the entire machinery together to enhance the efficiency of RNA replication.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/metabolismo , Replicação Viral/fisiologia , Animais , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/metabolismo , Exorribonucleases/química , Exorribonucleases/metabolismo , Genoma Viral/genética , Humanos , Metiltransferases/química , Metiltransferases/metabolismo , Pandemias , Conformação Proteica em alfa-Hélice , RNA Helicases/química , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo
3.
PLoS Pathog ; 16(2): e1008338, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32059034

RESUMO

Interferon (IFN) stimulates a whole repertoire of cellular genes, collectively referred to as ISGs (Interferon-stimulated genes). ISG20, a 3´-5´ exonuclease enzyme, has been previously shown to bind and degrade hepatitis B Virus (HBV) transcripts. Here, we show that the N6-methyladenosine (m6A)-modified HBV transcripts are selectively recognized and processed for degradation by ISG20. Moreover, this effect of ISG20 is critically regulated by m6A reader protein, YTHDF2 (YTH-domain family 2). Previously, we identified a unique m6A site within HBV transcripts and confirmed that methylation at nucleotide A1907 regulates HBV lifecycle. In this report, we now show that the methylation at A1907 is a critical regulator of IFN-α mediated decay of HBV RNA. We observed that the HBV RNAs become less sensitive to ISG20 mediated degradation when methyltransferase enzymes or m6A reader protein YTHDF2 are silenced in HBV expressing cells. By using an enzymatically inactive form ISG20D94G, we further demonstrated that ISG20 forms a complex with m6A modified HBV RNA and YTHDF2 protein. Due to terminal redundancy, HBV genomic nucleotide A1907 position is acquired twice by pregenomic RNA (pgRNA) during transcription and therefore the sites of methylation are encoded within 5´ and 3´ epsilon stem loops. We generated HBV mutants that lack m6A site at either one (5´ or 3´) or both the termini (5´& 3´). Using these mutants, we demonstrated that m6A modified HBV RNAs are subjected to ISG20-mediated decay and propose sequence of events, in which ISG20 binds with YTHDF2 and recognizes m6A-modified HBV transcripts to carry out the ribonuclease activity. This is the first study, which identifies a hitherto unknown role of m6A modification of RNA in IFN-α induced viral RNA degradation and proposes a new role of YTHDF2 protein as a cofactor required for IFN-α mediated viral RNA degradation.


Assuntos
Exorribonucleases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Antivirais/farmacologia , Exonucleases/metabolismo , Exorribonucleases/genética , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Humanos , Interferon-alfa/farmacologia , Interferons/metabolismo , Metiltransferases/metabolismo , Estabilidade de RNA/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Replicação Viral/fisiologia
4.
PLoS Genet ; 16(1): e1008580, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31940341

RESUMO

RNA-binding proteins (RBPs) associate with the primary, precursor, and mature microRNAs, which in turn control post-transcriptional gene regulation. Here, by small RNAseq, we show that RBP FXR1 controls the expression of a subset of mature miRNAs, including highly expressed miR301a-3p in oral cancer cells. We also confirm that FXR1 controls the stability of miR301a-3p. Exoribonuclease PNPT1 degrades miR301a-3p in the absence of FXR1 in oral cancer cells, and the degradation is rescued in the FXR1 and PNPT1 co-knockdown cells. In vitro, we show that PNPT1 is unable to bind and degrade the miRNA once the FXR1-miRNA complex forms. Both miR301a-3p and FXR1 cooperatively target the 3'-UTR of p21 mRNA to promote its degradation. Thus, our work illustrates the unique role of FXR1 that is critical for the stability of a subset of mature miRNAs or at least miR301a-3p to target p21 in oral cancer.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , MicroRNAs/genética , Neoplasias Bucais/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Neoplasias Bucais/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/genética
5.
Nat Commun ; 11(1): 156, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919354

RESUMO

Technical problems intrinsic to the purification of preribosome intermediates have limited our understanding of ribosome biosynthesis in humans. Addressing this issue is important given the implication of this biological process in human disease. Here we report a preribosome purification and tagging strategy that overcomes some of the existing technical difficulties. Using these tools, we find that the pre-40S precursors go through two distinct maturation phases inside the nucleolus and follow a regulatory step that precedes late maturation in the cytoplasm. This regulatory step entails the intertwined actions of both PARN (a metazoan-specific ribonuclease) and RRP12 (a phylogenetically conserved 40S biogenesis factor that has acquired additional functional features in higher eukaryotes). Together, these results demonstrate the usefulness of this purification method for the dissection of ribosome biogenesis in human cells. They also identify distinct maturation stages and metazoan-specific regulatory mechanisms involved in the generation of the human 40S ribosomal subunit.


Assuntos
Nucléolo Celular/metabolismo , Proteínas Ribossômicas/biossíntese , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Linhagem Celular Tumoral , Exorribonucleases/metabolismo , Células HCT116 , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Precursores de RNA/biossíntese , Precursores de RNA/metabolismo , RNA Ribossômico/biossíntese , Subunidades Ribossômicas Menores de Eucariotos/genética , Coloração e Rotulagem/métodos
6.
Nat Commun ; 11(1): 122, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31913314

RESUMO

The No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stacks of stalled ribosomes. Although an endoribonuclease has been proposed to initiate cleavages upstream of the stall sequence, the production of two RNA fragments resulting from a unique cleavage has never been demonstrated. Here we use mRNAs expressing a 3'-ribozyme to produce truncated transcripts in vivo to mimic naturally occurring truncated mRNAs known to trigger NGD. This technique allows us to analyse endonucleolytic cleavage events at single-nucleotide resolution starting at the third collided ribosome, which we show to be Hel2-dependent. These cleavages map precisely in the mRNA exit tunnel of the ribosome, 8 nucleotides upstream of the first P-site residue and release 5'-hydroxylated RNA fragments requiring 5'-phosphorylation prior to digestion by the exoribonuclease Xrn1, or alternatively by Dxo1. Finally, we identify the RNA kinase Trl1, alias Rlg1, as an essential player in the degradation of NGD RNAs.


Assuntos
RNA Ligase (ATP)/metabolismo , RNA Fúngico/química , RNA Mensageiro/química , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Exorribonucleases/genética , Exorribonucleases/metabolismo , Fosforilação , RNA Ligase (ATP)/genética , Estabilidade de RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
7.
Nucleic Acids Res ; 48(1): 349-358, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31777937

RESUMO

Modifications at the 5'-end of RNAs play a pivotal role in determining their fate. In eukaryotes, the DXO/Rai1 family of enzymes removes numerous 5'-end RNA modifications, thereby regulating RNA turnover. Mouse DXO catalyzes the elimination of incomplete 5'-end caps (including pyrophosphate) and the non-canonical NAD+ cap on mRNAs, and possesses distributive 5'-3' exoribonuclease activity toward 5'-monophosphate (5'-PO4) RNA. Here, we demonstrate that DXO also catalyzes the hydrolysis of RNAs bearing a 5'-hydroxyl group (5'-OH RNA). The crystal structure of DXO in complex with a 5'-OH RNA substrate mimic at 2.0 Å resolution provides elegant insight into the molecular mechanism of this activity. More importantly, the structure predicts that DXO first removes a dinucleotide from 5'-OH RNA. Our nuclease assays confirm this prediction and demonstrate that this 5'-hydroxyl dinucleotide hydrolase (HDH) activity for DXO is higher than the subsequent 5'-3' exoribonuclease activity for selected substrates. Fission yeast Rai1 also has HDH activity although it does not have 5'-3' exonuclease activity, and the Rat1-Rai1 complex can completely degrade 5'-OH RNA. An Arabidopsis DXO1 variant is active toward 5'-OH RNA but prefers 5'-PO4 RNA. Collectively, these studies demonstrate the diverse activities of DXO/Rai1 and expands the collection of RNA substrates that can undergo 5'-3' mediated decay.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Exorribonucleases/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/genética , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleases/química , Exorribonucleases/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Camundongos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Transativadores/química , Transativadores/genética
8.
RNA ; 26(2): 199-208, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31719125

RESUMO

The 3'-termini of tRNA are the point of amino acid linkage and thus crucial for their function in delivering amino acids to the ribosome and other enzymes. Therefore, to provide tRNA functionality, cells have to ensure the integrity of the 3'-terminal CCA-tail, which is generated during maturation by the 3'-trailer processing machinery and maintained by the CCA-adding enzyme. We developed a new tRNA sequencing method that is specifically tailored to assess the 3'-termini of E. coli tRNA. Intriguingly, we found a significant fraction of tRNAs with damaged CCA-tails under exponential growth conditions and, surprisingly, this fraction decreased upon transition into stationary phase. Interestingly, tRNAs bearing guanine as a discriminator base are generally unaffected by CCA-tail damage. In addition, we showed tRNA species-specific 3'-trailer processing patterns and reproduced in vitro findings on preferences of the maturation enzyme RNase T in vivo.


Assuntos
Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala , Processamento de Terminações 3' de RNA/genética , RNA Nucleotidiltransferases/metabolismo , RNA de Transferência/genética , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Nucleotídeos , RNA Nucleotidiltransferases/genética , Precursores de RNA/genética , Estabilidade de RNA , RNA Bacteriano/genética , Análise de Sequência de RNA
9.
Am J Physiol Cell Physiol ; 318(1): C48-C62, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31618077

RESUMO

We recently published that type 2 diabetes promotes cell centrosome amplification via upregulation of Rho-associated protein kinase 1 (ROCK1) and 14-3-3 protein-σ (14-3-3σ). This study further investigates the molecular mechanisms underlying diabetes-associated centrosome amplification. We found that treatment of cells with high glucose, insulin, and palmitic acid levels increased the intracellular and extracellular protein levels of Wingless-type MMTV integration site family member 6 (Wnt6) as well as the cellular level of ß-catenin. The treatment also activated ß-catenin and promoted its nuclear translocation. Treatment of cells with siRNA species for Wnt6, Frizzled-4 (FZD4), or ß-catenin as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture medium could all attenuate the treatment-triggered centrosome amplification. Moreover, we showed that secreted Wnt6-FZD4-ß-catenin was the signaling pathway that was upstream of ROCK1 and 14-3-3σ. We found that advanced glycation end products (AGEs) were also able to increase the cellular and extracellular levels of Wnt6, the cellular protein level of ß-catenin, and centrosome amplification. Treatment of the cells with siRNA species for Wnt6 or FZD4 as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture could all inhibit the AGEs-elicited centrosome amplification. In colon tissues from a diabetic mouse model, the protein levels of Wnt6 and 14-3-3σ were increased. In conclusion, our results showed that the pathophysiological factors in type 2 diabetes, including AGEs, were able to induce centrosome amplification. It is suggested that secreted Wnt6 binds to FZD4 to activate the canonical Wnt6 signaling pathway, which is upstream of ROCK1 and 14-3-3σ, and that this is the cell signaling pathway underlying diabetes-associated centrosome amplification.


Assuntos
Centrossomo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Receptores Frizzled/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Proteínas 14-3-3/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Glicemia/metabolismo , Centrossomo/patologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Exorribonucleases/metabolismo , Feminino , Receptores Frizzled/genética , Produtos Finais de Glicação Avançada/farmacologia , Células HCT116 , Humanos , Insulina/sangue , Camundongos Endogâmicos ICR , Ácido Palmítico/farmacologia , Ligação Proteica , Ratos , Proteínas Wnt/genética , Quinases Associadas a rho/metabolismo
10.
Genes Dev ; 34(1-2): 132-145, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31805520

RESUMO

The allosteric and torpedo models have been used for 30 yr to explain how transcription terminates on protein-coding genes. The former invokes termination via conformational changes in the transcription complex and the latter proposes that degradation of the downstream product of poly(A) signal (PAS) processing is important. Here, we describe a single mechanism incorporating features of both models. We show that termination is completely abolished by rapid elimination of CPSF73, which causes very extensive transcriptional readthrough genome-wide. This is because CPSF73 functions upstream of modifications to the elongation complex and provides an entry site for the XRN2 torpedo. Rapid depletion of XRN2 enriches these events that we show are underpinned by protein phosphatase 1 (PP1) activity, the inhibition of which extends readthrough in the absence of XRN2. Our results suggest a combined allosteric/torpedo mechanism, in which PP1-dependent slowing down of polymerases over termination regions facilitates their pursuit/capture by XRN2 following PAS processing.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Terminação da Transcrição Genética/fisiologia , Linhagem Celular , Fator de Especificidade de Clivagem e Poliadenilação/genética , Exorribonucleases/metabolismo , Deleção de Genes , Células HCT116 , Humanos , RNA/metabolismo , RNA Polimerase II/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Ribonuclease H/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(2): 982-992, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879344

RESUMO

The exoribonuclease Rrp6p is critical for RNA decay in the nucleus. While Rrp6p acts on a large range of diverse substrates, it does not indiscriminately degrade all RNAs. How Rrp6p accomplishes this task is not understood. Here, we measure Rrp6p-RNA binding and degradation kinetics in vitro at single-nucleotide resolution and find an intrinsic substrate selectivity that enables Rrp6p to discriminate against specific RNAs. RNA length and the four 3'-terminal nucleotides contribute most to substrate selectivity and collectively enable Rrp6p to discriminate between different RNAs by several orders of magnitude. The most pronounced discrimination is seen against RNAs ending with CCA-3'. These RNAs correspond to 3' termini of uncharged tRNAs, which are not targeted by Rrp6p in cells. The data show that in contrast to many other proteins that use substrate selectivity to preferentially interact with specific RNAs, Rrp6p utilizes its selectivity to discriminate against specific RNAs. This ability allows Rrp6p to target diverse substrates while avoiding a subset of RNAs.


Assuntos
Exorribonucleases/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Escherichia coli , Exorribonucleases/química , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Cinética , RNA/química , RNA de Transferência/metabolismo , Especificidade por Substrato
12.
BMC Cancer ; 19(1): 1157, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31779593

RESUMO

BACKGROUND: Cervical cancer (CC), causing significant morbidity and mortality worldwide, is one of the most common gynecological malignancies in women. SFN has been reported as a potential prognostic marker with apparent high expression in tumors. Nevertheless, the function mechanism of SFN is not clear yet in CC. METHODS: The relative expressions of RNAs were detected by real-time quantitative PCR (RT-qPCR). Colony formation assay, EdU stained assay and CCK-8 assay were to check cell proliferation ability in CC. Flow cytometry and apoptosis related proteins analysis were used to measure cells apoptosis capacity. Luciferase reporter assay and RNA pull down assay were to verify the molecular mechanism. RESULTS: SFN was highly expressed in CC tissues and CC cell lines compared with normal tissues and normal cell line. After interfering SFN, cell proliferation, migration and invasion ability was inhibited as well as cell apoptosis ability was promoted. In subsequence, miR-383-5p exhibited conspicuous low expression in CC tissues. And miR-383-5p was found to bind to SFN and have anti-cancerous effects in CC. Moreover, LINC01128 displayed remarkable high expression in CC tissues. Besides, LINC01128 shortage could reduce the expression of SFN at mRNA and protein levels. And the affinity between LINC01128 and miR-383-5p was verified. In the end, it was proved that LINC01128 could enhance cell proliferation, migration and invasion as well as inhibit cell apoptosis by binding with miR-383-5p and upregulating SFN. CONCLUSION: LINC01128 expedited cells cellular process in CC by binding with miR-383-5p to release SFN.


Assuntos
Proteínas 14-3-3/genética , Biomarcadores Tumorais/genética , Exorribonucleases/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Neoplasias do Colo do Útero/genética , Proteínas 14-3-3/metabolismo , Apoptose , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Exorribonucleases/metabolismo , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Neoplasias do Colo do Útero/patologia
13.
Mol Cell ; 76(6): 896-908.e4, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31677974

RESUMO

Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA polymerase II (Pol II) elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, Pol II decelerates from >2 kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a model for termination, the "sitting duck torpedo" mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert Pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homolog NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Proteína Fosfatase 1/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/metabolismo , Terminação da Transcrição Genética , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Exorribonucleases/genética , Células HEK293 , Humanos , Cinética , Proteínas Nucleares/genética , Fosforilação , Poli A/metabolismo , Ligação Proteica , Proteína Fosfatase 1/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Fatores de Elongação da Transcrição/genética
14.
Crit Rev Biochem Mol Biol ; 54(4): 385-398, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31656086

RESUMO

Transcription and RNA decay are key determinants of gene expression; these processes are typically considered as the uncoupled beginning and end of the messenger RNA (mRNA) lifecycle. Here we describe the growing number of studies demonstrating interplay between these spatially disparate processes in eukaryotes. Specifically, cells can maintain mRNA levels by buffering against changes in mRNA stability or transcription, and can also respond to virally induced accelerated decay by reducing RNA polymerase II gene expression. In addition to these global responses, there is also evidence that mRNAs containing a premature stop codon can cause transcriptional upregulation of homologous genes in a targeted fashion. In each of these systems, RNA binding proteins (RBPs), particularly those involved in mRNA degradation, are critical for cytoplasmic to nuclear communication. Although their specific mechanistic contributions are yet to be fully elucidated, differential trafficking of RBPs between subcellular compartments are likely to play a central role in regulating this gene expression feedback pathway.


Assuntos
Citoplasma/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Transcrição Genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Códon de Terminação/genética , Citoplasma/metabolismo , Exorribonucleases/metabolismo , Expressão Gênica , Homeostase/genética , Humanos , Infecções/genética , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética
15.
Mol Cell ; 76(5): 784-796.e6, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31588022

RESUMO

Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria.


Assuntos
Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/metabolismo , Exorribonucleases/metabolismo , Mitocôndrias/enzimologia , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas , Estabilidade de RNA , RNA Mitocondrial/metabolismo , Proteínas 14-3-3/deficiência , Proteínas 14-3-3/genética , Animais , Biomarcadores Tumorais/genética , Exorribonucleases/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mitocondrial/genética , Células Sf9 , Spodoptera
16.
FEBS Open Bio ; 9(10): 1674-1688, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31420950

RESUMO

The gene encoding MG Orn has been identified from a metagenomic library created from the intertidal zone in Svalbard and encodes a protein of 184 amino acid residues. The mg orn gene has been cloned, recombinantly expressed in Escherichia coli, and purified to homogeneity. Biochemical characterization of the enzyme showed that it efficiently degrades short RNA oligonucleotide substrates of 2mer to 10mer of length and has an absolute requirement for divalent cations for optimal activity. The enzyme is more heat-labile than its counterpart from E. coli and exists as a homodimer in solution. The crystal structure of the enzyme has been determined to a resolution of 3.15 Å, indicating an important role of a disulfide bridge for the homodimer formation and as such for the function of MG Orn. Substitution of the Cys110 residue with either Gly or Ala hampered the dimer formation and severely affected the enzyme's ability to act on RNA. A conserved loop containing His128-Tyr129-Arg130 in the neighboring monomer is probably involved in efficient binding and processing of longer RNA substrates than diribonucleotides.


Assuntos
Dissulfetos/química , Exorribonucleases/química , Exorribonucleases/genética , Metagenoma/genética , Cromatografia em Gel , Biologia Computacional , Cristalografia por Raios X , Dissulfetos/metabolismo , Exorribonucleases/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica
17.
Cells ; 8(8)2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31387300

RESUMO

Poly(A)-specific ribonuclease (PARN), a multifunctional multi-domain deadenylase, is crucial to the regulation of mRNA turnover and the maturation of various non-coding RNAs. Despite extensive studies of the well-folding domains responsible for PARN catalysis, the structure and function of the C-terminal domain (CTD) remains elusive. PARN is a cytoplasm-nucleus shuttle protein with concentrated nucleolar distribution. Here, we identify the nuclear and nucleolar localization signals in the CTD of PARN. Spectroscopic studies indicated that PARN-CTD is intrinsically disordered with loosely packed local structures/tertiary structure. Phosphorylation-mimic mutation S557D disrupted the local structure and facilitated the binding of the CTD with the well-folded domains, with no impact on PARN deadenylase activity. Under normal conditions, the nucleolus-residing PARN recruited CBP80 into the nucleoli to repress its deadenylase activity, while DNA damage-induced phosphorylation of PARN-S557 expelled CBP80 from the nucleoli to discharge activity inhibition and attracted nucleoplasm-located CstF-50 into the nucleoli to activate deadenylation. The structure switch-induced function switch of PARN reshaped the profile of small nuclear non-coding RNAs to respond to DNA damage. Our findings highlight that the structure switch of the CTD induced by posttranslational modifications redefines the subset of binding partners, and thereby the RNA targets in the nucleoli.


Assuntos
Núcleo Celular/metabolismo , Dano ao DNA , Exorribonucleases/metabolismo , Sinais de Localização Nuclear , Dobramento de Proteína , Transporte Ativo do Núcleo Celular , Animais , Células CHO , Cricetinae , Cricetulus , Exorribonucleases/química , Células HEK293 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/química
18.
Hum Mutat ; 40(12): 2414-2429, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31448843

RESUMO

PARN encodes poly(A)-specific ribonuclease. Biallelic and monoallelic PARN variants are associated with Hoyeraal-Hreidarsson syndrome/dyskeratosis congenita and idiopathic pulmonary fibrosis (IPF), respectively. The molecular features associated with incomplete penetrance of PARN-associated IPF have not been described. We report a family with a rare missense, p.Y91C, and a novel insertion, p.(I274*), PARN variant. We found PARN p.Y91C had reduced deadenylase activity and the p.(I274*) transcript was depleted. Detailed analysis of the consequences of these variants revealed that, while PARN protein was lowest in the severely affected biallelic child who had the shortest telomeres, it was also reduced in his mother with the p.(I274*) variant but telomeres at the 50th percentile. Increased adenylation of telomerase RNA, human telomerase RNA, and certain small nucleolar RNAs, and impaired ribosomal RNA maturation were observed in cells derived from the severely affected biallelic carrier, but not in the other, less affected biallelic carrier, who had less severely shortened telomeres, nor in the monoallelic carriers who were unaffected and had telomeres ranging from the 1st to the 50th percentiles. We identified hsa-miR-202-5p as a potential negative regulator of PARN. We propose one or more genetic modifiers influence the impact of PARN variants on its targets and this underlies incomplete penetrance of PARN-associated disease.


Assuntos
Disceratose Congênita/genética , Exorribonucleases/genética , Retardo do Crescimento Fetal/genética , Deficiência Intelectual/genética , MicroRNAs/genética , Microcefalia/genética , Mutagênese Insercional , Mutação de Sentido Incorreto , Adolescente , Linhagem Celular , Pré-Escolar , Regulação para Baixo , Exorribonucleases/metabolismo , Feminino , Humanos , Masculino , Linhagem , Penetrância , Encurtamento do Telômero
19.
EMBO Mol Med ; 11(7): e10201, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31273937

RESUMO

PARN, poly(A)-specific ribonuclease, regulates the turnover of mRNAs and the maturation and stabilization of the hTR RNA component of telomerase. Biallelic PARN mutations were associated with Høyeraal-Hreidarsson (HH) syndrome, a rare telomere biology disorder that, because of its severity, is likely not exclusively due to hTR down-regulation. Whether PARN deficiency was affecting the expression of telomere-related genes was still unclear. Using cells from two unrelated HH individuals carrying novel PARN mutations and a human PARN knock-out (KO) cell line with inducible PARN complementation, we found that PARN deficiency affects both telomere length and stability and down-regulates the expression of TRF1, TRF2, TPP1, RAP1, and POT1 shelterin transcripts. Down-regulation of dyskerin-encoding DKC1 mRNA was also observed and found to result from p53 activation in PARN-deficient cells. We further showed that PARN deficiency compromises ribosomal RNA biogenesis in patients' fibroblasts and cells from heterozygous Parn KO mice. Homozygous Parn KO however resulted in early embryonic lethality that was not overcome by p53 KO. Our results refine our knowledge on the pleiotropic cellular consequences of PARN deficiency.


Assuntos
Disceratose Congênita/metabolismo , Exorribonucleases/deficiência , Retardo do Crescimento Fetal/metabolismo , Deficiência Intelectual/metabolismo , Microcefalia/metabolismo , RNA Ribossômico/biossíntese , Homeostase do Telômero , Telômero/metabolismo , Animais , Pré-Escolar , Modelos Animais de Doenças , Disceratose Congênita/genética , Disceratose Congênita/patologia , Exorribonucleases/metabolismo , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/patologia , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Masculino , Camundongos , Camundongos Knockout , Microcefalia/genética , Microcefalia/patologia , RNA Ribossômico/genética , Telômero/genética , Telômero/patologia
20.
EMBO J ; 38(15): e100986, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31267554

RESUMO

Aberrant transcription-associated RNA:DNA hybrid (R-loop) formation often causes catastrophic conflicts during replication, resulting in DNA double-strand breaks and genomic instability. Preventing such conflicts requires hybrid dissolution by helicases and/or RNase H. Little is known about how such helicases are regulated. Herein, we identify DDX5, an RGG/RG motif-containing DEAD-box family RNA helicase, as crucial player in R-loop resolution. In vitro, recombinant DDX5 resolves R-loops in an ATP-dependent manner, leading to R-loop degradation by the XRN2 exoribonuclease. DDX5-deficient cells accumulate R-loops at loci with propensity to form such structures based on RNA:DNA immunoprecipitation (DRIP)-qPCR, causing spontaneous DNA double-strand breaks and hypersensitivity to replication stress. DDX5 associates with XRN2 and resolves R-loops at transcriptional termination regions downstream of poly(A) sites, to facilitate RNA polymerase II release associated with transcriptional termination. Protein arginine methyltransferase 5 (PRMT5) binds and methylates DDX5 at its RGG/RG motif. This motif is required for DDX5 interaction with XRN2 and repression of cellular R-loops, but not essential for DDX5 helicase enzymatic activity. PRMT5-deficient cells accumulate R-loops, resulting in increased formation of γH2AX foci. Our findings exemplify a mechanism by which an RNA helicase is modulated by arginine methylation to resolve R-loops, and its potential role in regulating transcription.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , RNA/química , Motivos de Aminoácidos , Arginina/metabolismo , Linhagem Celular , DNA/metabolismo , Exorribonucleases/metabolismo , Células HEK293 , Humanos , Metilação , Proteína-Arginina N-Metiltransferases/genética , RNA/metabolismo , RNA Polimerase II/metabolismo
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