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1.
Sci Signal ; 13(651)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994211

RESUMO

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/química , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Genes Virais/genética , Humanos , Metilação , Metiltransferases/antagonistas & inibidores , Modelos Moleculares , Fases de Leitura Aberta/genética , Pandemias , Ligação Proteica , Conformação Proteica , Análogos de Capuz de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Viral/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
2.
Nat Commun ; 11(1): 4455, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901005

RESUMO

Dysregulated alternative splicing (AS) driving carcinogenetic mitosis remains poorly understood. Here, we demonstrate that cancer metastasis-associated antigen 1 (MTA1), a well-known oncogenic chromatin modifier, broadly interacts and co-expresses with RBPs across cancers, contributing to cancerous mitosis-related AS. Using developed fCLIP-seq technology, we show that MTA1 binds abundant transcripts, preferentially at splicing-responsible motifs, influencing the abundance and AS pattern of target transcripts. MTA1 regulates the mRNA level and guides the AS of a series of mitosis regulators. MTA1 deletion abrogated the dynamic AS switches of variants for ATRX and MYBL2 at mitotic stage, which are relevant to mitosis-related tumorigenesis. MTA1 dysfunction causes defective mitotic arrest, leads to aberrant chromosome segregation, and results in chromosomal instability (CIN), eventually contributing to tumorigenesis. Currently, little is known about the RNA splicing during mitosis; here, we uncover that MTA1 binds transcripts and orchestrates dynamic splicing of mitosis regulators in tumorigenesis.


Assuntos
Carcinogênese/genética , Carcinogênese/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Mitose/fisiologia , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Processamento Alternativo , Animais , Sítios de Ligação/genética , Montagem e Desmontagem da Cromatina/genética , Instabilidade Cromossômica , Feminino , Células HCT116 , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Mitose/genética , Neoplasias/genética , Neoplasias/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transativadores/antagonistas & inibidores , Transativadores/genética
3.
Int J Mol Sci ; 21(19)2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32993015

RESUMO

The outbreak of a novel coronavirus SARS-CoV-2 responsible for the COVID-19 pandemic has caused a worldwide public health emergency. Due to the constantly evolving nature of the coronaviruses, SARS-CoV-2-mediated alterations on post-transcriptional gene regulations across human tissues remain elusive. In this study, we analyzed publicly available genomic datasets to systematically dissect the crosstalk and dysregulation of the human post-transcriptional regulatory networks governed by RNA-binding proteins (RBPs) and micro-RNAs (miRs) due to SARS-CoV-2 infection. We uncovered that 13 out of 29 SARS-CoV-2-encoded proteins directly interacted with 51 human RBPs, of which the majority of them were abundantly expressed in gonadal tissues and immune cells. We further performed a functional analysis of differentially expressed genes in mock-treated versus SARS-CoV-2-infected lung cells that revealed enrichment for the immune response, cytokine-mediated signaling, and metabolism-associated genes. This study also characterized the alternative splicing events in SARS-CoV-2-infected cells compared to the control, demonstrating that skipped exons and mutually exclusive exons were the most abundant events that potentially contributed to differential outcomes in response to the viral infection. A motif enrichment analysis on the RNA genomic sequence of SARS-CoV-2 clearly revealed the enrichment for RBPs such as SRSFs, PCBPs, ELAVs, and HNRNPs, suggesting the sponging of RBPs by the SARS-CoV-2 genome. A similar analysis to study the interactions of miRs with SARS-CoV-2 revealed functionally important miRs that were highly expressed in immune cells, suggesting that these interactions may contribute to the progression of the viral infection and modulate the host immune response across other human tissues. Given the need to understand the interactions of SARS-CoV-2 with key post-transcriptional regulators in the human genome, this study provided a systematic computational analysis to dissect the role of dysregulated post-transcriptional regulatory networks controlled by RBPs and miRs across tissue types during a SARS-CoV-2 infection.


Assuntos
Betacoronavirus/genética , Betacoronavirus/metabolismo , Infecções por Coronavirus/virologia , Redes Reguladoras de Genes , MicroRNAs/genética , Pneumonia Viral/virologia , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Regulação da Expressão Gênica , Genoma Viral , Humanos , MicroRNAs/metabolismo , Pandemias , Mapas de Interação de Proteínas , Proteínas de Ligação a RNA/genética
4.
Ecotoxicol Environ Saf ; 204: 111056, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32763566

RESUMO

Strontium (Sr) is an emerging environmental pollutant that has become a major global concern after the nuclear accident at the Fukushima Daiichi Nuclear Power Plant in 2011. Although many studies have demonstrated the harmful effects of Sr on plant growth and development at the physiological level, knowledge regarding how plants sense and respond to Sr stress at the molecular level is limited. Recent studies have suggested that microRNAs (miRNAs) function as key regulators of plant growth and development as well as in the responses of plants to environmental stresses, including salinity, drought, cold, nutrient starvation, and heavy metals. In this study, we examined the global expression profile of miRNAs under Sr stress using small RNA sequencing analysis in Arabidopsis to better understand the molecular basis of plant responses to Sr stress. To identify specific Sr-responsive miRNAs, we performed comparative miRNA expression profiling analysis using control, CaCl2-, and SrCl2-treated seedlings. Compared to the control treatment, the expressions of most miRNAs were considerably decreased in the Sr-treated seedlings. However, under Sr stress, the expressions of primary miRNAs (pri-miRNAs) and their target genes were significantly increased; the protein levels of HYPONASTIC LEAVES 1 (HYL1), one of the core components of the microprocessor complex, were strongly reduced despite the increased HYL1 mRNA expression. In addition, hyl1-2 mutant plants were shown to be more sensitive to Sr stress than wild-type plants. Collectively, our results strongly suggested that Sr stress may be associated with the disruption of miRNA biogenesis by reducing the protein level of HYL1, which is required to maintain proper growth and development for plants. Our findings further indicated that some miRNAs may play important roles in plant responses to Sr stress.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , MicroRNAs/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Poluentes do Solo/toxicidade , Estrôncio/toxicidade , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Estresse Oxidativo/genética , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Processamento Pós-Transcricional do RNA
5.
PLoS Genet ; 16(8): e1008893, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841241

RESUMO

All tRNAs are extensively modified, and modification deficiency often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of any of several tRNA body modifications results in rapid tRNA decay (RTD) of certain mature tRNAs by the 5'-3' exonucleases Rat1 and Xrn1. As tRNA quality control decay mechanisms are not extensively studied in other eukaryotes, we studied trm8Δ mutants in the evolutionarily distant fission yeast Schizosaccharomyces pombe, which lack 7-methylguanosine at G46 (m7G46) of their tRNAs. We report here that S. pombe trm8Δ mutants are temperature sensitive primarily due to decay of tRNATyr(GUA) and that spontaneous mutations in the RAT1 ortholog dhp1+ restored temperature resistance and prevented tRNA decay, demonstrating conservation of the RTD pathway. We also report for the first time evidence linking the RTD and the general amino acid control (GAAC) pathways, which we show in both S. pombe and S. cerevisiae. In S. pombe trm8Δ mutants, spontaneous GAAC mutations restored temperature resistance and tRNA levels, and the trm8Δ temperature sensitivity was precisely linked to GAAC activation due to tRNATyr(GUA) decay. Similarly, in the well-studied S. cerevisiae trm8Δ trm4Δ RTD mutant, temperature sensitivity was closely linked to GAAC activation due to tRNAVal(AAC) decay; however, in S. cerevisiae, GAAC mutations increased tRNA loss and exacerbated temperature sensitivity. A similar exacerbated growth defect occurred upon GAAC mutation in S. cerevisiae trm8Δ and other single modification mutants that triggered RTD. Thus, these results demonstrate a conserved GAAC activation coincident with RTD in S. pombe and S. cerevisiae, but an opposite impact of the GAAC response in the two organisms. We speculate that the RTD pathway and its regulation of the GAAC pathway is widely conserved in eukaryotes, extending to other mutants affecting tRNA body modifications.


Assuntos
Exorribonucleases/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA de Transferência/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , tRNA Metiltransferases/metabolismo , Aminoácidos/metabolismo , Evolução Molecular , Exorribonucleases/genética , RNA de Transferência/metabolismo , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética , tRNA Metiltransferases/genética
6.
Nat Commun ; 11(1): 4269, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859890

RESUMO

Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.


Assuntos
Processamento Pós-Transcricional do RNA , RNA Mitocondrial/química , RNA de Transferência/química , Feminino , Código Genético , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Estrutura Molecular , Nucleosídeo Q/biossíntese , Nucleosídeo Q/química , Fosforilação Oxidativa , Placenta , Gravidez , RNA Mitocondrial/isolamento & purificação , RNA Mitocondrial/metabolismo , RNA de Transferência/isolamento & purificação , RNA de Transferência/metabolismo , RNA-Seq
7.
Am J Hum Genet ; 107(3): 544-554, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32730804

RESUMO

RNA polymerase II interacts with various other complexes and factors to ensure correct initiation, elongation, and termination of mRNA transcription. One of these proteins is SR-related CTD-associated factor 4 (SCAF4), which is important for correct usage of polyA sites for mRNA termination. Using exome sequencing and international matchmaking, we identified nine likely pathogenic germline variants in SCAF4 including two splice-site and seven truncating variants, all residing in the N-terminal two thirds of the protein. Eight of these variants occurred de novo, and one was inherited. Affected individuals demonstrated a variable neurodevelopmental disorder characterized by mild intellectual disability, seizures, behavioral abnormalities, and various skeletal and structural anomalies. Paired-end RNA sequencing on blood lymphocytes of SCAF4-deficient individuals revealed a broad deregulation of more than 9,000 genes and significant differential splicing of more than 2,900 genes, indicating an important role of SCAF4 in mRNA processing. Knockdown of the SCAF4 ortholog CG4266 in the model organism Drosophila melanogaster resulted in impaired locomotor function, learning, and short-term memory. Furthermore, we observed an increased number of active zones in larval neuromuscular junctions, representing large glutamatergic synapses. These observations indicate a role of CG4266 in nervous system development and function and support the implication of SCAF4 in neurodevelopmental phenotypes. In summary, our data show that heterozygous, likely gene-disrupting variants in SCAF4 are causative for a variable neurodevelopmental disorder associated with impaired mRNA processing.


Assuntos
Deficiência Intelectual/genética , Transtornos do Neurodesenvolvimento/genética , Convulsões/genética , Fatores de Processamento de Serina-Arginina/genética , Animais , Criança , Drosophila melanogaster/genética , Feminino , Técnicas de Silenciamento de Genes , Variação Genética/genética , Heterozigoto , Humanos , Deficiência Intelectual/fisiopatologia , Locomoção/genética , Masculino , Mutação/genética , Transtornos do Neurodesenvolvimento/fisiopatologia , RNA Polimerase II/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Convulsões/fisiopatologia , Sequenciamento Completo do Exoma
8.
Nucleic Acids Res ; 48(14): 8035-8049, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32609822

RESUMO

Extracellular RNAs participate in intercellular communication, and are being studied as promising minimally invasive diagnostic markers. Several studies in recent years showed that tRNA halves and distinct Y RNA fragments are abundant in the extracellular space, including in biofluids. While their regulatory and diagnostic potential has gained a substantial amount of attention, the biogenesis of these extracellular RNA fragments remains largely unexplored. Here, we demonstrate that these fragments are produced by RNase 1, a highly active secreted nuclease. We use RNA sequencing to investigate the effect of a null mutation of RNase 1 on the levels of tRNA halves and Y RNA fragments in the extracellular environment of cultured human cells. We complement and extend our RNA sequencing results with northern blots, showing that tRNAs and Y RNAs in the non-vesicular extracellular compartment are released from cells as full-length precursors and are subsequently cleaved to distinct fragments. In support of these results, formation of tRNA halves is recapitulated by recombinant human RNase 1 in our in vitro assay. These findings assign a novel function for RNase 1, and position it as a strong candidate for generation of tRNA halves and Y RNA fragments in biofluids.


Assuntos
RNA de Transferência/metabolismo , RNA não Traduzido/metabolismo , Ribonucleases/metabolismo , Humanos , Células K562 , Mutação , Clivagem do RNA , Processamento Pós-Transcricional do RNA , RNA de Transferência/química , RNA não Traduzido/química , RNA-Seq
9.
Nat Commun ; 11(1): 3422, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647223

RESUMO

The Integrator complex processes 3'-ends of spliceosomal small nuclear RNAs (snRNAs). Furthermore, it regulates transcription of protein coding genes by terminating transcription after unstable pausing. The molecular basis for Integrator's functions remains obscure. Here, we show that INTS10, Asunder/INTS13 and INTS14 form a separable, functional Integrator module. The structure of INTS13-INTS14 reveals a strongly entwined complex with a unique chain interlink. Unexpected structural homology to the Ku70-Ku80 DNA repair complex suggests nucleic acid affinity. Indeed, the module displays affinity for DNA and RNA but prefers RNA hairpins. While the module plays an accessory role in snRNA maturation, it has a stronger influence on transcription termination after pausing. Asunder/INTS13 directly binds Integrator's cleavage module via a conserved C-terminal motif that is involved in snRNA processing and required for spermatogenesis. Collectively, our data establish INTS10-INTS13-INTS14 as a nucleic acid-binding module and suggest that it brings cleavage module and target transcripts into proximity.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ácidos Nucleicos/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Autoantígeno Ku/química , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA/química , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Homologia Estrutural de Proteína
10.
In Vivo ; 34(3 Suppl): 1593-1596, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: covidwho-536995

RESUMO

The Covid-19 pandemic is a world-wide crisis without an effective therapy. While most approaches to therapy are using repurposed drugs that were developed for other diseases, it is thought that targeting the biology of the SARS-CoV-2 virus, which causes Covid-19, can result in an effective therapeutic treatment. The coronavirus RNA cap structure is methylated by two viral methyltransferases that transfer methyl groups from S-adenosylmethionine (SAM). The proper methylation of the virus depends on the level of methionine in the host to form SAM. Herein, we propose to restrict methionine availability by treating the patient with oral recombinant methioninase, aiming to treat Covid-19. By restricting methionine we not only interdict viral replication, which depends on the viral RNA cap methyaltion, but also inhibit the proliferation of the infected cells, which have an increased requirement for methionine. Most importantly, the virally-induced T-cell- and macrophage-mediated cytokine storm, which seems to be a significant cause for Covid-19 deaths, can also be inhibited by restricting methionine, since T-cell and macrophrage activation greatly increases the methionine requirement for these cells. The evidence reviewed here suggests that oral recombinant methioninase could be a promising treatment for coronavirus patients.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Liases de Carbono-Enxofre/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Metionina/metabolismo , Pneumonia Viral/tratamento farmacológico , Capuzes de RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Viral/efeitos dos fármacos , Administração Oral , Antivirais/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/uso terapêutico , Betacoronavirus/fisiologia , Liases de Carbono-Enxofre/administração & dosagem , Ensaios Clínicos como Assunto , Infecções por Coronavirus/complicações , Infecções por Coronavirus/imunologia , Síndrome da Liberação de Citocina/prevenção & controle , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Metanálise como Assunto , Metilação/efeitos dos fármacos , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/imunologia , Pseudomonas putida/enzimologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , S-Adenosilmetionina/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Replicação Viral/efeitos dos fármacos
11.
RNA ; 26(10): 1345-1359, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32554553

RESUMO

Metazoan replication-dependent histone pre-mRNAs are cleaved at the 3' end by U7 snRNP, an RNA-guided endonuclease that contains U7 snRNA, seven proteins of the Sm ring, FLASH, and four polyadenylation factors: symplekin, CPSF73, CPSF100, and CstF64. A fully recombinant U7 snRNP was recently reconstituted from all 13 components for functional and structural studies and shown to accurately cleave histone pre-mRNAs. Here, we analyzed the activity of recombinant U7 snRNP in more detail. We demonstrate that in addition to cleaving histone pre-mRNAs endonucleolytically, reconstituted U7 snRNP acts as a 5'-3' exonuclease that degrades the downstream product generated from histone pre-mRNAs as a result of the endonucleolytic cleavage. Surprisingly, recombinant U7 snRNP also acts as an endonuclease on single-stranded DNA substrates. All these activities depend on the ability of U7 snRNA to base-pair with the substrate and on the presence of the amino-terminal domain (NTD) of symplekin in either cis or trans, and are abolished by mutations within the catalytic center of CPSF73, or by binding of the NTD to the SSU72 phosphatase of RNA polymerase II. Altogether, our results demonstrate that recombinant U7 snRNP functionally mimics its endogenous counterpart and provide evidence that CPSF73 is both an endonuclease and a 5'-3' exonuclease, consistent with the activity of other members of the ß-CASP family. Our results also raise the intriguing possibility that CPSF73 may be involved in some aspects of DNA metabolism in vivo.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/genética , Endonucleases/genética , Exonucleases/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U7/genética , Animais , Histonas/genética , Camundongos , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA/genética
12.
Mol Cell ; 78(5): 876-889.e6, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32502422

RESUMO

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.


Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , MicroRNAs/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Receptores Estrogênicos/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Sequências Repetidas Invertidas/genética , Sequências Repetidas Invertidas/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , MicroRNAs/genética , Proteínas Associadas à Matriz Nuclear/genética , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Estrogênicos/genética
13.
Nat Commun ; 11(1): 2834, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503981

RESUMO

Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by m5C at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote m5C induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing m5C-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-m5C axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Animais , Linhagem Celular Tumoral , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Resistencia a Medicamentos Antineoplásicos/genética , Técnicas de Silenciamento de Genes , Humanos , Metilação , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
In Vivo ; 34(3 Suppl): 1593-1596, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32503816

RESUMO

The Covid-19 pandemic is a world-wide crisis without an effective therapy. While most approaches to therapy are using repurposed drugs that were developed for other diseases, it is thought that targeting the biology of the SARS-CoV-2 virus, which causes Covid-19, can result in an effective therapeutic treatment. The coronavirus RNA cap structure is methylated by two viral methyltransferases that transfer methyl groups from S-adenosylmethionine (SAM). The proper methylation of the virus depends on the level of methionine in the host to form SAM. Herein, we propose to restrict methionine availability by treating the patient with oral recombinant methioninase, aiming to treat Covid-19. By restricting methionine we not only interdict viral replication, which depends on the viral RNA cap methyaltion, but also inhibit the proliferation of the infected cells, which have an increased requirement for methionine. Most importantly, the virally-induced T-cell- and macrophage-mediated cytokine storm, which seems to be a significant cause for Covid-19 deaths, can also be inhibited by restricting methionine, since T-cell and macrophrage activation greatly increases the methionine requirement for these cells. The evidence reviewed here suggests that oral recombinant methioninase could be a promising treatment for coronavirus patients.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Liases de Carbono-Enxofre/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Metionina/metabolismo , Pneumonia Viral/tratamento farmacológico , Capuzes de RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Viral/efeitos dos fármacos , Administração Oral , Antivirais/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/uso terapêutico , Betacoronavirus/fisiologia , Liases de Carbono-Enxofre/administração & dosagem , Ensaios Clínicos como Assunto , Infecções por Coronavirus/complicações , Infecções por Coronavirus/imunologia , Síndrome da Liberação de Citocina/prevenção & controle , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Metanálise como Assunto , Metilação/efeitos dos fármacos , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/imunologia , Pseudomonas putida/enzimologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , S-Adenosilmetionina/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Replicação Viral/efeitos dos fármacos
15.
Viruses ; 12(5)2020 05 10.
Artigo em Inglês | MEDLINE | ID: covidwho-209739

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causing the COVID-19 respiratory disease pandemic utilizes unique 2'-O-methyltransferase (2'-O-MTase) capping machinery to camouflage its RNA from innate immune recognition. The nsp16 catalytic subunit of the 2'-O-MTase is unusual in its requirement for a stimulatory subunit (nsp10) to catalyze the ribose 2'-O-methylation of the viral RNA cap. Here we provide a computational basis for drug repositioning or de novo drug development based on three differential traits of the intermolecular interactions of the SARS-CoV-2-specific nsp16/nsp10 heterodimer, namely: (1) the S-adenosyl-l-methionine-binding pocket of nsp16, (2) the unique "activating surface" between nsp16 and nsp10, and (3) the RNA-binding groove of nsp16. We employed ≈9000 U.S. Food and Drug Administration (FDA)-approved investigational and experimental drugs from the DrugBank repository for docking virtual screening. After molecular dynamics calculations of the stability of the binding modes of high-scoring nsp16/nsp10-drug complexes, we considered their pharmacological overlapping with functional modules of the virus-host interactome that is relevant to the viral lifecycle, and to the clinical features of COVID-19. Some of the predicted drugs (e.g., tegobuvir, sonidegib, siramesine, antrafenine, bemcentinib, itacitinib, or phthalocyanine) might be suitable for repurposing to pharmacologically reactivate innate immune restriction and antagonism of SARS-CoV-2 RNAs lacking 2'-O-methylation.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/virologia , Evasão da Resposta Imune/efeitos dos fármacos , Pneumonia Viral/virologia , Processamento Pós-Transcricional do RNA , RNA Viral/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Reposicionamento de Medicamentos , Humanos , Imunidade Inata , Metilação , Modelos Moleculares , Pandemias , Pneumonia Viral/imunologia
16.
Nucleic Acids Res ; 48(10): 5572-5590, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32365187

RESUMO

RNA decay is a key element of mitochondrial RNA metabolism. To date, the only well-documented machinery that plays a role in mtRNA decay in humans is the complex of polynucleotide phosphorylase (PNPase) and SUV3 helicase, forming the degradosome. REXO2, a homolog of prokaryotic oligoribonucleases present in humans both in mitochondria and the cytoplasm, was earlier shown to be crucial for maintaining mitochondrial homeostasis, but its function in mitochondria has not been fully elucidated. In the present study, we created a cellular model that enables the clear dissection of mitochondrial and non-mitochondrial functions of human REXO2. We identified a novel mitochondrial short RNA, referred to as ncH2, that massively accumulated upon REXO2 silencing. ncH2 degradation occurred independently of the mitochondrial degradosome, strongly supporting the hypothesis that ncH2 is a primary substrate of REXO2. We also investigated the global impact of REXO2 depletion on mtRNA, revealing the importance of the protein for maintaining low steady-state levels of mitochondrial antisense transcripts and double-stranded RNA. Our detailed biochemical and structural studies provide evidence of sequence specificity of the REXO2 oligoribonuclease. We postulate that REXO2 plays dual roles in human mitochondria, 'scavenging' nanoRNAs that are produced by the degradosome and clearing short RNAs that are generated by RNA processing.


Assuntos
Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/metabolismo , Exorribonucleases/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA de Cadeia Dupla/metabolismo , RNA Mitocondrial/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/fisiologia , Biomarcadores Tumorais/química , Biomarcadores Tumorais/fisiologia , Exorribonucleases/química , Exorribonucleases/fisiologia , Células HeLa , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Multimerização Proteica , Especificidade por Substrato
17.
Nucleic Acids Res ; 48(12): 6874-6888, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32427329

RESUMO

MicroRNAs (miRNAs) are predicted to regulate the expression of >60% of mammalian genes and play fundamental roles in most biological processes. Deregulation of miRNA expression is a hallmark of most cancers and further investigation of mechanisms controlling miRNA biogenesis is needed. The double stranded RNA-binding protein, NF90 has been shown to act as a competitor of Microprocessor for a limited number of primary miRNAs (pri-miRNAs). Here, we show that NF90 has a more widespread effect on pri-miRNA biogenesis than previously thought. Genome-wide approaches revealed that NF90 is associated with the stem region of 38 pri-miRNAs, in a manner that is largely exclusive of Microprocessor. Following loss of NF90, 22 NF90-bound pri-miRNAs showed increased abundance of mature miRNA products. NF90-targeted pri-miRNAs are highly stable, having a lower free energy and fewer mismatches compared to all pri-miRNAs. Mutations leading to less stable structures reduced NF90 binding while increasing pri-miRNA stability led to acquisition of NF90 association, as determined by RNA electrophoretic mobility shift assay (EMSA). NF90-bound and downregulated pri-miRNAs are embedded in introns of host genes and expression of several host genes is concomitantly reduced. These data suggest that NF90 controls the processing of a subset of highly stable, intronic miRNAs.


Assuntos
Sequências Repetidas Invertidas/genética , MicroRNAs/genética , Neoplasias/genética , Proteínas do Fator Nuclear 90/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano/genética , Humanos , MicroRNAs/biossíntese , Proteínas do Fator Nuclear 90/antagonistas & inibidores , Processamento Pós-Transcricional do RNA/genética
18.
J Plant Physiol ; 249: 153167, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32353606

RESUMO

Histone marks including histone modifications and histone variants may affect the processes of gene transcription and co-transcriptional RNA processing depending on their specific deposition patterns within genes. Here, we analyzed distribution patterns of rice histone marks and divided them into seven clusters according to their enrichment in promoter, transcription start site (TSS), and gene body regions. Expression levels of the genes in each cluster were explored to disclose the importance of histone marks in the processes of transcription. We show that: a) H3K4me3 and histone acetylation marks show locally different distributions at TSS, implying that they may play different roles in transcription initiation. b) H3K36me1 enriched at TSS has a negative effect on transcription. c) Genes with high level of expression were marked by H3K36me3 at both the TSS and body regions. In addition, we found that H3K4me2, H3K23ac, H3K4ac, and H2A.Z show exon-biased enrichment, suggesting they may be chromatin marks involved in co-transcriptional splicing. Finally, we identified histone marks that discriminate constitutive expression genes (CEGs) from tissue-specific expressed genes (TSEGs). Taken together, the analysis revealed distribution patterns of different histone marks in rice to infer their potential roles in transcription and RNA processing. The results lay foundation for further understanding the mechanism by which histone marks are involved in the regulation of these processes in plants.


Assuntos
Histonas/genética , Oryza/genética , Proteínas de Plantas/genética , RNA/metabolismo , Transcrição Genética , Histonas/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Processamento Pós-Transcricional do RNA
19.
Nucleic Acids Res ; 48(12): 6839-6854, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32449937

RESUMO

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.


Assuntos
Proteínas de Arabidopsis/genética , MicroRNAs/genética , RNA Helicases/genética , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genética , Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/genética , Exossomos/genética , Regulação da Expressão Gênica de Plantas/genética , Mutação/genética , Precursores de RNA/genética , Estabilidade de RNA/genética , Ribonuclease III/genética
20.
Viruses ; 12(5)2020 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-32397643

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causing the COVID-19 respiratory disease pandemic utilizes unique 2'-O-methyltransferase (2'-O-MTase) capping machinery to camouflage its RNA from innate immune recognition. The nsp16 catalytic subunit of the 2'-O-MTase is unusual in its requirement for a stimulatory subunit (nsp10) to catalyze the ribose 2'-O-methylation of the viral RNA cap. Here we provide a computational basis for drug repositioning or de novo drug development based on three differential traits of the intermolecular interactions of the SARS-CoV-2-specific nsp16/nsp10 heterodimer, namely: (1) the S-adenosyl-l-methionine-binding pocket of nsp16, (2) the unique "activating surface" between nsp16 and nsp10, and (3) the RNA-binding groove of nsp16. We employed ≈9000 U.S. Food and Drug Administration (FDA)-approved investigational and experimental drugs from the DrugBank repository for docking virtual screening. After molecular dynamics calculations of the stability of the binding modes of high-scoring nsp16/nsp10-drug complexes, we considered their pharmacological overlapping with functional modules of the virus-host interactome that is relevant to the viral lifecycle, and to the clinical features of COVID-19. Some of the predicted drugs (e.g., tegobuvir, sonidegib, siramesine, antrafenine, bemcentinib, itacitinib, or phthalocyanine) might be suitable for repurposing to pharmacologically reactivate innate immune restriction and antagonism of SARS-CoV-2 RNAs lacking 2'-O-methylation.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/virologia , Evasão da Resposta Imune/efeitos dos fármacos , Pneumonia Viral/virologia , Processamento Pós-Transcricional do RNA , RNA Viral/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Reposicionamento de Medicamentos , Humanos , Imunidade Inata , Metilação , Modelos Moleculares , Pandemias , Pneumonia Viral/imunologia
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