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1.
Mol Cell ; 81(16): 3339-3355.e8, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352206

RESUMO

Cancer cells selectively promote translation of specific oncogenic transcripts to facilitate cancer survival and progression, but the underlying mechanisms are poorly understood. Here, we find that N7-methylguanosine (m7G) tRNA modification and its methyltransferase complex components, METTL1 and WDR4, are significantly upregulated in intrahepatic cholangiocarcinoma (ICC) and associated with poor prognosis. We further reveal the critical role of METTL1/WDR4 in promoting ICC cell survival and progression using loss- and gain-of-function assays in vitro and in vivo. Mechanistically, m7G tRNA modification selectively regulates the translation of oncogenic transcripts, including cell-cycle and epidermal growth factor receptor (EGFR) pathway genes, in m7G-tRNA-decoded codon-frequency-dependent mechanisms. Moreover, using overexpression and knockout mouse models, we demonstrate the crucial oncogenic function of Mettl1-mediated m7G tRNA modification in promoting ICC tumorigenesis and progression in vivo. Our study uncovers the important physiological function and mechanism of METTL1-mediated m7G tRNA modification in the regulation of oncogenic mRNA translation and cancer progression.


Assuntos
Colangiocarcinoma/genética , Proteínas de Ligação ao GTP/genética , Metiltransferases/genética , Biossíntese de Proteínas , Animais , Carcinogênese/genética , Colangiocarcinoma/patologia , Progressão da Doença , Receptores ErbB/genética , Guanosina/análogos & derivados , Guanosina/genética , Humanos , Camundongos , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA de Transferência/genética
2.
Mol Cell ; 81(16): 3323-3338.e14, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352207

RESUMO

The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.


Assuntos
Carcinogênese/genética , Metiltransferases/genética , Neoplasias/genética , tRNA Metiltransferases/genética , Guanosina/análogos & derivados , Guanosina/genética , Humanos , Metilação , Neoplasias/patologia , Oncogenes/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA de Transferência/genética
3.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360765

RESUMO

Mitochondria, often referred to as the powerhouses of cells, are vital organelles that are present in almost all eukaryotic organisms, including humans. They are the key energy suppliers as the site of adenosine triphosphate production, and are involved in apoptosis, calcium homeostasis, and regulation of the innate immune response. Abnormalities occurring in mitochondria, such as mitochondrial DNA (mtDNA) mutations and disturbances at any stage of mitochondrial RNA (mtRNA) processing and translation, usually lead to severe mitochondrial diseases. A fundamental line of investigation is to understand the processes that occur in these organelles and their physiological consequences. Despite substantial progress that has been made in the field of mtRNA processing and its regulation, many unknowns and controversies remain. The present review discusses the current state of knowledge of RNA processing in human mitochondria and sheds some light on the unresolved issues.


Assuntos
Mitocôndrias/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Mitocôndrias/genética , RNA Mitocondrial/genética
4.
Int J Mol Sci ; 22(16)2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34445460

RESUMO

Yeast phenotypes associated with the lack of wobble uridine (U34) modifications in tRNA were shown to be modulated by an allelic variation of SSD1, a gene encoding an mRNA-binding protein. We demonstrate that phenotypes caused by the loss of Deg1-dependent tRNA pseudouridylation are similarly affected by SSD1 allelic status. Temperature sensitivity and protein aggregation are elevated in deg1 mutants and further increased in the presence of the ssd1-d allele, which encodes a truncated form of Ssd1. In addition, chronological lifespan is reduced in a deg1 ssd1-d mutant, and the negative genetic interactions of the U34 modifier genes ELP3 and URM1 with DEG1 are aggravated by ssd1-d. A loss of function mutation in SSD1, ELP3, and DEG1 induces pleiotropic and overlapping phenotypes, including sensitivity against target of rapamycin (TOR) inhibitor drug and cell wall stress by calcofluor white. Additivity in ssd1 deg1 double mutant phenotypes suggests independent roles of Ssd1 and tRNA modifications in TOR signaling and cell wall integrity. However, other tRNA modification defects cause growth and drug sensitivity phenotypes, which are not further intensified in tandem with ssd1-d. Thus, we observed a modification-specific rather than general effect of SSD1 status on phenotypic variation in tRNA modification mutants. Our results highlight how the cellular consequences of tRNA modification loss can be influenced by protein targeting specific mRNAs.


Assuntos
Transferases Intramoleculares/deficiência , Processamento Pós-Transcricional do RNA/genética , RNA Fúngico , RNA de Transferência , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Variação Biológica da População , Transferases Intramoleculares/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Mol Cell ; 81(16): 3243-3245, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34416137

RESUMO

Orellana et al. (2021) and Dai et al. (2021) demonstrate that increased m7G modification of a subset of tRNAs by the METTL1/WDR4 complex stabilizes these mRNAs against decay, increases translation efficiency, reduces ribosome pausing, is associated with poor survival in human cancers, and is directly transforming.


Assuntos
Neoplasias , Processamento Pós-Transcricional do RNA , Proteínas de Ligação ao GTP/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
6.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199774

RESUMO

Over a thousand nucleus-encoded mitochondrial proteins are imported from the cytoplasm; however, mitochondrial (mt) DNA encodes for a small number of critical proteins and the entire suite of mt:tRNAs responsible for translating these proteins. Mitochondrial RNase P (mtRNase P) is a three-protein complex responsible for cleaving and processing the 5'-end of mt:tRNAs. Mutations in any of the three proteins can cause mitochondrial disease, as well as mutations in mitochondrial DNA. Great strides have been made in understanding the enzymology of mtRNase P; however, how the loss of each protein causes mitochondrial dysfunction and abnormal mt:tRNA processing in vivo has not been examined in detail. Here, we used Drosophila genetics to selectively remove each member of the complex in order to assess their specific contributions to mt:tRNA cleavage. Using this powerful model, we find differential effects on cleavage depending on which complex member is lost and which mt:tRNA is being processed. These data revealed in vivo subtleties of mtRNase P function that could improve understanding of human diseases.


Assuntos
Mitocôndrias/enzimologia , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Ribonuclease P/metabolismo , Alelos , Animais , Drosophila melanogaster/genética , Mitocôndrias/patologia , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo
7.
Mol Cell ; 81(16): 3422-3439.e11, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34320405

RESUMO

Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). More than 1,800 miRNA loci are annotated in humans, but it remains largely unknown whether and at which sites pri-miRNAs are cleaved by DROSHA. Here, we performed in vitro processing on a full set of human pri-miRNAs (miRBase version 21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs on the basis of DROSHA dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing and unproductive cleavage events such as "nick" or "inverse" processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.


Assuntos
MicroRNAs/genética , Processamento Pós-Transcricional do RNA/genética , Ribonuclease III/genética , Fatores de Processamento de Serina-Arginina/genética , Sítios de Ligação/genética , Genoma Humano/genética , Células HEK293 , Humanos , Interferência de RNA
8.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298969

RESUMO

Cancer is a complex disease involving alterations of multiple processes, with both genetic and epigenetic features contributing as core factors to the disease. In recent years, it has become evident that non-coding RNAs (ncRNAs), an epigenetic factor, play a key role in the initiation and progression of cancer. MicroRNAs, the most studied non-coding RNAs subtype, are key controllers in a myriad of cellular processes, including proliferation, differentiation, and apoptosis. Furthermore, the expression of miRNAs is controlled, concomitantly, by other epigenetic factors, such as DNA methylation and histone modifications, resulting in aberrant patterns of expression upon the occurrence of cancer. In this sense, aberrant miRNA landscape evaluation has emerged as a promising strategy for cancer management. In this review, we have focused on the regulation (biogenesis, processing, and dysregulation) of miRNAs and their role as modulators of the epigenetic machinery. We have also highlighted their potential clinical value, such as validated diagnostic and prognostic biomarkers, and their relevant role as chromatin modifiers in cancer therapy.


Assuntos
Epigênese Genética , MicroRNAs/genética , Neoplasias/genética , RNA Neoplásico/genética , Pesquisa Médica Translacional , Biomarcadores Tumorais , Neoplasias da Mama/genética , Metilação de DNA , DNA de Neoplasias/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Terapia Genética , Código das Histonas , Humanos , Neoplasias Pulmonares/genética , MicroRNAs/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Neoplasias Ovarianas/genética , Prognóstico , Processamento Pós-Transcricional do RNA , RNA Neoplásico/biossíntese , Neoplasias Gástricas/genética
9.
Cell Death Dis ; 12(8): 732, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301919

RESUMO

Severe coronavirus disease 2019 (COVID-19) is characterized by symptoms of lymphopenia and multiorgan damage, but the underlying mechanisms remain unclear. To explore the function of N6-methyladenosine (m6A) modifications in COVID-19, we performed microarray analyses to comprehensively characterize the m6A epitranscriptome. The results revealed distinct global m6A profiles in severe and mild COVID-19 patients. Programmed cell death and inflammatory response were the major biological processes modulated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Further, RBM15, a major m6A methyltransferase, was significantly elevated and positively correlated with disease severity. Silencing RBM15 drastically reduced lymphocyte death in vitro. Knockdown of RBM15 remarkably suppressed the expression levels of multitarget genes related to programmed cell death and inflammatory response. This study shows that SARS-CoV-2 infection alters the m6A epitranscriptome of lymphocytes, particularly in the case of severe patients. RBM15 regulated host immune response to SARS-CoV-2 by elevating m6A modifications of multitarget genes. These findings indicate that RBM15 can serve as a target for the treatment of COVID-19.


Assuntos
Adenosina/análogos & derivados , COVID-19/genética , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Transcriptoma , Adenosina/metabolismo , COVID-19/patologia , Linhagem Celular Tumoral , Epigênese Genética , Feminino , Humanos , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Ligação a RNA/genética , Células THP-1
10.
Methods Mol Biol ; 2314: 513-531, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235668

RESUMO

Next-generation sequencing technologies facilitate the analysis of multiple important properties of transcriptomes in addition to gene expression levels. Here, we describe a method for mapping RNA 5' ends in Mycobacterium tuberculosis and Mycobacterium smegmatis, which allows the determination of transcription start sites (TSSs), comparative analysis of promoter usage under different conditions, and mapping of endoribonucleolytic cleavage sites. We describe in detail the procedures for constructing RNA sequencing libraries appropriate for RNA 5' end mapping using an Illumina sequencing platform, as well as bioinformatic procedures for data analysis.


Assuntos
Regiões 5' não Traduzidas/genética , Mycobacterium tuberculosis/genética , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , Análise de Sequência de RNA/métodos , Sítio de Iniciação de Transcrição , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Bacteriano/análise , Transcriptoma
11.
Int J Mol Sci ; 22(14)2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34299038

RESUMO

Ribosomal RNA is a major component of the ribosome. This RNA plays a crucial role in ribosome functioning by ensuring the formation of the peptide bond between amino acids and the accurate decoding of the genetic code. The rRNA carries many chemical modifications that participate in its maturation, the formation of the ribosome and its functioning. In this review, we present the different modifications and how they are deposited on the rRNA. We also describe the most recent results showing that the modified positions are not 100% modified, which creates a heterogeneous population of ribosomes. This gave rise to the concept of specialized ribosomes that we discuss. The knowledge accumulated in the yeast Saccharomyces cerevisiae is very helpful to better understand the role of rRNA modifications in humans, especially in ribosomopathies.


Assuntos
Modelos Biológicos , Processamento Pós-Transcricional do RNA , RNA Fúngico/genética , RNA Ribossômico/genética , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Humanos , Ribossomos/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
12.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209806

RESUMO

Pre-mRNA splicing is a key process in the regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, Nrl1 regulates splicing and expression of several genes and non-coding RNAs, and also suppresses the accumulation of R-loops. Here, we report analysis of interactions between Nrl1 and selected RNA-processing proteins and regulation of Nrl1 function by phosphorylation. Bacterial two-hybrid system (BACTH) assays revealed that the N-terminal region of Nrl1 is important for the interaction with ATP-dependent RNA helicase Mtl1 while the C-terminal region of Nrl1 is important for interactions with spliceosome components Ctr1, Ntr2, and Syf3. Consistent with this result, tandem affinity purification showed that Mtl1, but not Ctr1, Ntr2, or Syf3, co-purifies with the N-terminal region of Nrl1. Interestingly, mass-spectrometry analysis revealed that in addition to previously identified phosphorylation sites, Nrl1 is also phosphorylated on serines 86 and 112, and that Nrl1-TAP co-purifies with Cka1, the catalytic subunit of casein kinase 2. In vitro assay showed that Cka1 can phosphorylate bacterially expressed Nrl1 fragments. An analysis of non-phosphorylatable nrl1 mutants revealed defects in gene expression and splicing consistent with the notion that phosphorylation is an important regulator of Nrl1 function. Taken together, our results provide insights into two mechanisms that are involved in the regulation of the spliceosome-associated factor Nrl1, namely domain-specific interactions between Nrl1 and RNA-processing proteins and post-translational modification of Nrl1 by phosphorylation.


Assuntos
Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Caseína Quinase II/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Processamento Pós-Transcricional do RNA , Splicing de RNA , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/fisiologia , Spliceossomos/metabolismo , Técnicas do Sistema de Duplo-Híbrido
13.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201807

RESUMO

MicroRNAs (miRNAs) are small, non-coding RNAs that function as endogenous gene silencers. Soon after the discovery of miRNAs, a subset of brain-enriched and brain-specific miRNAs were identified and significant advancements were made in delineating miRNA function in brain development. However, understanding the molecular mechanisms that regulate miRNA biogenesis in normal and diseased brains has become a prevailing challenge. Besides transcriptional regulation of miRNA host genes, miRNA processing intermediates are subjected to multifaceted regulation by canonical miRNA processing enzymes, RNA binding proteins (RBPs) and epitranscriptomic modifications. Further still, miRNA activity can be regulated by the sponging activity of other non-coding RNA classes, namely circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs). Differential abundance of these factors in neuronal and glial lineages partly underlies the spatiotemporal expression and function of lineage-specific miRNAs. Here, we review the continuously evolving understanding of the regulation of neuronal and glial miRNA biogenesis at the transcriptional and posttranscriptional levels and the cooperativity of miRNA species in targeting key mRNAs to drive lineage-specific development. In addition, we review dysregulation of neuronal and glial miRNAs and the detrimental impacts which contribute to developmental brain disorders.


Assuntos
Encefalopatias/patologia , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Neuroglia/patologia , Neurônios/patologia , RNA Mensageiro/metabolismo , Animais , Encefalopatias/genética , Encefalopatias/metabolismo , Humanos , Neuroglia/metabolismo , Neurônios/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética
14.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206573

RESUMO

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the "spacer" sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3' end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5' end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5' end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5' end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA.


Assuntos
RNA Ribossômico 5,8S/genética , RNA Ribossômico 5,8S/metabolismo , DNA Espaçador Ribossômico , Endorribonucleases , Regulação Fúngica da Expressão Gênica , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , RNA Fúngico , RNA Ribossômico 5,8S/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência
15.
Nat Genet ; 53(8): 1156-1165, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34211177

RESUMO

The most prevalent post-transcriptional mRNA modification, N6-methyladenosine (m6A), plays diverse RNA-regulatory roles, but its genetic control in human tissues remains uncharted. Here we report 129 transcriptome-wide m6A profiles, covering 91 individuals and 4 tissues (brain, lung, muscle and heart) from GTEx/eGTEx. We integrate these with interindividual genetic and expression variation, revealing 8,843 tissue-specific and 469 tissue-shared m6A quantitative trait loci (QTLs), which are modestly enriched in, but mostly orthogonal to, expression QTLs. We integrate m6A QTLs with disease genetics, identifying 184 GWAS-colocalized m6A QTL, including brain m6A QTLs underlying neuroticism, depression, schizophrenia and anxiety; lung m6A QTLs underlying expiratory flow and asthma; and muscle/heart m6A QTLs underlying coronary artery disease. Last, we predict novel m6A regulators that show preferential binding in m6A QTLs, protein interactions with known m6A regulators and expression correlation with the m6A levels of their targets. Our results provide important insights and resources for understanding both cis and trans regulation of epitranscriptomic modifications, their interindividual variation and their roles in human disease.


Assuntos
Adenosina/análogos & derivados , Encéfalo/fisiologia , Pulmão/fisiologia , Músculo Esquelético/fisiologia , Locos de Características Quantitativas , Adenosina/genética , Adenosina/metabolismo , Estudo de Associação Genômica Ampla , Coração/fisiologia , Humanos , Metilação , Especificidade de Órgãos , Polimorfismo de Nucleotídeo Único , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Reprodutibilidade dos Testes
16.
Nat Methods ; 18(7): 711, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34239099
17.
Methods Mol Biol ; 2348: 71-90, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34160800

RESUMO

Mammalian genomes are pervasively transcribed and a small fraction of RNAs produced codify for proteins. The importance of noncoding RNAs for the maintenance of cell functions is well known (e.g., rRNAs, tRNAs), but only recently it was first demonstrated the involvement of microRNAs (miRNAs) in posttranscriptional regulation and then the activity of long noncoding RNAs (lncRNAs) in the regulation of miRNAs, DNA structure and protein function. LncRNAs have an expression more cell specific than other RNAs and basing on their subcellular localization exert different functions. In this book chapter we consider different protocols to evaluate the expression of lncRNAs at the single cell level using genome-wide approaches. We considered the skeletal muscle as example because the most abundant tissue in mammals involved in the regulation of metabolism and body movement. We firstly described how to isolate the smallest complete contractile system responsible for muscle metabolic and contractile traits (myofibers). We considered how to separate long and short RNAs to allow the sequencing of the full-length transcript using the SMART technique for the retrotranscription. Because of myofibers are multinucleated cells and because of it is better to perform single cell sequencing on fresh tissues we described the single-nucleus sequencing that can be applied to frozen tissues. The chapter concludes with a description of bioinformatics approaches to evaluate differential expression from single-cell or single-nucleus RNA sequencing.


Assuntos
Biologia Computacional/métodos , RNA Longo não Codificante/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Regulação da Expressão Gênica , Biblioteca Gênica , MicroRNAs/genética , Fibras Musculares Esqueléticas/metabolismo , Poliadenilação , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética
18.
Nat Commun ; 12(1): 3778, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145251

RESUMO

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.


Assuntos
Adenosina/análogos & derivados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Metiltransferases/metabolismo , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Adenosina/metabolismo , Animais , Linhagem Celular , Drosophila melanogaster , Células HeLa , Humanos , Metilação , Metiltransferases/genética , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética
19.
Genes Dev ; 35(13-14): 1005-1019, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34168039

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.


Assuntos
Coronavirus Humano OC43/fisiologia , Processamento Pós-Transcricional do RNA/genética , SARS-CoV-2/fisiologia , Replicação Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Proteínas do Nucleocapsídeo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacos
20.
Nucleic Acids Res ; 49(13): 7361-7374, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34181729

RESUMO

N6-methyladenosine (m6A) is a common modification on endogenous RNA transcripts in mammalian cells. Technologies to precisely modify the RNA m6A levels at specific transcriptomic loci empower interrogation of biological functions of epitranscriptomic modifications. Here, we developed a bidirectional dCasRx epitranscriptome editing platform composed of a nuclear-localized dCasRx conjugated with either a methyltransferase, METTL3, or a demethylase, ALKBH5, to manipulate methylation events at targeted m6A sites. Leveraging this platform, we specifically and efficiently edited m6A modifications at targeted sites, reflected in gene expression and cell proliferation. We employed the dCasRx epitranscriptomic editor system to elucidate the molecular function of m6A-binding proteins YTHDF paralogs (YTHDF1, YTHDF2 and YTHDF3), revealing that YTHDFs promote m6A-mediated mRNA degradation. Collectively, our dCasRx epitranscriptome perturbation platform permits site-specific m6A editing for delineating of functional roles of individual m6A modifications in the mammalian epitranscriptome.


Assuntos
Adenosina/análogos & derivados , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Metiltransferases/metabolismo , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Proteínas Associadas a CRISPR/genética , Proliferação de Células , Células Cultivadas , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Metiltransferases/genética , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcriptoma
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