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1.
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
2.
Nucleic Acids Res ; 49(5): e27, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33313824

RESUMO

Cellular RNAs are subject to a myriad of different chemical modifications that play important roles in controlling RNA expression and function. Dysregulation of certain RNA modifications, the so-called 'epitranscriptome', contributes to human disease. One limitation in studying the functional, physiological, and pathological roles of the epitranscriptome is the availability of methods for the precise mapping of individual RNA modifications throughout the transcriptome. 3-Methylcytidine (m3C) modification of certain tRNAs is well established and was also recently detected in mRNA. However, methods for the specific mapping of m3C throughout the transcriptome are lacking. Here, we developed a m3C-specific technique, Hydrazine-Aniline Cleavage sequencing (HAC-seq), to profile the m3C methylome at single-nucleotide resolution. We applied HAC-seq to analyze ribosomal RNA (rRNA)-depleted total RNAs in human cells. We found that tRNAs are the predominant m3C-modified RNA species, with 17 m3C modification sites on 11 cytoplasmic and 2 mitochondrial tRNA isoacceptors in MCF7 cells. We found no evidence for m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNAs in these cells. HAC-seq provides a novel method for the unbiased, transcriptome-wide identification of m3C RNA modification at single-nucleotide resolution, and could be widely applied to reveal the m3C methylome in different cells and tissues.


Assuntos
Citidina/análogos & derivados , RNA de Transferência/química , Análise de Sequência de RNA/métodos , Compostos de Anilina/química , Citidina/análise , Citidina/metabolismo , Humanos , Hidrazinas/química , Células MCF-7 , RNA de Transferência/metabolismo , Transcriptoma
3.
ACS Cent Sci ; 6(12): 2196-2208, 2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33376781

RESUMO

The fates of RNA species in a cell are controlled by ribonucleases, which degrade them by exploiting the universal structural 2'-OH group. This phenomenon plays a key role in numerous transformative technologies, for example, RNA interference and CRISPR/Cas13-based RNA editing systems. These approaches, however, are genetic or oligomer-based and so have inherent limitations. This has led to interest in the development of small molecules capable of degrading nucleic acids in a targeted manner. Here we describe click-degraders, small molecules that can be covalently attached to RNA species through click-chemistry and can degrade them, that are akin to ribonucleases. By using these molecules, we have developed the meCLICK-Seq (methylation CLICK-degradation Sequencing) a method to identify RNA modification substrates with high resolution at intronic and intergenic regions. The method hijacks RNA methyltransferase activity to introduce an alkyne, instead of a methyl, moiety on RNA. Subsequent copper(I)-catalyzed azide-alkyne cycloaddition reaction with the click-degrader leads to RNA cleavage and degradation exploiting a mechanism used by endogenous ribonucleases. Focusing on N6-methyladenosine (m6A), meCLICK-Seq identifies methylated transcripts, determines RNA methylase specificity, and reliably maps modification sites in intronic and intergenic regions. Importantly, we show that METTL16 deposits m6A to intronic polyadenylation (IPA) sites, which suggests a potential role for METTL16 in IPA and, in turn, splicing. Unlike other methods, the readout of meCLICK-Seq is depletion, not enrichment, of modified RNA species, which allows a comprehensive and dynamic study of RNA modifications throughout the transcriptome, including regions of low abundance. The click-degraders are highly modular and so may be exploited to study any RNA modification and design new technologies that rely on RNA degradation.

6.
Mol Cell ; 75(3): 620-630.e9, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31279659

RESUMO

mRNA modifications play important roles in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5' ends of mRNAs. Furthermore, PCIF1 catalyzes only 5' m6Am methylation of capped mRNAs but not internal m6A methylation in vitro and in vivo. To study the biological role of m6Am, we developed a robust methodology (m6Am-Exo-Seq) to map its transcriptome-wide distribution, which revealed no global crosstalk between m6Am and m6A under assayed conditions, suggesting that m6Am is functionally distinct from m6A. Importantly, we find that m6Am does not alter mRNA transcription or stability but negatively impacts cap-dependent translation of methylated mRNAs. Together, we identify the only human mRNA m6Am methyltransferase and demonstrate a mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Nucleares/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Transcrição Genética , Adenosina/genética , Regulação da Expressão Gênica/genética , Humanos , Metilação , Metiltransferases/genética , Fosforilação , Transcriptoma/genética
7.
Cell ; 162(2): 237-238, 2015 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-26186182

RESUMO

Throughout development, proliferative progenitors lose their mitotic potential, exit the cell cycle, and differentiate. In this issue, Ruijtenberg and van den Heuvel identify an important lineage-specific role for a SWI/SNF chromatin-remodeling complex that collaborates with core cell-cycle regulators to promote cell-cycle exit and terminal muscle cell differentiation.


Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/crescimento & desenvolvimento , Músculos/citologia , Animais
8.
Cell ; 161(4): 868-78, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25936839

RESUMO

In mammalian cells, DNA methylation on the fifth position of cytosine (5mC) plays an important role as an epigenetic mark. However, DNA methylation was considered to be absent in C. elegans because of the lack of detectable 5mC, as well as homologs of the cytosine DNA methyltransferases. Here, using multiple approaches, we demonstrate the presence of adenine N(6)-methylation (6mA) in C. elegans DNA. We further demonstrate that this modification increases trans-generationally in a paradigm of epigenetic inheritance. Importantly, we identify a DNA demethylase, NMAD-1, and a potential DNA methyltransferase, DAMT-1, which regulate 6mA levels and crosstalk between methylations of histone H3K4 and adenines and control the epigenetic inheritance of phenotypes associated with the loss of the H3K4me2 demethylase spr-5. Together, these data identify a DNA modification in C. elegans and raise the exciting possibility that 6mA may be a carrier of heritable epigenetic information in eukaryotes.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Metilação de DNA , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Adenina/metabolismo , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Fertilidade , Histonas/metabolismo , Mutação , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Filogenia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética
9.
Mol Cell Biol ; 29(10): 2889-98, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19289503

RESUMO

In Saccharomyces cerevisiae, transcriptional silencing occurs at the cryptic mating-type loci (HML and HMR), telomeres, and ribosomal DNA (rDNA; RDN1). Silencing in the rDNA is unusual in that polymerase II (Pol II) promoters within RDN1 are repressed by Sir2 but not Sir3 or Sir4. rDNA silencing unidirectionally spreads leftward, but the mechanism of limiting its spreading is unclear. We searched for silencing barriers flanking the left end of RDN1 by using an established assay for detecting barriers to HMR silencing. Unexpectedly, the unique sequence immediately adjacent to RDN1, which overlaps a prominent cohesin binding site (CARL2), did not have appreciable barrier activity. Instead, a fragment located 2.4 kb to the left, containing a tRNA(Gln) gene and the Ty1 long terminal repeat, had robust barrier activity. The barrier activity was dependent on Pol III transcription of tRNA(Gln), the cohesin protein Smc1, and the SAS1 and Gcn5 histone acetyltransferases. The location of the barrier correlates with the detectable limit of rDNA silencing when SIR2 is overexpressed, where it blocks the spreading of rDNA heterochromatin. We propose a model in which normal Sir2 activity results in termination of silencing near the physical rDNA boundary, while tRNA(Gln) blocks silencing from spreading too far when nucleolar Sir2 pools become elevated.


Assuntos
DNA Ribossômico/genética , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Heterocromatina/metabolismo , Histona Desacetilases/metabolismo , Saccharomyces cerevisiae , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuínas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Ribossômico/metabolismo , Genoma Fúngico , Histona Acetiltransferases/metabolismo , Histona Desacetilases/genética , Análise em Microsséries , RNA Polimerase III/metabolismo , RNA de Transferência de Glutamina/genética , RNA de Transferência de Glutamina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Sirtuína 2 , Sirtuínas/genética
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