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1.
Cell ; 175(7): 1872-1886.e24, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30449621

RESUMO

Generation of the "epitranscriptome" through post-transcriptional ribonucleoside modification embeds a layer of regulatory complexity into RNA structure and function. Here, we describe N4-acetylcytidine (ac4C) as an mRNA modification that is catalyzed by the acetyltransferase NAT10. Transcriptome-wide mapping of ac4C revealed discretely acetylated regions that were enriched within coding sequences. Ablation of NAT10 reduced ac4C detection at the mapped mRNA sites and was globally associated with target mRNA downregulation. Analysis of mRNA half-lives revealed a NAT10-dependent increase in stability in the cohort of acetylated mRNAs. mRNA acetylation was further demonstrated to enhance substrate translation in vitro and in vivo. Codon content analysis within ac4C peaks uncovered a biased representation of cytidine within wobble sites that was empirically determined to influence mRNA decoding efficiency. These findings expand the repertoire of mRNA modifications to include an acetylated residue and establish a role for ac4C in the regulation of mRNA translation.


Assuntos
Citidina/análogos & derivados , Acetiltransferase N-Terminal E/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Acetilação , Citidina/genética , Citidina/metabolismo , Células HeLa , Humanos , Acetiltransferase N-Terminal E/genética , Acetiltransferases N-Terminal , RNA Mensageiro/genética
2.
EMBO J ; 35(3): 335-55, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26711177

RESUMO

Intragenic 5-methylcytosine and CTCF mediate opposing effects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF-binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF-mediated alternative splicing through conversion of 5-methylcytosine to its oxidation derivatives. 5-hydroxymethylcytosine and 5-carboxylcytosine are enriched at an intragenic CTCF-binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5-methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5-hydroxymethylcytosine and 5-methylcytosine at downstream CTCF-binding sites is a general feature of alternative splicing in naïve and activated CD4(+) T cells. These findings significantly expand our current concept of the pre-mRNA "splicing code" to include dynamic intragenic DNA methylation catalyzed by the TET proteins.


Assuntos
5-Metilcitosina/metabolismo , Processamento Alternativo , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Fator de Ligação a CCCTC , Linhagem Celular , Dioxigenases , Humanos , Oxigenases de Função Mista , Oxirredução
3.
Nucleic Acids Res ; 45(22): 12780-12797, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29244186

RESUMO

Actively transcribed genes adopt a unique chromatin environment with characteristic patterns of enrichment. Within gene bodies, H3K36me3 and cytosine DNA methylation are elevated at exons of spliced genes and have been implicated in the regulation of pre-mRNA splicing. H3K36me3 is further responsive to splicing, wherein splicing inhibition led to a redistribution and general reduction over gene bodies. In contrast, little is known of the mechanisms supporting elevated DNA methylation at actively spliced genic locations. Recent evidence associating the de novo DNA methyltransferase Dnmt3b with H3K36me3-rich chromatin raises the possibility that genic DNA methylation is influenced by splicing-associated H3K36me3. Here, we report the generation of an isogenic resource to test the direct impact of splicing on chromatin. A panel of minigenes of varying splicing potential were integrated into a single FRT site for inducible expression. Profiling of H3K36me3 confirmed the established relationship to splicing, wherein levels were directly correlated with splicing efficiency. In contrast, DNA methylation was equivalently detected across the minigene panel, irrespective of splicing and H3K36me3 status. In addition to revealing a degree of independence between genic H3K36me3 and DNA methylation, these findings highlight the generated minigene panel as a flexible platform for the query of splicing-dependent chromatin modifications.


Assuntos
Metilação de DNA , Éxons/genética , Precursores de RNA/genética , Splicing de RNA , Animais , Cromatina/genética , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Camundongos , Modelos Genéticos , DNA Metiltransferase 3B
4.
iScience ; 19: 326-339, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31404833

RESUMO

The mechanisms supporting dynamic regulation of CTCF-binding sites remain poorly understood. Here we describe the TET-catalyzed 5-methylcytosine derivative, 5-carboxylcytosine (5caC), as a factor driving new CTCF binding within genomic DNA. Through a combination of in vivo and in vitro approaches, we reveal that 5caC generally strengthens CTCF association with DNA and facilitates binding to suboptimal sequences. Dramatically, profiling of CTCF binding in a cellular model that accumulates genomic 5caC identified ~13,000 new CTCF sites. The new sites were enriched for overlapping 5caC and were marked by an overall reduction in CTCF motif strength. As CTCF has multiple roles in gene expression, these findings have wide-reaching implications and point to induced 5caC as a potential mechanism to achieve differential CTCF binding in cells.

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