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1.
RNA ; 30(5): 548-559, 2024 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-38531647

RESUMEN

N 1-methyl adenosine (m1A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m1A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m1A. In this study, we introduce a reduction-based m1A sequencing (red-m1A-seq). We report that NaBH4 reduction of m1A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1A to m6A to provide good controls, allowing the detection of m1A with higher sensitivity and accuracy. We applied red-m1A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m1A sites, but also new m1A sites in mt-tRNAAsn-GTT and 5.8S rRNA.


Asunto(s)
ARN de Transferencia , ARN , Humanos , Metilación , ARN de Transferencia/química , ARN/genética , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismo , Metiltransferasas/metabolismo , ARN Mensajero/genética
2.
Nucleic Acids Res ; 52(8): 4257-4275, 2024 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-38366571

RESUMEN

Complex biological processes are regulated by both genetic and epigenetic programs. One class of epigenetic modifications is methylation. Evolutionarily conserved methyl-CpG-binding domain (MBD)-containing proteins are known as readers of DNA methylation. MBD5 is linked to multiple human diseases but its mechanism of action remains unclear. Here we report that the zebrafish Mbd5 does not bind to methylated DNA; but rather, it directly binds to 5-methylcytosine (m5C)-modified mRNAs and regulates embryonic development, erythrocyte differentiation, iron metabolism, and behavior. We further show that Mbd5 facilitates removal of the monoubiquitin mark at histone H2A-K119 through an interaction with the Polycomb repressive deubiquitinase (PR-DUB) complex in vivo. The direct target genes of Mbd5 are enriched with both RNA m5C and H2A-K119 ubiquitylation signals. Together, we propose that zebrafish MBD5 is an RNA m5C reader that potentially links RNA methylation to histone modification and in turn transcription regulation in vivo.


Asunto(s)
5-Metilcitosina , Histonas , Ubiquitinación , Proteínas de Pez Cebra , Pez Cebra , Animales , Histonas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , 5-Metilcitosina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Metilación de ADN , Desarrollo Embrionario/genética , Epigénesis Genética
3.
Nat Biotechnol ; 2024 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-38238480

RESUMEN

RNA fate and function are affected by their structures and interactomes. However, how RNA and RNA-binding proteins (RBPs) assemble into higher-order structures and how RNA molecules may interact with each other to facilitate functions remain largely unknown. Here we present KARR-seq, which uses N3-kethoxal labeling and multifunctional chemical crosslinkers to covalently trap and determine RNA-RNA interactions and higher-order RNA structures inside cells, independent of local protein binding to RNA. KARR-seq depicts higher-order RNA structure and detects widespread intermolecular RNA-RNA interactions with high sensitivity and accuracy. Using KARR-seq, we show that translation represses mRNA compaction under native and stress conditions. We determined the higher-order RNA structures of respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV) and identified RNA-RNA interactions between the viruses and the host RNAs that potentially regulate viral replication.

4.
Nat Methods ; 21(2): 247-258, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38200227

RESUMEN

RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA targets are still lacking, especially when the interaction is dynamic or samples are limited. Here we present an assay of reverse transcription-based RBP binding site sequencing (ARTR-seq), which relies on in situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet crosslinking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic RNA binding by RBPs over a short period of time, as demonstrated by the profiling of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 minutes.


Asunto(s)
ARN , Transcripción Reversa , ARN/genética , ARN/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Sitios de Unión/genética , Unión Proteica
5.
J Clin Invest ; 133(24)2023 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-38099498

RESUMEN

Activation of TGF-ß signaling serves as an extrinsic resistance mechanism that limits the potential for radiotherapy. Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) antagonizes TGF-ß signaling and is implicated in cancer progression. However, the molecular mechanisms of BAMBI regulation in immune cells and its impact on antitumor immunity after radiation have not been established. Here, we show that ionizing radiation (IR) specifically reduces BAMBI expression in immunosuppressive myeloid-derived suppressor cells (MDSCs) in both murine models and humans. Mechanistically, YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2) directly binds and degrades Bambi transcripts in an N6-methyladenosine-dependent (m6A-dependent) manner, and this relies on NF-κB signaling. BAMBI suppresses the tumor-infiltrating capacity and suppression function of MDSCs via inhibiting TGF-ß signaling. Adeno-associated viral delivery of Bambi (AAV-Bambi) to the tumor microenvironment boosts the antitumor effects of radiotherapy and radioimmunotherapy combinations. Intriguingly, combination of AAV-Bambi and IR not only improves local tumor control, but also suppresses distant metastasis, further supporting its clinical translation potential. Our findings uncover a surprising role of BAMBI in myeloid cells, unveiling a potential therapeutic strategy for overcoming extrinsic radioresistance.


Asunto(s)
Neoplasias , Factor de Crecimiento Transformador beta , Animales , Humanos , Ratones , Proteínas de la Membrana/metabolismo , Neoplasias/genética , Neoplasias/radioterapia , Proteínas de Unión al ARN/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral
6.
Nat Cell Biol ; 25(9): 1359-1368, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37640841

RESUMEN

N6-methyladenosine (m6A) methylation can be deposited on chromatin-associated RNAs (caRNAs) by the RNA methyltransferase complex (MTC) to regulate chromatin state and transcription. However, the mechanism by which MTC is recruited to distinct genomic loci remains elusive. Here we identify RBFOX2, a well-studied RNA-binding protein, as a chromatin factor that preferentially recognizes m6A on caRNAs. RBFOX2 can recruit RBM15, an MTC component, to facilitate methylation of promoter-associated RNAs. RBM15 also physically interacts with YTHDC1 and recruits polycomb repressive complex 2 (PRC2) to the RBFOX2-bound loci for chromatin silencing and transcription suppression. Furthermore, we found that this RBFOX2/m6A/RBM15/YTHDC1/PRC2 axis plays a critical role in myeloid leukaemia. Downregulation of RBFOX2 notably inhibits survival/proliferation of acute myeloid leukaemia cells and promotes their myeloid differentiation. RBFOX2 is also required for self-renewal of leukaemia stem/initiation cells and acute myeloid leukaemia maintenance. Our study presents a pathway of m6A MTC recruitment and m6A deposition on caRNAs, resulting in locus-selective chromatin regulation, which has potential therapeutic implications in leukaemia.


Asunto(s)
Leucemia Mieloide , Humanos , Diferenciación Celular/genética , Cromatina/genética , ARN , Factores de Empalme de ARN/genética , Proteínas Represoras/genética
7.
EMBO J ; 42(15): e113126, 2023 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-37345898

RESUMEN

N6 -methyladenosine (m6 A) in messenger RNA (mRNA) regulates immune cells in homeostasis and in response to infection and inflammation. The function of the m6 A reader YTHDF2 in the tumor microenvironment (TME) in these contexts has not been explored. We discovered that the loss of YTHDF2 in regulatory T (Treg) cells reduces tumor growth in mice. Deletion of Ythdf2 in Tregs does not affect peripheral immune homeostasis but leads to increased apoptosis and impaired suppressive function of Treg cells in the TME. Elevated tumor necrosis factor (TNF) signaling in the TME promotes YTHDF2 expression, which in turn regulates NF-κB signaling by accelerating the degradation of m6 A-modified transcripts that encode NF-κB-negative regulators. This TME-specific regulation of Treg by YTHDF2 points to YTHDF2 as a potential target for anti-cancer immunotherapy, where intratumoral Treg cells can be targeted to enhance anti-tumor immune response while avoiding Treg cells in the periphery to minimize undesired inflammations.


Asunto(s)
FN-kappa B , Neoplasias , Ratones , Animales , FN-kappa B/genética , Neoplasias/genética , Transducción de Señal , Inmunoterapia , Inflamación , Microambiente Tumoral
8.
Nat Neurosci ; 26(8): 1328-1338, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37365312

RESUMEN

Repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we show that N6-methyladenosine (m6A), the most prevalent internal mRNA modification, is downregulated in C9ORF72-ALS/FTD patient-derived induced pluripotent stem cell (iPSC)-differentiated neurons and postmortem brain tissues. The global m6A hypomethylation leads to transcriptome-wide mRNA stabilization and upregulated gene expression, particularly for genes involved in synaptic activity and neuronal function. Moreover, the m6A modification in the C9ORF72 intron sequence upstream of the expanded repeats enhances RNA decay via the nuclear reader YTHDC1, and the antisense RNA repeats can also be regulated through m6A modification. The m6A reduction increases the accumulation of repeat RNAs and the encoded poly-dipeptides, contributing to disease pathogenesis. We further demonstrate that, by elevating m6A methylation, we could significantly reduce repeat RNA levels from both strands and the derived poly-dipeptides, rescue global mRNA homeostasis and improve survival of C9ORF72-ALS/FTD patient iPSC-derived neurons.


Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Dipéptidos/genética , Dipéptidos/metabolismo , Expansión de las Repeticiones de ADN/genética , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , ARN , ARN Mensajero
9.
Cancer Cell ; 41(7): 1294-1308.e8, 2023 07 10.
Artículo en Inglés | MEDLINE | ID: mdl-37236197

RESUMEN

RNA N6-methyladenosine (m6A) modification is implicated in cancer progression. However, the impact of m6A on the antitumor effects of radiotherapy and the related mechanisms are unknown. Here we show that ionizing radiation (IR) induces immunosuppressive myeloid-derived suppressor cell (MDSC) expansion and YTHDF2 expression in both murine models and humans. Following IR, loss of Ythdf2 in myeloid cells augments antitumor immunity and overcomes tumor radioresistance by altering MDSC differentiation and inhibiting MDSC infiltration and suppressive function. The remodeling of the landscape of MDSC populations by local IR is reversed by Ythdf2 deficiency. IR-induced YTHDF2 expression relies on NF-κB signaling; YTHDF2 in turn leads to NF-κB activation by directly binding and degrading transcripts encoding negative regulators of NF-κB signaling, resulting in an IR-YTHDF2-NF-κB circuit. Pharmacological inhibition of YTHDF2 overcomes MDSC-induced immunosuppression and improves combined IR and/or anti-PD-L1 treatment. Thus, YTHDF2 is a promising target to improve radiotherapy (RT) and RT/immunotherapy combinations.


Asunto(s)
FN-kappa B , Neoplasias , Animales , Humanos , Ratones , Regulación de la Expresión Génica , Células Mieloides/metabolismo , Neoplasias/genética , Neoplasias/radioterapia , FN-kappa B/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal
10.
Protein Cell ; 14(9): 683-697, 2023 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-37030005

RESUMEN

METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m6A methyltransferase complex (MTC) that installs m6A. Surprisingly, depletion of METTL3 or METTL14 displayed distinct effects on stemness maintenance of mouse embryonic stem cell (mESC). While comparable global hypo-methylation in RNA m6A was observed in Mettl3 or Mettl14 knockout mESCs, respectively. Mettl14 knockout led to a globally decreased nascent RNA synthesis, whereas Mettl3 depletion resulted in transcription upregulation, suggesting that METTL14 might possess an m6A-independent role in gene regulation. We found that METTL14 colocalizes with the repressive H3K27me3 modification. Mechanistically, METTL14, but not METTL3, binds H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3. Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression. The effects of METTL14 on regulation of H3K27me3 is essential for the transition from self-renewal to differentiation of mESCs. This work reveals a regulatory mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m6A, and critically impacts transcriptional regulation, stemness maintenance, and differentiation of mESCs.


Asunto(s)
Cromatina , Histonas , Animales , Ratones , Metilación , Histonas/metabolismo , ARN Mensajero/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , ARN/metabolismo
11.
Science ; 379(6633): 677-682, 2023 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-36705538

RESUMEN

N6-methyladenosine (m6A) is the most abundant messenger RNA (mRNA) modification and plays crucial roles in diverse physiological processes. Using a massively parallel assay for m6A (MPm6A), we discover that m6A specificity is globally regulated by suppressors that prevent m6A deposition in unmethylated transcriptome regions. We identify exon junction complexes (EJCs) as m6A suppressors that protect exon junction-proximal RNA within coding sequences from methylation and regulate mRNA stability through m6A suppression. EJC suppression of m6A underlies multiple global characteristics of mRNA m6A specificity, with the local range of EJC protection sufficient to suppress m6A deposition in average-length internal exons but not in long internal and terminal exons. EJC-suppressed methylation sites colocalize with EJC-suppressed splice sites, which suggests that exon architecture broadly determines local mRNA accessibility to regulatory complexes.


Asunto(s)
Exones , Regulación de la Expresión Génica , Empalme del ARN , ARN Mensajero , ARN Mensajero/genética , ARN Mensajero/metabolismo , Humanos , Animales
12.
Transl Stroke Res ; 14(6): 806-810, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-35737185

RESUMEN

Methylation of adenosine at N1 position yields N1-methyladenosine (m1A), which is an epitranscriptomic modification that regulates mRNA metabolism. Recent studies showed that altered m1A methylation promotes acute and chronic neurological diseases. We currently evaluated the effect of focal ischemia on cerebral m1A methylome and its machinery. Adult male C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and the peri-infarct cortex was analyzed at 12 h and 24 h of reperfusion. The bulk abundance of m1A was measured by mass spectrometry and dot blot, and transcriptome-wide m1A alterations were profiled using antibody-independent m1A-quant-seq. Expression of the m1A writers and erasers was estimated by real-time PCR. Ischemia significantly decreased m1A levels and concomitantly upregulated m1A demethylase alkB homolog 3 at 24 h of reperfusion compared to sham. Transcriptome-wide profiling showed differential m1A methylation at 14 sites (8 were hypo- and 6 were hypermethylated). Many of those are located in the 3'-UTRs of unannotated transcripts proximal to the genes involved in regulating protein complex assembly, circadian rhythms, chromatin remodeling, and chromosome organization. Using several different approaches, we show for the first time that m1A epitranscriptomic modification in RNA is highly sensitive to cerebral ischemia.


Asunto(s)
Accidente Cerebrovascular Isquémico , Ratones , Animales , Masculino , Ratones Endogámicos C57BL , Metilación , Transcriptoma , Isquemia
13.
Nat Biotechnol ; 41(3): 344-354, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36302989

RESUMEN

Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different 'writer' proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.


Asunto(s)
Seudouridina , Procesamiento Postranscripcional del ARN , ARN Mensajero , Animales , Humanos , Ratones , Composición de Base , Mamíferos/genética , Seudouridina/genética , Seudouridina/metabolismo , ARN/genética , ARN/metabolismo , Procesamiento Postranscripcional del ARN/genética , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Sulfitos
14.
Science ; 376(6596): 968-973, 2022 05 27.
Artículo en Inglés | MEDLINE | ID: mdl-35511947

RESUMEN

N6-methyladenosine (m6A) is the most abundant internal modification on mammalian messenger RNA. It is installed by a writer complex and can be reversed by erasers such as the fat mass and obesity-associated protein FTO. Despite extensive research, the primary physiological substrates of FTO in mammalian tissues and development remain elusive. Here, we show that FTO mediates m6A demethylation of long-interspersed element-1 (LINE1) RNA in mouse embryonic stem cells (mESCs), regulating LINE1 RNA abundance and the local chromatin state, which in turn modulates the transcription of LINE1-containing genes. FTO-mediated LINE1 RNA m6A demethylation also plays regulatory roles in shaping chromatin state and gene expression during mouse oocyte and embryonic development. Our results suggest broad effects of LINE1 RNA m6A demethylation by FTO in mammals.


Asunto(s)
Adenosina/análogos & derivados , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Cromatina , Regulación del Desarrollo de la Expresión Génica , Elementos de Nucleótido Esparcido Largo , Células Madre Embrionarias de Ratones , Oocitos , ARN Mensajero , Adenosina/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Cromatina/metabolismo , Desmetilación , Elementos de Nucleótido Esparcido Largo/genética , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Oocitos/crecimiento & desarrollo , ARN Mensajero/genética , ARN Mensajero/metabolismo
16.
Protein Cell ; 12(6): 455-474, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33886094

RESUMEN

N6-methyladenosine (m6A) on chromosome-associated regulatory RNAs (carRNAs), including repeat RNAs, plays important roles in tuning the chromatin state and transcription, but the intrinsic mechanism remains unclear. Here, we report that YTHDC1 plays indispensable roles in the self-renewal and differentiation potency of mouse embryonic stem cells (ESCs), which highly depends on the m6A-binding ability. Ythdc1 is required for sufficient rRNA synthesis and repression of the 2-cell (2C) transcriptional program in ESCs, which recapitulates the transcriptome regulation by the LINE1 scaffold. Detailed analyses revealed that YTHDC1 recognizes m6A on LINE1 RNAs in the nucleus and regulates the formation of the LINE1-NCL partnership and the chromatin recruitment of KAP1. Moreover, the establishment of H3K9me3 on 2C-related retrotransposons is interrupted in Ythdc1-depleted ESCs and inner cell mass (ICM) cells, which consequently increases the transcriptional activities. Our study reveals a role of m6A in regulating the RNA scaffold, providing a new model for the RNA-chromatin cross-talk.


Asunto(s)
Adenosina/metabolismo , Células Madre Embrionarias de Ratones , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Transcripción Genética
18.
Mol Cell ; 78(3): 382-395.e8, 2020 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-32183942

RESUMEN

N6-Methyldeoxyadenosine (6mA) has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA (gDNA). However, the biological functions of 6mA in mammals have yet to be adequately explored, largely due to its low abundance in most mammalian genomes. Here, we report that mammalian mitochondrial DNA (mtDNA) is enriched for 6mA. The level of 6mA in HepG2 mtDNA is at least 1,300-fold higher than that in gDNA under normal growth conditions, corresponding to approximately four 6mA modifications on each mtDNA molecule. METTL4, a putative mammalian methyltransferase, can mediate mtDNA 6mA methylation, which contributes to attenuated mtDNA transcription and a reduced mtDNA copy number. Mechanistically, the presence of 6mA could repress DNA binding and bending by mitochondrial transcription factor (TFAM). Under hypoxia, the 6mA level in mtDNA could be further elevated, suggesting regulatory roles for 6mA in mitochondrial stress response. Our study reveals DNA 6mA as a regulatory mark in mammalian mtDNA.


Asunto(s)
ADN Mitocondrial/metabolismo , Desoxiadenosinas/metabolismo , Metiltransferasas/metabolismo , Animales , Metilación de ADN , ADN Mitocondrial/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxiadenosinas/genética , Regulación de la Expresión Génica , Células Hep G2 , Humanos , Hipoxia/genética , Metiltransferasas/genética , Ratones Endogámicos C57BL , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
19.
J Am Chem Soc ; 142(10): 4539-4543, 2020 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-32077696

RESUMEN

DNA 5-methylcytosine (5mC)-specific mapping has been hampered by severe DNA degradation and the presence of 5-hydroxymethylcytosine (5hmC) using the conventional bisulfite sequencing approach. Here, we present a 5mC-specific whole-genome amplification method (5mC-WGA), with which we achieved 5mC retention during DNA amplification from limited input down to 10 pg scale with limited interference from 5hmC signals, providing DNA 5mC methylome with high reproducibility and accuracy.


Asunto(s)
5-Metilcitosina/química , ADN/análisis , Técnicas de Amplificación de Ácido Nucleico/métodos , Análisis de Secuencia de ADN/métodos , Animales , ADN/química , Metilación de ADN , Humanos , Ratones , Sulfitos/química , Secuenciación Completa del Genoma
20.
Science ; 367(6477): 580-586, 2020 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-31949099

RESUMEN

N 6-methyladenosine (m6A) regulates stability and translation of messenger RNA (mRNA) in various biological processes. In this work, we show that knockout of the m6A writer Mettl3 or the nuclear reader Ythdc1 in mouse embryonic stem cells increases chromatin accessibility and activates transcription in an m6A-dependent manner. We found that METTL3 deposits m6A modifications on chromosome-associated regulatory RNAs (carRNAs), including promoter-associated RNAs, enhancer RNAs, and repeat RNAs. YTHDC1 facilitates the decay of a subset of these m6A-modified RNAs, especially elements of the long interspersed element-1 family, through the nuclear exosome targeting-mediated nuclear degradation. Reducing m6A methylation by METTL3 depletion or site-specific m6A demethylation of selected carRNAs elevates the levels of carRNAs and promotes open chromatin state and downstream transcription. Collectively, our results reveal that m6A on carRNAs can globally tune chromatin state and transcription.


Asunto(s)
Adenosina/análogos & derivados , Cromatina/metabolismo , Metiltransferasas/metabolismo , ARN Nuclear/metabolismo , Transcripción Genética , Adenosina/metabolismo , Animales , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Metilación , Metiltransferasas/genética , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo
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