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1.
Mol Cancer ; 20(1): 115, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34496885

RESUMO

BACKGROUND: Adenosine deaminases acting on RNA (ADARs) modify many cellular RNAs by catalyzing the conversion of adenosine to inosine (A-to-I), and their deregulation is associated with several cancers. We recently showed that A-to-I editing is elevated in thyroid tumors and that ADAR1 is functionally important for thyroid cancer cell progression. The downstream effectors regulated or edited by ADAR1 and the significance of ADAR1 deregulation in thyroid cancer remain, however, poorly defined. METHODS: We performed whole transcriptome sequencing to determine the consequences of ADAR1 deregulation for global gene expression, RNA splicing and editing. The effects of gene silencing or RNA editing were investigated by analyzing cell viability, proliferation, invasion and subnuclear localization, and by protein and gene expression analysis. RESULTS: We report an oncogenic function for CDK13 in thyroid cancer and identify a new ADAR1-dependent RNA editing event that occurs in the coding region of its transcript. CDK13 was significantly over-edited (c.308A > G) in tumor samples and functional analysis revealed that this editing event promoted cancer cell hallmarks. Finally, we show that CDK13 editing increases the nucleolar abundance of the protein, and that this event might explain, at least partly, the global change in splicing produced by ADAR1 deregulation. CONCLUSIONS: Overall, our data support A-to-I editing as an important pathway in cancer progression and highlight novel mechanisms that might be used therapeutically in thyroid and other cancers.

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.
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
4.
Methods Mol Biol ; 2298: 15-27, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085236

RESUMO

While over 150 distinct types of chemical modifications are known to occur on various cellular RNAs and can be dynamically controlled, the function of most of these modifications remains poorly defined. Collectively, these RNA modifications have been recently termed the "epitranscriptome". Identification and annotation of individual RNA modifications throughout the transcriptome are key for studying the role of the epitranscriptome in the regulation of gene expression and for elucidating the functional relevance of particular RNA modifications in diverse physiological and disease processes. In this protocol, we demonstrate how to identify and annotate RNA modifications based on the informatic analysis of methylated RNA immunoprecipitation and sequencing (MeRIP-seq) data, using RNAmod, a convenient one-stop online interactive platform for the annotation, analysis, and visualization of mRNA modifications.


Assuntos
Informática/métodos , Processamento Pós-Transcricional do RNA/genética , RNA/genética , Transcriptoma/genética , Linhagem Celular Tumoral , Células Hep G2 , Humanos , Análise de Sequência de RNA/métodos
5.
Nature ; 593(7860): 602-606, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33953397

RESUMO

MicroRNAs (miRNAs) have essential functions during embryonic development, and their dysregulation causes cancer1,2. Altered global miRNA abundance is found in different tissues and tumours, which implies that precise control of miRNA dosage is important1,3,4, but the underlying mechanism(s) of this control remain unknown. The protein complex Microprocessor, which comprises one DROSHA and two DGCR8 proteins, is essential for miRNA biogenesis5-7. Here we identify a developmentally regulated miRNA dosage control mechanism that involves alternative transcription initiation (ATI) of DGCR8. ATI occurs downstream of a stem-loop in DGCR8 mRNA to bypass an autoregulatory feedback loop during mouse embryonic stem (mES) cell differentiation. Deletion of the stem-loop causes imbalanced DGCR8:DROSHA protein stoichiometry that drives irreversible Microprocessor aggregation, reduced primary miRNA processing, decreased mature miRNA abundance, and widespread de-repression of lipid metabolic mRNA targets. Although global miRNA dosage control is not essential for mES cells to exit from pluripotency, its dysregulation alters lipid metabolic pathways and interferes with embryonic development by disrupting germ layer specification in vitro and in vivo. This miRNA dosage control mechanism is conserved in humans. Our results identify a promoter switch that balances Microprocessor autoregulation and aggregation to precisely control global miRNA dosage and govern stem cell fate decisions during early embryonic development.

7.
J Exp Med ; 218(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33156926

RESUMO

The mRNA N6-methyladenosine (m6A) modification has emerged as an essential regulator of normal and malignant hematopoiesis. Inactivation of the m6A mRNA reader YTHDF2, which recognizes m6A-modified transcripts to promote m6A-mRNA degradation, results in hematopoietic stem cell (HSC) expansion and compromises acute myeloid leukemia. Here we investigate the long-term impact of YTHDF2 deletion on HSC maintenance and multilineage hematopoiesis. We demonstrate that Ythdf2-deficient HSCs from young mice fail upon serial transplantation, display increased abundance of multiple m6A-modified inflammation-related transcripts, and chronically activate proinflammatory pathways. Consistent with the detrimental consequences of chronic activation of inflammatory pathways in HSCs, hematopoiesis-specific Ythdf2 deficiency results in a progressive myeloid bias, loss of lymphoid potential, HSC expansion, and failure of aged Ythdf2-deficient HSCs to reconstitute multilineage hematopoiesis. Experimentally induced inflammation increases YTHDF2 expression, and YTHDF2 is required to protect HSCs from this insult. Thus, our study positions YTHDF2 as a repressor of inflammatory pathways in HSCs and highlights the significance of m6A in long-term HSC maintenance.


Assuntos
Adenosina/análogos & derivados , Células-Tronco Hematopoéticas/metabolismo , Inflamação/genética , Proteínas de Ligação a RNA/metabolismo , Adenosina/metabolismo , Animais , Linhagem da Célula , Proliferação de Células , Senescência Celular , Deleção de Genes , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Inflamação/patologia , Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
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
9.
Sci Transl Med ; 12(566)2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087503

RESUMO

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.


Assuntos
Anemia de Diamond-Blackfan , Anemia de Diamond-Blackfan/tratamento farmacológico , Animais , Apoptose , Calmodulina , Eritropoese , Humanos , Proteína Supressora de Tumor p53 , Peixe-Zebra
10.
Cell Cycle ; 19(18): 2314-2326, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32816599

RESUMO

TRIM71 is an important RNA-binding protein in development and disease, yet its direct targets have not been investigated globally. Here we describe a number of disease and developmentally-relevant TRIM71 RNA targets such as the MBNL family, LIN28B, MDM2, and TCF7L2. We describe a new role for TRIM71 as capable of positive or negative RNA regulation depending on the RNA target. We found that TRIM71 co-precipitated with IMP1 which could explain its multiple mechanisms of RNA regulation, as IMP1 is typically thought to stabilize RNAs. Deletion of the NHL domain of TRIM71 impacted its ability to bind to RNA and RNAs bound by congenital hydrocephalus-associated point mutations in the RNA-binding NHL domain of TRIM71 clustered closely with RNAs bound by the NHL deletion mutant. Our work expands the possible mechanisms by which TRIM71 may regulate RNAs and elucidates further potential RNA targets.

11.
Nat Commun ; 11(1): 2619, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457326

RESUMO

DIS3L2-mediated decay (DMD) is a surveillance pathway for certain non-coding RNAs (ncRNAs) including ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), and RMRP. While mutations in DIS3L2 are associated with Perlman syndrome, the biological significance of impaired DMD is obscure and pathological RNAs have not been identified. Here, by ribosome profiling (Ribo-seq) we find specific dysregulation of endoplasmic reticulum (ER)-targeted mRNA translation in DIS3L2-deficient cells. Mechanistically, DMD functions in the quality control of the 7SL ncRNA component of the signal recognition particle (SRP) required for ER-targeted translation. Upon DIS3L2 loss, sustained 3'-end uridylation of aberrant 7SL RNA impacts ER-targeted translation and causes ER calcium leakage. Consequently, elevated intracellular calcium in DIS3L2-deficient cells activates calcium signaling response genes and perturbs ESC differentiation. Thus, DMD is required to safeguard ER-targeted mRNA translation, intracellular calcium homeostasis, and stem cell differentiation.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Exorribonucleases/metabolismo , Macrossomia Fetal/microbiologia , RNA Mensageiro/metabolismo , Tumor de Wilms/microbiologia , Animais , Sinalização do Cálcio/genética , Diferenciação Celular , Células-Tronco Embrionárias , Exorribonucleases/deficiência , Exorribonucleases/genética , Macrossomia Fetal/enzimologia , Macrossomia Fetal/genética , Regulação da Expressão Gênica , Humanos , Insulina/metabolismo , Camundongos , Biossíntese de Proteínas , RNA Citoplasmático Pequeno/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Uridina Monofosfato/metabolismo , Tumor de Wilms/enzimologia , Tumor de Wilms/genética
12.
Nucleic Acids Res ; 48(W1): W218-W229, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32427338

RESUMO

Ribosome profiling (Ribo-seq) is a powerful technology for globally monitoring RNA translation; ranging from codon occupancy profiling, identification of actively translated open reading frames (ORFs), to the quantification of translational efficiency under various physiological or experimental conditions. However, analyzing and decoding translation information from Ribo-seq data is not trivial. Although there are many existing tools to analyze Ribo-seq data, most of these tools are designed for specific or limited functionalities and an easy-to-use integrated tool to analyze Ribo-seq data is lacking. Fortunately, the small size (26-34 nt) of ribosome protected fragments (RPFs) in Ribo-seq and the relatively small amount of sequencing data greatly facilitates the development of such a web platform, which is easy to manipulate for users with or without bioinformatic expertise. Thus, we developed RiboToolkit (http://rnabioinfor.tch.harvard.edu/RiboToolkit), a convenient, freely available, web-based service to centralize Ribo-seq data analyses, including data cleaning and quality evaluation, expression analysis based on RPFs, codon occupancy, translation efficiency analysis, differential translation analysis, functional annotation, translation metagene analysis, and identification of actively translated ORFs. Besides, easy-to-use web interfaces were developed to facilitate data analysis and intuitively visualize results. Thus, RiboToolkit will greatly facilitate the study of mRNA translation based on ribosome profiling.


Assuntos
Códon , Biossíntese de Proteínas , Ribossomos , Software , Células 3T3 , Animais , Estresse do Retículo Endoplasmático/genética , Camundongos , Anotação de Sequência Molecular , Fases de Leitura Aberta , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
14.
Nat Protoc ; 14(11): 3220-3242, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31619810

RESUMO

Precise identification of sites of RNA modification is key to studying the functional role of such modifications in the regulation of gene expression and for elucidating relevance to diverse physiological processes. tRNA reduction and cleavage sequencing (TRAC-Seq) is a chemically based approach for the unbiased global mapping of 7-methylguansine (m7G) modification of tRNAs at single-nucleotide resolution throughout the tRNA transcriptome. m7G TRAC-Seq involves the treatment of size-selected (<200 nt) RNAs with the demethylase AlkB to remove major tRNA modifications, followed by sodium borohydride (NaBH4) reduction of m7G sites and subsequent aniline-mediated cleavage of the RNA chain at the resulting abasic sites. The cleaved sites are subsequently ligated with adaptors for the construction of libraries for high-throughput sequencing. The m7G modification sites are identified using a bioinformatic pipeline that calculates the cleavage scores at individual sites on all tRNAs. Unlike antibody-based methods, such as methylated RNA immunoprecipitation and sequencing (meRIP-Seq) for enrichment of methylated RNA sequences, chemically based approaches, including TRAC-Seq, can provide nucleotide-level resolution of modification sites. Compared to the related method AlkAniline-Seq (alkaline hydrolysis and aniline cleavage sequencing), TRAC-Seq incorporates small RNA selection, AlkB demethylation, and sodium borohydride reduction steps to achieve specific and efficient single-nucleotide resolution profiling of m7G sites in tRNAs. The m7G TRAC-Seq protocol could be adapted to chemical cleavage-mediated detection of other RNA modifications. The protocol can be completed within ~9 d for four biological replicates of input and treated samples.


Assuntos
Guanosina/análogos & derivados , RNA de Transferência/química , Análise de Sequência de RNA/métodos , Animais , Genômica , Guanosina/análise , Guanosina/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metilação , Camundongos , RNA de Transferência/genética , Software , Transcriptoma
15.
Nat Struct Mol Biol ; 26(6): 490-500, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160785

RESUMO

Ribosomal RNA (rRNA) biogenesis is a multistep process requiring several nuclear and cytoplasmic exonucleases. The exact processing steps for mammalian 5.8S rRNA remain obscure. Here, using loss-of-function approaches in mouse embryonic stem cells (mESCs) and deep sequencing of rRNA intermediates, we investigate the requirements of exonucleases known to be involved in 5.8S maturation at nucleotide resolution and explore the role of the Perlman syndrome-associated 3'-5' exonuclease Dis3l2 in rRNA processing. We uncover a novel cytoplasmic intermediate that we name '7SB' rRNA that is generated through sequential processing by distinct exosome complexes. 7SB rRNA can be oligoadenylated by an unknown enzyme and/or oligouridylated by TUT4/7 and subsequently processed by Dis3l2 and Eri1. Moreover, exosome depletion triggers Dis3l2-mediated decay (DMD) as a surveillance pathway for rRNAs. Our data identify previously unknown 5.8S rRNA processing steps and provide nucleotide-level insight into the exonuclease requirements for mammalian rRNA processing.


Assuntos
Exorribonucleases/metabolismo , RNA Ribossômico 5,8S/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Macrossomia Fetal/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Transporte de RNA , Ribossomos/metabolismo , Uridina/metabolismo , Tumor de Wilms/metabolismo
16.
Nucleic Acids Res ; 47(W1): W548-W555, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31147718

RESUMO

Dynamic and reversible RNA modifications such as N6-methyladenosine (m6A) can play important roles in regulating messenger RNA (mRNA) splicing, export, stability and translation. Defective mRNA modification through altered expression of the methyltransferase and/or demethylases results in developmental defects and cancer progression. Identifying modified mRNAs, annotating the distribution of modification sites across the mRNA, as well as characterizing and comparing other modification features are essential for studying the function and elucidating the mechanism of mRNA modifications. Several methods including methylated RNA immunoprecipitation and sequencing (MeRIP-seq) are available for the detection of mRNA modifications. However, a convenient and comprehensive tool to annotate diverse kinds of mRNA modifications in different species is lacking. Here, we developed RNAmod (https://bioinformatics.sc.cn/RNAmod), an interactive, one-stop, web-based platform for the automated analysis, annotation, and visualization of mRNA modifications in 21 species. RNAmod provides intuitive interfaces to show outputs including the distribution of RNA modifications, modification coverage for different gene features, functional annotation of modified mRNAs, and comparisons between different groups or specific gene sets. Furthermore, sites of known RNA modification, as well as binding site data for hundreds of RNA-binding proteins (RBPs) are integrated in RNAmod to help users compare their modification data with known modifications and to explore the relationship with the binding sites of known RBPs. RNAmod is freely available and meets the emerging need for a convenient and comprehensive analysis tool for the fast-developing RNA modification field.


Assuntos
Anotação de Sequência Molecular , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Software , Animais , Sítios de Ligação , Humanos , Camundongos , RNA Mensageiro/química , Proteínas de Ligação a RNA/metabolismo , Ratos , Integração de Sistemas
17.
Cancer Cell ; 35(4): 664-676.e7, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991026

RESUMO

Resistance to asparaginase, an antileukemic enzyme that depletes asparagine, is a common clinical problem. Using a genome-wide CRISPR/Cas9 screen, we found a synthetic lethal interaction between Wnt pathway activation and asparaginase in acute leukemias resistant to this enzyme. Wnt pathway activation induced asparaginase sensitivity in distinct treatment-resistant subtypes of acute leukemia, but not in normal hematopoietic progenitors. Sensitization to asparaginase was mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits glycogen synthase kinase 3 (GSK3)-dependent protein ubiquitination and proteasomal degradation, a catabolic source of asparagine. Inhibiting the alpha isoform of GSK3 phenocopied this effect, and pharmacologic GSK3α inhibition profoundly sensitized drug-resistant leukemias to asparaginase. Our findings provide a molecular rationale for activation of Wnt/STOP signaling to improve the therapeutic index of asparaginase.


Assuntos
Antineoplásicos/farmacologia , Asparaginase/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia/tratamento farmacológico , Polietilenoglicóis/farmacologia , Mutações Sintéticas Letais , Via de Sinalização Wnt/genética , Proteína Wnt3A/genética , Animais , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Células Jurkat , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Masculino , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estabilidade Proteica , Proteólise , Células THP-1 , Ubiquitinação , Proteína Wnt3A/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Cell Stem Cell ; 25(1): 137-148.e6, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31031138

RESUMO

Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation, generating self-renewing leukemic stem cells (LSCs). Here, we show that the mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m6A transcripts that contribute to the overall integrity of LSC function, including the tumor necrosis factor receptor Tnfrsf2, whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly, YTHDF2 is not essential for normal HSC function, with YTHDF2 deficiency actually enhancing HSC activity. Thus, we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion.


Assuntos
Leucemia Mieloide Aguda/terapia , Células-Tronco Neoplásicas/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Autorrenovação Celular , Hematopoese , Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Células THP-1
19.
Methods ; 155: 10-19, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30395968

RESUMO

Post-transcriptional modification of RNA, the so-called 'Epitranscriptome', can regulate RNA structure, stability, localization, and function. Numerous modifications have been identified in virtually all classes of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), and other noncoding RNAs (ncRNAs). These modifications may occur internally (by base or sugar modifications) and include RNA methylation at different nucleotide positions, or by the addition of various nucleotides at the 3'-end of certain transcripts by a family of terminal nucleotidylyl transferases. Developing methods to specifically and accurately detect and map these modifications is essential for understanding the molecular function(s) of individual RNA modifications and also for identifying and characterizing the proteins that may read, write, or erase them. Here, we focus on the characterization of RNA species targeted by 3' terminal uridylyl transferases (TUTases) (TUT4/7, also known as Zcchc11/6) and a 3'-5' exoribonuclease, Dis3l2, in the recently identified Dis3l2-mediated decay (DMD) pathway - a dedicated quality control pathway for a subset of ncRNAs. We describe the detailed methods used to precisely identify 3'-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3'-end modifications.


Assuntos
Biologia Computacional/métodos , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Uridina/metabolismo , Região 3'-Flanqueadora , Animais , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/deficiência , Exorribonucleases/genética , Edição de Genes/métodos , Camundongos , Células-Tronco Embrionárias Murinas , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Estabilidade de RNA , RNA Guia/genética , RNA Guia/metabolismo , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo
20.
Nature ; 561(7724): 556-560, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30232453

RESUMO

N6-methyladenosine (m6A) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered m6A homeostasis is linked to cancer1-5. m6A modification is catalysed by METTL3 and enriched in the 3' untranslated region of a large subset of mRNAs at sites close to the stop codon5. METTL3 can promote translation but the mechanism and relevance of this process remain unknown1. Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon, supporting a mechanism of mRNA looping for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci in close proximity to 5' cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs-including bromodomain-containing protein 4-that is also m6A-modified in human primary lung tumours. The METTL3-eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mechanism of translation control that is based on mRNA looping and identify METTL3-eIF3h as a potential therapeutic target for patients with cancer.


Assuntos
Carcinogênese , Fator de Iniciação 3 em Eucariotos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Metiltransferases/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Animais , Linhagem Celular Tumoral , Ciclização , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Conformação de Ácido Nucleico , Polirribossomos/química , Polirribossomos/metabolismo , Ligação Proteica , RNA Mensageiro/genética
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