RESUMEN
N(6)-methyladenosine (m(6)A) is the most abundant internal modification of messenger RNA. While the presence of m(6)A on transcripts can impact nuclear RNA fates, a reader of this mark that mediates processing of nuclear transcripts has not been identified. We find that the RNA-binding protein HNRNPA2B1 binds m(6)A-bearing RNAs in vivo and in vitro and its biochemical footprint matches the m(6)A consensus motif. HNRNPA2B1 directly binds a set of nuclear transcripts and elicits similar alternative splicing effects as the m(6)A writer METTL3. Moreover, HNRNPA2B1 binds to m(6)A marks in a subset of primary miRNA transcripts, interacts with the microRNA Microprocessor complex protein DGCR8, and promotes primary miRNA processing. Also, HNRNPA2B1 loss and METTL3 depletion cause similar processing defects for these pri-miRNA precursors. We propose HNRNPA2B1 to be a nuclear reader of the m(6)A mark and to mediate, in part, this mark's effects on primary microRNA processing and alternative splicing. PAPERCLIP.
Asunto(s)
Adenosina/análogos & derivados , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Procesamiento Postranscripcional del ARN , Adenosina/metabolismo , Empalme Alternativo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Metilación , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , TranscriptomaRESUMEN
The first step in the biogenesis of microRNAs is the processing of primary microRNAs (pri-miRNAs) by the microprocessor complex, composed of the RNA-binding protein DGCR8 and the type III RNase DROSHA. This initial event requires recognition of the junction between the stem and the flanking single-stranded RNA of the pri-miRNA hairpin by DGCR8 followed by recruitment of DROSHA, which cleaves the RNA duplex to yield the pre-miRNA product. While the mechanisms underlying pri-miRNA processing have been determined, the mechanism by which DGCR8 recognizes and binds pri-miRNAs, as opposed to other secondary structures present in transcripts, is not understood. Here we find in mammalian cells that methyltransferase-like 3 (METTL3) methylates pri-miRNAs, marking them for recognition and processing by DGCR8. Consistent with this, METTL3 depletion reduced the binding of DGCR8 to pri-miRNAs and resulted in the global reduction of mature miRNAs and concomitant accumulation of unprocessed pri-miRNAs. In vitro processing reactions confirmed the sufficiency of the N(6)-methyladenosine (m(6)A) mark in promoting pri-miRNA processing. Finally, gain-of-function experiments revealed that METTL3 is sufficient to enhance miRNA maturation in a global and non-cell-type-specific manner. Our findings reveal that the m(6)A mark acts as a key post-transcriptional modification that promotes the initiation of miRNA biogenesis.
Asunto(s)
Adenosina/análogos & derivados , MicroARNs/química , MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN , Adenosina/metabolismo , Secuencia de Bases , Línea Celular , Regulación de la Expresión Génica , Humanos , Metilación , Metiltransferasas/deficiencia , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas de Unión al ARN/metabolismo , Especificidad por SustratoRESUMEN
A fundamental feature of cell signaling is the conversion of extracellular signals into adaptive transcriptional responses. The role of RNA modifications in this process is poorly understood. The small nuclear RNA 7SK prevents transcriptional elongation by sequestering the cyclin dependent kinase 9/cyclin T1 (CDK9/CCNT1) positive transcription elongation factor (P-TEFb) complex. We found that epidermal growth factor signaling induces phosphorylation of the enzyme methyltransferase 3 (METTL3), leading to METTL3-mediated methylation of 7SK. 7SK methylation enhanced its binding to heterogeneous nuclear ribonucleoproteins, causing the release of the HEXIM1 P-TEFb complex subunit1 (HEXIM1)/P-TEFb complex and inducing transcriptional elongation. Our findings establish the mechanism underlying 7SK activation and uncover a previously unknown function for the m6A modification in converting growth factor signaling events into a regulatory transcriptional response via an RNA methylation-dependent switch.
Asunto(s)
Factor B de Elongación Transcripcional Positiva , Proteínas de Unión al ARN , Humanos , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The developmental transcription factor SOX4 contributes to the metastatic spread of multiple solid cancer types, but its direct target genes that mediate cancer progression are not well defined. Using a systematic molecular and genomic approach, we identified the TMEM2 transmembrane protein gene as a direct transcriptional target of SOX4. TMEM2 was transcriptionally activated by SOX4 in breast cancer cells where, like SOX4, TMEM2 was found to mediate proinvasive and promigratory effects. Similarly, TMEM2 was sufficient to promote metastatic colonization of breast cancer cells and its expression in primary breast tumors associated with a higher likelihood of metastatic relapse. Given earlier evidence that genetic inactivation of SOX4 or TMEM2 yield similar defects in cardiac development, our findings lead us to propose that TMEM2 may not only mediate the pathologic effects of SOX4 on cancer progression but also potentially its contributions to embryonic development. Cancer Res; 76(17); 4994-5005. ©2016 AACR.