RESUMEN
Over the past decade, mRNA modifications have emerged as important regulators of gene expression control in cells. Fueled in large part by the development of tools for detecting RNA modifications transcriptome wide, researchers have uncovered a diverse epitranscriptome that serves as an additional layer of gene regulation beyond simple RNA sequence. Here, we review the proteins that write, read, and erase these marks, with a particular focus on the most abundant internal modification, N6-methyladenosine (m6A). We first describe the discovery of the key enzymes that deposit and remove m6A and other modifications and discuss how our understanding of these proteins has shaped our views of modification dynamics. We then review current models for the function of m6A reader proteins and how our knowledge of these proteins has evolved. Finally, we highlight important future directions for the field and discuss key questions that remain unanswered.
Asunto(s)
Adenosina , Regulación de la Expresión Génica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Adenosina/genética , Adenosina/metabolismo , Proteínas/genética , Proteínas/metabolismo , TranscriptomaRESUMEN
RNA-binding proteins (RBPs) regulate nearly every aspect of mRNA processing and are important regulators of gene expression in cells. However, current methods for transcriptome-wide identification of RBP targets are limited, since they examine only a single RBP at a time and do not provide information on the individual RNA molecules that are bound by a given RBP. Here, we overcome these limitations by developing TRIBE-STAMP, an approach for single-molecule detection of the target RNAs of two RNA binding proteins simultaneously in cells. We applied TRIBE-STAMP to the cytoplasmic m6A reader proteins YTHDF1, YTHDF2, and YTHDF3 and discovered that individual mRNA molecules can be bound by more than one YTHDF protein throughout their lifetime, providing new insights into the function of YTHDF proteins in cells. TRIBE-STAMP is a highly versatile approach that enables single-molecule analysis of the targets of RBP pairs simultaneously in the same cells.
Asunto(s)
Proteínas de Unión al ARN , ARN , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcriptoma , Procesamiento Postranscripcional del ARNRESUMEN
N6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are incompletely understood. RBM45 acts as an m6A-binding protein that is highly expressed during neurodevelopment. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings show a role for RBM45 in controlling mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.
Asunto(s)
Proteínas Portadoras , Proteínas de Unión al ARN , Proteínas Portadoras/metabolismo , Unión Proteica , ARN/metabolismo , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The transport of mRNAs to distal subcellular compartments is an important component of spatial gene expression control in neurons. However, the mechanisms that control mRNA localization in neurons are not completely understood. Here, we identify the abundant base modification, m6A, as a novel regulator of this process. Transcriptome-wide analysis following genetic loss of m6A reveals hundreds of transcripts that exhibit altered subcellular localization in hippocampal neurons. Additionally, using a reporter system, we show that mutation of specific m6A sites in select neuronal transcripts diminishes their localization to neurites. Single molecule fluorescent in situ hybridization experiments further confirm our findings and identify the m6A reader proteins YTHDF2 and YTHDF3 as mediators of this effect. Our findings reveal a novel function for m6A in controlling mRNA localization in neurons and enable a better understanding of the mechanisms through which m6A influences gene expression in the brain.
Asunto(s)
Metiltransferasas/metabolismo , Proteínas de Unión al ARN , Adenina/metabolismo , Encéfalo/metabolismo , Hibridación Fluorescente in Situ , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
N6-methyladenosine (m6A) is an abundant RNA modification that plays critical roles in RNA regulation and cellular function. Global m6A profiling has revealed important aspects of m6A distribution and function, but to date such studies have been restricted to large populations of cells. Here, we develop a method to identify m6A sites transcriptome-wide in single cells. We uncover surprising heterogeneity in the presence and abundance of m6A sites across individual cells and identify differentially methylated mRNAs across the cell cycle. Additionally, we show that cellular subpopulations can be distinguished based on their RNA methylation signatures, independent from gene expression. These studies reveal fundamental features of m6A that have been missed by m6A profiling of bulk cells and suggest the presence of cell-intrinsic mechanisms for m6A deposition.
Asunto(s)
Adenosina/análogos & derivados , Perfilación de la Expresión Génica , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transcriptoma , Adenosina/metabolismo , Células HEK293 , Humanos , Metilación , ARN Mensajero/genéticaRESUMEN
microRNA (miRNA)-mediated gene silencing is enacted through the recruitment of effector proteins that direct translational repression or degradation of mRNA targets, but the relative importance of their activities for animal development remains unknown. Our concerted proteomic surveys identified the uncharacterized GYF-domain encoding protein GYF-1 and its direct interaction with IFE-4, the ortholog of the mammalian translation repressor 4EHP, as key miRNA effector proteins in Caenorhabditis elegans. Recruitment of GYF-1 protein to mRNA reporters in vitro or in vivo leads to potent translation repression without affecting the poly(A) tail or impinging on mRNA stability. Loss of gyf-1 is synthetic lethal with hypomorphic alleles of embryonic miR-35-42 and larval (L4) let-7 miRNAs, which is phenocopied through engineered mutations in gyf-1 that abolish interaction with IFE-4. GYF-1/4EHP function is cascade-specific, as loss of gyf-1 had no noticeable impact on the functions of other miRNAs, including lin-4 and lsy-6. Overall, our findings reveal the first direct effector of miRNA-mediated translational repression in C. elegans and its physiological importance for the function of several, but likely not all miRNAs.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , MicroARNs/genética , Biosíntesis de Proteínas , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Genes Letales , MicroARNs/metabolismo , Dominios Proteicos , Proteómica , Complejo Silenciador Inducido por ARN/metabolismoRESUMEN
Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational epigenetic inheritance (TEI) of gene silencing. We identified and characterized a family of nuclear Argonaute-interacting proteins (ENRIs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. ENRI-1/2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, they are negative regulators of nuclear RNAi triggered from exogenous sources. Loss of ENRI-3, an unstable protein expressed mostly in the male germline, misdirects the RNAi response to transposable elements and impairs TEI. The ENRIs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Silenciador del Gen/fisiología , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Células Germinativas/metabolismo , Proteínas Nucleares/metabolismo , Interferencia de ARN/fisiología , ARN Bicatenario/metabolismo , ARN Nuclear/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genéticaRESUMEN
RNA modifications, collectively referred to as 'the epitranscriptome,' have recently emerged as a pervasive feature of cellular mRNAs which have diverse impacts on gene expression. In the last several years, technological advances improving our ability to identify mRNA modifications, coupled with the discovery of proteins that add and remove these marks, have substantially expanded our knowledge of how the epitranscriptome shapes gene expression. Efforts to uncover functional roles for mRNA modifications have begun to reveal important roles for some marks within the nervous system, and animal models have emerged which demonstrate severe neurodevelopmental and neurocognitive abnormalities resulting from the loss of mRNA modification machinery. Here, we review the recent advances in the field of neuroepitranscriptomics, with a particular emphasis on how modifications to mRNAs within the brain contribute to synaptic activity.
Asunto(s)
Encéfalo , Plasticidad Neuronal , Animales , ARN MensajeroRESUMEN
Apoptosis is a genetically programmed cell death process with profound roles in development and disease. MicroRNAs modulate the expression of many proteins and are often deregulated in human diseases, such as cancer. C. elegans germ cells undergo apoptosis in response to genotoxic stress by the combined activities of the core apoptosis and MAPK pathways, but how their signalling thresholds are buffered is an open question. Here we show mir-35-42 miRNA family play a dual role in antagonizing both NDK-1, a positive regulator of MAPK signalling, and the BH3-only pro-apoptotic protein EGL-1 to regulate the magnitude of DNA damage-induced apoptosis in the C. elegans germline. We show that while miR-35 represses EGL-1 by promoting transcript degradation, repression of NDK-1 may be through sequestration of the transcript to inhibit translation. Importantly, dramatic increase in NDK-1 expression was observed in cells about to die. In the absence of miR-35, increased NDK-1 activity enhanced MAPK signalling that lead to significant increases in germ cell death. Our findings demonstrate that NDK-1 acts upstream of (or in parallel to) EGL-1, and that miR-35 targets both egl-1 and ndk-1 to fine-tune cell killing in response to genotoxic stress.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Sistema de Señalización de MAP Quinasas , MicroARNs/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Nucleósido-Difosfato Quinasa/metabolismo , Regiones no Traducidas 3' , Animales , Apoptosis/fisiología , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Daño del ADN , Regulación hacia Abajo , Células Germinativas , MicroARNs/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Nucleósido-Difosfato Quinasa/biosíntesis , Nucleósido-Difosfato Quinasa/genética , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Although strong evidence supports the importance of their cooperative interactions, microRNA (miRNA)-binding sites are still largely investigated as functionally independent regulatory units. Here, a survey of alternative 3ÎUTR isoforms implicates a non-canonical seedless site in cooperative miRNA-mediated silencing. While required for target mRNA deadenylation and silencing, this site is not sufficient on its own to physically recruit miRISC. Instead, it relies on facilitating interactions with a nearby canonical seed-pairing site to recruit the Argonaute complexes. We further show that cooperation between miRNA target sites is necessary for silencing in vivo in the C. elegans embryo, and for the recruitment of the Ccr4-Not effector complex. Using a structural model of cooperating miRISCs, we identified allosteric determinants of cooperative miRNA-mediated silencing that are required for both embryonic and larval miRNA functions. Our results delineate multiple cooperative mechanisms in miRNA-mediated silencing and further support the consideration of target site cooperation as a fundamental characteristic of miRNA function.
Asunto(s)
Caenorhabditis elegans/genética , Silenciador del Gen , MicroARNs/genética , Complejo Silenciador Inducido por ARN/química , Factores de Transcripción/química , Regiones no Traducidas 3' , Empalme Alternativo , Animales , Proteínas Argonautas/química , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Secuencia de Bases , Sitios de Unión , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Embrión no Mamífero , MicroARNs/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
MicroRNAs (miRNAs) impinge on the translation and stability of their target mRNAs, and play key roles in development, homeostasis and disease. The gene regulation mechanisms they instigate are largely mediated through the CCR4NOT deadenylase complex, but the molecular events that occur on target mRNAs are poorly resolved. We observed a broad convergence of interactions of germ granule and P body mRNP components on AIN-1/GW182 and NTL-1/CNOT1 in Caenorhabditis elegans embryos. We show that the miRISC progressively matures on the target mRNA from a scanning form into an effector mRNP particle by sequentially recruiting the CCR4NOT complex, decapping and decay, or germ granule proteins. Finally, we implicate intrinsically disordered proteins, key components in mRNP architectures, in the embryonic function of lsy-6 miRNA. Our findings define dynamic steps of effector mRNP assembly in miRNA-mediated silencing, and identify a functional continuum between germ granules and P bodies in the C. elegans embryo.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , MicroARNs/metabolismo , Interferencia de ARN , Ribonucleoproteínas/metabolismo , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Gránulos Citoplasmáticos/metabolismo , Embrión no Mamífero/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , ARN Mensajero/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , Ribonucleasas/metabolismoRESUMEN
Cytoplasmic poly(A)-binding proteins (PABPs) link mRNA 3' termini to translation initiation factors, but they also play key roles in mRNA regulation and decay. Reports from mice, zebrafish and Drosophila further involved PABPs in microRNA (miRNA)-mediated silencing, but through seemingly distinct mechanisms. Here, we implicate the two Caenorhabditis elegans PABPs (PAB-1 and PAB-2) in miRNA-mediated silencing, and elucidate their mechanisms of action using concerted genetics, protein interaction analyses, and cell-free assays. We find that C. elegans PABPs are required for miRNA-mediated silencing in embryonic and larval developmental stages, where they act through a multi-faceted mechanism. Depletion of PAB-1 and PAB-2 results in loss of both poly(A)-dependent and -independent translational silencing. PABPs accelerate miRNA-mediated deadenylation, but this contribution can be modulated by 3'UTR sequences. While greater distances with the poly(A) tail exacerbate dependency on PABP for deadenylation, more potent miRNA-binding sites partially suppress this effect. Our results refine the roles of PABPs in miRNA-mediated silencing and support a model wherein they enable miRNA-binding sites by looping the 3'UTR poly(A) tail to the bound miRISC and deadenylase.