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RNA-binding proteins (RBPs) are central players in RNA homeostasis and the control of gene expression. The identification of RBP targets, interactions, and the regulatory networks they control is crucial for understanding their cellular functions. Traditional methods for identifying RBP targets across the transcriptome have been insightful but are limited by their focus on a single RBP at a time and their general inability to identify individual RNA molecules that are bound by RBPs of interest. Recently, we overcame these limitations by developing TRIBE-STAMP, a method which enables concurrent identification of the RNA targets of two RBPs of interest with single-molecule resolution. TRIBE-STAMP works by tagging desired RBPs with either the ADAR or APOBEC1 RNA editing enzymes and expressing them in cells, followed by RNA-seq. Subsequent computational identification of A-to-I and C-to-U editing events enables the simultaneous identification of the ADAR- and APOBEC1-fused RBP target RNAs, respectively. Here, we present a detailed protocol for TRIBE-STAMP, including considerations for fusion protein expression in cells and step-by-step computational analysis of sequencing data. TRIBE-STAMP is a simple and highly versatile approach for single-molecule identification of the targets of RBPs which enables unprecedented insights into the biological interplay between RBP pairs in cells., (Copyright © 2024. Published by Elsevier Inc.)

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, N 6 -methyladenosine (m 6 A). We first describe the discovery of the key enzymes that deposit and remove m 6 A 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 m 6 A 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.

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 m 6 A 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., (© 2022 Flamand et al.; Published by Cold Spring Harbor Laboratory Press.)

N 6 -methyladenosine (m 6 A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m 6 A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m 6 A contributes to brain development are incompletely understood. RBM45 acts as an m 6 A-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 m 6 A-dependent and m 6 A-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., Competing Interests: Declaration of interests K.D.M. has filed a patent application for the DART-seq technology through Duke University., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

N 6 -methyladenosine (m 6 A) is an abundant RNA modification that plays critical roles in RNA regulation and cellular function. Global m 6 A profiling has revealed important aspects of m 6 A distribution and function, but to date such studies have been restricted to large populations of cells. Here, we develop a method to identify m 6 A sites transcriptome-wide in single cells. We uncover surprising heterogeneity in the presence and abundance of m 6 A 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 m 6 A that have been missed by m 6 A profiling of bulk cells and suggest the presence of cell-intrinsic mechanisms for m 6 A deposition., Competing Interests: Declaration of interests K.D.M. has filed a patent application for the DART-seq technology through Duke University., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: Declaration of Interests E.A.M. is a founder and director of STORM Therapeutics. This company has not contributed to this research in any way. The authors declare no competing interests., (Copyright © 2020. Published by Elsevier Inc.)

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.

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 3΄UTR 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., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 CCR4–NOT 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 CCR4–NOT 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.

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., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)