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      Single-cell analysis of the epitranscriptome: RNA modifications under the microscope

      review-article
      a , a , a , b , c , d
      RNA Biology
      Taylor & Francis
      Single-cell, epitranscriptome, RNA modifications, m6A, Inosine

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          ABSTRACT

          The identification of mechanisms capable of modifying genetic information by the addition of covalent RNA modifications distinguishes a level of complexity in gene expression which challenges key long-standing concepts of RNA biology. One of the current challenges of molecular biology is to properly understand the molecular functions of these RNA modifications, with more than 170 different ones having been identified so far. However, it has not been possible to map specific RNA modifications at a single-cell resolution until very recently. This review will highlight the technological advances in single-cell methodologies aimed at assessing and testing the biological function of certain RNA modifications, focusing on m 6A. These advances have allowed for the development of novel strategies that enable the study of the ‘epitranscriptome’. Nevertheless, despite all these improvements, many challenges and difficulties still need fixing for these techniques to work efficiently.

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          Most cited references74

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          Massively parallel digital transcriptional profiling of single cells

          Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3′ mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.
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            Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.

            An extensive repertoire of modifications is known to underlie the versatile coding, structural and catalytic functions of RNA, but it remains largely uncharted territory. Although biochemical studies indicate that N(6)-methyladenosine (m(6)A) is the most prevalent internal modification in messenger RNA, an in-depth study of its distribution and functions has been impeded by a lack of robust analytical methods. Here we present the human and mouse m(6)A modification landscape in a transcriptome-wide manner, using a novel approach, m(6)A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12,000 m(6)A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks--around stop codons and within long internal exons--and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m(6)A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signalling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m(6)A has a fundamental role in regulation of gene expression.
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              Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.

              Methylation of the N(6) position of adenosine (m(6)A) is a posttranscriptional modification of RNA with poorly understood prevalence and physiological relevance. The recent discovery that FTO, an obesity risk gene, encodes an m(6)A demethylase implicates m(6)A as an important regulator of physiological processes. Here, we present a method for transcriptome-wide m(6)A localization, which combines m(6)A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq). We use this method to identify mRNAs of 7,676 mammalian genes that contain m(6)A, indicating that m(6)A is a common base modification of mRNA. The m(6)A modification exhibits tissue-specific regulation and is markedly increased throughout brain development. We find that m(6)A sites are enriched near stop codons and in 3' UTRs, and we uncover an association between m(6)A residues and microRNA-binding sites within 3' UTRs. These findings provide a resource for identifying transcripts that are substrates for adenosine methylation and reveal insights into the epigenetic regulation of the mammalian transcriptome. Copyright © 2012 Elsevier Inc. All rights reserved.
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                Author and article information

                Journal
                RNA Biol
                RNA Biol
                RNA Biology
                Taylor & Francis
                1547-6286
                1555-8584
                18 February 2024
                2024
                18 February 2024
                : 21
                : 1
                : 1-8
                Affiliations
                [a ]Cancer Epigenetics Group, Josep Carreras Leukaemia Research Institute (IJC); , Badalona, Spain
                [b ]Centro de Investigación Biomédica en Red Cancer (CIBERONC); , Madrid, Spain
                [c ]Institució Catalana de Recerca i Estudis Avançats (ICREA); , Barcelona, Spain
                [d ]Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB); , Barcelona, Spain
                Author notes
                CONTACT Manel Esteller mesteller@ 123456carrerasresearch.org Cancer Epigenetics Group, Josep Carreras Leukaemia Research Institute (IJC); , Badalona, Barcelona Spain
                Article
                2315385
                10.1080/15476286.2024.2315385
                10877985
                38368619
                67002f11-1044-4098-a0c6-3f155dcd3a00
                © 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

                History
                Page count
                Figures: 3, Tables: 1, References: 75, Pages: 8
                Categories
                Review Article
                Review

                Molecular biology
                single-cell,epitranscriptome,rna modifications,m6a,inosine
                Molecular biology
                single-cell, epitranscriptome, rna modifications, m6a, inosine

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