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Going global: the new era of mapping modifications in RNA

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The post‐transcriptional modification of RNA by the addition of one or more chemical groups has been known for over 50 years. These chemical modifications, once thought to be static, are now being discovered to play key regulatory roles in gene expression. The advent of massive parallel sequencing of RNA (RNA‐seq) now allows us to probe the complexity of cellular RNA and how chemically altering RNA structure expands the RNA vocabulary. Here we present an overview of the various strategies and technologies that are available to profile RNA chemical modifications at the cellular level. These strategies can be characterized as targeted and untargeted approaches: targeted strategies are developed for one single chemical modification while untargeted strategies are more broadly applicable to a range of such chemical changes. Key for all of these approaches is the ability to locate modifications within the RNA sequence. While most of these methods are built upon an RNA‐Seq pipeline, alternative approaches based on mass spectrometry or conventional DNA sequencing retain value in the overall analysis process. We also look forward toward future opportunities and technologies that may expand the types of modifications that can be globally profiled. Given the ever increasing recognition that these RNA chemical modifications play important biological roles, a variety of methods, preferably orthogonal approaches, will be required to globally identify, validate and quantify RNA chemical modifications found in the transcriptome. WIREs RNA 2017, 8:e1367. doi: 10.1002/wrna.1367 This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Processing > RNA Editing and Modification RNA Methods > RNA Analyses In Vitro and In Silico
Reversible chemical modifications that regulate the flow of genetic information. In the central dogma, genetic information is passed from DNA to RNA and then to protein. Epigenetic DNA modifications (e.g., the formation of 5‐methylcytosine (m5C; also known as 5mC) and 5‐hydroxymethylcytosine (hm5C; also known as 5hmC)) and histone modifications (e.g., methylation (me) and acetylation (ac)) are known to have important roles in regulating cell differentiation and development. Reversible RNA modifications (e.g., the formation of N6‐methyladenosine (m6A) and N6‐hydroxymethyladenosine (hm6A)) add an additional layer of dynamic regulation of biological processes. (Reprinted with permission from Ref . Copyright 2014 Nature Publishing Group)
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Schematic representation of DNA‐based exclusion list for enhanced detection of modified RNAs by LC‐MS/MS. (Reprinted with permission from Ref . Copyright 2015 American Chemical Society)
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Schematic representation of demethylase‐thermostable group II intron RT tRNA sequencing (DM‐tRNA‐seq). (Reprinted with permission from Ref . Copyright 2015 Nature Publishing Group)
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Chemistry and outline of ICE‐seq. (a) Chemistry of inosine a cyanoethylation. Inosine (I) on an RNA strand is cyanoethylated with acrylonitrile to form N1‐cyanoethylinosine (ce1I). (b) ICE‐seq procedure. O Schemes without (CE− condition) or with (CE+ condition) cyanoethylation of RNA are shown on the left and right, respectively. RNA and cDNA are O indicated by gray and black arrows, respectively. The I in the RNA strand Inosine (I) is specifically cyanoethylated to form ce1I (CE+). In both conditions, RNA bearing A at the editing site is converted to T in the cDNA during the first‐strand synthesis. In the CE− condition, RNA bearing I is transcribed to C in the cDNA. In the CE+ condition, first‐strand cDNA extension is arrested at the ce1I site (red arrow). Second strands of cDNA are synthesized to obtain double‐stranded cDNA that is then subjected to the end‐repair reaction and adapter ligation. The amplified cDNA with 400–450 bp is gel‐purified. The cDNAs for the CE− and CE+ conditions are sequenced from both ends using next‐generation sequencing. After data processing of sequence reads, A‐to‐I RNA editing sites can be identified by detecting erased G‐containing reads upon cyanoethylation. (Reprinted with permission from Ref . Copyright 2015 Nature Publishing Group)
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Outline of MeRIP‐Seq protocol and distribution of sequencing reads. (a) Schematic representation of MeRIP‐Seq. Total RNA is subjected to RiboMinus treatment to remove rRNA species. RNAs containing m6A are then immunoprecipitated by mixing the RNA with m6A antibody‐coupled Dynabeads. m6A‐containing RNAs are then eluted from the antibody‐coupled beads and subjected to a second round of m6A immunoprecipitation. The resulting RNA pool, which is highly enriched for m6A‐containing RNAs, is then subjected to next‐generation sequencing. (b) Schematic of sequencing reads and their alignment to locations in the genome surrounding an m6A site. (Top) An mRNA that contains a single m6A residue along its length. (Middle) Individual 100 nt wide mRNA fragments that are isolated following m6A immunoprecipitation, each of which contains the same m6A residue from the mRNA depicted above. (Bottom) Histogram showing predicted frequency of MeRIP‐Seq reads obtained by sequencing individual immunoprecipitated fragments. Read frequency is predicted to increase with closer proximity to the m6A site, forming a ‘peak’ that is roughly 200 nt wide at its base and 100 nt wide at its midpoint. (c) Sequencing reads from MeRIP‐Seq converge over m6A sites. Representative UCSC Genome Browser plot from MeRIP‐Seq data, which demonstrates typical read frequency peak formation surrounding a site of m6A (shown here is the 30 UTR of Pax6). Peak height is displayed as reads per base per million mapped reads (BPM). (Reprinted with permission from Ref . Copyright 2012 Cell Press)
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Common themes in global profiling by RNA‐Seq approaches. (a) Chemical modification derivatization strategies are used to enhance reverse transcriptase (RT) stops. (b) Differential analysis takes advantage of varying sensitivity of RNA‐Seq to the presence of chemical modifications. (c) Affinity purification—usually through antibodies targeting specific chemically modified nucleosides—enriches the RNA pool prior to next‐generation sequencing.
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Representative RNA chemical modifications. [m5C] 5‐methylcytidine; [hm5C] 5‐hydroxymethylcytidine; [m6A] N6‐methyladenosine; [m6Am] N6,2′‐O‐dimethyladenosine; [Ψ] pseudouridine; [I] inosine.
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RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry
RNA Methods > RNA Analyses In Vitro and In Silico
RNA Processing > RNA Editing and Modification

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