Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

Abstract

The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m6A methylation in mRNAs, dozens of deep sequencing-based methods and protocols were proposed for the analysis of various RNA modifications, allowing us to considerably extend the list of detectable modified residues. Many of the currently used methods rely on the particular reverse transcription signatures left by RNA modifications in cDNA; these signatures may be naturally present or induced by an appropriate enzymatic or chemical treatment. The newest approaches also include labeling at RNA abasic sites that result from the selective removal of RNA modification or the enhanced cleavage of the RNA ribose-phosphate chain (perhaps also protection from cleavage), followed by specific adapter ligation. Classical affinity/immunoprecipitation-based protocols use either antibodies against modified RNA bases or proteins/enzymes, recognizing RNA modifications. In this survey, we review the most recent achievements in this highly dynamic field, including promising attempts to map RNA modifications by the direct single-molecule sequencing of RNA by nanopores.

Changes of the tRNA Modification Pattern during the Development of Dictyostelium discoideum

“…To investigate tRNA modification patterns in D. discoideum in a systematic manner, individual tRNA pools were analyzed for candidate modification sites by their RT signatures (accumulation of base misincorporations and/or apparent RTa sites) extracted from LOTTEseq data. To this end, we elaborated on the knowledge about modification-specific RT signals provided by a series of previous studies [3,[11][12][13]16,18,25,26,28,34,48], adding our own observations as well as published information on the TGIRT enzyme [19,49,50]. This group II intron-derived thermostable reverse transcriptase exhibits a high fidelity and processivity [51].…”

Long Non-Coding RNA Epigenetics

“…NGS methods based on short-read sequencing have difficulty in determining the modification patterns of the entire transcript sequence. Recently, a direct modification detection method, called nanopore sequencing, has been developed [ 155 , 156 , 157 ]. The Oxford Nanopore Technologies (ONT) sequencer can directly sequence individual native RNA or DNA molecules.…”

RNA Modifications in Pathogenic Bacteria: Impact on Host Adaptation and Virulence

“…A detailed description of the available methods is outside the scope of the review. We will summarize the most employed approaches with their limits and advantages (Table 1), and details will be found in more specific articles [30][31][32][33].…”

“…This limits the ability to characterize the plasticity of the epitranscriptome in a systematic and unbiased manner. A recent review described in details the RNAseq based approaches [31]. Noteworthy, the very recent development of single molecule direct RNA sequencing method by Oxford Nanopore Technologies (ONT, Oxford, UK), is promising for the analysis of modification landscape on a specific RNA sequence, including Ψs [48][49][50][51].…”