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Detecting the epitranscriptome

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Abstract RNA modifications and their corresponding epitranscriptomic writer and eraser enzymes regulate gene expression. Altered RNA modification levels, dysregulated writers, and sequence changes that disrupt epitranscriptomic marks have been linked to mitochondrial and neurological diseases, cancer, and multifactorial disorders. The detection of epitranscriptomics marks is challenging, but different next generation sequencing (NGS)‐based and mass spectrometry‐based approaches have been used to identify and quantitate the levels of individual and groups of RNA modifications. NGS and mass spectrometry‐based approaches have been coupled with chemical, antibody or enzymatic methodologies to identify modifications in most RNA species, mapped sequence contexts and demonstrated the dynamics of specific RNA modifications, as well as the collective epitranscriptome. While epitranscriptomic analysis is currently limited to basic research applications, specific approaches for the detection of individual RNA modifications and the epitranscriptome have potential biomarker applications in detecting human conditions and diseases. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Processing > tRNA Processing RNA in Disease and Development > RNA in Disease
The cellular epitranscriptome. Epitranscriptomic marks are found on many RNA molecules, including mRNA, tRNA, lnRNA, snRNA, rRNA, and miRNA. N6‐methyladenosine (m6A) and pseudouridine (ψ) can be found on mRNA, lncRNA, and rRNAs. 2′‐O methyladenosine (Am) is found on snRNA and other RNAs. N1‐methyladenosine (m1A) and N6‐N6‐dimethyladenosine (m66A) have been identified on rRNA, with m1A also found on mRNA, 7‐methylguanosine (m7G) is found on mRNA and tRNA and queuosine (Q) on tRNA. Additionally, inosine (I), N2,N2,‐dimethylguanosine (m22Gm), N4‐acetylcytidine (ac4C), 5‐methylcytidine (m5C), and 5‐hydroxymethylcytidine (hm5C) are RNA modifications found in the epitranscriptome
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tRNA modification defects can be linked to some diseases. Defects in the modification of tRNA are associated with mitochondrial (infantile liver failure, MERFF, MELAS), neurological (intellectual disability, familial, dysautonomia, amyotrophic lateral sclerosis, Rolandic epilepsy, and Dubowitz‐like syndrome), cancers (colorectal, skin, breast, urothelial, bladder, cervix, and testicular), type II diabetes, and bronchial asthma. Diseases linked to modifications at specific positions of tRNA are detailed
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RNA modifications can be directly analyzed by mass spectrometry. RNA can be fragmented to (a) nucleosides or nucleotides. The nucleosides can be further purified using liquid chromatography, ionized by electrospray ionization and analyzed by tandem mass spectrometry. The nucleotides owing to their overall negative charge, can be purified by ion‐pair reversed phase HPLC. The purification using LC can be avoided all together by all analyzing the nucleotides by ion‐mobility MS. Alternatively, the nucleotides can be analyzed using direct‐infusion electrospray ionization (ESI)‐MS. (b) Oligonucleotides can be generated from RNA enzymatically digesting with RNase. The digested products can then be purified using reversed phase HPLC. The purified sample can then be analyzed using ESI‐MS or MALDI
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Some RNA modifications have the potential to be directly analyzed by nanopores. (a) Schematic of the nanopore‐seq library preparation for sequencing. Specific primers are annealed and ligated to the poly‐A tailed RNA which can be followed by an RT‐step or omitted. A motor sequencing adaptor protein is then ligated to the prepared RNA. (b) Protein I guides the RNA to the nanopore, unzips the RNA if double‐stranded, and controls the speed as the molecule translocates through the nanopore. Protein II, a sensor, resides in the narrowest part within the pore and detects the nucleotide specific disruption in electrical current and its retention time while the molecule passes through the pore. Comparing the signals obtained from the possible modified nucleosides to standards, base calling software can determine the sequence of the molecule in real‐time. (c) Identification of a m6A in nanopores has been achieved by comparing two sequences, one without (top) and one with the modification (bottom). An RNA modification in a sequence is generally characterized by a non‐standard drop in the ionic current and altered retention time in the pore, relative to standards (A, C, G, and U)
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Some RNA modifications can be identified by NGS of amplified products. (a) When treated with a bisulfite reagent, native C's are converted to U's whereas 5‐methylcytidine (m5C) remain unchanged. When the treated sample is subjected to reverse transcription, the bisulfite‐converted U base pairs with A whereas m5C base pairs with G creating a signature pattern detailing the locations of the m5C. Alternatively, m5C can also be detected using Aza‐IP as described in Section 3.1. (b) ARM‐seq relies on enzyme‐induced demethylation of methyl‐modified A, G (outlined here), and C, as the methylated forms promote reverse transcription errors. In ARM‐seq, demethylases are used to remove the methyl group which upon reverse transcription yields a mutation signature. (c) MeRIP‐seq relies on enrichment of modified RNA by antibody selection prior to sequencing, with this example specific to 1‐methyladenosine (m1A). The enrichment approach has also been also applied to 5‐hydroxymethylcytidine (hm5C), N4‐acetylcytidine (ac4C), m5C, and m6A (Amort et al., 2017; N. Liu et al., 2015; Shen et al., 2020)
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tRNA can be modified at many different positions. Some major sites of tRNA post‐transcriptional modification are indicated by bordered circles (in black). Position 34 in the tRNA anticodon loop contains a wide array of modifications that includes 5‐methoxycarbonylmethyluridine (mcm5U) and 2′O‐methylguanosine (Gm). Another hotspot for modification on the anticodon loop is position 37 which can have diverse set of modifications that includes N6‐threonylcarbamoyladenosine (t6A)
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RNA in Disease and Development > RNA in Disease
RNA Processing > tRNA Processing
RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems

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