Methods for RNA Modification Mapping Using Deep Sequencing: Established and New Emerging Technologies

Abstract

New analytics of post-transcriptional RNA modifications have paved the way for a tremendous upswing of the biological and biomedical research in this field. This especially applies to methods that included RNA-Seq techniques, and which typically result in what is termed global scale modification mapping. In this process, positions inside a cell`s transcriptome are receiving a status of potential modification sites (so called modification calling), typically based on a score of some kind that issues from the particular method applied. The resulting data are thought to represent information that goes beyond what is contained in typical transcriptome data, and hence the field has taken to use the term "epitranscriptome". Due to the high rate of newly published mapping techniques, a significant number of chemically distinct RNA modifications have become amenable to mapping, albeit with variegated accuracy and precision, depending on the nature of the technique. This review gives a brief overview of known techniques, and how they were applied to modification calling.

Keywords: Next Generation Sequencing; RNA modification; RNA-Seq; chemical treatment; deep sequencing; engineered Reverse Transcriptase enzymes; epitranscriptome.

Figure 1

Detection of 2′-O-methylation, m⁶A and AlkB-sensitive modifications in RNA (a) Engineered polymerase arrests reverse transcription at 2′-O-methylated residue at normal [dNTP]; (b) Ab-independent detection of m⁶A residue using an engineered polymerase or a 4-Se derivative of dTTP (4Se-dTTP). The presence of an m⁶A residue in the RNA template is reflected by a misincorporation signature in the cDNA; (c) Improved confidence in the detection of AlkB-sensitive RNA modifications (m³C, m¹A, m¹G, and m²₂G) comes from comparison of RT-signatures (including misincorporation and RT-arrest) before and after AlkB treatment.

Figure 2

Use of specific chemical reagents for detection of s⁴U, inosine (I) and m⁷G/m³C: (a) 4-thiouridine (s⁴U) readily reacts with iodoacetamide, with subsequent change of base-paring properties. After iodoacetamide treatment, derivatized s⁴U is read as C during RT step and this U- > C transition is detected in the sequencing data; (b) Detection of inosine (I) in RNA by acrylonitrile derivatization (ICE-Seq). Unmodified inosine gives an A- > G misincorporation signature, which can be distinguished from SNPs by additional treatment with acrylonitrile. The resulting derivatized inosine creates abortive products during RT-primer extension; (c) AlkAniline-Seq protocol for detection of m⁷G and m³C residues in RNA. Mild alkaline hydrolysis creates an RNA abasic site at the sensitive residue and subsequent aniline treatment releases 5′-phosphate at the N + 1 nucleotide. These 5′-phosphates are used for selective adapter ligation during library preparation step. AlkAniline-Seq signal is detected as a substantial proportion of sequencing reads starting at the same position.