Discovering and Mapping the Modified Nucleotides That Comprise the Epitranscriptome of mRNA

Abstract

An important mechanism of gene expression regulation is the regulated modification of nucleotides in messenger RNA (mRNA). These modified nucleotides affect mRNA translation, stability, splicing, and other processes. A cluster of nucleotide modifications is found adjacent to the mRNA cap structure and another set can be found internally within transcripts. The most prominent modifications are methylations of adenosine to form either N 6-methyladenosine (m 6 A), an internal modified nucleotide, or N 6 ,2 ′-O-dimethyladenosine (m 6 Am), which is found exclusively at the first templated nucleotide of certain mRNAs. In addition, other rare modified nucleotides have been identified and together these form the epitranscriptomic code of mRNA. In the case of some modified nucleotides, the presence, location, or abundance is a subject of debate. Here, we review the methods that enable the discovery of modified nucleotides and how these approaches can be used to map epitranscriptomic modifications in mRNA. Outline 1 Introduction 2 The history of the detection of methylated nucleotides in mRNA 3 Advantages and challenges of biochemical methods 4 Next-generation sequencing-based technologies for mapping internal nucleotide modifications 5 Mapping the cap epitranscriptome 6 Concluding remarks and further directions References

Multiple links between 5-methylcytosine content of mRNA and translation

“…The merits of using bsRNA-seq to discover m 5 C sites transcriptome-wide have been controversially discussed (Legrand et al 2017;Linder and Jaffrey 2019;Motorin and Helm 2019;Trixl and Lusser 2019). We contend that the bsRNA-seq approach as presented here is fit-for-purpose as it is based on a combination of efficient bisulfite reaction conditions, bespoke read mapping and conservative site calling from replicate data.…”

“…Expansion in scope and refinement of such methods (Schaefer et al 2017;Linder and Jaffrey 2019; Motorin and Helm 2019) have now produced maps of several modifications in tissues and cells of diverse origins (Fray and Simpson 2015;Burgess et al 2016;Marbaniang and Vogel 2016;Kennedy et al 2017;Shen et al 2019), spawning the term 'epitranscriptomics' to mainly (but not exclusively) refer to research into the function of modified nucleosides in mRNA ). Challenges exist not only in the accurate detection of these generally sparse modifications but also in ascribing molecular, cellular and organismic functions to these 'epitranscriptomic marks'-in mRNA (Sibbritt et al 2013;Peer et al 2017;Nachtergaele and He 2018) as well as in ncRNA Jacob et al 2017).…”

“…it also detects hm 5 C) and is affected by incomplete conversion of unmodified cytosines due to RNA structure and the variability in reaction conditions. Stringency criteria to balance false negative against false positive site calls have also been set differently between studies, leading to, for example, drastically different estimates for sites in mRNA from a handful to thousands (Legrand et al 2017;Linder and Jaffrey 2019). Regardless of chosen method, a redeeming feature might be that with greater insight into experimental limitations and with refinement of bioinformatic methods, new, better quality m 5 C epitranscriptomic maps can supersede early, pioneering attempts.…”

Occurrence and Functions of m6A and Other Covalent Modifications in Plant mRNA

“…Although chemically modified nucleotides have been known to exist in RNA, even in mRNA, since more than 40 years (Holley et al, 1965;Desrosiers et al, 1974;Perry and Kelley, 1974;Boccaletto et al, 2018), the mRNA modification field has only taken off with the recent introduction of high-throughput sequencingbased methods to map modified nucleotides in specific mRNAs transcriptome wide (see Box 1 for a historical context of the development of the field). Three main categories of such methods enjoy widespread use (see Linder and Jaffrey [2019] for a detailed review focused on methods). (1) Antibodies specific to a modification are used to immunoprecipitate fragmented mRNA, and RNA sequencing (RNAseq) is applied to input and immunoprecipitated RNA fractions.…”

Multiple links between 5-methylcytosine content of mRNA and translation

“…Expansion in scope and refinement of such methods [10][11][12] have now produced maps of several modifications in tissues and cells of diverse origins [13][14][15][16][17], spawning the term 'epitranscriptomics' to mainly (but not exclusively) refer to research into the function of modified nucleosides in mRNA [18]. Challenges exist not only in the accurate detection of these generally sparse modifications but also in ascribing molecular, cellular and organismic functions to these 'epitranscriptomic marks'-in mRNA [19][20][21] as well as in ncRNA [22,23].…”