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Epitranscriptomic Research

Why Is Epitranscriptomic Stoichiometry Vital for RNA Modification Study?


RNA modifications, such as m6A, m1A, m5C, and pseudouridine, together form the epitranscriptome and collectively encode a new layer of gene expression regulation. m6A, the most abundant internal modification in mRNAs and lncRNAs, impacts all aspects of post-transcriptional mRNA/lncRNA metabolism and functions [1]. In addition, m6A are also involved in many other ncRNA functions, including cap-independent translation initiation of circRNA[2], and pri-miRNA processing[3].

The potential effects of RNA modifications depend on not only which gene transcripts, but also the percentage of transcripts that are modified. However, current transcriptome-wide RNA modification profiling methods deal mostly with mapping the modified sites but are unable to quantify the percentage of modified RNA for that transcript. The lack of such quantitative information has been a major concern for scientists (Text box).

The scientists’ top concerns

"The potential effect of an mRNA modification depends on both the molecular consequences and the percentage of transcripts that are modified. For example, a modification that leads to accelerated mRNA decay is unlikely to have much biological effect if only 1% of transcripts are modified, whereas a modification that causes an alternative protein variant to be produced could be functionally important, even at very low levels. A limitation of current m6A and pseudouridine profiling methods is the lack of quantitative information about the extent of modification. Changes in the relative enrichment of a particular sequence in m6A pulldowns from different growth states have been used to infer regulation of modification, but the absolute fraction of mRNA that is modified cannot be determined from these data… High-throughput methods to quantify site-specific m6A and pseudouridine would considerably advance the field.”

[1] Wendy V. Gilbert, Tristan A. Bell, Cassandra Schaening. Science (2016)

"Another important aspect that is yet to be addressed is the dynamics of RNA modification stoichiometry. Current epitranscriptome studies deal mostly with identifying which sites are modified and not the fraction of RNAs in which each site is modified. Low-throughput analysis of m6A modification sites in mRNA and viral RNA shows that no m6A site is modified in 100% of transcripts. Changes in modification stoichiometry may also represent a dynamic parameter of RNA modification biology. As modifications can affect mRNA structure and/or the recruitment of RBPs, modification of a fraction of transcripts at any specific site would generate two distinct mRNA species that differ only in their structures or the readers that bind to them. Therefore, changing modification stoichiometry could represent another mechanism to generate functional diversity from the same RNA transcript. High-throughput methods that can determine modification stoichiometry are needed to address this aspect of the epitranscriptome.”

[4] Cole J.T. Lewis, Tao Pan, Auinash Kalsotra. Nat Rev Mol Cell Biol (2017)

Arraystar Epitranscriptomic Microarrays combine the two-color channel microarray technology with RNA modification immunoprecipitation to quantify the percentage of RNA that are modified at transcript isoform-specific level. The microarrays cover the epitranscriptomes of mRNA, lncRNA, circRNA, pre-miRNA, pri-miRNA, snoRNA, and snRNA classes. The quantitative epitranscriptomic profiling provides the vital information to study the regulatory impacts of RNA modifications.

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1.  Gilbert WV, Bell TA, Schaening C: Messenger RNA modifications: Form, distribution, and function. Science 2016, 352(6292):1408-1412.[PMID: 27313037]
2.  Yang Y et al: Extensive translation of circular RNAs driven by N(6)-methyladenosine. Cell Res 2017, 27(5):626-641.[PMID: 28281539]
3.  Alarcon CR et al: HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events. Cell 2015, 162(6):1299-1308.[PMID: 26321680]
4.  Lewis CJ, Pan T, Kalsotra A: RNA modifications and structures cooperate to guide RNA-protein interactions. Nat Rev Mol Cell Biol 2017, 18(3):202-210.[PMID: 28144031]
5.  Qin Y et al: TRIM9 short isoform preferentially promotes DNA and RNA virus-induced production of type I interfe*** by recruiting GSK3beta to TBK1. Cell Res 2016, 26(5):613-628.[PMID: 26915459]

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