First genome-wide identification of short cyclic phosphate-containing RNAs

Our comprehensive identification of 2′,3′-cyclic phosphate-containing RNAs (cP-RNAs) unraveled a novel, abundant class of non-coding RNAs which may play a role in aging.

Cells express immensely diverse species of RNA molecules that play essential roles in numerous biological processes. With the advent of next-generation sequencing (NGS) technology, efforts to identify the expressed RNA molecules have greatly advanced our understanding of RNA biology. Although NGS-based RNA-sequencing (RNA-seq) has become a ubiquitous tool in biological and medical research, the current standard RNA-seq methods, particularly those targeting short non-coding RNAs (ncRNAs), do not fully capture all of the RNAs expressed but allow for some “escapers” to slip by undetected. RNAs generated by ribonuclease cleavage sometimes harbor a 2′,3′-cyclic phosphate (cP) at their 3′-ends, and those cP-containing RNAs (cP-RNAs) are one such escaper that are not ligated to a 3′-adapter and thus uncaptured by standard RNA-seq.

An increasing number of studies has been suggesting biological significances of the expression of cP-RNAs. Yet, cP-RNAs remained a hidden component in the transcriptome, infrequently recognized and characterized. Given that cP-RNAs are expressed as functional molecules, capturing the entire repertoire of cP-RNAs would broaden the catalog of functional ncRNAs and reveal significant biological events that have been eluding standard RNA-seq. We previously developed “cP-RNA-seq,” which is able to specifically sequence cP-RNAs (Honda et al., PNAS 2015; Honda et al., Nat Protoc 2016).

Here we provide first genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. Using cP-RNA-seq that can exclusively sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from cytoplasmic transfer RNAs (tRNAs), messenger RNAs (mRNAs), and ribosomal RNAs (rRNAs). Determination of the processing sites of substrate RNAs for cP-RNA generation revealed highly-specific RNA cleavage events between cytidine and adenosine in cP-RNA biogenesis. cP-RNAs were not evenly derived from the overall region of substrate RNAs but rather from specific sites, implying that cP-RNAs are not from random degradation but are produced through a regulated biogenesis pathway. The identified cP-RNAs were abundantly accumulated in mouse tissues, and the expression levels of cP-RNAs showed age-dependent reduction. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles.

This paper was also covered on EurekAlert! Click here to read more.

References

  1. Shigematsu, M, Morichika, K, Kawamura, T, Honda, S, Kirino, Y. Genome-wide identification of short 2',3'-cyclic phosphate-containing RNAs and their regulation in aging. PLoS Genet. 2019;15 (11):e1008469. doi: 10.1371/journal.pgen.1008469. PubMed PMID:31721758 PubMed Central PMC6853296.

Comments are closed