Genome-wide profiling of the C. elegans dsRNAome

Abstract

Recent studies hint that endogenous dsRNA plays an unexpected role in cellular signaling. However, a complete understanding of endogenous dsRNA signaling is hindered by an incomplete annotation of dsRNA-producing genes. To identify dsRNAs expressed in Caenorhabditis elegans, we developed a bioinformatics pipeline that identifies dsRNA by detecting clustered RNA editing sites, which are strictly limited to long dsRNA substrates of Adenosine Deaminases that act on RNA (ADAR). We compared two alignment algorithms for mapping both unique and repetitive reads and detected as many as 664 editing-enriched regions (EERs) indicative of dsRNA loci. EERs are visually enriched on the distal arms of autosomes and are predicted to possess strong internal secondary structures as well as sequence complementarity with other EERs, indicative of both intramolecular and intermolecular duplexes. Most EERs were associated with protein-coding genes, with ∼1.7% of all C. elegans mRNAs containing an EER, located primarily in very long introns and in annotated, as well as unannotated, 3 ′ UTRs. In addition to numerous EERs associated with coding genes, we identified a population of prospective noncoding EERs that were distant from protein-coding genes and that had little or no coding potential. Finally, subsets of EERs are differentially expressed during development as well as during starvation and infection with bacterial or fungal pathogens. By combining RNA-seq with freely available bioinformatics tools, our workflow provides an easily accessible approach for the identification of dsRNAs, and more importantly, a catalog of the C. elegans dsRNAome.

Identification of the long, edited dsRNAome of LPS-stimulated immune cells

“…2A,B). Consistent with the fact that mice and humans do not have holocentric chromosomes, EERs did not cluster on chromosome arms as observed for Caenorhabditis elegans (Whipple et al 2015), but we did observe an abundance of EERs on human Chromosome 19. Chromosome 19 is dense with genes and repeats (Grimwood et al 2004), likely driving the higher abundance of EERs.…”

“…It is possible that deeper sequencing of the mouse transcriptome will reveal additional EERs. Previous work from our laboratory using the nematode C. elegans also revealed many EERs located within intronic sequences, suggesting that structured regions in the mouse may serve additional or different regulatory functions than those in the worm and human (Whipple et al 2015). Although the human transcriptome contained a lower fraction of EERs within 3 ′ UTRs relative to introns, the absolute number of EERs within 3 ′ UTRs is higher in human than mouse (307 hEERs versus 109 mEERs).…”

“…1B; Supplemental Fig. S1; Whipple et al 2015). EERs were first defined as 50-nt windows containing ≥3 editing sites.…”

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…Among the known substrates are mRNAs with hairpin structures in 3' UTRs; editing however does not necessarily prevent nuclear export and presence on polysomes (Morse and Bass, 1999;Morse et al, 2002;Hundley et al, 2008). Approximately 1.7% of C. elegans mRNAs contained such editing-enriched regions (Whipple et al, 2015). A-to-I editing also affects small RNAs.…”

Trans and cis factors affecting A-to-I RNA editing efficiency of a noncoding editing target in C. elegans

“…Similarly, downstream dsRNA hairpins within the introns of the human Gabra-3 and Neil1 transcripts have also been shown to increase editing efficiency of adjacent editing sites in coding regions (Daniel et al 2012(Daniel et al , 2014. While all of these examples involved regulating editing efficiency in coding regions, it is unclear whether this mechanism also extends to editing events in noncoding regions, which harbor the vast majority of editing sites identified to date (Bazak et al 2014;Washburn et al 2014;Whipple et al 2015).…”