Mapping the dsRNA World.

Long double-stranded RNAs (dsRNAs) are abundantly expressed in animals, in which they frequently occur in introns and 3' untranslated regions of mRNAs. Functions of long, cellular dsRNAs are poorly understood, although deficiencies in adenosine deaminases that act on RNA, or ADARs, promote their recognition as viral dsRNA and an aberrant immune response. Diverse dsRNA-binding proteins bind cellular dsRNAs, hinting at additional roles. Understanding these roles is facilitated by mapping the genomic locations that express dsRNA in various tissues and organisms. ADAR editing provides a signature of dsRNA structure in cellular transcripts. In this review, we detail approaches to map ADAR editing sites and dsRNAs genome-wide, with particular focus on high-throughput sequencing methods and considerations for their successful application to the detection of editing sites and dsRNAs.

Inverted repeat structures are associated with essential and highly expressed genes on C. elegans autosome distal arms.

Complementary sequences in cellular transcripts base-pair to form double-stranded RNA (dsRNA) structures. Because transposon-derived repeats often give rise to self-complementary sequences, dsRNA structures are prevalent in eukaryotic genomes, typically occurring in gene introns and untranslated regions (UTRs). However, the regulatory impact of double-stranded structures within genes is not fully understood. We used three independent methods to define loci in Caenorhabditis elegans predicted to form dsRNA and correlated these structures with patterns of gene expression, gene essentiality, and genome organization. As previously observed, dsRNA loci are enriched on distal arms of C. elegans autosomes, where genes typically show less conservation and lower overall expression. In contrast, we find that dsRNAs are associated with essential genes on autosome arms, and dsRNA-associated genes exhibit higher-than-expected expression and histone modification patterns associated with transcriptional elongation. Genes with significant repetitive sequence content are also highly expressed, and, thus, observed gene expression trends may relate either to dsRNA structures or to repeat content. Our results raise the possibility that as-yet-undescribed mechanisms promote expression of loci that produce dsRNAs, despite their well-characterized roles in gene silencing.

C. elegans ADARs antagonize silencing of cellular dsRNAs by the antiviral RNAi pathway.

Cellular dsRNAs are edited by adenosine deaminases that act on RNA (ADARs). While editing can alter mRNA-coding potential, most editing occurs in noncoding sequences, the function of which is poorly understood. Using dsRNA immunoprecipitation (dsRIP) and RNA sequencing (RNA-seq), we identified 1523 regions of clustered A-to-I editing, termed editing-enriched regions (EERs), in four stages of Caenorhabditis elegans development, often with highest expression in embryos. Analyses of small RNA-seq data revealed 22- to 23-nucleotide (nt) siRNAs, reminiscent of viral siRNAs, that mapped to EERs and were abundant in adr-1;adr-2 mutant animals. Consistent with roles for these siRNAs in silencing, EER-associated genes (EAGs) were down-regulated in adr-1;adr-2 embryos, and this was dependent on associated EERs and the RNAi factor RDE-4. We observed that ADARs genetically interact with the 26G endogenous siRNA (endo-siRNA) pathway, which likely competes for RNAi components; deletion of factors required for this pathway (rrf-3 or ergo-1) in adr-1;adr-2 mutant strains caused a synthetic phenotype that was rescued by deleting antiviral RNAi factors. Poly(A)+ RNA-seq revealed EAG down-regulation and antiviral gene induction in adr-1;adr-2;rrf-3 embryos, and these expression changes were dependent on rde-1 and rde-4 Our data suggest that ADARs restrict antiviral silencing of cellular dsRNAs.