Mapping the dsRNA World

Abstract

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.

Figure 1.

Representative double-stranded RNAs (dsRNAs) from three organisms. UNAfold-predicted RNA structures are shown for mouse Sppl2a 3′ untranslated region (3′ UTR), human SPPL2A 3′ UTR, and Caenorhabditis elegans eif-2α pre-mRNA. Approximate lengths of highly base-paired regions are shown for scale, and minimum (most stable) predicted folding free energies (ΔG) are reported beneath structures.

Figure 2.

Mouse, human, and Caenorhabditis elegans dsRNAomes. Vertical black lines denote positions of editing enriched regions (EERs) on chromosomes of mouse, human, and C. elegans. Chromosomes are not drawn to scale, so the horizontal black bars at the bottom display relative chromosome length. Maps of mouse and human dsRNAomes were generated with Idiographica and that for C. elegans with PhenoGram (Kin and Ono 2007; Wolfe et al. 2013). BMDMs, bone marrow–derived macrophages.

Figure 3.

Repeat content of mouse, human, and Caenorhabditis elegans editing enriched regions (EERs). Pie charts depict percentage of total EER sequences that overlap RepeatMasker-annotated repeats (mouse: mm10; human: hg19; C. elegans: ce10). Major classes of repetitive elements (>2% of total EER sequence) are labeled, and classes comprising <2% are grouped as “Other.” Nonrepetitive sequences did not overlap any sequences annotated as repetitive by RepeatMasker. SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; LTR, long terminal repeat; DNA TE, DNA transposable element.

Figure 4.

Expression of Caenorhabditis elegans editing enriched region (EER)-associated genes (EAGs). Tukey box plot shows gene expression, in fragments per kilobase*million reads (FPKM), for all expressed genes or EAGs in RNA-seq of four C. elegans developmental stages: embryo, early larval (E. larval; L1–L2), late larval (L. larval; L3–L4), and young adult (Y. adult) stages. ****, P < 0.0001, Mann–Whitney U-test.

Figure 5.

Predicted intron structure along chromosome III for two nematode species. Length-normalized UNAFold-predicted folding free energies are plotted by relative position on chromosome III of Caenorhabditis elegans (blue) and Caenorhabditis briggsae (red). Trends observed on chromosome III are representative of all C. elegans autosomes. Average intronic ΔG/nt values were calculated by splitting chromosomes into 1000 equal-length segments and averaging ΔG/nt values of introns in each segment. Lower ΔG/nt values indicate presence of more stable intronic structures. ΔG, predicted folding free energy; nt, nucleotide.