Effects of ADARs on small RNA processing pathways in C. elegans

Abstract

Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). To evaluate effects of ADARs on small RNAs that derive from dsRNA precursors, we performed deep-sequencing, comparing small RNAs from wild-type and ADAR mutant Caenorhabditis elegans. While editing in small RNAs was rare, at least 40% of microRNAs had altered levels in at least one ADAR mutant strain, and miRNAs with significantly altered levels had mRNA targets with correspondingly affected levels. About 40% of siRNAs derived from endogenous genes (endo-siRNAs) also had altered levels in at least one mutant strain, including 63% of Dicer-dependent endo-siRNAs. The 26G class of endo-siRNAs was significantly affected by ADARs, and many altered 26G loci had intronic reads and histone modifications associated with transcriptional silencing. Our data indicate that ADARs, through both direct and indirect mechanisms, are important for maintaining wild-type levels of many small RNAs in C. elegans.

Figure 1.

ADARs affect miRNA and mRNA target levels, but few miRNAs are edited. (A,B) Number of loci with increased (A) or decreased (B) reads in ADAR mutants compared to WT were plotted on the y-axis. miRNA loci were binned (x-axis) according to their fold-change in a mutant strain compared to WT. All miRNA loci altered ≥2-fold were affected in at least one mutant strain; loci altered ≥1.2-fold but <2-fold were affected in at least two mutant strains. (C) Representative Northern blot of mirVana enriched RNA (2 μg) from indicated strains, probed for miRNAs, with U6 as a loading control. Fold-change determined by sequencing (illum) and Northern blot (north) were tabulated, comparing each mutant to WT. Blanks indicate sequencing did not predict a significant difference. (Error) Standard deviation (STD), adr-1(−/−);adr-2(−/−) abbreviated adr-1;2. (D) Fold-change of pri-miRNA in ADAR mutants compared to WT, as determined by qRT-PCR, was compared to change in miRNA. Levels of miR-800 and miR-1830 were determined by Northern blot, miR-253 by sequencing; the latter did not show a significant change in single mutants. (E) Fold-change of miR-800 (see Fig. 1C, Northern blot) compared to fold-change of predicted mRNA targets, as determined by microarray analyses (adr-1[−/−];adr-2[−/−] vs. WT worms) (Supplemental Table S2). (Error bars) Standard error of the mean (SEM) for mRNAs, STD for miR-800. (F) qRT-PCR validation of miR-800 mRNA target levels in adr-1(−/−);adr-2(−/−) worms compared to WT. (Error bars) SEM for microarray, STD for qRT-PCR. (G) Structure of pri-mir-800, with miRNA sequence underlined and bold and miRNA* sequence italicized. Percent editing at indicated adenosine (double arrowhead) is tabulated for each strain. (Single arrowheads) Drosha and Dicer cleavage sites.

Figure 2.

Many endo-siRNA loci show altered reads in ADAR mutant strains. Plots show number of loci (y-axis) with decreased (A) or increased (B) antisense reads in indicated strains compared to WT, or decreased (C) or increased (D) sense reads in indicated strains compared to WT. Loci were binned (x-axis) according to observed fold-change. Loci altered ≥2-fold were affected in at least one mutant strain; loci altered ≥1.2-fold but <2-fold were affected in a least two mutant strains.

Figure 3.

Overview of endo-siRNA loci affected by ADARs. (Pie charts) Number and percent of loci affected by different ADARs or Dicer's helicase domain. Loci affected in both adr-1(−/−) and adr-2(−/−) are abbreviated ADR-1 + 2.

Figure 4.

Many endo-siRNA loci have levels affected by both Dicer and ADARs. (A,B) Plots show change in read numbers (as % of WT) observed in the dcr-1(−/−)K39A strain compared to adr-1(−/−) (A) or adr-2(−/−) (B) strains. Points are differentiated based on the strand affected in the ADAR mutant strain (sense or antisense) and whether the affected locus gives rise to 26G endo-siRNAs. Of the 1173 loci with decreased reads in the dcr-1(−/−)K39A strain compared to WT, 299 had altered sense reads, 791 had altered antisense reads, and 99 had changes in both. (C,D) Distributions of antisense and sense reads from the Inverse and Correlated classes, differentiating reads based on length.

Figure 5.

ADAR-affected 26G loci have small RNA reads that can base-pair in specific structures and have histone modification patterns associated with transcriptional silencing. (A) Bar height indicates number of termini with a given terminus from putative Inverse 26G structures, relative to the antisense strand. If multiple reads could form different structures at a given location, the structure with the predominate read was analyzed. (B) Consensus structure of predominant structures from 26G loci. Nearly all reads had a 5′ guanosine, while other positions varied. (C) Histone modification and histone variant patterns for various groups of endo-siRNA loci. χ² tests compare patterns from each group to the random data set, with P-values < 0.05 (*) or < 1 × 10⁻¹⁰ (**). (cor) Correlated loci, (inv) Inverse loci, (inv 26) Inverse 26G loci.

Figure 6.

Reads from the F43E2.6 locus are edited. Putative dsRNA is shown with the predominant sense read (underlined, bold) pairing with the predominant antisense read (italicized). (Double arrowheads) Edited adenosines, with percent editing tabulated for each strain. (Single arrowheads) Termini of predominant reads.

Figure 7.

Model of ADARs' effects on the biogenesis of C. elegans small RNAs. Direct (red X) and indirect (blue X) effects are indicated. In WT cells (top), ADAR binding to certain dsRNAs competes with binding by other dsRBPs, in some cases inhibiting processing (e.g., pri-miRNAs, Inverse endo-siRNA precursors), while ADAR editing (red *) inhibits processing of other RNAs (e.g., rncs-1). Indirect effects arise in mutant strains when dsRNAs normally bound or edited by ADARs (e.g., miRNAs, rncs-1) are released to compete with dsRNA in other pathways.