How does RNA editing affect dsRNA-mediated gene silencing?

Abstract

In general, double-stranded RNA (dsRNA)-binding proteins (dsRBPs) are not sequence-specific. A dsRNA molecule in a cell will interact with any dsRBP it comes in contact with, suggesting that different dsRNA-mediated pathways intersect and affect each other. This paper analyzes evidence that the ADAR RNA editing enzymes, which act on dsRNA, affect dsRNA-mediated gene silencing pathways. Examples of how ADARs alter gene silencing pathways such as RNA interference, as well as mechanisms that allow the pathways to coexist and maintain their unique functions, are discussed.

Figure 1

(A) The illustration depicts a pre-mRNA that contains examples of structures that are selectively (upper) and nonselectively (lower) edited. Coding exons are depicted in blue, an intron in gray, and 5′ and 3′ UTRs as black lines. Parallel lines represent base-paired regions with dots representing unpaired nucleotides. The selectively edited hairpin involves pairing between exon and intron sequences and is patterned after the R/G editing site, shown as a white A, found in certain mammalian glutamate receptor pre-mRNAs; the hairpin contains 28 bps, a loop, and mismatches as indicated (Aruscavage and Bass 2000). The nonselectively edited structure is patterned after the 3′UTR of the C. elegans gene, C35E7.6 (Morse et al. 2002). The edited structures are scaled relative to their actual lengths (20 nucleotides indicated), but 520 nucleotides of each strand of the 3′UTR structure were omitted as indicated. (B) The cartoon illustrates that the number of adenosines deaminated by an ADAR at reaction completion, or the selectivity of the enzyme, increases with the thermodynamic stability of the RNA structure. The top structure represents a long, completely base-paired dsRNA ≥ 50 bp which is deaminated nonselectively, showing 50–60% of its adenosines converted to inosines (red diamonds) at reaction completion. As indicated, structures that are less stable because they are shorter, or interrupted by mismatches, bulges, or loops, contain fewer inosines at the end of the reaction. Blue lines represent a specific sequence that exists as a separate molecule or between two internal loops of a longer structure.

Figure 2

The cartoon illustrates the proposed pathway leading to transgene silencing in C. elegans strains that lack ADARs (adr(−/−)). Starting at the upper left corner, DNA encoding GFP (green rectangle) is injected into a worm, whereby it is covalently linked to form repetitive arrays that are maintained as extrachromosomal elements. In wild-type animals, GFP mRNA is expressed, as evidenced by strong GFP fluorescence (bottom left corner). Black arrows indicate the direction of transcription, and as shown, readthrough transcription of the repetitive array also gives rise to sense and antisense RNAs that hybridize to form dsRNA. In a wild-type animal, this dsRNA is deaminated by ADARs and thus does not lead to silencing. In animals lacking ADARs (adr(-/-)), the dsRNA enters the RNAi pathway, leading to silencing. Photographs of worms were taken by Jeff Habig, and strains are as described (Knight and Bass 2002).

Figure 3

The lowest free-energy secondary structure is shown for four miRNA precursors, as predicted by mfold (Mathews et al. 1999; Zuker 2003). Nucleotides shown in red represent mature miRNA sequences as experimentally determined (Lagos-Quintana et al. 2002; Houbaviy et al. 2003; Poy et al. 2004). When incubated with recombinant ADAR, inosine can be detected within the two pri-miRNA sequences above the dotted line (edited), whereas inosine cannot be detected in those below the line (unedited; as reported in Yang et al. 2006).