Evolutionary analysis reveals regulatory and functional landscape of coding and non-coding RNA editing

Abstract

Adenosine-to-inosine RNA editing diversifies the transcriptome and promotes functional diversity, particularly in the brain. A plethora of editing sites has been recently identified; however, how they are selected and regulated and which are functionally important are largely unknown. Here we show the cis-regulation and stepwise selection of RNA editing during Drosophila evolution and pinpoint a large number of functional editing sites. We found that the establishment of editing and variation in editing levels across Drosophila species are largely explained and predicted by cis-regulatory elements. Furthermore, editing events that arose early in the species tree tend to be more highly edited in clusters and enriched in slowly-evolved neuronal genes, thus suggesting that the main role of RNA editing is for fine-tuning neurological functions. While nonsynonymous editing events have been long recognized as playing a functional role, in addition to nonsynonymous editing sites, a large fraction of 3’UTR editing sites is evolutionarily constrained, highly edited, and thus likely functional. We find that these 3’UTR editing events can alter mRNA stability and affect miRNA binding and thus highlight the functional roles of noncoding RNA editing. Our work, through evolutionary analyses of RNA editing in Drosophila, uncovers novel insights of RNA editing regulation as well as its functions in both coding and non-coding regions.

Adaptation of A-to-I RNA editing in Drosophila

“…melanogaster and D . pseudoobscura , we found 416 (41.8%) and 78 (17.3%) of them are located outside the coding regions, respectively (Tables 2 and 3), which is consistent with a recent study [84] and suggests a possible functional role for these sites, such as influencing alternative splicing [26–29], microRNA targeting [30–32], or other cellular processes related to RNAs [14]. …”

“…Meanwhile, we conducted the same analysis on the 171 sites that have editing events fixed in both females and males, however, we did not observe any of those sites have editing levels associated with SNPs under the same criteria. These comparisons suggest cis-regulatory elements affect the levels or status of editing across strains, as previously observed [41, 84, 92]. …”

Learning cis-regulatory principles of ADAR-based RNA editing from CRISPR-mediated mutagenesis

“…The structure-similarity score of variants compared to WT feature ("sim_nor_score") was positively correlated with editing levels. These four features corroborate with previous results that the overall structural stability of RNA substrate is positively correlated with the editing activity 18 . The seventh ranking feature was the position of the mutation along the RNA molecule ("mut_pos").…”

“…We reasoned that features affecting RNA thermodynamics could also affect editing efficiency 18 . We observed significant differences in the Free Energy ( Fig.…”

“…It is proposed that both the primary sequence and secondary structure (i.e., cis-acting regulatory elements) guide the preference and specificity of ADARs 7, [14][15][16][17][18][19] . ADAR has a preferred sequence motif, in particular the 5' and 3' nearest neighboring positions (-1 and +1 nt) to the editing site [14][15][16][17][18][19] . Editing can be enhanced or suppressed by deviations from perfect base-pairing (i.e., mismatches, bulges and loops), suggesting complex structural contributions to editing specificity [14][15][16] .…”