RNA editing contributes to epitranscriptome diversity in chronic lymphocytic leukemia

RNA editing—primarily conversion of adenosine to inosine (A > I)—is a widespread posttranscriptional mechanism, mediated by Adenosine Deaminases acting on RNA (ADAR) enzymes to alter the RNA sequence of primary transcripts. Hence, in addition to somatic mutations and alternative RNA splicing, RNA editing can be a further source for recoding events. Although RNA editing has been detected in many solid cancers and normal tissue, RNA editing in chronic lymphocytic leukemia (CLL) has not been addressed so far. We determined global RNA editing and recurrent, recoding RNA editing events from matched RNA-sequencing and whole exome sequencing data in CLL samples from 45 untreated patients. RNA editing was verified in a validation cohort of 98 CLL patients and revealed substantially altered RNA editing profiles in CLL compared with normal B cells. We further found that RNA editing patterns were prognostically relevant. Finally, we showed that ADAR knockout decreased steady state viability of MEC1 cells and made them more susceptible to treatment with fludarabine and ibrutinib in vitro. We propose that RNA editing contributes to the pathophysiology of CLL and targeting the RNA editing machinery could be a future strategy to maximize treatment efficacy

Elevated RNA Editing Activity Is a Major Contributor to Transcriptomic Diversity in Tumors

Genomic mutations in key genes are known to drive tumorigenesis and have been the focus of much attention in recent years. However, genetic content also may change farther downstream. RNA editing alters the mRNA sequence from its genomic blueprint in a dynamic and flexible way. A few isolated cases of editing alterations in cancer have been reported previously. Here, we provide a transcriptome-wide characterization of RNA editing across hundreds of cancer samples from multiple cancer tissues, and we show that A-to-I editing and the enzymes mediating this modification are significantly altered, usually elevated, in most cancer types. Increased editing activity is found to be associated with patient survival. As is the case with somatic mutations in DNA, most of these newly introduced RNA mutations are likely passengers, but a few may serve as drivers that may be novel candidates for therapeutic and diagnostic purposes

Dynamic hyper-editing underlies temperature adaptation in <i>Drosophila</i>

<div><p>In <i>Drosophila</i>, A-to-I editing is prevalent in the brain, and mutations in the editing enzyme ADAR correlate with specific behavioral defects. Here we demonstrate a role for ADAR in behavioral temperature adaptation in <i>Drosophila</i>. Although there is a higher level of editing at lower temperatures, at 29°C more sites are edited. These sites are less evolutionarily conserved, more disperse, less likely to be involved in secondary structures, and more likely to be located in exons. Interestingly, hypomorph mutants for ADAR display a weaker transcriptional response to temperature changes than wild-type flies and a highly abnormal behavioral response upon temperature increase. In sum, our data shows that ADAR is essential for proper temperature adaptation, a key behavior trait that is essential for survival of flies in the wild. Moreover, our results suggest a more general role of ADAR in regulating RNA secondary structures <i>in vivo</i>.</p></div

EvoFold analysis of hyper-editing sites.

<p>EvoFold analysis of hyper-editing sites.</p

Editing sites at lower temperatures are edited more frequently and are more commonly flanked by complementary sequences.

<p><b>(A)</b> Mean conservation (PhastCons) score of hyper-edited sites. Position 0 indicates the position of editing site. Blue line denotes conservation mean for editing sites supported by more than one event, red line denoted conservation mean for editing sites supported by only one event, and black line represents background conservation of chosen randomly adenosines. Left figure represents all genome wide hyper-editing sites, while the right figure represents hyper-editing sites in coding regions (CDS). The information from the non-hyper-edited reads was included. <b>(B)</b> RNA secondary structure prediction using BLAST[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006931#pgen.1006931.ref050" target="_blank">50</a>] tool (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006931#sec009" target="_blank">Methods</a>). Blue bars donate for predicted dsRNA structure involving the hyper-editing site, as we succeeded to match the editing regions with their anti-sense sequence. Red bars denote for matches found in the sense sequence, representing the control. Green bars denote for predicted dsRNA structure involving the hyper-editing site after converting the adenosine (A) to its edited form, guanosine (G). Violet bars represents the control for the converted adenosines. <b>(C)</b> Genomic locations of detected hyper-editing sites show increase in the number of exonic sites at 29°C.</p

Functional analysis of hyper-edited genes.

<p>Functional analysis of hyper-edited genes.</p

ADAR hypomorph flies display temperature dependent behavioral abnormalities.

<p><b>(A)</b> ADAR hypomorph flies (red) are less active than control flies (blue) both at 18°C and 29°C. Total activity per day obtained by adding the average activity during the light and dark periods (8 days). N = 32 and 29 for hypomorph flies at 18°C and 29°C respectively and N = 27 for control flies at both temperatures. Statistical significance was assessed by Student-t test. Error bars represents SEM. <b>(B)</b> Although less active than their controls, at 18°C, the pattern of day-night activity of ADAR hypomorph and control flies is similar, with higher activity during the day. We calculated and ploted the average activity during the light (9 days) or dark periods (8 nights). Statistical significance was assessed by Student-t test. Error bars represents SEM. <b>(C)</b> At 29°C, control flies increase their night activity whereas the ADAR hypomorph flies remaine active mostly during the day. Statistical significance was assessed by Student-t test. Error bars represents SEM. <b>(D)</b> Behavioral activity assay for control (left) and ADAR hypomorph flies (right) that were exposed to 12:12h light:dark (L:D) cycles at 29°C for 4 days and then transferred to 18°C (L:D cycles) for 5 days. N = 29 for control and N = 32 for Adar hypomorph flies. An arrow marks the transition time point. Error bars represent SEM. <b>(E)</b> same as in (D), with the opposite temperature transfer, from 18 to 29°C. N = 30 for control and N = 31 for ADAR hypomorph flies. An arrow marks the transition time point.</p

The degree and prevalence of A-to-I RNA editing are dynamically affected by temperature.

<p><b>(A)</b> Generation of editing list by combining the RADAR database (2,697 sites), Rennan's and Rosbash's datasets[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006931#pgen.1006931.ref011" target="_blank">11</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006931#pgen.1006931.ref032" target="_blank">32</a>] (3,580 and 1,341 sites respectively) with novel hyper-editing sites detected by our method (30,190 sites). This resulted in a list of 32,974 unique sites, containing 11,097 editing sites in CDS. <b>(B)</b> Hyper-editing motif. The sequence near the hyper-editing sites is depleted of Gs upstream and enriched with Gs downstream as expected from ADAR targets. <b>(C)</b> Editing index, fraction of inosines among all expressed adenosines of all detected editing sites, show lower editing levels at 29°C. <b>(D)</b> Editing levels of significantly altered 55 editing sites in CDS. Each site is presented by a number which indicates its position in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006931#pgen.1006931.s006" target="_blank">S1 Table</a>. <b>(E)</b> The distribution of hyper-editing detected sites, shows higher number of sites found at elevated temperature. <b>(F)</b> Average hyper-editing events per detected sites. Statistical significance between 18°C and 29°C was assessed by Student-t test (p<10⁻⁴). <b>(G)</b> Editing cluster's difference between temperatures. Left panel presents the average cluster length for each temperature. Right panel presents the average unique number of detected editing-sites for each temperature.</p

Identification of ADAR1 adenosine deaminase dependency in a subset of cancer cells

Systematic exploration of cancer cell vulnerabilities can inform the development of novel cancer therapeutics. Here, through analysis of genome-scale loss-of-function datasets, we identify adenosine deaminase acting on RNA (ADAR or ADAR1) as an essential gene for the survival of a subset of cancer cell lines. ADAR1-dependent cell lines display increased expression of interferon-stimulated genes. Activation of type I interferon signaling in the context of ADAR1 deficiency can induce cell lethality in non-ADAR1-dependent cell lines. ADAR deletion causes activation of the double-stranded RNA sensor, protein kinase R (PKR). Disruption of PKR signaling, through inactivation of PKR or overexpression of either a wildtype or catalytically inactive mutant version of the p150 isoform of ADAR1, partially rescues cell lethality after ADAR1 loss, suggesting that both catalytic and non-enzymatic functions of ADAR1 may contribute to preventing PKR-mediated cell lethality. Together, these data nominate ADAR1 as a potential therapeutic target in a subset of cancers