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

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

EvoFold analysis of hyper-editing sites.

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

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

Fmrp Interacts with Adar and Regulates RNA Editing, Synaptic Density and Locomotor Activity in Zebrafish

<div><p>Fragile X syndrome (FXS) is the most frequent inherited form of mental retardation. The cause for this X-linked disorder is the silencing of the fragile X mental retardation 1 (<i>fmr1</i>) gene and the absence of the fragile X mental retardation protein (Fmrp). The RNA-binding protein Fmrp represses protein translation, particularly in synapses. In <i>Drosophila</i>, Fmrp interacts with the adenosine deaminase acting on RNA (Adar) enzymes. Adar enzymes convert adenosine to inosine (A-to-I) and modify the sequence of RNA transcripts. Utilizing the <i>fmr1</i> zebrafish mutant (<i>fmr1</i>-/-), we studied Fmrp-dependent neuronal circuit formation, behavior, and Adar-mediated RNA editing. By combining behavior analyses and live imaging of single axons and synapses, we showed hyperlocomotor activity, as well as increased axonal branching and synaptic density, in <i>fmr1</i>-/- larvae. We identified thousands of clustered RNA editing sites in the zebrafish transcriptome and showed that Fmrp biochemically interacts with the Adar2a protein. The expression levels of the <i>adar</i> genes and Adar2 protein increased in <i>fmr1</i>-/- zebrafish. Microfluidic-based multiplex PCR coupled with deep sequencing showed a mild increase in A-to-I RNA editing levels in evolutionarily conserved neuronal and synaptic Adar-targets in <i>fmr1</i>-/- larvae. These findings suggest that loss of Fmrp results in increased Adar-mediated RNA editing activity on target-specific RNAs, which, in turn, might alter neuronal circuit formation and behavior in FXS.</p></div

Targeted resequencing by mmPCR revealed differential RNA editing levels in <i>fmr1</i>-/- larvae.

<p><b>A.</b> Schematic representation of the three major steps in the amplification and quantification of multiple RNA editing sites by next-generation sequencing. 1. A microfluidic-based PCR using the Fluidigm Access Array platform on the IFC chip (sample and primer inlets, black arrows) generates 48 targeted amplicons from 48 different samples. Schematic representation of the “on-chip” PCR; target regions (blue lines) contain a targeted RNA editing site (red circle) amplified by PCR with forward and reverse target-specific primers (TSP-F/TSP-R) fused to common sequences (CS1/CS2). 2. “Off-chip” PCR generates mini-library tagging, and the addition of IT-adaptor sequences creates 48 fully tagged and sequencer-compatible mini-libraries. Fusion primers containing CS1 and CS2 (red line primers) and the Ion Torrent PGM adaptor sequences P1 (green) and Aseq (orange) are used to generate completed amplicons (blue lines flanked by red lines). Barcode sequences (yellow) for sample indexing are fused to the Aseq-CS2 primer. 3. Parallel sequencing of the combined libraries on Ion Torrent-PGM. All mini-libraries are pooled together. <b>B.</b> Dot plot represents all calculated values of A/G ratios [percentages (dots) and means (black horizontal lines)] in the set of target sites that met all selection criteria in WT (blue circles) and <i>fmr1</i>-/- (green circles) RNA. <b>C.</b> The ten editing sites that exhibited significant differential RNA editing levels between <i>fmr1</i>-/- and WT larvae (<i>n</i> = 20 batches of 10 larvae for each genotype, *<i>p</i><0.05, **<i>p</i><0.005). <b>D.</b> Representative RNA editing sites showed increased editing levels in the brains of <i>fmr1</i>-/- zebrafish. <i>gria3b</i> showed a 14% increase, <i>grik2</i> showed an 8% increase and <i>ache</i> showed an 18% increase (<i>gria3b</i> and <i>grik2</i>: WT, <i>n</i> = 4; <i>fmr1</i>-/-, <i>n</i> = 5; <i>ache</i>, <i>n</i> = 3 per genotype, one brain per sample, *<i>p</i><0.05, **<i>p</i><0.005). <b>E-F.</b> Genes with multiple editing sites located in close proximity in the same amplified target region, were analyzed to quantify the relative abundance of all possible protein combinations formed by the editing pattern. Grey bars represent differences in the relative abundance of mRNA transcripts between WT and <i>fmr1</i>-/- larvae. <b>E.</b> In <i>gria2a</i>, LR (Leucine, Arginine) represents the genomically encoded unedited version that exhibited a 2.6% difference in relative abundance (*<i>p</i><0.05). <b>F.</b> In <i>gria3a</i>, AV (Alanine, Valine) represents the double-edited form that exhibited a 1.6% difference in relative abundance (*<i>p</i><0.05). Values are represented as means ± SEM. Statistical significance was determined by two-sample <i>t</i>-test assuming unequal variances.</p

Fmrp-Adar interaction in zebrafish.

<p><b>A.</b> Phylogenetic tree of zebrafish and human Adar proteins. Sequences are labeled with gene names, chromosomal locations, and accession numbers. To standardize and simplify the nomenclature, we named the genes Adar1-3, as indicated on the right side of each clade. Similarity values of each Adar member appear on top of each clade. <b>B.</b> Sequence conservation and motif distribution of Adar proteins in zebrafish and humans. Protein domains: adenosine deaminase domain (deaminase, white), double-stranded RNA binding motif (dsRBM, black) and zDNA binding domain (z_alpha, light grey). <b>C-R.</b> <i>In situ</i> hybridization showing lateral (<b>C</b>, <b>E</b>, <b>F</b>, <b>H</b>, <b>I</b>, <b>K</b>, <b>L</b>, <b>N</b>, <b>O</b>, <b>Q</b>) and dorsal (<b>D</b>, <b>G</b>, <b>J</b>, <b>M</b>, <b>P</b>, <b>R</b>) views of the spatial expression pattern of all four <i>adar</i> genes in 2 dpf <b>(C-D, F-G, I-J, L-M)</b> and 6 dpf <b>(E, H, K, N)</b> WT larvae. Expression is detected primarily in the nervous system. <b>O-R.</b> Selected regions (black frames in <b>L</b> and <b>M</b>) show <i>adar2b</i> (<b>O-P)</b> and <i>adar3</i> (<b>Q-R)</b> expression in the spinal cord of 2 dpf WT embryo. <b>S.</b> HEK-293T cells were transiently transfected with the zebrafish proteins Adar2a and Fmrp fused to EGFP and MYC, respectively (EGFP-Adar2a and MYC-Fmrp). Co-immunoprecipitation was used to detect Adar2a and Fmrp interaction. Actin was used as a negative control. The cell lysate was immunoprecipitated with anti-actin, anti-MYC, or anti-EGFP. Proteins were purified from the complexes and separated by SDS-PAGE. <b>T.</b> Western blot shows the protein content following the transfection prior to the immunoprecipitation. The proteins were detected with specific antibodies against MYC, EGFP, and actin. <b>U.</b> Computational sequence homology predicted the number of RNA recognition elements (RREs) in the CDS of <i>adar</i> genes that are recognized by Fmrp. <b>V.</b> RNA immunoprecipitation (RIP) assays show that Fmrp binds <i>adar1</i>. PCR amplification of <i>adar1</i> on RNA extracted from a RIP experiment conducted with anti-Actin and anti-MYC antibodies, and on total RNA extracted from HEK293T cells. <b>W.</b> RT-PCR assays showed that the mRNA expression levels of all four <i>adar</i> genes increased in 6 dpf <i>fmr1</i>-/- larvae (grey bars) when compared with WT larvae (white bars). Values are represented as means ± SEM. *<i>p</i><0.05, **<i>p</i><0.005, two-way <i>t</i>-test assuming unequal variances. <b>X.</b> Adar2 protein expression was analyzed by Western blot with specific antibodies against Adar2 and actin as a loading control. Elevated Adar2 protein levels of approximately 30% are present in <i>fmr1</i>-/- brains.</p

Increased synaptic density and axon branching in the CNS of <i>fmr1</i>-/- embryos.

<p><b>A.</b> Wide-angled view of a confocal generated image of a single motor neuron in a 2 dpf embryo, which transiently expresses <i>mnx1X3</i>:<i>GAL4</i> and <i>uas</i>:<i>tRFP</i> constructs. DLAV, dorsal lateral anastomotic vessel; N, notochord; NT, neural tube; PVC, posterior cardinal vein; S, somite. <b>B-C.</b> Confocal imaging of motor neurons in 2 dpf <i>fmr1</i>-/- and WT embryos, which transiently express <i>mnx1X3</i>:<i>GAL4</i> and <i>uas</i>:<i>memYFP</i> constructs. <b>D-E.</b> Total arbor length <b>(D)</b> and number of branches <b>(E)</b> were measured in <i>fmr1</i>-/- (grey bars) and WT (white bars) embryos (WT, n = 17; <i>fmr1</i>-/-, n = 27, *<i>p<0</i>.<i>05</i>). Values are represented as means ± SEM. <b>F-G.</b> Confocal imaging of motor neurons in 2 dpf <i>fmr1</i>-/- and WT embryos, which transiently express <i>mnx1X3</i>:<i>GAL4</i>, <i>uas</i>:<i>SYP-EGFP</i> and <i>uas</i>:<i>tRFP</i> constructs. <b>H.</b> Total synaptic density was measured along the last 10 μm of a single branch of motor neurons in <i>fmr1</i>-/- and WT embryos (WT, n = 11; <i>fmr1</i>-/-, n = 17, *<i>p<0</i>.<i>05</i>). Scale bar = 10 μm. Values are represented as means ± SEM. <b>I-J.</b> Confocal imaging of spinal Rohon-Beard (RB) sensory neurons that project dorsally in 2 dpf <i>fmr1</i>-/- and WT embryos, which transiently express <i>huc</i>:<i>GAL4</i> and <i>uas</i>:<i>memYFP</i> constructs. <b>K-L.</b> The total arbor length (<b>K</b>) and number of branches (<b>L</b>) in the arbor of RB neurons were quantified in <i>fmr1</i>-/- and WT embryos (WT, n = 9; <i>fmr1</i>-/-, n = 10, *<i>p<0</i>.<i>05</i>). <b>M-N.</b> Confocal imaging of RB neurons in 2 dpf <i>fmr1</i>-/- and WT embryos, which transiently express <i>huc</i>:<i>GAL4</i>, <i>uas</i>: SYP-EGFP and <i>uas</i>:<i>tRFP</i> constructs. <b>O.</b> Total synaptic density was measured along the last 30 μm of a single branch of RB neurons in <i>fmr1</i>-/- (grey bars) and WT (white bars) embryos (WT, n = 10; <i>fmr1</i>-/-, n = 13). Scale bar = 30 μm. Values are represented as means ± SEM. Statistical significance was determined by two-sample <i>t</i>-test assuming unequal variances. <b>P.</b> Dorsal view of Hcrt neuron axons in a 2 dpf embryo, which transiently expresses the <i>hcrt</i>:<i>SYP-EGFP</i> construct. White arrow indicates the area analyzed. <b>Q-R.</b> Representative confocal imaging of Hcrt axons in 2 dpf <i>fmr1</i>-/- and WT embryos, which transiently express the <i>hcrt</i>:<i>SYP-EGFP</i> construct. <b>S.</b> Total synaptic density was measured along the last 10 μm of a single axonal branch of <i>hcrt</i> neurons in <i>fmr1</i>-/- and WT embryos (WT, n = 8; <i>fmr1</i>-/-, n = 9, *<i>p<0</i>.<i>05</i>). Scale bar = 10 μm. Values are represented as means ± SEM.</p