Profiling neural editomes reveals a molecular mechanism to regulate RNA editing during development

Abstract

Adenosine (A) to inosine (I) RNA editing contributes to transcript diversity and modulates gene expression in a dynamic, cell type-specific manner. During mammalian brain development, editing of specific adenosines increases, whereas the expression of A-to-I editing enzymes remains unchanged, suggesting molecular mechanisms that mediate spatiotemporal regulation of RNA editing exist. Herein, by using a combination of biochemical and genomic approaches, we uncover a molecular mechanism that regulates RNA editing in a neural- and development-specific manner. Comparing editomes during development led to the identification of neural transcripts that were edited only in one life stage. The stage-specific editing is largely regulated by differential gene expression during neural development. Proper expression of nearly one-third of the neurodevelopmentally regulated genes is dependent on adr-2, the sole A-to-I editing enzyme in C. elegans However, we also identified a subset of neural transcripts that are edited and expressed throughout development. Despite a neural-specific down-regulation of adr-2 during development, the majority of these sites show increased editing in adult neural cells. Biochemical data suggest that ADR-1, a deaminase-deficient member of the adenosine deaminase acting on RNA (ADAR) family, is competing with ADR-2 for binding to specific transcripts early in development. Our data suggest a model in which during neural development, ADR-2 levels overcome ADR-1 repression, resulting in increased ADR-2 binding and editing of specific transcripts. Together, our findings reveal tissue- and development-specific regulation of RNA editing and identify a molecular mechanism that regulates ADAR substrate recognition and editing efficiency.

Figure 1.

De novo identification of editing sites in adult neurons. (A) The adult neural editing sites identified were cross-referenced with previously reported sites to identify novel editing sites. (B) Genomic distribution of adult neural sites. (CDS) Coding region; (UTR) untranslated region; (ncRNA) noncoding RNA. (C) A Two Sample Logo analysis of editing sites identified in adult neural cells compared with adenosines present in the same dsRNA region. Enriched and depleted nucleotides are shown above and below the axis, respectively. The level of conservation is represented by letter height. Logos were generated using a Student's t-test with P < 0.05 and no Bonferroni correction.

Figure 2.

C. elegans neural editome change during development. (A) Comparison of the RNA-seq data sets from L1 and adult neural cells. The number of input reads for each step of the SAILOR bioinformatic pipeline is listed along with the number of de novo identified edited sites. (B) Genomic distribution of the identified L1 neural editing sites was determined using the WormBase annotations (WS275). (C) A Two Sample Logo analysis of editing sites identified in L1 neural cells compared with adenosines present in the same dsRNA region. Enriched and depleted nucleotides are shown above and below the axis, respectively. The level of conservation is represented by letter height. Logos were generated using a Student's t-test with P < 0.05 and no Bonferroni correction. (D) qPCR was used to quantify adr-2 expression in neural cells (left) and in whole-worm lysate (right) for both L1 and adults. The average of three independent replicates is plotted; error bars, SEM. Statistical significance was calculated using a Student's t-test. (***) P < 0.001; (ns) P > 0.05.

Figure 3.

Differential expression of many transcripts during neural development contributes to the stage-specific neural editomes. (A) Workflow for identifying stage-specific edited sites following the quantification of all the neural editing sites. (B) Distribution of stage-specific edited sites. (C) Genomic distribution of L1-specific edited transcripts based on the genomic location of identified edited sites. (CDS) Coding region; (UTR) untranslated region; (ncRNA) noncoding RNA. (D) Dots represent individual genes that are down-regulated (red; 4476, log₂fold < −0.5), up-regulated (blue; 5171, log₂fold > 0.5), or not significantly different (gray; 7697, P-adj > 0.05) between three biological replicates of wild-type neural L1 and adult RNA-seq data. (E) The average of three independent replicates of qPCR validation of neurodevelopmentally, differentially expressed genes is plotted; error bars, SEM. Statistical significance was calculated by Student's t-test. (*)P < 0.05; (***) P < 0.001. (F) Representation of the L1-specific edited transcripts based on differential expression during neural development. Dark blue represents transcripts differentially expressed in neural development and ADR-2 dependent (dotted) or independent (solid). Light blue represents transcripts that are not differentially expressed in neural development.

Figure 4.

Editing level increases in majority of the sites during neural development. (A) Heatmap representing the percentage of editing in L1 and adult neural cells at the sites (901) with more than five reads in neural RNA-seq from both life stages. (B) The 837 sites mapped to annotated transcripts were categorized into five groups depending on differential editing between L1 and adult neural cells. Bar graph represents the number of editing sites in each group. The number of unique transcripts in each group are listed above each bar.

Figure 5.

The inhibitory role of adr-1 on editing of neural transcripts is developmentally regulated. (A) Representation of neural RNA-seq reads covering the Y75B8A.8 transcript. Reads from adr-2(-) are negative controls. Green represents adenosine and brown represents guanosine at the marked chromosomal positions. (B) Sanger sequencing chromatograms of cDNA amplified from Y75B8A.8 reporter RNA. Editing sites are listed below the chromatogram. Sites 227 and 228 correspond to chromosomal positions of Chr III: 12,171,074 and 12,171,075. The nucleotides at each position are represented with a different color (green, adenosine; black, guanosine; blue, cytidine; red, thymidine). (C,D) The average of three independent replicates was plotted; error bars, SEM. One-way ANOVA was performed to determine the statistical significance. (****) P < 0.0001; (*) P < 0.05; (ns) P > 0.05. (C) Editing levels of the Y75B8A.8 neural reporter were measured in wild-type and adr-1(-) worms in L1 (left) and adult (right) stages using Sanger sequencing. (D) qPCR was performed to measure the absolute mRNA levels of adr-2 relative to adr-1 in both life stages. (E) Neural RNA-seq data were used to plot the L1 editing levels of representative sites from the group of neural RNAs that showed increased editing in development (P < 0.01, pairwise comparison using two-way ANOVA).

Figure 6.

ADR-1 inhibits editing at transcripts that show increased editing in neural development by binding. (A) Sanger sequencing chromatograms of cDNA amplified from Y75B8A.8 reporter RNA isolated from the indicated worms at the L1 stage. (B) Editing in A was quantitatively measured. The average of two independent replicates is plotted; error bars, SEM. Two-way ANOVA was used to determine statistical significance. (***) P < 0.001. (C) Lysates from the indicated worm strains were incubated with FLAG magnetic beads. A portion of the lysates before immunoprecipitation and the IPs was subjected to immunoblotting with a FLAG antibody (Sigma-Aldrich F1804). (D) Bar graph represents the fold enrichment of cDNA present in the IPs divided by cDNA present in the input lysates from the indicated worm strains and normalized to the same ratio in adr-1; adr-2(-) worms. The average of three (Y75B8A.8, WO7G4.3 and lam-2) and two (daam-1 and uba-2) independent replicates were plotted; error bars, SEM. One-way ANOVA was used to determine statistical significance. (*) P < 0.05; (ns) P > 0.05. (E) Proposed model for editing regulation of neural transcripts that show increased editing during development.