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. 2019 Apr 23;27(4):1244-1253.e4.
doi: 10.1016/j.celrep.2019.03.095.

Disruption in A-to-I Editing Levels Affects C. elegans Development More Than a Complete Lack of Editing

Nabeel S Ganem  1 , Noa Ben-Asher  1 , Aidan C Manning  2 , Sarah N Deffit  2 , Michael C Washburn  3 , Emily C Wheeler  4 , Gene W Yeo  4 , Orna Ben-Naim Zgayer  1 , Einav Mantsur  1 , Heather A Hundley  2 , Ayelet T Lamm  5
Affiliations

Disruption in A-to-I Editing Levels Affects C. elegans Development More Than a Complete Lack of Editing

Nabeel S Ganem et al. Cell Rep. .

Abstract

A-to-I RNA editing, catalyzed by ADAR proteins, is widespread in eukaryotic transcriptomes. Studies showed that, in C. elegans, ADR-2 can actively deaminate dsRNA, whereas ADR-1 cannot. Therefore, we set out to study the effect of each of the ADAR genes on the RNA editing process. We performed comprehensive phenotypic, transcriptomics, proteomics, and RNA binding screens on worms mutated in a single ADAR gene. We found that ADR-1 mutants exhibit more-severe phenotypes than ADR-2, and some of them are a result of non-editing functions of ADR-1. We also show that ADR-1 significantly binds edited genes and regulates mRNA expression, whereas the effect on protein levels is minor. In addition, ADR-1 primarily promotes editing by ADR-2 at the L4 stage of development. Our results suggest that ADR-1 has a significant role in the RNA editing process and in altering editing levels that affect RNA expression; loss of ADR-1 results in severe phenotypes.

Keywords: ADAR; C. elegans; gene expression; organism development; transcriptomics.

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Schematic View of adr-1 and adr-2 Genes and Isoforms and the Deletions That Were Used in This Study
The genes and known isoforms are represented in their relative lengths. Double-strand RNA binding motifs (dsRBMs) regions are shown in red, and the deamination catalytic domains are shown in blue. The deletion alleles (green) are scaled and positioned to reflect the area of deletion in the genes; adr-1 gv6 (1,560-bp deletion) and tm668 (967-bp deletion), adr-2 gv42 (1,013-bp deletion), and ok735 (1,371-bp deletion). See also Figures S1 and S2 and Data S1.
Figure 2.
Figure 2.. adr-1 and adr-2 Mutants Have Opposite Life-Span Phenotypes
(A) The life span of the mutant worms was followed, and the mean survival curves are presented for N2 (wild type [WT]), adr-1(gv6)I, adr-1(tm668)I, adr-2(gv42)III, adr-2(ok735)III, adr-1(gv6)I; adr-2(gv42)III and adr-1(tm668)I; adr-2(ok735)III. Mutations in adr-1 gene (adr-1(gv6)I or adr-1(tm668)I) reduce the life span of the worm compared with that of the WT worms, whereas mutation in adr-2 gene (adr-2(gv42)III or adr-2(ok735)III) extend the life span of the worm compared with that of the WT worms. Mutants for both genes have the same pattern as the WT worms. (B) The mean survival curves are presented for WT, adr-1 (tm668), and the rescue strains adr-1 (tm668) FLAG-ADR-1: adr-1(tm668) I blmEx1[ 3XFLAG-adr-1 genomic, rab3::gfp::unc-54] and adr-1 (tm668) FLAG-ADR-1 ds1+2 mutant: adr-1(tm668) I blmEx1 (3XFLAG-adr-1 genomic with mutations in dsRBD1 [K223E, K224A, and K227A], and dsRBD2 [K583E, K584A, and K587A], rab3::gfp::unc-54). Both transgenic adr-1 rescue strains have life spans extended more than adr-1(tm668). Each experiment was repeated at least five times.
Figure 3.
Figure 3.. adr-1 Mutants Have High Frequency of Vulva Abnormalities
(A) ADAR-mutant strains were scored for pvl phenotype, and the fraction of worms presenting the phenotype from total worms is presented. The p value was calculated with a two-sample, unequal-variance, heteroscedastic t test; *p < 0.01 compared with WT; Ψp < 0.01 compared with adr-1(tm668)I. (B) Worms were subjected to lin-1 RNAi. Multivulva phenotypes were scored at the first day of egg laying, and the fraction of worms exhibiting the phenotype from total worms is presented. Each experiment was repeated at least three times, and the standard deviation is presented by error bars. The p value was calculated with a two-sample, unequal-variance, heteroscedastic t test; *p < 0.01 compared with WT. NS, nonsignificant p value. (C) Worms were subjected to unc-22 RNAi, and the fraction of worms presenting the bag of worms phenotype is presented. The p value was calculated by two-sample, unequal-variance, heteroscedastic t test; *p < 0.01 compared with WT. Each experiment was repeated at least three times, and the standard deviation is shown by error bars.
Figure 4.
Figure 4.. Genes Edited at Their 3′ UTR Are Downregulated in adr-1 and adr-2 Mutants at the Embryo Stage
Log-scale plots presenting gene expression in wild-type (N2) worms versus adr-1 mutant worms (A and C) or adr-2 mutant worms (B and D) at the embryo stage (A and B) and at the L4 stage (C and D). Every dot in the graphs represents a gene. Red line is the regression line for all genes. The 3′ UTR-edited genes with significant padj value are in purple, and their regression line is presented in purple. Downregulated genes found by the proteomics analysis are in orange, and upregulated genes are in green. Alh-7 gene is downregulated in adr-1 mutants at the embryo stage in all analyses (blue in A). Adbp-1 gene is downregulated in adr-2 mutants in all analyses at the embryo stage (orange in B). See Figures S3, S6, and S7 and Tables S1, S2, and S5.
Figure 5.
Figure 5.. Significant Portions of Genes That Are Edited at L4 Stage Are Upregulated in adr-1 Mutants
Venn diagrams presenting the intersections between edited genes at the embryo or L4 developmental stages, and (A) genes with their expression downregulated at embryo stage in adr-1 mutants. (B) Genes with their expression downregulated at the L4 stage in adr-1 mutants. (C) Genes with their expression upregulated at embryo stage in adr-1 mutants. (D) Genes with their expression upregulated at the L4 stage in adr-1 mutants. See Figure S4.
Figure 6.
Figure 6.. Most of the Editing Sites That Appear Only in adr-1 Mutants Are in Genes with Editing Sites in Wild-Type Worms
(A) A bar graph representing nucleotide changes found in wild-type and adr-1 mutant worms at the L4 and embryo developmental stages. Also presented are nucleotide changes that were found in adr-1 mutants but not in wild-type worms. (B–D) Venn diagrams presenting the intersection between genes with editing sites that were detected in adr-1 mutants and not in wild-type worms and (B) Genes edited in wild-type worms at the embryo or L4 developmental stages. (C) Genes with their expression upregulated or downregulated at the embryo stage in adr-1 mutants. (D) Genes with their expression upregulated or downregulated at the L4 stage in adr-1 mutants. See Table S3.
Figure 7.
Figure 7.. ADR-1 Binds Edited Genes
Venn diagrams presenting the intersections between edited genes, genes identified as bound by ADR-1 using RIP-seq analysis and (A) Genes that their expression is downregulated at embryo stage in adr-1 mutants. (B) Genes that their expression is downregulated at L4 stage in adr-1 mutants. (C) Genes that are upregulated at the embryo stage in adr-1 mutants. (D) Genes that are upregulated at the L4 stage in adr-1 mutants. See Figure S5 and Table S4.
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