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A-to-I RNA editing affects lncRNAs expression after heat shock

ABSTRACT: Adenosine to inosine (A-to-I) RNA editing is a highly conserved regulatory process carried out by adenosine deaminases (ADARs) on dsRNAs. Although a significant fraction of the transcriptome is edited, the function of most editing sites is unknown. Previous studies indicated changes in A-to-I RNA editing frequencies following exposure to several stress types. However, the overall effect of stress on the expression of ADAR targets is not fully understood. Here we performed high-throughput RNA sequencing of wildtype and ADAR mutant C. elegans worms after heat shock, to analyze the effect of heat shock stress on the expression pattern of genes. We found that ADAR regulation following heat shock does not involve directly heat shock related genes. Our analysis also revealed that, lncRNAs and pseudogenes, which have a tendency for secondary RNA structures, are enriched among upregulated genes upon heat shock in ADAR mutant worms, while they are downregulated in ADAR mutant worms under permissive conditions. Therefore, temperature increases may destabilize dsRNA structures and protect them from RNAi degradation, despite the lack of ADAR function. These findings shade a new light on the dynamics of gene expression under heat shock in relation to ADAR function.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE122015 | GEO | 2018/12/12

REPOSITORIES: GEO

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Project description:A-to-I RNA editing is widespread in eukaryotic transcriptomes and plays an essential role in the creation of proteomic and phenotypic diversity. Loss of ADARs, the proteins responsible for A-to-I editing, results in lethality in mammals. In C. elegans, knocking out both ADARs, ADR-1 and ADR-2, results in aberrant behavior and abnormal development. Studies have shown that ADR-2 can actively deaminate dsRNA while ADR-1 cannot. However, as most studies of C. elegans ADARs were performed on worms mutated in both ADAR genes, the effects observed cannot be attributed to a single ADAR or to the interactions between ADAR genes. Therefore, we set to study the effects of each C. elegans ADAR on RNA editing, gene expression, protein levels and the contribution of each of ADAR to the phenotypes observed in worms mutated in both genes, in order to elucidate their distinct functions. We found significant differences in the phenotypes observed in worms mutated in a single ADAR gene. Worms harboring adr-1 mutations have a significant reduction in their lifespan, while worms harboring adr-2 mutations have extended lifespan. We also observed severe abnormalities in vulva formation mainly in adr-1 mutants, and we suggest that these phenotypes are a result of an RNA editing independent function of ADR-1. Mutations in each ADAR resulted in expressional changes in hundreds of genes, and a significant downregulation of edited genes. However, very few changes in the protein levels were observed. In addition, we found that ADR-1 binds many edited genes. Our results suggest that ADR-1 has a significant function in the RNA editing process and by altering editing levels it causes the severe phenotypes that we observed. In contrast, a complete lack of RNA editing is less harmful to the worms. Furthermore, our results indicate that the effect of RNA editing on the protein content in the cell is minor and probably the main purpose of these modifications is to antagonize or enhance other gene regulatory mechanisms that act on RNA.

Project description:Background: Adenosine deaminases that act on RNA (ADARs) bind to double-stranded and structured RNAs and deaminate adenosines to inosines. This A to I editing is widespread and required for normal life and development. Besides mRNAs and repetitive elements, ADARs can target miRNA precursors. Editing of miRNA precursors can affect processing efficiency and alter target specificity. Interestingly, ADARs can also influence miRNA abundance independent of RNA-editing. In mouse embryos where editing levels are low, ADAR2 was found to be the major ADAR protein that affects miRNA abundance. Here we extend our analysis to adult mouse brains where high editing levels are observed. Results: Using Illumina deep sequencing we compare the abundances of mature miRNAs and editing events within them, between wild-type and ADAR2 knockout mice in the adult mouse brain. Reproducible changes in abundance of specific miRNAs are observed in ADAR2 deficient mice. Most of these quantitative changes seem unrelated to A to I editing events. However, many A to G transitions in cDNAs prepared from mature miRNA sequences, reflecting A to I editing events in the RNA, are observed with frequencies reaching up to 80%. About half of these editing events are primarily caused by ADAR2 while a few miRNAs show increased editing in the absence of ADAR2, suggesting preferential editing by ADAR1. Moreover, novel, previously unknown editing events were identified in several miRNAs. In general 64% of all editing events are located within the seed region of mature miRNAs. In one of these cases retargeting of the edited miRNA could be verified in reporter assays. Also, altered processing efficiency upon editing near a processing site could be experimentally verified. Conclusions: ADAR2 can significantly influence the abundance of certain miRNAs in the brain. Only in a few cases changes in miRNA abundance can be explained by miRNA editing. Thus, ADAR2 binding to miRNA precursors, without editing them, may influence their processing and thereby abundance. ADAR1 and ADAR2 have both overlapping and distinct specificities for editing of miRNA editing sites. Over 60% of editing occurs in the seed region possibly changing target specificities for many edited miRNAs. Examination of the effect of ADAR2 on mature miRNA abundance and sequence in adult mouse brain.

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A-to-I RNA editing affects lncRNAs expression after heat shock

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