Project description:Cellular RNAs containing double-stranded RNA (dsRNA) structures are subject to A-to-I RNA editing by the adenosine deaminases that act on RNA (ADARs). While A-to-I editing can alter mRNA coding potential, most editing is observed in non-coding sequences, the function of which remains poorly characterized. Using a dsRNA immunoprecipitation and high-thoughput sequencing (dsRIP-Seq) approach, we identify 1523 expressed A-to-I edited regions and characterize their expression during Caenorhabditis elegans development. We observe that edited regions are highly expressed in early development and are closely associated with protein-coding genes. Edited dsRNA structures give rise to abundant small interfering RNAs (siRNAs) that are negatively correlated with ADAR expression and regulate the developmental expression of associated genes.
Project description:Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). To evaluate effects of ADARs on small RNAs that derive from dsRNA precursors, we performed deep-sequencing, comparing small RNAs from wildtype and ADAR mutant C. elegans. While editing in small RNAs was rare, at least 40% of microRNAs had altered levels in at least one ADAR mutant strain, and miRNAs with significantly altered levels had mRNA targets with correspondingly affected levels. About 40% of siRNAs derived from endogenous genes (endo-siRNAs) also had altered levels in at least one mutant strain, including 63% of Dicer-dependent endo-siRNAs. The 26G class of endo-siRNAs was significantly affected by ADARs, and many altered 26G loci had intronic reads, and histone modifications associated with transcriptional silencing. Our data indicate ADARs, through both direct and indirect mechanisms, are important for maintaining wildtype levels of many small RNAs in C. elegans.
Project description:Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). To evaluate effects of ADARs on small RNAs that derive from dsRNA precursors, we performed deep-sequencing, comparing small RNAs from wildtype and ADAR mutant C. elegans. While editing in small RNAs was rare, at least 40% of microRNAs had altered levels in at least one ADAR mutant strain, and miRNAs with significantly altered levels had mRNA targets with correspondingly affected levels. About 40% of siRNAs derived from endogenous genes (endo-siRNAs) also had altered levels in at least one mutant strain, including 63% of Dicer-dependent endo-siRNAs. The 26G class of endo-siRNAs was significantly affected by ADARs, and many altered 26G loci had intronic reads, and histone modifications associated with transcriptional silencing. Our data indicate ADARs, through both direct and indirect mechanisms, are important for maintaining wildtype levels of many small RNAs in C. elegans. Deep sequencing of small RNAs in wild-type (N2), adr-1 null, adr-2 null and adr-1;adr-2 null mixed stage C. elegans
Project description:Cellular RNAs containing double-stranded RNA (dsRNA) structures are subject to A-to-I RNA editing by the adenosine deaminases that act on RNA (ADARs). While A-to-I editing can alter mRNA coding potential, most editing is observed in non-coding sequences, the function of which remains poorly characterized. To correlate small RNA population with expression patterns of ADARs and hyperedited RNAs (editing-enriched regions: EERs) defined and characterized in a separate RNAseq analysis, we re-analyzed existing smallRNAseq datasets of a wildtype strain and a strain lacking ADARs (adr-1;adr-2). Analysis of primary siRNAs from mixed-stage worms revealed that ADARs impact siRNA biogenesis from EERs. We then analyzed primary and secondary RNAs mapping to EERs from embryo-stage or L4-stage worms and observed that ADAR effects on siRNA levels are dependent on developmental stage.
Project description:Small RNAs recognize, bind, and regulate other complementary cellular RNAs. The introduction of small RNAs to eukaryotic cells frequently results in unintended silencing of related, but not identical, RNAs: a process termed off-target gene silencing. Off-target gene silencing is one of the major concerns during the application of small RNA-based technologies for gene discovery and the treatment of human disease. Off-target gene silencing is commonly thought to be due to inherent biochemical limitations of the RNAi machinery. Here we show that, following the introduction of exogenous sources of dsRNA, the nuclear RNAi pathway, but not its cytoplasmic counterparts, is the primary source of off-target silencing in C. elegans. In addition, we show that during the normal course of growth and development the nuclear RNAi pathway regulates repetitive gene families. Therefore, we speculate that RNAi off-target effects might not be “mistakes” but, rather, an intentional and genetically programmed aspect of small RNA-mediated gene silencing, which might allow small RNAs to silence rapidly evolving parasitic nucleic acids. Finally, reducing off-target effects by manipulating the nuclear RNAi pathway in vivo might improve the efficacy of small RNA-based technologies. (The Zhou X, Xu F, Mao H, Ji J, Meng Y, Feng X, and Shouhong Guang (2014) Nuclear RNAi Contributes to the Silencing of Off-Target Genes and Repetitive Sequences in Caenorhabditis elegans. Genetics 2014 May;197(1):121-32. doi: 10.1534/genetics.113.159780.)