ADARs affect the biogenesis of many small RNAs in C. elegans, but few mature small RNAs are edited
ABSTRACT: 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:Adenosine deaminases that act on RNA (ADARs) catalyze the conversion of adenosine to inosine in dsRNA. C. elegans ADARs, ADR-1 and ADR-2, promote the expression of genes containing dsRNA structures by preventing their processing into siRNAs and silencing by RNAi. The 26G endogenous siRNA (endo-siRNA) pathway generates a subset of siRNAs distinct from those made in adr-1;adr-2 mutants, but using many of the same factors. We found that adr-1;adr-2;rrf-3 mutants, lacking both ADARs and the RNA-dependent RNA polymerase RRF-3 required for the 26G pathway, display a bursting phenotype rescued by the RNAi factors RDE-1 and RDE-4. To determine what gene expression changes underlie the synthetic phenotype of adr-1;adr-2;rrf-3 mutants, we sequenced poly(A)+ RNA from adr-1;adr-2;rrf-3 embryos, their parent strains, and strains rescued with mutations in rde-1 and rde-4. We found that genes associated with edited structures were robustly downregulated in adr-1;adr-2;rrf-3 mutants in a manner partially dependent on rde-1 and rde-4. Additionally, genes induced during Orsay virus infections were induced in rrf-3 mutants and further upregulated in adr-1;adr-2;rrf-3 mutants, again dependent in part on rde-1 and rde-4. Overall design: RNAseq of poly(A)+ RNA from C.elegans mixed-stage embryos, four biological replicates per genotype, six genotypes: wildtype (Bristol N2), adr-1(uu49);adr-2(uu28), rrf-3(uu56), adr-1(uu49);adr-2(uu28);rrf-3(uu56), adr-1(uu49);adr-2(uu28);rrf-3(uu56);rde-1(uu51), and adr-1(uu49);adr-2(uu28);rrf-3(uu56);rde-4(uu53).
Project description:ADAR proteins alter gene expression both via catalyzing adenosine-to-inosine RNA editing and in an editing-independent manner by binding to target RNAs. Loss of ADARs affects neuronal function in all animals studied to date. To identify important neuronal targets in C. elegans, we performed the first unbiased assessment of the effects of ADR-2, the C. elegans editing enzyme, on the neural transcriptome. We identified the neural editome and gene expression changes associated with the loss of adr-2. As C. elegans lacking adr-2 exhibit reduced chemotaxis, our studies focused on targets that regulate this process. We identified an edited mRNA, clec-41, whose expression is dependent on ADR-2. Expressing clec-41 in adr-2 deficient neural cells restored chemotaxis. This study is the first of its kind in the RNA editing field to span from developing novel methodology for tissue-specific target identification to organismal behavior, significantly advancing our understanding of ADAR functions in neural cells. Overall design: Strand-specific editing sites and differential expression analysis was done on triplicate WT (wildtype) and Adr2- (control) C. elegans neural cells.
Project description:The nematode Caenorhabditis elegans contains each of the broad classes of eukaryotic small RNAs, including microRNAs (miRNAs), endogenous small-interfering RNAs (endo-siRNAs) and piwi-interacting RNAs (piRNAs). To better understand the evolution of these regulatory RNAs, we deep sequenced small RNAs from C. elegans and three closely related nematodes: C. briggsae, C. remanei and C. brenneri. The results reveal a fluid landscape of small RNA pathways with essentially no conservation of individual sequences aside from a subset of miRNAs. We identified 52 miRNA families that are conserved in each of the four species as well as numerous miRNAs that are species specific or shared between only two or three species. Despite a lack of conservation of individual piRNAs and siRNAs many of the features of each pathway, including genomic distribution, are conserved. We show that in each species, 26G siRNAs trigger stage-specific secondary siRNA formation. We also observe that piRNAs trigger siRNA formation from targets containing up to three mismatches in each species. Finally, we show that nematodes produce two distinct sex-specific classes of piRNAs, suggesting different roles for piRNAs in male and female germlines. Sequencing small RNAs from four Caenorhabditis species: C. elegans, C. briggsae, C. remanei and C. brenneri
Project description:Eukaryotic cells express several classes of small RNAs that regulate gene expression and ensure genome maintenance. Endogenous siRNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) mainly control gene and transposon expression in the germline, while microRNAs (miRNAs) generally function in post-transcriptional gene silencing in both somatic and germline cells. To provide an evolutionary and developmental perspective on small RNA pathways in nematodes, we identified and characterized known and novel small RNA classes through gametogenesis and embryo development in the parasitic nematode Ascaris suum and compared them with known small RNAs of Caenorhabditis elegans. piRNAs, Piwi-clade Argonautes, and other proteins associated with the piRNA pathway have been lost in Ascaris. miRNAs are synthesized immediately following fertilization in utero, prior to pronuclear fusion, and before the first cleavage of the zygote. This is the earliest expression of small RNAs ever described at a developmental stage long thought to be transcriptionally quiescent. A comparison of the two classes of Ascaris endo-siRNAs, 22G-RNAs and 26G-RNAs, to those in C. elegans, suggests great diversification and plasticity in the use of small RNA pathways during spermatogenesis in different nematodes. Our data reveal conserved characteristics of nematode small RNAs as well as features unique to Ascaris that illustrate significant flexibility in the use of small RNAs pathways, some of which are likely an adaptation to Ascaris’ life cycle and parasitism. We generated transcriptomes from Ascaris germline and embryos for de-novo assembly as well as cDNA expression profiles. Two types of libraries were prepared: 1) sheared, full-length cDNA synthesized using a combination of oligo-dT and random hexamer priming and 2) cDNA prepared from RNA first chemically sheared and then double-stranded cDNA prepared using ramom hexamer priming.
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. Overall design: RNA-Seq of total RNA and RNA immunoprecipitated with a dsRNA-specific antibody (dsRIP) from whole C. elegans at four stages of development: embryos, early stage larvae (L1-L2), late stage larvae (L3-L4), and young adults.
Project description:Small RNAs, including piRNAs, miRNAs and endogenous siRNAs, bind Argonaute proteins to form RNA-silencing complexes that target coding genes, transposons and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in C. elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the Argonaute ergo-1 and the 3' methyltransferase henn-1. Quantitative RT-PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans. This SuperSeries is composed of the following subset Series: GSE34320: Analysis of 22G siRNA triggered siRNA amplification in Caenorhabditis elegans GSE34321: Analysis of 3' 2'-O-methylated small RNAs in Caenorhabditis elegans Refer to individual Series
Project description:Eukaryotic cells express several classes of small RNAs that regulate gene expression and ensure genome maintenance. Endogenous siRNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) mainly control gene and transposon expression in the germline, while microRNAs (miRNAs) generally function in post-transcriptional gene silencing in both somatic and germline cells. To provide an evolutionary and developmental perspective on small RNA pathways in nematodes, we identified and characterized known and novel small RNA classes through gametogenesis and embryo development in the parasitic nematode Ascaris suum and compared them with known small RNAs of Caenorhabditis elegans. piRNAs, Piwi-clade Argonautes, and other proteins associated with the piRNA pathway have been lost in Ascaris. miRNAs are synthesized immediately following fertilization in utero, prior to pronuclear fusion, and before the first cleavage of the zygote. This is the earliest expression of small RNAs ever described at a developmental stage long thought to be transcriptionally quiescent. A comparison of the two classes of Ascaris endo-siRNAs, 22G-RNAs and 26G-RNAs, to those in C. elegans, suggests great diversification and plasticity in the use of small RNA pathways during spermatogenesis in different nematodes. Our data reveal conserved characteristics of nematode small RNAs as well as features unique to Ascaris that illustrate significant flexibility in the use of small RNAs pathways, some of which are likely an adaptation to Ascaris’ life cycle and parasitism. We constructed and analyzed 51 libraries from discrete regions of gametogenesis and synchronized stages of embryo development in the parasitic nematode Ascaris, using three types of small RNA libraries preparation methods: 1) 28 libraries of 18-34 or 18-40 nt RNAs with a 5’ monophosphate (5’ monophosphate libraries), 2) 4 libraries of 18-34 nt RNAs with a 5’ monophosphate but treated with periodate to enrich for small RNAs with 3’ end modifications (5’ monophosphate, 3’ end modified), and 3) 19 libraries of 18-28 or 18-40 nt RNAs with a 5’ triphosphate, diphosphate, or monophosphate (5’ allphosphate). These libraries enabled us to identify different small RNAs, characterize their different 5’ and 3’ ends, and their expression profiles through gametogenesis and embryogenesis.
Project description:Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that in the germ-line, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germ-line nuage structures called P-granules. WAGO-1 silences certain genes, transposons, pseudogenes and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest novel regulatory functions for small RNAs. Overall design: Total RNA was extracted from C. elegans drh-3(ne4253) and wild type animals
Project description:We analyzed the C. elegans small RNA response to high copy transgene sequences expressed in the soma in a wild type and an eri-6/7 mutant background. We also analyzed small RNA defects in the arl-8(tm2472) mutant. Transgene siRNAs are 22 nt long, mostly antisense, and correspond to the promoter, coding regions, the 3'UTR and plamsid sequences present on the transgene. Transgene siRNAs are decreased in the eri-6/7 mutant. In the arl-8 mutant, 26G siRNAs in the ALG-3/4 dependent endogenous RNAi pathway are decreased. Sequencing small RNAs from C. elegans transgenic strains and mutants.
Project description:Years after the discovery that Dicer is a key enzyme in gene-silencing, the role of its helicase domain remains enigmatic. Here we show that this domain is critical for accumulation of certain endogenous small interfering RNAs (endo-siRNAs) in C. elegans. The domain is required for the production of the direct products of Dicer, or primary endo-siRNAs, and consequently, affects levels of downstream intermediates, the secondary endo-siRNAs. Consistent with the role of endo-siRNAs in silencing, their loss correlates with an increase in cognate mRNA levels. We find that the helicase domain of Dicer is not required for microRNA (miRNA) processing, or RNA interference following exposure to exogenous double-stranded RNA. Comparisons of wildtype and helicase-defective strains using deep-sequencing analyses show that the helicase domain is required by a subset of annotated endo-siRNAs, in particular, those associated with the slightly longer 26 nucleotide small RNA species containing a 5’ guanosine. Overall design: We reintroduced either wildtype Dicer, or Dicer harboring a mutation (K39A) in it's helicase domain, into dcr-1(ok247) mutant worms via transgene rescue. We then used high-throughput sequencing to compare levels of small RNAs present in each of these strains.