Small-RNAs in L4 and young adult stages
Ontology highlight
ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
SUBMITTER: Jin-Wu Nam
PROVIDER: E-GEOD-36394 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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 AscarisM-bM-^@M-^Y 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 5M-bM-^@M-^Y monophosphate (5M-bM-^@M-^Y monophosphate libraries), 2) 4 libraries of 18-34 nt RNAs with a 5M-bM-^@M-^Y monophosphate but treated with periodate to enrich for small RNAs with 3M-bM-^@M-^Y end modifications (5M-bM-^@M-^Y monophosphate, 3M-bM-^@M-^Y end modified), and 3) 19 libraries of 18-28 or 18-40 nt RNAs with a 5M-bM-^@M-^Y triphosphate, diphosphate, or monophosphate (5M-bM-^@M-^Y allphosphate). These libraries enabled us to identify different small RNAs, characterize their different 5M-bM-^@M-^Y and 3M-bM-^@M-^Y ends, and their expression profiles through gametogenesis and embryogenesis.
2011-06-16 | E-GEOD-26955 | biostudies-arrayexpress
Project description:In animals, piRNAs, and their associated Piwi proteins, guard germ cell genomes against mobile genetic elements via an RNAi-like mechanism. In C. elegans, 21U-RNAs comprise the piRNA class and these collaborate with 22G RNAs, via unclear mechanisms, to discriminate self from non-self and selectively and heritably silence the latter. Recent work indicates that 21U-RNAs are post-transcriptional processing products of individual transcription units that produce ~26 nucleotide capped precursors. Yet, nothing is known of how the expression of precursors is controlled or of how primary transcripts give rise to mature small RNAs. We conducted a genome-wide RNAi screen to identify components of the 21U biogenesis machinery. Screening by direct, qPCR-based measurements of mature 21U-RNA levels, we identified 22 genes important for 21U-RNA production, termed TOFUs (Twenty-One-u Fouled Up). We also identified 7 genes that normally repress 21U production. By measuring mature 21U-RNA and precursor levels for the 7 strongest hits from the screen, we have assigned factors to discrete stages of 21U-RNA production. Our work has identified factors separately required for the transcription of 21U precursors, and the processing of these precursors into mature 21U-RNAs, thereby providing an essential resource for studying the biogenesis of this important small RNA class. Small RNA and capped small RNA sequencing from total RNA of C. elegans subjected to different RNAi and different C. elegans mutants
2014-04-01 | E-GEOD-53970 | biostudies-arrayexpress
Project description:MicroRNAs (miRNA), discovered in C. elegans, are short non-coding RNAs that bind and regulate the expression of target mRNAs in animals and plants. C. elegans miRNAs bind to partially complementary sequences in the 3' UTR of the target mRNA, which results in translational repression through mRNA destabilization. The high-throughput sequencing of RNA cleavage fragments was performed to directly detect cleaved miRNA targets in C. elegans. Based on this analysis, miR-249 was identified as a potential miRNA that regulates a ZK637.6 pseudogene, paralogous to asna-1 (ZK637.5), by a cleavage mechanism with extensive, evolutionary conserved complementarity. Additionally, we validated miR-249-directed cleavage of the ZK637.6 by a gene-specific 5M-bM-^@M-^Y rapid amplification of cDNA ends and observed notable difference in expression of ZK637.6 in wild-type versus mir-249 mutant C. elegans. Furthermore, phosphate-independent small-RNA sequencing analysis revealed that miR-249 target genes, including ZK637.6 pseudogene, showed 22G-RNA productions dependent on miR-249 targeting. These findings may lead to a better understanding of the biological roles of miRNAs for pseudogenes in C. elegans. Total small RNAs from wild-type and mir-249 mutant in adult stage worms were subjected to small RNA sequencing using an Illumina platform with Tobacco Acid Pyrophosphatase (TAP) treatment, allowing detection of secondary siRNAs carrying 5M-bM-^@M-^Y-tri-phosphate.
2013-08-08 | E-GEOD-41900 | biostudies-arrayexpress
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 5M-bM-^@M-^Y guanosine. 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.
2010-04-01 | E-GEOD-20336 | biostudies-arrayexpress
Project description:We report the comprehensive sequencing of small RNA libraries created from different developmental stages of the basal chordate, Ciona intestinalis. Surprisingly, half of the identified miRNA loci encode not two, but up to four distinct small RNAs. These additional RNAs, termed M-bM-^@M-^\moRsM-bM-^@M-^], are generated from sequences adjacent to the predicted ~60-nt pre-miRNA. moRs appear to be produced by RNAse-III-like processing, are ~20-nt in length, have 5M-bM-^@M-^Y-monophosphate and 3M-bM-^@M-^Y-hydroxl termini, and like miRNAs, are developmentally regulated. Sequencing of small RNA libraries from 4 stages of Ciona intestinalis. Ciona_LarvaMir309_RNASeq and Dmel_Toll10b_RNASeq are sequencing runs to detect if the expression of these RNA products is specific to Ciona or not. An ectopic transcript of the Drosophila melanogaster miR-309 locus was expressed in Ciona to see if it processed the miRs differently than Drosophila melanogaster. The Dmel sequence is from the endogenous case and is a control for comparison.
2009-01-18 | E-GEOD-13625 | biostudies-arrayexpress
Project description:High-throughput sequencing of small RNAs from Xenopus tropicalis (adult liver, adult skin, oocytes stage I, II, III, IV, V, VI). total RNA, ~18-42 nt RNAs isolated using PAGE, ligation to adapters requires 5' monophosphate and 3' OH Illumina/Solexa sequencing of adult liver, adult skin, oocytes stage I, II, III, IV, V, VI
2009-07-09 | E-GEOD-14952 | biostudies-arrayexpress
Project description:High-throughput pyrosequencing of endogenous small RNAs from CSR-1 IP complexes and csr-1(tm892) and ego-1(om97) mutants with corresponding controls. RNAi-related pathways regulate diverse processes, from developmental timing to transposon silencing. Here, we show that in C. elegans the Argonaute CSR-1, the RNA-dependent RNA polymerase EGO-1, the Dicer-related helicase DRH-3, and the Tudor-domain protein EKL-1 localize to chromosomes and are required for proper chromosome segregation. In the absence of these factors chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Surprisingly, the CSR-1 interacting small RNAs (22G-RNAs) are antisense to thousands of germline-expressed protein-coding genes. Nematodes assemble holocentric chromosomes in which continuous kinetochores must span the expressed domains of the genome. We show that CSR-1 interacts with chromatin at target loci, but does not down-regulate target mRNA or protein levels. Instead, our findings support a model in which CSR-1 complexes target protein-coding domains to promote their proper organization within the holocentric chromosomes of C. elegans. 5 samples examined. Small RNAs that co-immunopercipitate with CSR-1 protein and input sample. Small RNAs from csr-1(tm892) and ego-1(om97) mutants and corresponding congenic wild type strain.
2009-10-02 | E-GEOD-18165 | biostudies-arrayexpress
Project description:Organisms exhibit a fascinating array of gene-silencing pathways, which have evolved in part, to confront invasive nucleic acids such as transposons and viruses. A key question raised by the existence of these pathways is how do they distinguish M-bM-^@M-^\selfM-bM-^@M-^] from M-bM-^@M-^\non-selfM-bM-^@M-^] nucleic acids? Evidence exists for a number of mechanisms that might facilitate detection of foreign sequences including mechanisms that sense copy-number, unpaired DNA, or aberrant RNA (e.g.dsRNA). Here we describe an RNA-induced epigenetic silencing pathway, RNAe, that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate RNAe, while maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences, while two other Argonaute pathways serve as epigenetic memories of "self" and "non-self" RNAs. These findings suggest how organisms may utilize RNAi-related mechanisms not only to recognize and silence foreign genes, but also to keep inventory of all genes expressed in the germ-line. Examine small RNA population changes in different transgene lines. FLAG::WAGO-9 was immunoprecipitated from 20 mg of lysate essentially as described (Gu et al., 2009). Small RNAs were extracted from WAGO-9 immune complexes as well as a portion of the input lysate, gel-purified, pre-treated with TAP, cloned and sequenced as described (Gu et al., 2009).
2012-07-17 | E-GEOD-38724 | biostudies-arrayexpress
Project description:Diverse naturally-occurring small RNA species interact with Argonaute proteins to mediate sequence-specific regulation in animals. In addition to micro-RNAs (miRNAs), which collectively regulate thousands of target mRNAs, other endogenous small RNA species include the Piwi-associated piRNAs that are important for fertility and a less well-characterized class of small RNAs often referred to simply as endo-siRNAs. Here we have utilized deep-sequencing technology and C. elegans genetics to explore the biogenesis and function of endo-siRNAs. We describe conditional alleles of the dicer-related helicase, drh-3, that implicate DRH-3 in both the response to foreign dsRNA as well as the RNA-dependent RNA Polymerase (RdRP)-dependent biogenesis of a diverse class of endogenous small RNAs, termed 22G-RNAs. We show that 22G-RNAs are abundantly expressed in the germline and maternally inherited and are the products of at least two distinct 22G-RNA systems. One system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage-related structures termed P-granules. The WAGO 22G-RNA system silences transposons, pseudogenes and cryptic loci as well as a number of genes. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one of the multiple, distinct WAGO surveillance pathways. These findings broaden our understanding of the biogenesis and diversity of 22G-RNA species and suggest potential novel regulatory functions for these small RNAs. 18 samples examined. Small RNA libraries generated from: C. elegans animals with mutations in the WAGO pathway and a WAGO-1 immunopercipitate.
2009-10-01 | E-GEOD-18215 | biostudies-arrayexpress
Project description:During each life cycle germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute (AGO) proteins and their small-RNA cofactors are expressed during male gametogenesis and promote male fertility. Here we show that the CSR-1 AGO functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal that represents an epigenetic memory of male-specific gene expression, while CSR-1, which is abundant in mature sperm, transmits this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic binary signals in the form of Argonaute/small-RNA complexes that constitute a memory of which genes were active in preceding generations. Examine small RNA changes in WT and alg-3/4 mutant males cultured at 20M-BM-0C and 25M-BM-0C, as well as determine the small RNAs enriched in a FLAG::CSR-1 IP from male worms grown at 25M-BM-0C. mRNA sequencing was also performed to determine how transcripts targeted by small RNAs change in mutant background at 20M-BM-0C and 25M-BM-0C.
2013-12-19 | E-GEOD-49672 | biostudies-arrayexpress