Genome-wide analysis of siRNA accumulation in Arabidopsis treated with bacteria
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE19694 | GEO | 2010/07/23
SECONDARY ACCESSION(S): PRJNA122227
REPOSITORIES: GEO
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Project description:To identify endogenous siRNAs in response to bacterial pathogen at a whole genome level, we performed small RNA profiling on Pseudomonas syringae-challenged Arabidopsis and obtained more than 24.6 million (M) reads with more than 3.8 M unique small RNAs that perfectly matched Arabidopsis genome. We found some new miRNAs and some miRNA induced by pathogen infection. We also identified more than 20K unique siRNAs from the NAT mRNAs and 22K siRNAs from the introns or intron-exon junction of the NATs. Sequencing of small RNAs from Arabidopsis infected by control and 3 bacterias in 6h and 14h.
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Project description:The development of high-throughput genomic technologies has revealed that a large fraction of the genomes of eukaryotes is associated with the expression of noncoding RNAs. One class of noncoding RNA, the cis-natural antisense transcripts (cis-NATs), are particularly interesting as they are at least partially complementary to the protein-coding mRNAs. Although most studies described cis-NATs involved in the regulation of transcription, a few reports have shown recently that cis-NATs can also regulate translation of the cognate sense coding genes in plants and mammals. In order to identify novel examples of translation regulator cis-NATs in Arabidopsis thaliana, we designed a high-throughput experiment based on polysome profiling and RNA-sequencing. Expression of cis-NATs and translation efficiency of the cognate coding mRNAs were measured in roots and shoots in response to various conditions, including phosphate deficiency and treatment with phytohormones. We identified several promising candidates, and validated a few of them experimentally, in Arabidopsis thaliana transgenic lines over-expressing in trans the translation regulator candidate cis-NATs.
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Project description:Arabidopsis thaliana (Arabidopsis) encodes five DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1 to DRB5, that predominantly act as non-catalytic cofactors for DICER-LIKE (DCL) proteins in the double-stranded RNA (dsRNA) processing stages of small RNA (sRNA) production pathways. In the nucleus, DRB1 is required for microRNA (miRNAs) processing from imperfectly dsRNA precursors by DCL1. Similarly, DRB4 is required by DCL4 for small-interfering RNAs (siRNAs) production from endogenous or exogenous perfectly dsRNA templates. DRB2 has been recently demonstrated to be required for miRNA and siRNA production in developmentally-important tissues of Arabidopsis while the requirement of either DRB3 or DRB5 in sRNA production remains unclear. Here, we analyse in parallel, the contribution of all five DRB protein family members to the global sRNA landscape of Arabidopsis floral tissues. In depth bioinformatic analysis of sRNA sequencing datasets generated from floral tissues of DRB knockout mutant (drb) plant lines, drb1, drb2, drb4, drb12, drb14, drb24, and drb35 and their comparison to the floral sRNA profile of wild-type Arabidopsis, has enabled confident assignment of the requirement of DRB1, DRB2 and DRB4 for the production of specific miRNA and siRNA subclasses in this tissue. Our analyses have additionally identified novel and/or expanded roles for DRB2 in miRNA, trans-acting siRNAs (tasiRNAs) and natural antisense transcript siRNAs (natsiRNAs) production.
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Project description:Most endogenous siRNAs in Arabidopsis are dependent on RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) for their biogenesis. Recent work has demonstrated that the maize MEDIATOR OF PARAMUTATION1 (mop1) gene is a predicted ortholog of the Arabidopsis RDR2 gene. The mop1 gene is required for establishment of paramutation and maintenance of transcriptional silencing of transposons and transgenes, suggesting the potential involvement of small RNAs. We analyzed small RNAs in wildtype maize and in the isogenic mop1-1 loss-of-function mutant using Illumina’s sequencing-by-synthesis (SBS) technology, which allowed us to characterize the complement of maize small RNAs to considerable depth. Similar to rdr2 in Arabidopsis, in mop1-1, the 24 nt (nucleotide) endogenous heterochromatic short-interfering siRNAs were dramatically reduced resulting in an enrichment of miRNAs and trans-acting siRNAs (ta-siRNAs). In contrast to the Arabidopsis rdr2 mutant, the mop1-1 plants retained a highly abundant heterochromatic ~22 nt class of small RNAs. These data suggest that maize, unlike Arabidopsis, has a second mechanism for heterochromatic siRNA production. The enrichment of miRNAs and loss of 24 nt heterochromatic siRNAs in mop1-1 should be advantageous for miRNA discovery as the maize genome becomes more fully sequenced.
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Project description:Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals that endogenous small interfering RNAs (siRNAs) are produced via bidirectional transcription. >100 cis-NATs with overlapping 3' exons generate 21-nt, Dicer-2 (Dcr-2)Âdependent, 3'-end modified siRNAs. To determine whether any co-expressed cisNATs are denied entry into the RNAi pathway, we analyzed the gene expression profile of S2 cells. The analysis suggested that the processing of cis-NATs by RNAi are actively restricted, and the selected loci are enriched for nucleic acidÂbased functions and include Argonaute-2 (AGO2) itself. Experiment Overall Design: Drosophila Schneider cells (S2) were treated with dsRNA against GFP for 8 days. The treatment was done in duplicate. Total RNA was extracted from the dsRNA treated cells using trizol. Experiment Overall Design: Gene expression analysis was conducted using Drosophila Genome 2.0 Genechip® arrays (Affymetrix, Santa Clara, CA). Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3¹ Amplification One-Cycle Target labeling kit according to manufacturer¹s protocol. For each array, 10ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000 and data was obtained using the Genechip® Operating Software (Version 1.2.0.037).
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Project description:The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is approximately 155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1-7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs. Keywords: small RNA sequences generated by 454 sequencing
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