Unknown,Transcriptomics,Genomics,Proteomics

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Novel microRNA-target RNA pairs revealed by Parallel analysis of RNA ends

ABSTRACT: MicroRNAs are important regulatory molecules in most eukaryotes and the identification of their mRNA targets is essential for their functional analysis. From inflorescence tissue of Arabidopsis, >28,000,000 signatures were sequenced from 5’ ends of polyadenylated products of mRNA decay. Within the set of ~27,000 transcripts included in the 3,500,000 non-redundant signatures, several previously predicted but non-validated miRNA targets were found. Like validated targets, most showed a single abundant signature at the miRNA cleavage site, particularly in libraries from a mutant deficient in the 5’ to 3’ exonuclease AtXRN4. Among the most unexpected miRNA targets discovered were miRNA precursor transcripts that are self-targeted by their own mature miRNA. Although the miRNAs of Arabidopsis have been extensively investigated, working in reverse from the cleaved targets, additional novel miRNAs were identified and validated. This deep and versatile approach will impact the study of other aspects of RNA processing beyond miRNA-target RNA pair analyses. Keywords: miRNA-target RNA pairs, Palallel analysis of RNA ends, PARE, SBS RNA from inflorescence tissue from wildtype Arabidopsis and xrn4 mutants was extracted. Following polyA RNA extraction, libraries for PARE were constructed. RNA from inflorescence tissue from wildtype Arabidopsis (Col-0), and rdr2 and dcl2,3,4 mutants was extracted. The material was submitted to Illumina and libraries were constructed for small RNAs. Individual small RNA libraries from inflorescences of Arabidopsis wildtype, rdr2, and dcl2/dcl3/dcl4 were sequenced with SBS at Illumina, Inc. The distribution of small RNA sizes in each library is in agreement with that published previously for the wild type or the respective mutants sequenced with MPSS or 454; however, the presence and abundances of some small RNAs may differ. This may be attributed to the use of different sequencing technologies with different depths, biological differences in the tissue used, or other unknown reasons. raw data requested but not provided for GSM280226 and GSM280227

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Marcelo German

PROVIDER: E-GEOD-11094 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends.

German Marcelo A MA Pillay Manoj M Jeong Dong-Hoon DH Hetawal Amit A Luo Shujun S Janardhanan Prakash P Kannan Vimal V Rymarquis Linda A LA Nobuta Kan K German Rana R De Paoli Emanuele E Lu Cheng C Schroth Gary G Meyers Blake C BC Green Pamela J PJ

Nature biotechnology 20080609 8

MicroRNAs (miRNAs) are important regulatory molecules in most eukaryotes and identification of their target mRNAs is essential for their functional analysis. Whereas conventional methods rely on computational prediction and subsequent experimental validation of target RNAs, we directly sequenced >28,000,000 signatures from the 5' ends of polyadenylated products of miRNA-mediated mRNA decay, isolated from inflorescence tissue of Arabidopsis thaliana, to discover novel miRNA-target RNA pairs. With ...[more]

PMID: 18542052

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Project description:Maize LEAFBLADELESS1 (LBL1) and Arabidopsis SUPPRESSOR OF GENE SILENCING3 (SGS3) play orthologous roles in the biogenesis of 21 nucleotide trans-acting short-interfering RNAs (tasiRNAs). The phenotypes conditioned by mutation of lbl1 and SGS3 are, however, strikingly different, suggesting that the activities of these small RNA biogenesis components, or the tasiRNAs and their targets might not be entirely conserved. To investigate the basis for this phenotypic variation, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects all major classes of small RNAs, and reveal unexpected crosstalk between tasiRNA biogenesis and other small RNA pathways regulating miRNAs, retrotransposons, and DNA transposons. We further identified genomic regions generating phased siRNAs, including numerous loci generating 22-nt phased small RNAs from long hairpin RNAs or overlapping antisense transcripts not previously described in other plant species. By combining both analyses, we identified nine TAS loci, all belonging to the conserved TAS3 family. Contrary to other plant species, no TAS loci targeted by a single miRNA were identified. Information from target prediction, RNAseq, and PARE analyses identified the tasiARFs as the major functional tasiRNAs in the maize vegetative apex where they regulate expression of ARF3 homologs. As such, divergence in TAS pathways is unlikely to account for the distinct phenotypes of tasiRNA biogenesis mutants in Arabidopsis and maize. Instead, the data suggests variation in the spatiotemporal regulation of ARF3, or divergence in its function, as a plausible basis for the dramatic phenotypic differences observed upon mutation of SGS3/lbl1 in Arabidopsis and maize. An analysis of tasiRNA biogenesis, activity, and contribution to developmental phenotypes in the maize leaf. Data generated includes small RNA sequencing data and mRNA sequencing data. All data was generated in both wild type and lbl1 mutant maize leaf apices. Three replicates were generated for each genotype for the small RNA data. Two of these replicates were also used for the RNA-seq data.

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Novel microRNA-target RNA pairs revealed by Parallel analysis of RNA ends

Publications

Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends.

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