High-throughput SOLEXA sequencing of endogenous small RNAs from Arabidopsis thaliana wild-type
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE35335 | GEO | 2014/01/25
SECONDARY ACCESSION(S): PRJNA152651
REPOSITORIES: GEO
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Project description:The length of Arabidopsis microRNA genes and their precursors (pri-miRNAs) can very between several hundreds bp to several thousands bp. They may contain introns, precursors may undergo alternative splicing, and often there are more than one polyadenylation site. An intriguing question arise: why to transcribe thousands of base pairs to produce a small RNA molecule of about 21 nt in length? We decided to sequence small RNAs from 35-day-old rosette leaves and to map the obtained reads of 18-26 nt in length to Arabidopsis pri-miRNAs. Our analyses revealed the presence of sRNAs that can derive only from miRNA precursors. An example is the identification of a substantial number of miR319b.2 reads. This sRNA is a product of pri-miR319b processing. Our further experimental analyses show that it carries multiple signatures of a functional microRNA 2 samples examined: wild type
2014-01-25 | E-GEOD-35335 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) regulate plant development by post-transcriptional regulation of target genes. In Arabidopsis thaliana, DCL1 processes precursors (pri-miRNAs) to miRNA duplexes, which associate with AGO1. Additional proteins act in concert with DCL1 (e.g. HYL1 and SERRATE) or AGO1, respectively, to facilitate efficient and precise pri-miRNA processing and miRNA loading. In this study, we show that the accumulation of plant microRNAs depends on RECEPTOR FOR ACTIVATED C KINASE 1 (RACK1), a scaffold protein found in all higher eukaryotes. miRNA levels are reduced in rack1 mutants and our data suggest that RACK1 affects the microRNA pathway via several distinct mechanisms involving direct interactions with known microRNA factors: RACK1 ensures the accumulation and processing of some pri-miRNAs, directly interacts with SERRATE and is part of an AGO1 complex. As a result, mutations in RACK1 lead to misregulation of miRNA target genes, which is important for ABA responses and phyllotaxy. In conclusion, our study discovered complex functioning of RACK1 proteins in the Arabidopsis miRNA pathway, which are important for miRNA production and therefore plant development.
2013-07-01 | GSE40579 | GEO
Project description:In this study, in order to identify miRNA targets, a degradome library derived from anthers of the WT and GMS (Genetic Male Sterility) mutant representing three stages of development was constructed and sequenced, resulting in the generation of 24.6 million raw reads. After removal of low quality sequences and adapter sequences, 24.4 million clean reads were obtained and 98% were 20 or 21 nt in length as expected in that normally length distribution peak of degradome fragment is between 20 and 21 nt [Addo-Quaye C, Eshoo TW, Bartel DP, Axtell MJ: Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr Biol 2008, 18:758-762].
2013-07-28 | GSE43389 | GEO
Project description:The Chinese surf clam (Mactra chinensis) is an economically important clam, distributed in Liaoning and Shandong province. In recently years，because of coastal environmental deterioration and overfishing, the natural population of M. chinensis have considerably declined . In this paper, we study the microRNA transcriptome of gills, including control and experimental group was sequenced through Illumina Hi-seq 2500 CE. And the differential expression was used to find the functional microRNA response to the Cd2+ exposure. Through Illumina Hi-seq 2500, a total of 14,415,256 clean reads and 15,570,111 clean reads were yielded in the gill of control and experimental group respectively. A total of 14,584,077 sRNA, in which there are 12,505,055 sRNA shared by S01 & S02, including 187,859 unique sRNA. The distribution of the sRNA length in the two library was similar, most of them were 26-27 nt. 27 nt was the most abundant length in S01, followded by 28 nt, 26 nt, and 23 nt; 26 nt was the most abundant length in S02, and followed by 27 nt, 28 nt and 23 nt. 50 miRNA was found in unique sRNA, including 38 conserved and 12 novel genes. The most abundant length of microRNA in the two library was the same, 23 nt. Through the analyze of differential expression analysis, the expression of 5 miRNA was induced with significantly difference, and 17 miRNA was down regulated and 28 miRNA was up regulated without significantly difference. So the miRNA in gill of M. chinensis might involve the environmental stress. 542 target genes were yielded when the 50 miRNA were hit to mRNA genome. And the target genes of differential expression miRNA were annotated by hitting to the NCBI database, and 4 genes hit to the COG, 1 genes hit to the GO, 5 genes hit to the KEGG and 11 genes hit to the nr database. The genes hit to the NCBI database include E3 ubiquitin-protein ligase, Wnt signaling pathway and Regulator of G-protein signaling 22.
2018-08-02 | GSE85561 | GEO
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2011-01-29 | E-GEOD-26909 | biostudies-arrayexpress
Project description:A battery of spliceosome-associated proteins has been identified in microRNA (miRNA) biogenesis; however, the underlying mechanisms remain elusive. The intron lariat spliceosome (ILS) complex is highly conserved among eukaryotes and its disassembly marks the end of a canonical splicing cycle. In this study, we show that two conserved disassembly factors of the ILS complex, ILP1 and NTR1, positively regulate microRNA biogenesis through facilitating transcriptional elongation in Arabidopsis. ILP1 and NTR1 form a stable complex and co-regulate alternative splicing of more than a hundred genes across the genome including the core circadian gene LHY and some pri-miRNAs. Dysfunction in either ILP1 or NTR1 result in reduced RNA polymerase II occupancy at elongated regions of MIR chromatins, without affecting MIR promoter activity, pri-miRNA decay and DCL1 processing. Our results provide insights into the molecular mechanisms of spliceosomal machineries in non-coding RNA regulation.
2019-11-12 | PXD011542 | Pride
Project description:MicroRNA (miRNA)-guided target RNA expression is vital for a wide variety of biological processes in eukaryotes. The integration of miRNAs in diverse biological networks relies upon the confirmation of their RNA targets. Most miRNA targets in Arabidopsis are validated, but those in rice are yet to be characterized. To identify transcriptome-wide small RNA targets in rice, we generated 20-nt small cDNA library and obtained nearly 40 million reads. Sequence analysis yielded 11,552,007 unique reads that can be perfectly mapped to the rice genome. Sequence analysis not only found homologous targets for conserved miRNAs but also many novel targets. Besides miRNA atregts, the rice degradome contained fragments derived from MIRNA precursors. A closer inspection of these fragments revealed a unique pattern distinct from siRNA producing loci. This attribute can serve as one of the ancillary criteria for separating miRNAs from siRNAs in plants. Keywords: high throughout sequencing of rice degradome Identify transcriptome-wide small RNA targets in rice
2010-05-03 | E-GEOD-17398 | biostudies-arrayexpress
Project description:MicroRNA-offset RNAs (moRs) were first identified in simple chordates and subsequently in mouse and human cells by deep sequencing of short RNAs. MoRs are derived from sequences located immediately adjacent to microRNAs (miRs) in the primary miR (pri-miR). Currently moRs are considered to be simply a by-product of miR biosynthesis that lack biological activity. Here we show for the first time that a moR is biologically active. We now demonstrate that endogenous and over-expressed moR-21 significantly alters gene expression and inhibits the proliferation of vascular smooth muscle cells (VSMC). We report that the "seed region" of moR-21 as well as the "seed match region" in the target gene 3'UTR are indispensable for moR-21-mediated gene down-regulation. We further demonstrated that moR-21-mediated gene repression is Argonaute 2 (Ago2) dependent. In addition, we find that miR-21 and moR-21 may regulate different genes in a given pathway and can oppose each other in regulating certain genes. Taken together, these findings provide the first evidence that microRNA offset RNA regulates gene expression and is biologically active. Small RNAs were harvested from mouse aortic smooth muscle cells (AoSMCs), with three biological replicates, using miReasy kits (Qiagen) according to the manufacturerâ??s instructions. RNA was quantified using a Nanodrop spectrophotometer. Small RNA library construction and illumina HiSeq 75bp single read sequencing were performed at the Yale Center for Genome Analysis. Reads were trimmed to remove adaptor sequences, and inserts longer than 13 bases were mapped to the mouse mm10 genome using Bowtie (with settings â??n 0 -m 5 --best --strata, allowing 0 mismatches and up to 5 genomic loci with a perfect match, {Genome Biol. 2009;10(3):R25, Langmead B, Trapnell C, Pop M, Salzberg SL}). Reads that mapped to within 50 bp of more than one annotated mouse pri-miR (as defined by miRBase {Kozomara A, Griffiths-Jones S. Nucelic Acids Res 2014 42:D68-D73}), were allotted evenly between these locations. Reads that fell entirely within established miRbase 3p or 5p miR coordinates, plus or minus 3 bp, were counted as 3p or 5p miRs, respectively. Reads that fell entirely within the range of 35 bp 3â?? to 3 bp 5â?? of the last base of a 3p miR were counted as 3p moR reads. Those that fell entirely between 3bp 3â?? and 35 bp 5â?? of the first base of a 5p miR were counted as 5p moR reads. On average each library contained 17.4 million qualifying inserts, of which 13.8 million (79.3%) mapped to one of these four regions, indicating that the large majority of inserts represented miR or moR sequences. Reads were divided by the number of million reads mapping to these regions in each library to give normalized reads per million reads (RPMR) values.
2016-06-01 | E-GEOD-75113 | biostudies-arrayexpress
Project description:RNA interference (RNAi) technology is widely used in basic and translational research. By mimicking natural primary microRNA (pri-miRNA) cluster, multiple engineered hairpins can be transcribed as a single transcript from the same Pol-II promoter, enabling multiplex RNAi in mammalian cells 1-5. However, constructing synthetic miRNA cluster is still time-consuming, and the processing and function of miRNA cluster are incompletely understood. Here, we identified a miRNA precursor architecture that allowed precise miRNA maturation. We established a hierarchical cloning method for efficient construction of synthetic miRNA cluster harboring up to 18 miRNA precursors. We demonstrated that maturation and function of individual miRNA precursors were independent on their positions in the cluster. We then analyzed the integration efficiency of miRNA clusters with varying number of miRNA precursors by using CRISPR/Cas9 mediated integration, piggyBac transposon system, and lentiviral system. This synthetic miRNA cluster system provides an important tool for multiplex RNAi in mammalian cells. Different kinds of synthetic miRNAs or miRNA clusters, including the same synthetic miRNA precursor embedded in a series of derivative backbones, 18 synthetic miRNA precursors, miRNA precursors with various stem lengths and miRNA clusters carrying different numbers of different pri-miRNA hairpins, were expressed in HEK293 cells. The microRNA maturation pattern of individual miRNA precursors was detected by small RNA sequencing.
2016-09-01 | E-GEOD-71088 | biostudies-arrayexpress
Project description:MicroRNA (miRNA)-guided target RNA expression is vital for a wide variety of biological processes in eukaryotes. The integration of miRNAs in diverse biological networks relies upon the confirmation of their RNA targets. Most miRNA targets in Arabidopsis are validated, but those in rice are yet to be characterized. To identify transcriptome-wide small RNA targets in rice, we generated 20-nt small cDNA library and obtained nearly 40 million reads. Sequence analysis yielded 11,552,007 unique reads that can be perfectly mapped to the rice genome. Sequence analysis not only found homologous targets for conserved miRNAs but also many novel targets. Besides miRNA atregts, the rice degradome contained fragments derived from MIRNA precursors. A closer inspection of these fragments revealed a unique pattern distinct from siRNA producing loci. This attribute can serve as one of the ancillary criteria for separating miRNAs from siRNAs in plants. Keywords: high throughout sequencing of rice degradome
2010-05-04 | GSE17398 | GEO