Small RNA profiling of the allelic series of miR-17~92 mutant embryos
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Doron Betel
PROVIDER: E-GEOD-63660 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:We report the generation of an allelic series of miR-17~92 mutant mice, each carrying the targeted deletion of specific miRNA families encoded by the cluster. We integrate the phenotypic characterization of these strains with RNA-seq analysis showing the consequences of selective miRNA inactivation on gene expression in the developing embryo. We generated and analyzed RNA-seq data from 8 different miR-17~92 alleles, in three different microdissected tissues collected from E9.5 mouse embryos. Datasets contain biological triplicates (except for duplicates in one case, Delta_17_18_92) for a total of 71 samples.
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Project description:The study sought to determine whether deletion of individual seed families within the miR-17~92 cluster affected the expression of the remaining microRNAs of the cluster.
2015-05-22 | GSE63660 | GEO
Project description:We report the generation of an allelic series of miR-17~92 mutant mice, each carrying the targeted deletion of specific miRNA families encoded by the cluster. We integrate the phenotypic characterization of these strains with RNA-seq analysis showing the consequences of selective miRNA inactivation on gene expression in the developing embryo.
2015-02-02 | GSE63813 | GEO
Project description:RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and as RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5' to 3' in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3' UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5' to 3' on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3' UTRs. Flp-In T-REx HEK293 cells expressing FLAG/HA-tagged MOV10 WT, MOV10 K530A, MOV10 D645N and UPF1 were sequenced. mRNA half-life data under GSE56751.
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Project description:Dicer is a deeply conserved endoribonuclease with key functions in small RNA biogenesis. Here we employed PAR-CLIP/iPAR-CLIP to identify direct Dicer binding sites in the transcriptomes of human cells and human. We found hundreds of novel miRNAs and non-canonical Dicer substrates with high sensitivity. Small RNA production depended on structure of the binding site and is globally biased towards the 5' arm of hairpins. Unexpectedly, in both species Dicer bound numerous hairpins inside mRNAs without observable small RNA production. Our data revealed ~100 mRNAs of protein coding genes to be targeted in both human and worm. These mRNAs significantly overlapped with the RNAi pathway. We also, unexpectedly, found that mitochondrial transcripts are Dicer targets in both species. We demonstrate functional consequences of Dicer binding by perturbation analysis. Taken together,we provide the first genome-wide catalog of direct Dicer targets. Our results suggest widespread function outside of miRNA biogenesis. PAR-CLIP basically as described previously (Hafner et al. 2010).
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Project description:To exert regulatory function, miRNAs guide Argonaute (AGO) proteins to partially complementary sites on target RNAs. Crosslinking and immunoprecipitation (“re state-of-the-art to map AGO binding sites, but assigning the targeting miRNA to these sites relies on bioinformatics predictions and is therefore indirect. To directly and unambiguously identify miRNA:target site interactions, we modified our CLIP methodology in C. elegans to experimentally ligate miRNAs to their target sites. Unexpectedly, ligation reactions also occurred in absence of the exogenous ligase. Our in vivo dataset and re-analysis of published mammalian AGO-CLIP data for miRNA-chimeras yielded >17,000 miRNA:target site interactions. Analysis of interactions and extensive experimental validation of chimera-discovered targets of viral miRNAs suggest that our strategy identifies canonical, non-canonical, and non-conserved miRNA interactions. Our data suggest that ~80% of miRNA:targets have perfect or partial seed complementarity. In summary, analysis of miRNA:target chimeras enables the systematic, context-specific, in vivo discovery of miRNA interactions. In vivo PAR-CLIP basically as described previously (Jungkamp et al. 2011) using GFP-tagged ALG-1 expressing worms in L3 stage. Worm lysate was treated with RNase T1. Following immunoprecipitation and a second RNase T1 digest, it was proceeded as described in Hafner et al. 2010. For the modified iPAR-CLIP ligation samples and its control samples immuno-purified complexes were treated with PNK phospathase minus, subjected to ligation with T4 RNA ligase/no ligase added and subsequently phosphorylated with PNK. Protein purification and RNA library preparation essentially as described in Hafner et al., but with the selection of longer RNA products.
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Project description:The precise control of miR-17~92 microRNA (miRNA) is essential for normal development and overexpression of certain miRNAs from this cluster is oncogenic. Here we find the relative expression of the six miRNAs processed from the primary (pri-miR-17~92) transcript is dynamically regulated during embryonic stem cell differentiation. We identify a new miRNA biogenesis intermediate, termed ‘progenitor-miRNA’ (pro-miRNA), that is an efficient substrate for Microprocessor. An autoinhibitory 5’ RNA fragment is cleaved to generate pro-miRNA and selectively license Microprocessor-mediated production of pre-miR-17, -18a, -19a, 20a, and -19b. Using genetic, biochemical, and structural methods we define two complementary cis-regulatory repression domains required for the formation of this inhibitory RNA conformation. We find the endonuclease CPSF3 (CPSF73), and the Spliceosome-associated ISY1 are required for pro-miRNA biogenesis and expression of all miRNAs within the cluster except miR-92. Thus, developmentally regulated generation of pro-miRNA explains the posttranscriptional control of miR-17~92 expression in development. Illumina RNAseq in WT, dgcr8-/- and dicer-/- mESCs and small RNA seq in WT mESCs
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Project description: Not available
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Project description:The Piwi protein Mili and the Piwi-interacting RNA (piRNA) pathway are required to establish L1 DNA methylation during epigenetic reprogramming that accompanies male germ cell development. Although Mili is expressed throughout most of adult spermatogenesis, its function therein remains unknown. We find that Mili displays a hitherto unappreciated dynamic expression profile during spermatogenesis, expressed in the mitotic spermatogonia but absent at the early meiotic stages only to reappear in late zygotene spermatocytes. Here we show using conditional mutagenesis that Mili's endonuclease activity post-transcriptionally cleaves L1 transcripts commencing upon its reappearance in late zygotene spermatocytes even in the presence of normal L1 DNA methylation
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Project description:The miR-17-92 cluster targets mRNAs involved in distinct pathways which either promote or inhibit tumor progression. However, the cellular and molecular mechanisms underlying miR-17~92 cluster mediated pro- or anti- tumorigenic effects has not been studied. In this study, we found that inhibition of colon cancer progression is dictated by quantitatively controlling expression of the miR-17~92 cluster. To identify molecular mechanisms giving rise to the different growth/metastasis patterns, we profiled gene expression in miR-Ctrl, miR-17~92Med and miR-17~92Hi cells using Affymetrix Murine GeneST Arrays.
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