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MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila.

ABSTRACT: microRNAs (miRNAs) are approximately 22 nucleotide regulatory RNAs derived from hairpins generated either by Drosha cleavage (canonical substrates) or by splicing and debranching of short introns (mirtrons). The 5' end of the highly conserved Drosophila mirtron-like locus mir-1017 is coincident with the splice donor, but a substantial "tail" separates its hairpin from the 3'splice acceptor. Genetic and biochemical studies define a biogenesis pathway involving splicing, lariat debranching, and RNA exosome-mediated "trimming," followed by conventional dicing and loading into AGO1 to yield a miRNA that can repress seed-matched targets. Analysis of cloned small RNAs yielded six additional candidate 3' tailed mirtrons in D. melanogaster. Altogether, these data reveal an unexpected role for the exosome in the biogenesis of miRNAs from hybrid mirtron substrates.

SUBMITTER: Flynt AS

PROVIDER: S-EPMC2904328 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila.

Flynt Alex S AS Greimann Jaclyn C JC Chung Wei-Jen WJ Lima Christopher D CD Lai Eric C EC

Molecular cell 20100601 6

microRNAs (miRNAs) are approximately 22 nucleotide regulatory RNAs derived from hairpins generated either by Drosha cleavage (canonical substrates) or by splicing and debranching of short introns (mirtrons). The 5' end of the highly conserved Drosophila mirtron-like locus mir-1017 is coincident with the splice donor, but a substantial "tail" separates its hairpin from the 3'splice acceptor. Genetic and biochemical studies define a biogenesis pathway involving splicing, lariat debranching, and RN ...[more]

PMID: 20620959

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Project description:The precise mechanism of intercellular communication among cancer cells after radiation exposure remains unclear. Exosomes are membrane-enclosed small vesicles constituted by lipid bilayers and are recognized as mediators of intercellular communication that transport a variety of intracellular components, including microRNA (miRNA). Here we identified novel roles of exosomes released from irradiated cells to neighboring cancer cells. To confirm the presence of exosomes in the culture media of the human pancreatic cancer cell line MIAPaCa-2, ultracentrifugation was performed followed by transmission electron microscopy and nanoparticle tracking analysis (NanoSight) using the exosome-specific surface markers CD9 and CD63. Subsequent endocytosis of exosomes was confirmed by fluorescent microscopy. Cell survival after irradiation and following the addition of exosomes was evaluated by a colony-forming assay. Expression of miRNAs in exosomes was then quantified by microarray analysis, while those of Cu/Zn superoxide dismutase (SOD1) and Mn-superoxide dismutase (SOD2) enzymes in MIAPaCa-2 cells were evaluated by western blotting. Results showed that the uptake of irradiated exosomes was significantly higher than that of non-irradiated exosomes. Notably, irradiated exosomes induced higher intracellular levels of ROS and a higher frequency of DNA damage in MIAPaCa-2 cells, as determined by fluorescent microscopy and immunocytochemistry, respectively. Moreover, six upregulated and five downregulated miRNAs were identified in 5 Gy- and 8 Gy-irradiated cells using miRNA microarray analyses. Further analyses using miRNA-mimics and real time reverse transcription PCR identified miR-6823-5p as a possible candidate for SOD1 inhibition, leading to increased intracellular ROS level and DNA damage. This is the first study to demonstrate that irradiated exosomes can enhance the radiation effect via increased intracellular ROS levels in cancer cells, potentially leading to improved understanding of the bystander effect of neighboring cancer cells.

Dataset Information

MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila.

Publications

MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila.

Similar Datasets

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