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Gene Silencing In Vitro and In Vivo Using Intronic MicroRNAs

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1218)

Abstract

MicroRNAs (miRNAs) are small, single-stranded noncoding RNAs important in many biological processes through posttranscriptional modification of complementary intracellular messenger RNAs (mRNAs). MiRNAs have been reported to induce RNA interference (RNAi), by utilizing the miRNA-induced silencing complex (miRISC) to target mRNAs. They were first discovered in Caenorhabditis elegans as native RNA fragments that modulate a wide range of genetic regulatory pathways during embryonic development, and are now recognized as small gene silencers transcribed from the noncoding regions of a genome. In humans, nearly 97 % of the genome is noncoding DNA and changes in these sequences are frequently noted to manifest in clinical and circumstantial malfunction; for example, type 2 myotonic dystrophy and fragile X syndrome were found to be associated with miRNAs derived from introns. Intronic miRNA (mirtrons) is a class of miRNAs derived from the processing of non-protein-coding regions of gene transcripts. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of RNA polymerase (Pol)-II and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in C. elegans, mouse, and human cells; however, their functions and applications have not been reported. It is notable that there are different, but still highly conserved, mirtrons in mammalian than in invertebrates, and could be an indication that mirtrons are an evolutionary precursor to existing miRNA biogenesis pathways. Here, we show that intron-derived miRNA is not only able to induce RNAi in mammalian cells but also in fish, chicken embryos, and adult mice cells, demonstrating the evolutionary preservation of this gene regulation system in vivo. These miRNA-mediated animal models provide artificial means to reproduce the mechanisms of miRNA-induced disease in vivo and will shed further light on miRNA-related therapies.

Key words

MicroRNA (miRNA) RNA interference (RNAi) RNA polymerase type II (Pol II) RNA splicing Intron Intronic miRNA (mirtrons) RNA-induced gene-silencing complex (RISC) Gene silencing in vivo 
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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Cell and Neurobiology, Keck School of MedicineUniversity of Southern CaliforniaLos AngelesUSA
  2. 2.Division of Regenerative MedicineWJWU & LYNN (W&L) Institute for Stem Cell ResearchSanta Fe SpringsUSA

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