Isolation and Identification of Gene-Specific MicroRNAs.

Abstract

Computer programming has identified hundreds of genomic hairpin sequences, many with functions yet to be determined. Because transfection of hairpin-like microRNA precursors (pre-miRNAs) into mammalian cells is not always sufficient to trigger RNA-induced gene silencing complex (RISC) assembly, a key step for inducingRNA interference (RNAi)-related gene silencing, we havedeveloped an intronic miRNA expressionsystem to overcome this problem by inserting a hairpin-like pre-miRNA structure into the intron region of a gene, and hencesuccessfully increase the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. This intronic miRNA biogenesis mechanismhas been found to depend on a coupled interaction of nascent messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNAso obtained is transcribed by type-II RNApolymerases, coexpressed within a primary gene transcript, and thenexcised out of the gene transcript by intracellular RNA splicing and processing machineries. After that, ribonuclease III (RNaseIII)endonucleases further process the spliced introns into mature miRNAs. Using this intronicmiRNA expression system, we have shown for the first time that the intron-derived miRNAs are able to elicit strong RNAi effects in not only human and mouse cells in vitrobut also in zebrafishes, chicken embryos, and adult micein vivo. We have also developed a miRNA isolation protocol, based on the complementarity between the designed miRNA and its targeted gene sequence, to purify and identify the mature miRNAs generated. As a result, several intronic miRNA identities and structures have been confirmed. According to this proof-of-principle methodology, we now have full knowledge to design variousintronicpre-miRNA inserts that are more efficient and effective for inducing specific gene silencing effects in vitro and in vivo.