title: The biogenesis and characterization of mammalian microRNAs of mirtron origin.
abstract: Mirtrons, short hairpin pre-microRNA (miRNA) mimics directly produced by intronic splicing, have recently been identified and experimentally confirmed in invertebrates. While there is evidence to suggest several mammalian miRNAs have mirtron origins, this has yet to be experimentally demonstrated. Here, we characterize the biogenesis of mammalian mirtrons by ectopic expression of splicing-dependent mirtron precursors. The putative mirtrons hsa-miR-877, hsa-miR-1226 and mmu-miR-1224 were designed as introns within eGFP. Correct splicing and function of these sequences as introns was shown through eGFP fluorescence and RT-PCR, while all mirtrons suppressed perfectly complementary luciferase reporter targets to levels similar to that of corresponding independently expressed pre-miRNA controls. Splicing-deficient mutants and disruption of key steps in miRNA biogenesis demonstrated that mirtron-mediated gene knockdown was splicing-dependent, Drosha-independent and had variable dependence on RNAi pathway elements following pre-miRNA formation. The silencing effect of hsa-miR-877 was further demonstrated to be mediated by the generation of short anti-sense RNA species expressed with low abundance. Finally, the mammalian mirtron hsa-miR-877 was shown to reduce mRNA levels of an endogenous transcript containing hsa-miR-877 target sites in neuronal SH-SY5Y cells. This work confirms the mirtron origins of three mammalian miRNAs and suggests that they are a functional class of splicing-dependent miRNAs which are physiologically active.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Sibley
authors.forename: Yiqi
authors.initials: Y
authors.lastname: Seow
authors.forename: Sheena
authors.initials: S
authors.lastname: Saayman
authors.forename: Krijn K
authors.initials: KK
authors.lastname: Dijkstra
authors.forename: Samir
authors.initials: S
authors.lastname: El Andaloussi
authors.forename: Marc S
authors.initials: MS
authors.lastname: Weinberg
authors.forename: Matthew J A
authors.initials: MJ
authors.lastname: Wood
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D007438:Introns; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D034622:RNA Interference; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D043244:Ribonuclease III / Q000502:physiology
chemical_list: C558413:MIRN1224 microRNA, mouse; C570298:MIRN877 microRNA, human; D035683:MicroRNAs; D012322:RNA Precursors; C517918:DROSHA protein, human; D043244:Ribonuclease III