microRNAs (miRNAs) are an abundant class of small RNAs that derive from distinctive hairpin precursor transcripts. Their biogenesis typically involves cleavage by the Drosha and Dicer RNase III enzymes, and the resultant mature miRNAs mediate extensive networks of post-transcriptional regulation. In the course of analyzing the miRNA pathway in Drosophila, we uncovered atypical miRNAs derived from short hairpin introns that we term mirtrons. Their biogenesis substitutes a seemingly obligate processing event by the Drosha RNase III enzyme with the splicing machinery. We characterized the biogenesis and evolutionary range of mirtrons across four animal species, using biochemical, molecular, genetic, and computational methods. This work led to a series of unexpected avenues that further expanded the diversity of non- canonical miRNAs, including a hybrid pathway that marries splicing with the RNA exosome to generate hairpins that serve as Dicer substrates, as well as short hairpins that generate miRNAs in a Dicer-independent mechanism in vertebrates. In addition, this work served as a foundation of our biological studies of miRNA functions. For our renewal application, we selected several directions on understanding enzymes and mechanisms that mediate the biogenesis of mirtrons and Dicer-independent miRNAs. We present extensive unpublished data for our proposed Aims, including the discovery of hundreds of mammalian mirtrons, including a novel 3'tailed subtype in mammals, the first demonstration of Dicer-independent miRNA function in Drosophila, and establishment of in vitro assays for Dicer-independent miRNA production. These preliminary data are the basis of individual gene studies to understand detailed mechanisms, which we will extend with genomewide perspectives. In addition, we wish to relate these findings to their broader implications with respect to other small RNAs (i.e. canonical miRNAs) and discovery of novel substrates. Finally, we are concerned with relating our research to larger evolutionary questions, including the evolutionary emergence of miRNAs and of RNAi itself, and with exploiting our mechanistic knowledge to advance RNA silencing as a technological tool.

Public Health Relevance

microRNAs are an extensive class of ~22 nucleotide RNAs that control the activity of messenger RNAs, the templates for protein synthesis. microRNAs dysfunction is linked to disease and cancer, while reciprocally, there is great potential for exploiting microRNAs and related regulatory RNAs as research tools and therapeutic strategies. This proposal extends our long-standing commitment to dissecting mechanisms of microRNA biogenesis by exploring new pathways that generate microRNAs and investigating enzymes that influence microRNA function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083300-07
Application #
8538420
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
2007-09-21
Project End
2016-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
7
Fiscal Year
2013
Total Cost
$464,755
Indirect Cost
$210,652
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
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Garaulet, Daniel L; Sun, Kailiang; Li, Wanhe et al. (2016) miR-124 Regulates Diverse Aspects of Rhythmic Behavior in Drosophila. J Neurosci 36:3414-21
Bortolamiol-Becet, Diane; Hu, Fuqu; Jee, David et al. (2015) Selective Suppression of the Splicing-Mediated MicroRNA Pathway by the Terminal Uridyltransferase Tailor. Mol Cell 59:217-28
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Wen, Jiayu; Ladewig, Erik; Shenker, Sol et al. (2015) Analysis of Nearly One Thousand Mammalian Mirtrons Reveals Novel Features of Dicer Substrates. PLoS Comput Biol 11:e1004441
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Wen, Jiayu; Duan, Hong; Bejarano, Fernando et al. (2015) Adaptive regulation of testis gene expression and control of male fertility by the Drosophila hairpin RNA pathway. [Corrected]. Mol Cell 57:165-78
Lin, Ching-Jung; Smibert, Peter; Zhao, Xiaoyu et al. (2015) An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis. RNA 21:2103-18

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