RNA interference (RNAi) is a post-transcriptional/transcriptional gene silencing mechanism conserved from fungi to humans. In RNAi pathways, small non-coding RNAs (sRNAs) with sizes ranging from 20-30 nucleotides (nt), including microRNAs (miRNAs) and various small interfering RNAs (siRNAs), associate with and guide Argonaute family proteins to messenger RNA targets, resulting in the silencing of gene expression in diverse biological processes. The filamentous fungus Neurospora crassa, an organism that broadly employs gene silencing in regulation of gene expression, offers a unique and powerful system for understanding of RNAi pathways. We have previously established the biochemical mechanism for the dsRNA-induced activation of the RNAi pathway. By purifying the Argonaute QDE-2- associated sRNAs from Neurospora, we recently discovered three new types of sRNAs in this organism, including the DNA damage-induced qiRNA, first fungal miRNA-like sRNAs (milRNAs) and Dicer- independent small interfering RNAs (disiRNAs). Importantly, our studies demonstrated the existence of diverse sRNA biogenesis pathways in this organism, including Dicer-dependent and Dicer-independent mechanisms, and the roles of the Argonaute protein QDE-2 in sRNA biogenesis.
In Specific Aim 1, we determine the mechanism of how DNA damage induces of qiRNA production and quelling.
In Specific Aim 2, we will determine how different pri-milRNAs are processed into mature milRNAs by different pathways and will uncover the "design" principles that steer different milRNAs into different pathways.
In Specific Aim 3, we will determine the biogenesis pathway and function of disiRNAs. Together, these studies address several fundamental questions in small RNA biogenesis and will expand our current knowledge of sRNA biogenesis pathways and sRNA function.

Public Health Relevance

RNA interference (RNAi) and related pathways use small non-coding RNAs, such as miRNAs, to play important roles in numerous biological processes, including development, antiviral defense, gene regulation and maintenance of genomic integrity. In addition to its wide use as a tool for study of gene function and gene identification, development and adoption of RNAi technologies have been extensively used in pharmaceutical target validation and in therapeutic development. Our goal is to understand the biogenesis pathways of small RNAs using a simple eukaryotic model system, which will potentially lead to new therapeutic approaches for treating human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM084283-05
Application #
8503458
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
2009-04-01
Project End
2017-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
5
Fiscal Year
2013
Total Cost
$286,200
Indirect Cost
$106,200
Name
University of Texas Sw Medical Center Dallas
Department
Physiology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
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Dang, Yunkun; Li, Liande; Guo, Wei et al. (2013) Convergent transcription induces dynamic DNA methylation at disiRNA loci. PLoS Genet 9:e1003761
Xue, Zhihong; Yuan, Haiyan; Guo, Jinhu et al. (2012) Reconstitution of an Argonaute-dependent small RNA biogenesis pathway reveals a handover mechanism involving the RNA exosome and the exonuclease QIP. Mol Cell 46:299-310
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