The discovery that regulatory RNAs control biological pathways have revolutionized our understanding of gene expression over the past decade. At the forefront, microRNAs (miRNAs) have proven to be an abundant and essential class of RNA molecules in plants and animals. The importance of miRNAs in human biology is highlighted by the new recognition that mis-regulation of specific miRNA pathways underlies complex diseases, including cancer, heart ailments and neuronal pathologies. To understand the role of miRNAs under normal and disease conditions, two basic questions must be addressed: (1) how is miRNA expression regulated and how is this altered in the disease state, and (2) what are the biologically relevant miRNA targets, whose mis-regulation causes the disease phenotypes? These problems are the foundation of the work proposed here to elucidate a novel mechanism for controlling miRNA biogenesis and to determine how the miRNA complex recognizes and regulates specific targets in vivo. Typically, miRNAs recruit Argonaute (AGO) and its co-factors to specific mRNAs to trigger decay or translational repression. Here, an entirely new role for AGO in regulating miRNA biogenesis will be investigated.
In Aim 1, the mechanism used by AGO to enhance the processing of let-7, and potentially other miRNAs, will be determined in C. elegans and human cells. These studies will reveal a novel pathway for controlling miRNA expression that may be broadly relevant for understanding how miRNA levels fluctuate during development and disease progression.
Aim 2 will tackle the daunting problem of how imperfect base-pairing suffices for the regulation of specific targets by miRNAs. Three complementary methods will be employed to decipher how AGO recognizes and regulates specific targets in the endogenous context. These strategies take advantage of sensitive biochemical methods, unique worm strains and robust computational pipelines to assess the genome wide targeting properties of AGO guided by specific miRNAs in C. elegans. The consequences of AGO binding to its targets will also be analyzed to provide a comprehensive view of miRNA function in a live animal. Ultimately, these studies will yield unprecedented datasets for deciphering the rules used in vivo for miRNA target recognition and regulation in a multicellular animal.

Public Health Relevance

In the relatively short time since their discovery, microRNAs (miRNAs) have emerged as key regulators of gene expression, and mis-regulation of miRNA pathways has been linked to numerous human diseases, including cancer, heart ailments and neurological disorders. This proposal aims to elucidate a novel mechanism for regulating miRNA expression and to help solve the complicated problem of how miRNAs recognize and regulate specific target genes in vivo.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01GM071654-10
Application #
8694583
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Broughton, James P; Pasquinelli, Amy E (2016) A tale of two sequences: microRNA-target chimeric reads. Genet Sel Evol 48:31
Pasquinelli, Amy E (2016) A sense-able microRNA. Genes Dev 30:2019-2020
Broughton, James P; Lovci, Michael T; Huang, Jessica L et al. (2016) Pairing beyond the Seed Supports MicroRNA Targeting Specificity. Mol Cell 64:320-333
Mondol, Vanessa; Ahn, Byoung Chan; Pasquinelli, Amy E (2015) Splicing remodels the let-7 primary microRNA to facilitate Drosha processing in Caenorhabditis elegans. RNA 21:1396-403
Pasquinelli, Amy E (2015) MicroRNAs: heralds of the noncoding RNA revolution. RNA 21:709-10
Van Wynsberghe, Priscilla M; Pasquinelli, Amy E (2014) Period homolog LIN-42 regulates miRNA transcription to impact developmental timing. Worm 3:e974453
Van Wynsberghe, Priscilla M; Finnegan, Emily F; Stark, Thomas et al. (2014) The Period protein homolog LIN-42 negatively regulates microRNA biogenesis in C. elegans. Dev Biol 390:126-35
Lima, Sarah Azoubel; Pasquinelli, Amy E (2014) Identification of miRNAs and their targets in C. elegans. Adv Exp Med Biol 825:431-50
Fonslow, Bryan R; Moresco, James J; Tu, Patricia G et al. (2014) Mass spectrometry-based shotgun proteomic analysis of C. elegans protein complexes. WormBook :1-18
Broughton, James P; Pasquinelli, Amy E (2013) Identifying Argonaute binding sites in Caenorhabditis elegans using iCLIP. Methods 63:119-25

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