How do small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) regulate gene expression and protect the genome? RNAi encompasses the cellular response to exogenous double-stranded RNA (dsRNA)-delivered experimentally or generated during a viral infection-and the response to parasitic genetic elements, such as transposons and highly repeated sequences. Our experimental strategy uses Drosophila melanogaster as a model system, because flies combine outstanding genetic, biochemical, and phenotypic tools.
Aim 1 will explore why Drosophila Dicer-1 and Dicer-2 use ATP. Dicer enzymes make siRNAs and miRNAs. Dicer enzymes contain protein motifs that suggest they are ATPases. The function of Dicer proteins is modified by their dsRNA-binding partner proteins-Loquacious (Loqs) and R2D2 in flies. How do R2D2 and the three different isoforms of Loqs extend or restrict the function of Dicer-1 and Dicer- 2? siRNAs associate with the protein Argonaute2 to form the RNA-induced silencing complex (RISC), which mediates RNAi.
In Aim 2, new technologies will be used to study the function of RISC, including single-molecule fluorescence techniques to study the cycle of RISC function, and deep sequencing methods that allow us to compare millions of different sequences in a single experiment to test the contribution of each siRNA nucleotide to binding and catalysis.
Aim 3 proposes to define the biological functions of endogenous siRNAs. One function of endo-siRNAs is to protect the somatic genome from transposon expression, but endo-siRNAs also map to convergently transcribed mRNAs, and at least one endo-siRNAs acts in trans to regulate expression of an highly complementary mRNA. A combination of genetics, high throughput sequencing, and bioinformatic analysis will be employed to define the target repertoire of Drosophila endogenous siRNAs.
Aim 4 seeks to identify the role of Drosophila piRNAs in epigenetic silencing. piRNAs provide the primary genomic defense in the germ line against transposons and repetitive sequence elements, yet how they are produced or how they suppress transposon or cellular gene expression remains unknown. Of particular interest is testing the idea that piRNAs acting early in development can set the epigenetic state of genes whose expression and phenotypic consequences can be observed only in the adult fly.

Public Health Relevance

The Drosophila RNAi and piRNA pathways are highly similar to their mammalian counterparts, so what is learned in these studies will advance our understanding of RNA silencing in humans. Moreover, RNAi has become an essential laboratory tool and siRNA therapeutics are currently in human trials. Thus, the proposed studies promise to contribute to the development of practical applications of RNAi as a gene discovery technology and as a human therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM062862-12
Application #
8294634
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Bender, Michael T
Project Start
2001-04-01
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
12
Fiscal Year
2012
Total Cost
$496,354
Indirect Cost
$194,619
Name
University of Massachusetts Medical School Worcester
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
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Wang, Wei; Han, Bo W; Tipping, Cindy et al. (2015) Slicing and Binding by Ago3 or Aub Trigger Piwi-Bound piRNA Production by Distinct Mechanisms. Mol Cell 59:819-30
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Wang, Wei; Yoshikawa, Mayu; Han, Bo W et al. (2014) The initial uridine of primary piRNAs does not create the tenth adenine that Is the hallmark of secondary piRNAs. Mol Cell 56:708-16
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