The broad, long-term objective is to genetically dissect RNA interference in Drosophila and identify the molecular components mediating this important process. Double-stranded RNA (dsRNA) induces potent cellular responses that compose an essential defense against deleterious RNAs, including viruses, viral replication intermediates and transposable elements. In many systems, dsRNA triggers RNA interference (RNAi); a dramatic and sequence specific destabilization of transcripts homologous to the dsRNA trigger. Normal cellular genes can be silenced using RNAi making this technique an important new tool to elucidate function of orphan gene products. Although the phenomenon is remarkably similar in diverse organisms, the precise mechanisms mediating RNAi are not fully understood. Genetic screens have identified several important components required for RNAi in Arabidopsis, Neurospora and C. elegans. However, these screens have not been saturating and recovered only viable mutations. Mutant screens for RNAi defective mutants in Drosophila have not been possible because of variable penetrance of RNAi suppression. Recently we solved this difficulty with a novel transgene design that effectively silences genes in adult tissues. These studies set the groundwork for the genetic screen proposed here designed to elucidate the molecular basis for RNAi in Drosophila. In .
Specific Aim 1 we will isolate mutants defective and enhanced for RNAi suppression of the eye color pigment transporter WHITE using the FLP/FRT recombination system. The advantages of this screen are: 1. mutants affecting RNAi will be easily identified as eye color mutants, 2. homozygous mutant clones will be restricted to the compound eye, therefore we will recover mutations in viable and in potentially lethal genes required for RNAi not identified in other systems, 3. We will recover both suppressor and enhancer mutations affecting RNAi.
In Specific Aim 2 we will use complementation, deficiency mapping, sequence analysis of mutated candidate genes and germline transformation rescue to identify the genes responsible for the mutant phenotypes observed in our screen. Completion of the studies will enhance our understanding of RNAi in Drosophila and broaden our understanding of RNAi in general. Recovery of enhancer of RNAi mutants may provide enhanced genetic backgrounds to facilitate research on orphan gene function in Drosophila in the post-genomic era. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070684-03
Application #
7032257
Study Section
Genetics Study Section (GEN)
Program Officer
Rhoades, Marcus M
Project Start
2004-04-01
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
3
Fiscal Year
2006
Total Cost
$312,285
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Pharmacology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Sanders, Charcacia; Smith, Dean P (2011) LUMP is a putative double-stranded RNA binding protein required for male fertility in Drosophila melanogaster. PLoS One 6:e24151
Kalidas, Savitha; Sanders, Charcacia; Ye, Xuecheng et al. (2008) Drosophila R2D2 mediates follicle formation in somatic tissues through interactions with Dicer-1. Mech Dev 125:475-85