Article

Combined use of RNAi and quantitative proteomics to study gene function in Drosophila.

Max-Planck-Institute of Biochemistry, Department of Proteomics and Signal Transduction, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Molecular cell (impact factor: 14.61). 10/2008; 31(5):762-72. DOI:10.1016/j.molcel.2008.07.018 pp.762-72
Source: PubMed

ABSTRACT RNA interference is a powerful way to study gene function and is frequently combined with microarray analysis. Here we introduce a similar technology at the protein level by simultaneously applying Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and RNA interference (RNAi) to Drosophila SL2 cells. After knockdown of ISWI, an ATP-hydrolyzing motor of different chromatin remodeling complexes, we obtained a quantitative proteome of more than 4,000 proteins. ISWI itself was reduced 10-fold as quantified by SILAC. Several hundred proteins were significantly regulated and clustered into distinct functional categories. Acf-1, a direct interaction partner of ISWI, is severely depleted at the protein, but not the transcript, level; this most likely results from reduced protein stability. We found little overall correlation between changes in the transcriptome and proteome with many protein changes unaccompanied by message changes. However, correlation was high for those mRNAs that changed significantly by microarray.

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Keywords

applying Stable Isotope Labeling
 
Cell culture
 
different chromatin
 
direct interaction partner
 
distinct functional categories
 
Drosophila SL2 cells
 
hundred proteins
 
ISWI
 
likely results
 
message changes
 
mRNAs
 
protein changes unaccompanied
 
protein level
 
protein stability
 
proteome
 
quantitative proteome
 
RNA interference
 
RNAi
 
similar technology