Combined use of RNAi and quantitative proteomics to study gene function in Drosophila.
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.
Bonaldi
Tiziana
T
Max-Planck-Institute of Biochemistry, Department of Proteomics and Signal Transduction, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Straub
Tobias
T
Cox
Jürgen
J
Kumar
Chanchal
C
Becker
Peter B
PB
Mann
Matthias
M
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D020543:Proteome; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
C120419:Acf protein, Drosophila; D029721:Drosophila Proteins; D010455:Peptides; D020543:Proteome; D014157:Transcription Factors