A global approach combining proteome analysis and phenotypic screening with RNA interference yields novel apoptosis regulators.
Global approaches like proteome or transcriptome analyses have been performed extensively to identify candidate genes or proteins involved in biological and pathological processes. Here we describe the identification of proteins implicated in the regulation of apoptosis using proteome analysis and the functional validation of targets by RNA interference. A high-throughput platform for the validation of synthetic small interfering RNAs (siRNAs) by quantitative real-time PCR was established. Genes of the identified factors were silenced by automated siRNA transfection, and their role in apoptotic signaling was investigated. Using this strategy, nine new modulators of apoptosis were identified. A subsequent detailed study demonstrated that hepatoma-derived growth factor (HDGF) is required for TNFalpha-induced release of pro-apoptotic factors from mitochondria. The strategy described here may be used for hypothesis-free, global gene function analysis.
Machuy
Nikolaus
N
RNAx GmbH, Breddiner Weg 5, 13591 Berlin, Germany.
Thiede
Bernd
B
Rajalingam
Krishnaraj
K
Dimmler
Christiane
C
Thieck
Oliver
O
Meyer
Thomas F
TF
Rudel
Thomas
T
D017209:Apoptosis / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology; D019169:Jurkat Cells; D008928:Mitochondria / Q000378:metabolism; D010641:Phenotype; D020543:Proteome / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; D020543:Proteome; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C051610:hepatoma-derived growth factor