Screening for novel protein-protein interactions in living human cells with the help of the split-ubiquitin system

Abstract

The protein-protein interaction between human Suppressor of RNA polymerase B (hSrb7p), a component of the Mammalian Mediator complex, and human S-phase kinase associated protein (hSkp1p), a component of the Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complex, was observed; the biological significance of this interaction is that hSkp1 may be involved in transcription. This was examined using RNA-interference to knock-down hSkp1 transcripts in HeLa cells, and its effects on Heat-shock protein gene transcription using reverse-transcription polymerase chain reaction (RT-PCR). It was found that hSkp1 is necessary for the activation of Hsp70B' by heat-shock, and these results strengthened the link between ubiquitin-proteasome pathway and transcription. In the second part of this study, a cDNA library was cloned and the yeast split-ubiquitin system using hSkp1 as bait isolated cyclin-dependent kinase 2-interacting protein (CINP) as a novel interacting protein. This indicates that hSkp1p, and possibly the ubiquitin-proteasome pathway may be involved in DNA replication.