Identification of the Requiem 3' UTR Binding Proteins

초록

Requiem is a novel zinc finger protein that is essential for apoptosis in myeloid cells. We identified G8 that encodes a part of the 3' untranslated region (3'-UTR) of requiem gene as a putative globin gene switching factor, showing that transient over-expression of clone G8 induces the - to -globin switching in K562 cells. Our observations support the notion that the expression of requiem gene is regulated by the 3'-UTR-mediated translational control. To understand the RNA-protein interactions in the G8 region, we mapped factor-binding sequences by gel mobility shift assay using the serial deletion mutants of G8. Indeed, the point mutations in stem-loop structure of the core RNA sequences inhibited RNA-protein interactions. In addition, these RNA-protein interactions were decreased or interrupted in terminally differentiating MEL cells or apoptotic S49.1 cells, possibly by caspase-3. On the contrary, the stem-loop structure in the core RNA binding element was not sufficient for translational repression of the cis-linked genes, suggesting that the translational repression of requiem is controlled by multiple regulatory elements. As an effort to further our understanding on G8-mediated translational repression mechanism, we have attempted to identify the requiem 3'-UTR binding proteins (RUBPs) by affinity purification and mass spectrometry. Affinity purified proteins were separated by 2-dimensional gel electrophoresis and each protein spot was identified by MALDI-TOF mass spectrometery. At the end, we identified a bunch of proteins, including insulin-like growth factor II leader 3 mRNA binding protein (IMP-1) which is known to cause a dose-dependent translational repression of IGF-II leader 3 mRNA and also binds to the 3'-UTR of H19 mRNA (Nielsen et al., 1999). Their functional roles on translational repression as well as structural integrity are under investigation.