Identification of the requiem 3' UTR binding proteins

초록

Requiem is a novel zinc finger protein which 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 in K562 cells induces the γ- to β-globin switching. 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 with the G8 region, we mapped core factor binding sequences by gel mobility shift assay using the serial deletion mutants of G8. Indeed, point-mutations in stem-loop structure of the core RNA binding element 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 along with their trans-acting proteins. 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 of factors and mass spectrometry. To purify RUBPs, RNA template of whole sequences of the requiem 3'-UTR (G8) was employed as a ligand. 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 the translational repression as well as a structural integrity of RUBPs are under investigation.