role of polypyrimidine tract binding protein in mediating internal initiation of translation of interferon regulatory factor 2 rna段位的角色结合蛋白在调节内部的翻译起始干扰素调节因子2 rna.pdfVIP

Role of Polypyrimidine Tract Binding Protein in Mediating Internal Initiation of Translation of Interferon Regulatory Factor 2 RNA Debojyoti Dhar¤a, Musturi Venkataramana¤b, Anand Ponnuswamy, Saumitra Das* Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka, India Abstract Background: Earlier we have reported translational control of interferon regulatory factor 2 (IRF2) by internal initiation (Dhar et al, Nucleic Acids Res, 2007). The results implied possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general. Here we have investigated the secondary structure of the Internal Ribosome Entry Site of IRF2 RNA and demonstrated the role of PTB protein in ribosome assembly to facilitate internal initiation. Methodology/Principal Findings: We have probed the putative secondary structure of the IRF2 5 9UTR RNA using various enzymatic and chemical modification agents to constrain the secondary structure predicted from RNA folding algorithm Mfold. The IRES activity was found to be influenced by the interaction of trans-acting factor, polypyrimidine tract binding protein (PTB). Deletion of 25 nts from the 39terminus of the 5 9untranslated region resulted in reduced binding with PTB protein and also showed significant decrease in IRES activity compared to the wild type. We have also demonstrated putative contact points of PTB on the IRF2–59UTR using primer extension inhibition assay. Majority of the PTB toe-prints were found to be restricted to the 3 9end of the IRES. Additionally, Circular Dichroism (CD) spectra analysis suggested change in the conformation of the RNA upon PTB binding. Further, binding studies using S10 extract from HeLa cells, partially silenced for PTB gene expression, resulted in reduced binding by other trans-acting factors. Final