Understanding the role of pea enation mosaic virus mRNA 3’ untranslated region in translation initiation (566.5)

Abstract

Most eukaryotic mRNAs use two key elements at opposite ends of the mRNA, the 5’ m7GpppN cap and the 3’ poly adenosine (A) tail, to recruit translation initiation machinery and circularize mRNA. Pea Enation Mosaic Virus (PEMV) mRNA lacks both a 5’ m7GpppN cap and a 3’ poly (A) tail. A novel type of cap independent translation elements (PTE) is found in the 3’UTR and is essential for translation initiation, the detailed mechanisms of initiation are not well known. Direct protein fluorescence quenching and labeled RNA fluorescence anisotropy were used to investigate the binding affinity of PTE fragment with two important eukaryotic initiation factors, eIF4E (Kd=~7.2nM) and eIF4F (Kd=~8.4nM). These binding affinities are as high as those for eIF4E and eIF4F binding with the cap analogue m7GTP and much higher than those for m7GpppG. With the fluorescence anisotropy assays, we also demonstrated that eIF4A, eIF4B and Poly A tail binding protein (PABP), which can form a stable complex with eIF4F in vivo, only slightly increased the binding affinity of eIF4F with the PTE RNA (Kd<5nM). These binding affinities suggest the PTE element can sequester eIFs from the host cell. The role of eIF/PTE binding in ribosome assembly is being investigated.Grant Funding Source: NSF MCB 1157632