IRES 의존적 번역에서의 RNA-binding 인자의 기능연구

Abstract

Eukaryotic cells have many strategies to control the translation of mRNA for the synthesis of protein. Beyond the canonical cap-dependent model of cap recognition followed by ribosomal scanning, an alternative method of cap-independent translation was first described for the picornavirus family and subsequently for a growing subset of viral and cellular mRNAs. Translation of many cellular and viral mRNAs is directed by internal ribosomal entry sites (IRESs). Several proteins that enhance IRES activity through interactions with IRES elements have been discovered. However, the molecular basis for the IRES-activating function of the IRES-binding proteins remains unknown. Here, I report that NS1-associated protein 1 (NSAP1), which augments several cellular and viral IRES activities, enhances hepatitis C viral (HCV) IRES function by facilitating the formation of translation-competent 48S ribosome-mRNA complex. NSAP1, which is associated with the solvent side of the 40S ribosomal subunit, enhances 80S complex formation through correct positioning of HCV mRNA on the 40S ribosomal subunit. NSAP1 seems to accomplish this positioning function by directly binding to both a specific site in the mRNA downstream of the initiation codon and a 40S ribosomal protein (or proteins). Cellular IRES have pivotal role modulating protein synthesis in several stress condition that cap-dependent translation is inhibited. However, unlike viral IRES, basic module to be required for IRES activity in cellular IRES is fully unknown and to find such a module is challenging task. Here, I provide the evidence that ACR, an A rich sequence originated from HCV IRES, can function as artificial IRES in vitro and in vivo. The efficiency of ACR-driven translation is proportional to the number of ACR in reporters, and A to G substitution impaired ACR-dependent translation enhancement. NF45 is identified as ACR interacting protein and ACR-dependent translation was decreased in NF45 knockdown cells. These data indicate that ACR is one of the basic modules of cellular IRES modulated by NF45. In summary, I investigated molecular basis of RNA-binding protein in IRES-dependent translation of eukaryotic mRNAs. Especially, I showed that NSAP1 enhances HCV IRES-dependent translation by facilitating the formation of a translation-competent 48S complex that is ready to form the 80S complex through association of with the HCV IRES and 40S ribosomal subunit. This is the first report showing that a protein that interacts with an IRES element enhances translation through a direct interaction with the 40S ribosomal subunit. In addition, I also showed that ACR sequence originated from HCV IRES can function as an artificial IRES modulated by another cellular protein NF45. These studies shed light on the molecular mechanism of cellular factor on IRES-dependent translation that is still challenging.