Research Projects

Internal ribosomal entry sites (IRES) are special cis-acting RNA sequences that recruit ribosomes to initiate translation, typically in an m7G cap-independent manner (1). This mechanism allows protein synthesis in (+) SS RNA viruses, endogenous circular RNAs and cell stress conditions when cap-independent translation machinery is shut down (2,3). IRESs were first discovered in picornaviruses and later other viruses like Coxsackievirus B3 (CVB3), Hepatitis C (HCV), Foot-and-mouth virus (FMD), Cricket paralysis virus (CrPV) and Human immunodeficiency virus (HIV) (4-6). These IRES sequences are typically 400-800 nucleotide length and have highly structured RNA sequences that can bind translation initiation factors to recruit ribosomes. In addition, IRESs have also been discovered to function in cellular genes; they are of different structures and sizes (7). Flavivirus encompasses a large group of enveloped-positive strains of RNA viruses, which lead to 400 million annual infections globally. Dengue (DENV) and ZIKA are more closely related flaviviruses, and their genome is nearly 60-70% at the genome level (8). It is well documented that flavivirus control their translation by a cap-dependent mechanism, and recently identified that they can translate their genome in non-canonical cap-indecent mechanisms that do not require 5’cap (9). The mechanism underlying the alternative translation initiation used by DENV and ZIKA remains uncharacterized. Hence, investigating the IRES elements in these viruses, as well as the RNA-binding proteins involved in this process, will have significant implications for understanding the biology and evolution of mosquito-borne flaviviruses. Despite the importance of IRES, a currently available method for identifying IRES sequences relies on the bicistronic report assays, which can produce false positive signals if IRES has a cryptic promoter. In vitro engineered circular RNAs (CircRNA) have emerged as a promising alternative for screening IRES function as circRNA lack a 5’ m7G cap. In addition, circRNA construction with a splicesome-independent circularisation method could eliminate false back-splicing and forward trans-splicing reactions (10). In this proposed study, my lab will screen IRES elements in the 5' UTR and across the DENV and ZIKA genome employing in silico deep IRES scanning methods and experimentally validate their IRES efficiency using the circular split nLuc reporter. Next, we aim to determine the molecular mechanisms by which the identified IRES facilitate cap-independent circRNA translation by investigating their trans-acting factors. Finally, we investigate the efficiency of newly identified IRES elements in initiating the translation of artificially engineered circRNAs.