Poster abstracts
Poster number 22 submitted by Joe Kanlong
Investigating the role of transcription start site choice in translational control by the HIV-1 5ʹ UTR
Joseph G. Kanlong (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retrovirus Research, The Ohio State University, Columbus, OH), Crystal Stackhouse (Department of Structural Biology, Stanford University School of Medicine, Stanford, CA), Ryan Denniston (Center for RNA Biology, The Ohio State University, Columbus, OH), Joseph D. Puglisi, Elisabetta Viani Puglisi (Department of Structural Biology, Stanford University School of Medicine, Stanford, CA), Wei-Shau Hu (Viral Recombination Section, HIV DRP, NCI, Frederick, MD), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for Retrovirus Research, Center for RNA Biology, The Ohio State University, Columbus OH)
Abstract:
HIV-1 transcribes a 9.2 kb viral unspliced RNA that serves as both an mRNA, encoding the viral polyproteins Gag and Gag-pol, and as the viral genomic RNA, packaged into budding virions as a dimer. Heterogeneous transcription start site (TSS) usage by RNA pol II produces unspliced HIV-1 RNAs with one (1G) or three (3G) terminal guanosines and a modified 5ʹ cap. 1G RNAs are selectively packaged into viral particles as genomic RNA while 3G RNAs serve as the primary mRNA template for viral proteins. We and others have shown that the 5ʹ untranslated region (UTR) of 1G and 3G RNAs adopt distinct conformational ensembles that direct RNA fate. Although both can be translated, 3G RNAs are translated more efficiently than 1G RNAs, both in vitro and during infection. The precise mechanisms that govern efficient 3G translation are unclear. Eukaryotic translation is principally regulated at initiation, beginning with mRNA 5ʹ-end binding by eukaryotic initiation factor (eIF) 4F, a heterotrimeric protein complex consisting of eIF4G (scaffold), eIF4E (cap-binding), and eIF4A (helicase). To understand how differences in the HIV-1 RNA conformational ensembles affect translation initiation, we performed both single molecule FRET-based and gel-shift assays to interrogate the binding of eIF4F, as well as the ability of eIF4F to extend these RNAs for ribosome loading. While preliminary data show both RNAs are bound by eIF4F with similar affinities, the kinetics of RNA extension and 40S loading are slower on the 1G 5ʹ UTR. Recent studies suggest that HIV-1 contains multiple upstream open reading frames (uORFs) within the 5ʹ UTR that initiate from non-AUG start codons . Translation initiation at non-AUG start codons can be regulated by RNA structure. Ribosome profiling experiments in cells expressing mutant proviruses that generate predominantly 1G or 3G RNA were performed to investigate differences in uORF usage between the 1G and 3G 5ʹ UTRs. Preliminary data reveal that both 5ʹ UTRs contain multiple putative uORFs that suggest 5ʹ UTR conformation may drive differential uORF usage. The new insights gained from this work may be leveraged for the development of RNA-targeted therapeutic strategies.
Keywords: HIV-1, Translation, RNA structure