Poster abstracts
Poster number 32 submitted by Gabby Lee
Nanopore direct RNA sequencing reveals site-specific m6As crucial for regulating HIV-1 RNA and protein expression
Ga-Eun Lee (Department of Veterinary Biosciences, Ohio State University), Alice Baek, Sarah Golconda (Department of Veterinary Biosciences, Ohio State University), Asif Rayhan (Rieveschl Laboratory for Mass Spectrometry, Department of Chemistry, University of Cincinnati), Anastasios A. Manganaris (Department of Computer Sciences and Engineering, Ohio State University), Sanggu Kim (Department of Veterinary Biosciences, Ohio State University)
Abstract:
While the significance of chemical modifications on RNA has been demonstrated, the specific roles of these modifications in HIV-1 replication remain unclear and even controversial. Previous studies provide only low-resolution, type-specific RNA modification site mapping and relied on indirect analyses of phenotypic effects through overexpression or knockdown (knockout) of host effectors. These approaches overlook site-specific and context-dependent functions of viral RNA modifications. In this study, we introduce a novel Nanopore direct RNA sequencing (DRS) library preparation method for full-length HIV-1 RNAs. Our analysis unveiled a surprisingly simple HIV-1 modification landscape, with three dominant site-specific N6-methyladenosine (m6A) modifications located near the 3’ end. Confirmation of these m6A sites was achieved through site-directed mutagenesis, oligonucleotide LC–MS/MS, and in vitro treatments with a m6A eraser, ALKBH5. Interestingly, in our m6A-knockout-virus experiments, all the single mutants – lacking one of these three m6As – showed no notable reductions in any of the examined replication steps, including total HIV-1 RNA production, viral protein expression, virion production, and infectivity. However, the removal of all three dominant m6As (triple mutation) showed a significant reduction in full-length HIV-1 RNA and protein translation. To further investigate the defects of triple mutant in producing full-length RNA, we analyzed alternative splicing patterns of HIV-1 RNAs by quantifying the splicing donor and acceptor usage rates. The triple mutants exhibited a significantly lower fraction of unspliced RNAs and a higher utilization of splicing donors and acceptors compared to the WT, suggesting the crucial roles of the three m6As in the regulation of HIV-1 RNA isoforms. Our HIV-1-specific m6As knockout and full-length DRS analyses have shed light on the functional and evolutionary significance of these site-specific m6As in HIV-1 replication. The technological innovations presented here will also serve as a useful reference for future explorations of the complex RNA biology.
Keywords: Human immunodeficiency virus, Viral epitranscriptome, RNA modifications