Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As
Alice Baek1,2,3#, Ga-Eun Lee1,2,3,4#, Sarah Golconda1,2,3, Asif Rayhan5, Anastasios A. Manganaris4,6, Shuliang Chen1,2, Nagaraja Tirumuru1,2, Hannah Yu1,2,3, Shihyoung Kim1,2,3, Christopher Kimmel2,4, Olivier Zablocki7,8, Matthew B Sullivan7,8,9, Balasubrahmanyam Addepalli5, Li Wu10, and Sanggu Kim1,2,3,4,11*
1Center for Retrovirus Research, The Ohio State University, Columbus, OH, USA
2Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, USA
3Infectious Diseases Institute, The Ohio State University, Columbus, OH, USA
4Translational Data Analytics Institute, The Ohio State University, Columbus, OH, USA
5Rieveschl laboratories for mass spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA
6Department of Computer Science and Engineering, The Ohio State University, Columbus, OH, USA
7Center of Microbiome Science, Ohio State University, Columbus, OH, USA
8Department of Microbiology, Ohio State University, Columbus, OH, USA
9Department of Civil, Environmental and Geodetic Engineering, Ohio State University, Columbus, OH, USA
10Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
11Center for RNA Biology, The Ohio State University, Columbus, OH, USA.
#These authors contributed equally: Alice Baek, Ga-Eun Lee
*Corresponding author: Sanggu Kim,✉e-mail:kim.6477@osu.edu
SUMMARY
Although the significance of chemical modifications on RNA is acknowledged, the evolutionary benefits and specific roles in HIV-1 replication remain elusive. Most studies have provided only population-averaged values of modifications for fragmented RNAs at low resolution and have relied on indirect analyses of phenotypic effects by perturbing host effectors. Here, we analyzed chemical modifications on HIV-1 RNAs at the full-length, single-RNA level, and nucleotide resolution using novel direct RNA sequencing methods. Our data reveal an unexpectedly simple HIV-1 modification landscape, highlighting three predominant N6-methyladenosine (m6A) modifications near the 3' end. More densely installed in spliced viral mRNAs than in genomic RNAs, these m6As play a crucial role in maintaining normal levels of RNA splicing and translation. HIV-1 generates diverse RNA subspecies with distinct m6A ensembles, and maintaining multiple of these m6As on its RNAs provides additional stability and resilience to HIV-1 replication—an unexplored RNA-level evolutionary strategy.
USAGE
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