Journal Article

Determination of RNA structural diversity and its role in HIV-1 RNA splicing

Lan, Tammy C. T., Edwards, Matthew D., Tomezsko, Phillip J., Tsibris, Athe, Papenfuss, Anthony T., Emery, Ann, Kuritzkes, Daniel R., Gupta, Paromita, Swanstrom, Ronald, Mcintosh, Lachlan, Corbin, Vincent D. A., Glasgow, Margalit, Swaminathan, Harish, Bieniasz, Paul, Rouskin, Silvi, Persad, Sitara, Zang, Trinity

2020

Overview

Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes alternative splicing to produce diverse protein products that include structural proteins and regulatory factors 1 , 2 . Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis -regulatory elements 3 . Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name ‘detection of RNA folding ensembles using expectation–maximization’ (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages 4 , our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized 5 alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis 6 – 8 that heterogeneity in RNA conformation regulates splice-site use and viral gene expression. Dimethyl sulfate mutational profiling with sequencing, combined with the newly developed DREEM algorithm, reveals that heterogeneity of RNA structure in HIV-1 regulates the use of splice sites and expression of viral genes.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 45 / 45/23 / 45/90 / 631/1647/48 / 631/326/596/1787 / 631/45/500 / Algorithms / Alternative splicing / Alternative Splicing - genetics / Analysis / Base Sequence / Clustering / Dimethyl sulfate / Gene expression / Gene Expression Regulation, Viral / Genetic aspects / Genomes / Genomic structural variations / HEK293 Cells / Heterogeneity / HIV / HIV (Viruses) / HIV-1 - genetics / Human immunodeficiency virus / Humanities and Social Sciences / Humans / Influence / Isoforms / multidisciplinary / Mutation / Nucleic Acid Conformation / Nucleotide sequence / Population / Proteins / Regulatory sequences / Reproducibility of Results / Ribonucleic acid / RNA / RNA Folding / RNA Splice Sites - genetics / RNA splicing / RNA viruses / RNA, Viral - chemistry / RNA, Viral - genetics / Science / Science (multidisciplinary) / Sequence Analysis, RNA / Splicing / Structural proteins / Structure / Sulfates / Sulfuric Acid Esters / Thermodynamics / Transcription