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

Human immunodeficiency virus-1 (HIV-1) is a retrovirus with a 10-kb single-stranded RNA genome. HIV-1 must express all of its gene products from the same primary transcript, which undergoes alternative splicing to produce diverse protein products, including structural proteins and regulatory factors(1,2). Despite the critical role of alternative splicing, the mechanisms driving splice-site choice 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). We use DMS-MaPseq to probe the structure of HIV-1 RNA in cells and develop an algorithm called Detection of RNA folding Ensembles using Expectation-Maximization (DREEM), which reveals alternative conformations assumed by the same RNA sequence. Contrary to previous models, which analyzed population averages(4), our results reveal the widespread heterogeneous nature of HIV-1 RNA structure. In addition to confirming that in vitro characterized alternative structures for the HIV-1 Rev Responsive Element (RRE) 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(5–7) that RNA conformation heterogeneity regulates splice site usage and viral gene expression.