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

Abstract

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 D etection of R NA folding E nsembles using E xpectation- M aximization (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.

The short- and long-range RNA-RNA Interactome of SARS-CoV-2

“…Yet, the complex life cycle of viral RNA genomes, i.e. their engagement with multiple cellular and viral machineries such as the ones for replication, transcription, and translation, suggests a dynamic RNA structure, as we and others have reported for Zika virus (Huber et al, 2019;Li et al, 2018;Ziv et al, 2018) and for HIV-1 (Tomezsko et al, 2020) . Our structural analysis of SARS-CoV-2 reveals a high level of structural dynamics whereby alternative high-order conformations, some of which involve long-distance base-pairing, co-exist in vivo (Figure 2).…”

Perspectives on Viral RNA Genomes and the RNA Folding Problem

“…Detection of RNA folding Ensembles using Expectation Maximization (DREEM) presents a new approach to studying nucleotide chemical modification with Illumina sequencing [ 6 ]. Dimethyl sulfate (DMS) chemically modifies adenines and cytosine bases that are not Watson–Crick paired in the middle of a helix, and this information can be used to constrain RNA folding predictions [ 25 ].…”

“…The final weight and distribution of each cluster provides an estimate of the relative abundance of each structural conformation. The details of the DREEM algorithms are described in the supporting information of Tomeszko et al [ 6 ], and the authors make the code publicly available at .…”

“…Since the first observations of encapsidated RNA genomes, there have been many approaches to determining and modelling the structures of viral RNA genomes. This review will focus on two new techniques, asymmetric reconstructions from high resolution cryoelectron microscopy [ 4 , 5 ] and DREEM (Detection of RNA folding Ensembles using Expectation Maximization) [ 6 ], that probe the structures of encapsidated RNA. These techniques provide positive evidence that encapsidated RNA forms more than one structure inside a virus particle.…”

“…The different models for encapsidated viral RNA genomes in several viruses have different assumptions about the degree of order underlying the approach to structure elucidation. For example, three models have been proposed for the RNA genome of Human Immunodeficiency Virus: a model focused on a structured 5′UTR [ 7 ], an ensemble of RNA structures [ 6 , 8 ], and a single minimum free energy structure model [ 9 ]. Similarly, models for Dengue virus genomes have been proposed as a model with regions of well-ordered and dynamic structures [ 10 ], or as a single minimum free energy structure [ 11 ].…”