Encoded Conformational Dynamics of the HIV Splice Site A3 Regulatory Locus: Implications for Differential Binding of hnRNP Splicing Auxiliary Factors

• Published in: Journal of molecular biology Vol. 434; no. 18; p. 167728
• Main Authors: Chiu, Liang-Yuan, Emery, Ann, Jain, Niyati, Sugarman, Andrew, Kendrick, Nashea, Luo, Le, Ford, William, Swanstrom, Ronald, Tolbert, Blanton S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 30-09-2022; Elsevier
• Subjects: Alternative Splicing; calorimetry; conformational dynamics; Heterogeneous Nuclear Ribonucleoprotein A1 - metabolism; Heterogeneous-Nuclear Ribonucleoprotein Group F-H - genetics; Heterogeneous-Nuclear Ribonucleoprotein Group F-H - metabolism; HIV Alternative splicing; HIV Infections - virology; HIV-1 - genetics; hnRNPs; Humans; Mutation; NMR spectroscopy; Nucleic Acid Conformation; phylogenetic conservation; Regulatory Sequences, Ribonucleic Acid; RNA Splice Sites; RNA Splicing Factors - metabolism; RNA structural heterogeneity; RNA, Viral - chemistry; RNA, Viral - metabolism; calorimetry; conformational dynamics; HIV Alternative splicing; hnRNPs; NMR spectroscopy; RNA structural heterogeneity; phylogenetic conservation
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2022.167728

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•The HIV splice site A3 regulatory locus intrinsically adopts multiple structures in solution, which are phylogenetically conserved across different viral strains.•The major structure observed in vitro folds to completely sequester the PPyT in base pairs with the ESS2p element and a downstream Linker.•The 3D structure of A3SL1 reveals that the PPyT is completely engaged in tertiary interactions including with the A3 cleavage site, which undergoes slow conformational exchange.•Mutations designed to destabilize the PPyT:ESS2p helix correlate with increasing splicing to acceptor site A3 within HIV infected cells, validating the functional significance of this interaction.•Biophysical characterizations provide evidence that hnRNP A1 but not hnRNP H binds with high affinity and specificity to A3SL1.
Alternative splicing of the HIV transcriptome is controlled through cis regulatory elements functioning as enhancers or silencers depending on their context and the type of host RNA binding proteins they recruit. Splice site acceptor A3 (ssA3) is one of the least used acceptor sites in the HIV transcriptome and its activity determines the levels of tat mRNA. Splice acceptor 3 is regulated by a combination of cis regulatory sequences, auxiliary splicing factors, and presumably RNA structure. The mechanisms by which these multiple regulatory components coordinate to determine the frequency in which ssA3 is utilized is poorly understood. By NMR spectroscopy and phylogenetic analysis, we show that the ssA3 regulatory locus is conformationally heterogeneous and that the sequences that encompass the locus are conserved across most HIV isolates. Despite the conformational heterogeneity, the major stem loop (A3SL1) observed in vitro folds to base pair the Polypyrimdine Tract (PPyT) to the Exon Splicing Silencer 2p (ESS2p) element and to a conserved downstream linker. The 3D structure as determined by NMR spectroscopy further reveals that the A3 consensus cleavage site is embedded within a unique stereochemical environment within the apical loop, where it is surrounded by alternating base–base interactions. Despite being described as a receptor for hnRNP H, the ESS2p element is sequestered by base pairing to the 3′ end of the PPyT and within this context it cannot form a stable complex with hnRNP H. By comparison, hnRNP A1 directly binds to the A3 consensus cleavage site located within the apical loop, suggesting that it can directly modulate U2AF assembly. Sequence mutations designed to destabilize the PPyT:ESS2p helix results in an increase usage of ssA3 within HIV-infected cells, consistent with the PPyT becoming more accessible for U2AF recognition. Additional mutations introduced into the downstream ESS2 element synergize with ESS2p to cause further increases in ssA3 usage. When taken together, our work provides a unifying picture by which cis regulatory sequences, splicing auxiliary factors and RNA structure cooperate to provide stringent control over ssA3. We describe this as the pair-and-lock mechanism to restrict access of the PPyT, and posit that it operates to regulate a subset of the heterogenous structures encompassing the ssA3 regulatory locus.