Thermodynamic and Phylogenetic Insights into hnRNPA1 Recognition of the HIV-1 Exon Splicing Silencer 3 Element

摘要

Complete expression of the HIV-1 genome requires balanced usage of suboptimal splice sites. The 3′ acceptor site A7 (ssA7) is negatively regulated in part by an interaction between the host hnRNP A1 protein and a viral splicing silencer (ESS3). Binding of hnRNP A1 to ESS3 and other upstream silencers is sufficient to occlude spliceosome assembly. Efforts to understand the splicing repressive properties of hnRNP A1 on ssA7 have revealed hnRNP A1 binds specific sites within the context of a highly folded RNA structure; however, biochemical models assert hnRNP A1 disrupts RNA structure through cooperative spreading. In an effort to improve our understanding of the ssA7 binding properties of hnRNP A1, herein we have performed a combined phylogenetic and biophysical study of the interaction of its UP1 domain with ESS3. Phylogenetic analyses of group M sequences ($\bar{x}$ = 2860) taken from the Los Alamos HIV database reveal the ESS3 stem loop (SL3^ESS3) structure has been conserved throughout HIV-1 evolution, despite variations in primarysequence. Calorimetric titrations with UP1 clearly show the SL3^ESS3 structure is a critical binding determinant because deletionof the base-paired region reduces the affinity by ∼150-fold(Kd values of 27.8 nM and 4.2 μM).Cytosine substitutions of conserved apical loop nucleobases show UP1preferentially binds purines over pyrimidines, where site-specificinteractions were detected via saturation transfer difference nuclearmagnetic resonance. Chemical shift mapping of the UP1–SL3^ESS3 interface by ¹H–¹⁵N heteronuclearsingle-quantum coherence spectroscopy titrations reveals a broad interactionsurface on UP1 that encompasses both RRM domains and the inter-RRMlinker. Collectively, our results describe a UP1 binding mechanismthat is likely different from current models used to explain the alternativesplicing properties of hnRNP A1.