Nmr Studies Of Conformational Selection Of Hnrnp H On Rna Recognition And Its Interaction With The Hiv Exonic Splicing Silencer Ess2p Rna

Alternative splicing in human immunodeficiency virus type 1 (HIV-1) is tightly regulated by the equilibrium between the spliced and unspliced primary transcripts. This equilibrium is maintained by the presence of inefficient splice sites that are further regulated by hnRNP and SR proteins. The A3 3’-splice site of HIV-1 is required for Tat mRNA production. The inefficient utilization of this splice site has been attributed to the presence of a second exonic splicing silencer (ESS2p), which acts to repress splice site A3. HnRNP H binds to ESS2P element to inhibit the splicing process; however, there is no structural and mechanistic information about how hnRNP H recognizes ESS2P to inhibit the splicing. In our studies, we aim to characterize the three-dimensional structure of hnRNP H and ESS2P. The domain organization of hnRNP H/F proteins is modular consisting of N-terminal tandem quasi-RNA Recognition Motifs (HqRRM1,2) and a third C-terminal qRRM3 embedded within glycine-rich repeats. The tandem qRRMs are connected through a 10-residue linker with most of the amino acids strictly conserved between hnRNP H and F. We probed the structural dynamics of its HqRRM1,2 domain with x-ray crystallography, NMR spectroscopy, and Small Angle X-ray Scattering (SAXS). We observed that HqRRM12 contains multiple structures in solution by SAXS. These exchangeable conformations are located on the linker region and RNA recognition sites. Moreover, the three-dimensional structure of ESS2P has been determined using NMR spectroscopy and SAXS. Collectively, this work provides structural insight how hnRNP H recognizes the HIV ESS2P element.