Structure and Conformational Dynamics of the Splicing Factor hnRNP H

Abstract

Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) H/F family are multi purpose RNA binding proteins that participate in most stages of RNA metabolism. Despite having similar RNA sequence preferences, hnRNP H/F proteins function in overlapping but distinct cellular processes. The domain organization of hnRNP H/F proteins is modular consisting of N-terminal tandem quasi-RNA Recognition Motifs (H/FqRRM12) 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. A significant difference occurs at position 99 of the linker where hnRNP H contains a proline and hnRNP F an alanine. To investigate the influence of P99 on the conformational properties of hnRNP H, we probed the structural dynamics of its HqRRM12 domain with x-ray crystallography, NMR spectroscopy, and Small Angle X-ray Scattering (SAXS). We observed in the HqRRM12 contains multiple structures in solution by SAXS. These exchangeable conformations that located on the linker region and RNA recognition sites reach certain equilibrium in different temperature by NMR relaxation dispersion. The collective results best describe that HqRRM12 exists in a conformational equilibrium between compact and extended structures. The compact structure displays an electropositive surface formed at the qRRM1-qRRM2 interface; the surface is abrogated within the extended structure. Comparison of NMR relaxation parameters between HqRRM12 and FqRRM12 indicate that FqRRM12 primarily adopts an extended conformation. Introducing the P99A mutation into HqRRM12 alters its conformational dynamics to favor the more extended structure. Thus, our work demonstrates that the linker compositions confer different structural properties between hnRNP H/F family members that might contribute to their functional diversity.