Differential Conformational Dynamics Encoded by the Linker between Quasi RNA Recognition Motifs of Heterogeneous Nuclear Ribonucleoprotein H

Abstract

Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) F/H family are multipurpose RNA binding proteins that participate in most stages of RNA metabolism. Despite having similar RNA sequence preferences, hnRNP F/H proteins function in overlapping and, in some cases, distinct cellular processes. The domain organization of hnRNP F/H proteins is modular, consisting of N-terminal tandem quasi-RNA recognition motifs (F/HqRRM1,2) and a third C-terminal qRRM3 embedded between glycine-rich repeats. The tandem qRRMs are connected through a 10-residue linker, with several amino acids strictly conserved between hnRNP H and F. A significant difference occurs at position 105 of the linker, where hnRNP H contains a proline and hnRNP F an alanine. To investigate the influence of P105 on the conformational properties of hnRNP H, we probed the structural dynamics of its HqRRM1,2 domain with X-ray crystallography, NMR spectroscopy, and small-angle X-ray scattering. The collective results best describe that HqRRM1,2 exists in a conformational equilibrium between compact and extended structures. The compact structure displays an electropositive surface formed at the qRRM1-qRRM2 interface. Comparison of NMR relaxation parameters, including Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion, between HqRRM1,2 and FqRRM1,2 indicates that FqRRM1,2 primarily adopts a more extended and flexible conformation. Introducing the P105A mutation into HqRRM1,2 alters its conformational dynamics to favor an extended structure. Thus, our work demonstrates that the linker compositions confer different structural properties between hnRNP F/H family members that might contribute to their functional diversity.

Genome-Wide Analysis of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) Binding to HIV-1 RNA Reveals a Key Role for hnRNP H1 in Alternative Viral mRNA Splicing

“…Heteronuclear single quantum coherence (HSQC) titrations, wherein uniformly 15 N-labled HqRRM1,2 was titrated with unlabeled 5=-AGGGC-3=, revealed broadening of the NMR signals for HqRRM1,2 and indicated that binding specificity is governed by both the qRRM1 and qRRM2 domains ( Fig. 9B and C), similar to our previous observations with 5=-AGGGU-3= (54).…”

“…This notion is consistent with the model that hnRNPs can loop out entire exons (60), which would require a minimum of two binding sites surrounding the exon. Previously, we showed that the N-terminal HqRRM1,2 didomain of hnRNP H1 exists in a conformational equilibrium between compact and extended structures (54). The compact structure has both qRRMs juxtaposed, generating a continuous RNAbinding surface that would accommodate only a single isolated G-tract element.…”

Heterogeneous Nuclear Ribonucleoproteins Involved in the Functioning of Telomeres in Malignant Cells

“…These proteins do not contain the typical RNP-1 and RNP-2 consensus sequences but are capable of binding some nucleotide sequences [88,89]. Nevertheless, qRRM domains bind to G-tract RNA and participate in the regulation of the alternative splicing of pre-mRNAs [90,91].…”

HNRNPH1 destabilizes the G-quadruplex structures formed by G-rich RNA sequences that regulate the alternative splicing of an oncogenic fusion transcript

“…X-ray-crystallography and NMR studies show that the qRRM1-qRRM2 domains of HNRNPH1/H2 switch between a compact state, resulting in RNA recognition by both qRRMs of a single G-rich sequence and an extended state where it can bind to two different G-tracts. HNRNPH1/H2 primarily form compact states, and the linker between the two qRRMs remains rigid, differing from HNRNPF, which exhibits a flexible and extended state (44).…”

“…X-ray-crystallography and NMR studies show that the qRRM1-qRRM2 domains of HNRNPH1/H2 switch between a compact state, resulting in RNA recognition by both qRRMs of a single G-rich sequence and an extended state where it can bind to two different G-tracts. HNRNPH1/H2 primarily form compact states, and the linker between the two qRRMs remains rigid, differing from HNRNPF, which exhibits a flexible and extended state (44). Consistent with the assertion that the binding of HNRNPH/F proteins inhibits rG4 formation (16), Dominguez and co-workers showed that a single qRRM domain of HNRNPF could alter the splicing of the BCL-X pre-RNA by inhibiting the formation of the intramolecular bonds required for the formation of a secondary structure (43).…”