The high kinetic stability of a G-quadruplex limits hnRNP F qRRM3 binding to G-tract RNA

The RNA binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) F is involved in telomeres maintenance and pre-mRNA processing, such as alternative splicing and polyadenylation. It specifically recognizes RNA containing three consecutive guanines (G-tracts) that have the potential to assemble into G-quadruplexes. We have proposed recently that hnRNP F could regulate alternative splicing by remodeling RNA structures, such as G-quadruplexes. However, the exact mechanism of hnRNP F binding to such RNA sequences remains unknown. Here, we have studied the binding of the third RNA binding domain of hnRNP F [quasi-RNA recognition motif 3 (qRRM3)] to G-tract RNA using isothermal titration calorimetry, circular dichroism and nuclear magnetic resonance spectroscopy. Our results show that qRRM3 binds specifically exclusively to single-stranded G-tracts (ssRNA), in contrast to previous reports stating that the G-quadruplex was recognized as well. Furthermore, we demonstrate that the pre-existent ssRNA/G-quadruplex equilibrium slows down the formation of the protein-ssRNA complex. Based on in vitro transcription assays, we show that the rate of the protein-RNA complex formation is faster than that of the G-quadruplex. We propose a model according to which hnRNP F could bind RNA co-transcriptionally and prevents G-quadruplex formatio

Comparative analyses of the thermodynamic RNA binding signatures of different types of RNA recognition motifs

RNA recognition motifs (RRMs) are structurally versatile domains important in regulation of alternative splicing. Structural mechanisms of sequence-specific recognition of single-stranded RNAs (ssRNAs) by RRMs are well understood. The thermodynamic strategies are however unclear. Therefore, we utilized microcalorimetry and semi-empirical analyses to comparatively analyze the cognate ssRNA binding thermodynamics of four different RRM domains, each with a different RNA binding mode. The different binding modes are: canonical binding to the β-sheet surface; canonical binding with involvement of N- and C-termini; binding to conserved loops; and binding to an α-helix. Our results identify enthalpy as the sole and general force driving association at physiological temperatures. Also, networks of weak interactions are a general feature regulating stability of the different RRM-ssRNA complexes. In agreement, non-polyelectrolyte effects contributed between ∼75 and 90% of the overall free energy of binding in the considered complexes. The various RNA binding modes also displayed enormous heat capacity differences, that upon dissection revealed large differential changes in hydration, conformations and dynamics upon binding RNA. Altogether, different modes employed by RRMs to bind cognate ssRNAs utilize various thermodynamics strategies during the association process

The high kinetic stability of a G-quadruplex limits hnRNP F qRRM3 binding to G-tract RNA

The RNA binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) F is involved in telomeres maintenance and pre-mRNA processing, such as alternative splicing and polyadenylation. It specifically recognizes RNA containing three consecutive guanines (G-tracts) that have the potential to assemble into G-quadruplexes. We have proposed recently that hnRNP F could regulate alternative splicing by remodeling RNA structures, such as G-quadruplexes. However, the exact mechanism of hnRNP F binding to such RNA sequences remains unknown. Here, we have studied the binding of the third RNA binding domain of hnRNP F [quasi-RNA recognition motif 3 (qRRM3)] to G-tract RNA using isothermal titration calorimetry, circular dichroism and nuclear magnetic resonance spectroscopy. Our results show that qRRM3 binds specifically exclusively to single-stranded G-tracts (ssRNA), in contrast to previous reports stating that the G-quadruplex was recognized as well. Furthermore, we demonstrate that the pre-existent ssRNA/G-quadruplex equilibrium slows down the formation of the protein-ssRNA complex. Based on in vitro transcription assays, we show that the rate of the protein-RNA complex formation is faster than that of the G-quadruplex. We propose a model according to which hnRNP F could bind RNA co-transcriptionally and prevents G-quadruplex formation

The RING domain of the scaffold protein Ste5 adopts a molten globular character with high thermal and chemical stability

Ste5 is a scaffold protein that controls the pheromone response of the MAP-kinase cascade in yeast cells. Upon pheromone stimulation, Ste5 (through its RING-H2 domain) interacts with the β and γ subunits of an activated heterodimeric G protein and promotes activation of the MAP-kinase cascade. With structural and biophysical studies, we show that the Ste5 RING-H2 domain exists as a molten globule under native buffer conditions, in yeast extracts, and even in denaturing conditions containing urea (7 M). Furthermore, it exhibits high thermal stability in native conditions. Binding of the Ste5 RING-H2 domain to the physiological Gβ/γ (Ste4/Ste18) ligand is accompanied by a conformational transition into a better folded, more globular structure. This study reveals novel insights into the folding mechanism and recruitment of binding partners by the Ste5 RING-H2 domain. We speculate that many RING domains may share a similar mechanism of substrate recognition and molten-globule-like character