The role of the C-terminal helix of U1A protein in the interaction with U1hpII RNA

M J Law,I A Laird-Offringa,D S Lee,P P Anglim,C S Lee,I S Haworth

doi:10.1093/nar/gkt326

Abstract

Previous kinetic investigations of the N-terminal RNA Recognition Motif (RRM) domain of spliceosomal A protein of the U1 small nuclear ribonucleoprotein particle (U1A) interacting with its RNA target U1 hairpin II (U1hpII) provided experimental evidence for a ‘lure and lock’ model of binding. The final step of locking has been proposed to involve conformational changes in an α-helix immediately C-terminal to the RRM domain (helix C), which occludes the RNA binding surface in the unbound protein. Helix C must shift its position to accommodate RNA binding in the RNA–protein complex. This results in a new hydrophobic core, an intraprotein hydrogen bond and a quadruple stacking interaction between U1A and U1hpII. Here, we used a surface plasmon resonance-based biosensor to gain mechanistic insight into the role of helix C in mediating the interaction with U1hpII. Truncation, removal or disruption of the helix exposes the RNA-binding surface, resulting in an increase in the association rate, while simultaneously reducing the ability of the complex to lock, reflected in a loss of complex stability. Disruption of the quadruple stacking interaction has minor kinetic effects when compared with removal of the intraprotein hydrogen bonds. These data provide new insights into the mechanism whereby sequences C-terminal to an RRM can influence RNA binding.

Highlights

RNA recognition motifs (RRMs) are the most commonly found RNA-binding domain in eukaryotes and are implicated in many critical RNA–protein interactions in the cell [1,2,3,4]
Circular dichroism (CD) spectra (Supplementary Figure S1) indicate that the fold of RRM1 is maintained in U1A90 compared with U1 small nuclear ribonucleoprotein particle (U1A) and that the reduced affinity of U1A90 for U1 hairpin II (U1hpII) is not caused by perturbation of the RRM structure owing to complete removal of helix immediately C-terminal to the RRM domain (helix C)
The U1A–U1hpII interaction is among the strongest non-covalent interactions described, and determining its molecular basis is of interest with respect to both understanding the U1A–U1snRNP complex as well as understanding how tight biological interactions can be formed and maintained

Summary

Introduction

RNA recognition motifs (RRMs) are the most commonly found RNA-binding domain in eukaryotes and are implicated in many critical RNA–protein interactions in the cell [1,2,3,4]. RRM-containing proteins interact with their RNA targets with dynamics that reflect their biological function. RRMs are characterized by a b-a-b-b-a-b secondary structure that results in the formation of an antiparallel b-sheet comprising the RNA-binding platform [5]. RNP-1 and -2 are critical for highaffinity RNA binding by RRM-containing proteins, as evidenced by the dramatic loss in affinity on their mutation [8,9,10,11,12,13,14]. The distinct binding ‘specificity’ of different RRM-containing proteins is strongly influenced by the more variable regions of the domain

Methods

Results

Conclusion