Oligomeric transition and dynamics of RNA binding by the HuR RRM1 domain in solution

• Published in: Journal of biomolecular NMR Vol. 72; no. 3-4; pp. 179 - 192
• Main Authors: Lixa, Carolina, Mujo, Amanda, de Magalhães, Mariana T. Q., Almeida, Fabio C. L., Lima, Luis Mauricio T. R., Pinheiro, Anderson S.
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.12.2018; Springer Nature B.V
• Subjects: Adenine; Binding Sites; Biochemistry; Biological and Medical Physics; Biophysics; Cyclooxygenase-2; Deoxyribonucleic acid; Dimerization; Dimers; DNA; Domains; ELAV-Like Protein 1 - metabolism; Gene expression; Humans; HuR protein; Ionic mobility; Ionization; Mass Spectrometry; Mass spectroscopy; Monomers; NMR; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular; Physics; Physics and Astronomy; Post-transcription; Protein Binding; Recognition; Ribonucleic acid; RNA; RNA - chemistry; RNA - metabolism; RNA-Binding Proteins - chemistry; Scientific imaging; Spectroscopy; Spectroscopy/Spectrometry; Titration; Tumor Suppressor Proteins - chemistry; Uracil; Ion-mobility; NMR; HuR; RNA; Dynamics; RRM; Structure
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1007/s10858-018-0217-y

Human antigen R (HuR) functions as a major post-transcriptional regulator of gene expression through its RNA-binding activity. HuR is composed by three RNA recognition motifs, namely RRM1, RRM2, and RRM3. The two N-terminal RRM domains are disposed in tandem and contribute mostly to HuR interaction with adenine and uracil-rich elements (ARE) in mRNA. Here, we used a combination of NMR and electrospray ionization–ion mobility spectrometry–mass spectrometry (ESI–IMS–MS) to characterize the structure, dynamics, RNA recognition, and dimerization of HuR RRM1. Our solution structure reveals a canonical RRM fold containing a 19-residue, intrinsically disordered N-terminal extension, which is not involved in RNA binding. NMR titration results confirm the primary RNA-binding site to the two central β-strands, β1 and β3, for a cyclooxygenase 2 (Cox2) ARE I-derived, 7-nucleotide RNA ligand. We show by 15 N relaxation that, in addition to the N- and C-termini, the β2–β3 loop undergoes fast backbone dynamics (ps–ns) both in the free and RNA-bound state, indicating that no structural ordering happens upon RNA interaction. ESI–IMS–MS reveals that HuR RRM1 dimerizes, however dimer population represents a minority. Dimerization occurs via the α-helical surface, which is oppositely orientated to the RNA-binding β-sheet. By using a DNA analog of the Cox2 ARE I, we show that DNA binding stabilizes HuR RRM1 monomer and shifts the monomer–dimer equilibrium toward the monomeric species. Altogether, our results deepen the current understanding of the mechanism of RNA recognition employed by HuR.