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 invo... More
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 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.
