Lixa Carolina, Mujo Amanda, de Magalhães Mariana T Q, Almeida Fabio C L, Lima Luis Mauricio T R, Pinheiro Anderson S
Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-909, Brazil.
Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, 31270-901, Brazil.
J Biomol NMR. 2018 Dec;72(3-4):179-192. doi: 10.1007/s10858-018-0217-y. Epub 2018 Dec 8.
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.
人类抗原R（HuR）通过其RNA结合活性作为基因表达的主要转录后调节因子发挥作用。HuR由三个RNA识别基序组成，即RRM1、RRM2和RRM3。两个N端RRM结构域串联排列，主要促成HuR与mRNA中富含腺嘌呤和尿嘧啶的元件（ARE）相互作用。在此，我们结合使用核磁共振（NMR）和电喷雾电离-离子淌度质谱（ESI-IMS-MS）来表征HuR RRM1的结构、动力学、RNA识别和二聚化。我们的溶液结构揭示了一种典型的RRM折叠，包含一个19个残基的内在无序N端延伸，其不参与RNA结合。NMR滴定结果证实，对于源自环氧化酶2（Cox2）ARE I的7核苷酸RNA配体，主要RNA结合位点位于两个中央β链β1和β3上。我们通过N弛豫表明，除了N端和C端外，β2-β3环在游离状态和RNA结合状态下均经历快速的主链动力学（皮秒-纳秒），表明RNA相互作用时没有发生结构有序化。ESI-IMS-MS表明HuR RRM1会二聚化，然而二聚体群体占少数。二聚化通过α螺旋表面发生，该表面与RNA结合β片层方向相反。通过使用Cox2 ARE I的DNA类似物，我们表明DNA结合可稳定HuR RRM1单体并使单体-二聚体平衡向单体物种移动。总之，我们的结果加深了目前对HuR所采用的RNA识别机制的理解。
