The structure of the ARE-binding domains of Hu antigen R (HuR) undergoes conformational changes during RNA binding

Abstract

Human RNA-binding protein (HuR), a ubiquitously expressed member of the Hu protein family, plays an important role in mRNA degradation and has been implicated as a key post-transcriptional regulator. HuR contains three RNA-recognition motif (RRM) domains. The two N-terminal tandem RRM domains can selectively bind AU-rich elements (AREs), while the third RRM domain (RRM3) contributes to interactions with the poly-A tail of target mRNA and other ligands. Here, the X-ray structure of two methylated tandem RRM domains (RRM1/2) of HuR in their RNA-free form was solved at 2.9 Å resolution. The crystal structure of RRM1/2 complexed with target mRNA was also solved at 2.0 Å resolution; comparisons of the two structures show that HuR RRM1/2 undergoes conformational changes upon RNA binding. Fluorescence polarization assays (FPA) were used to study the protein-RNA interactions. Both the structure and the FPA analysis indicated that RRM1 is the primary ARE-binding domain in HuR and that the conformational changes induce subsequent contacts of the RNA substrate with the inter-domain linker and RRM2 which greatly improve the RNA-binding affinity of HuR.

Post-transcriptional Regulation of Programmed Cell Death 4 (PDCD4) mRNA by the RNA-binding Proteins Human Antigen R (HuR) and T-cell Intracellular Antigen 1 (TIA1)

“…Although RRM1 is the primary ARE recognition domain within HuR, the conformational change that takes place upon RNA binding leads to additional interactions between RRM2 and the target mRNA, ultimately increasing RNA binding affinity (11). This structure, along with other studies investigating the structural basis of HuR/RNA interactions (62), reveals a number of key residues that are predicted to be important for high affinity RNA binding by HuR (11,62).…”

“…Two N-terminal RNA recognition motifs (RRMs) within HuR mediate recognition of AREs located primarily within the 3ЈUTR of target mRNA transcripts (11,12). A third RRM in the C terminus of HuR has affinity for polyadenosine RNA and is thought to bind to the poly(A) tail of target mRNAs (13,14).…”

A fly trap mechanism provides sequence-specific RNA recognition by CPEB proteins

“…While most of the tandem RRMs are separated by a flexible interdomain linker and therefore do not interact in the free state, such as sex-lethal (Sxl), Nucleolin, PTB RRM12, Hrp1, Npl3, and HuR (Crowder et al 1999;Allain et al 2000;Perez-Canadillas 2006;Vitali et al 2006;Skrisovska and Allain 2008;Wang et al 2013), only a few tandem RRMs show interactions; namely, PTB RRM34, Prp24, FIR, and, more recently U2AF65, hnRNPA1, and hnRNPL (Vitali et al 2006;Bae et al 2007;Cukier et al 2010;Mackereth et al 2011;Barraud and Allain 2013;Zhang et al 2013). Such fixed orientation of two RRMs relative to each other often has a direct implication in the mechanism of action of the proteins.…”

Identification of novel proteins binding the AU-rich element of α-prothymosin mRNA through the selection of open reading frames (RIDome)

“…These RRMs are present as a single module in RBM38 35 or as a bipartite module in ELAVL1, where 2 tandem N-terminal domains (RRM1 and RRM2) selectively bind ARE, while RRM3 interacts with the poly(A) tail and other proteins. 36 Furthermore, when the ORF inserts of tested clones were sequenced, we observed that these inserts faithfully represented the predicted RNA-binding domains (Fig. 4B).…”