HuR thermal stability is dependent on domain binding and upon phosphorylation

Abstract

Abstract:Human antigen R (HuR) is a multitasking RNA binding protein involved in posttranscriptional regulation by recognizing Adenine and uracile Rich Elements (AREs) placed at the 3′ untranslated regions of mRNAs. The modular architecture of the protein, which consists of two N-terminal RNA recognition motifs (RRMs) in tandem spaced from a third one by a nuclear-cytoplasmic shuttling sequence, controls stability of many mRNA targets, as well as their translation rates. A higher level of regulation comes from the fact that both localization and function of HuR is strictly regulated by phosphorylation. Here, we report how the thermal stability of RRM2 is decreased by the presence of RRM1, indicating that both domains are interacting in solution. In addition, even though no significant structural changes are observed among mutants of HuR RRM12 mimicking phosphorylated species, slight differences in stability are appreciable, which may explain the RNA binding activity of HuR.Response to Reviewers: See attachment. Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions

“…As previous studies have described, the three domains of the TIA-1 protein contribute to RNA binding, with the RRM2 and RRM3 modules as the main recognition platforms (16), as in other RNA-binding proteins, such as the central domain units of KSRP (KH-type splicing regulatory protein) (29) and the N terminus of HuR (human antigen R) RRM modules (25). Whereas TIA-1 RRM2 dominates the interaction, RRM3 enhances it, playing an auxiliary role.…”

“…A temperature of 10°C was chosen to optimize the signal change upon protein binding. The integral of the CD signal between 255 and 265 nm was plotted against the ratio [RRM23]/[24-mer AU-rich RNA] and fitted to a 1:1 binding site model as reported previously (25).…”

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

“…Taking into account that HuR RRM1 and RRM2 domains behave as a functional unit, 54 we investigated whether RRM3 is also part of this domain arrangement or is independent, so as to characterize the overall structure of HuR full-length (FL). By recording 15 N-HSQC Figure S1, panel B), no significant changes in the chemical shifts or the line widths of the NMR resonances were detected.…”

“…The HuR RRM12 tandem-construct containing the 2 Nterminal RRM1 and RRM2 modules along with the 6xHis-tag was used as previously described. 54 The RRM3 domain comprises the amino acid sequence from W244 to K326, which was cloned into the pETM-11 vector using EcoRI and NotI restriction sites. Further site-directed mutagenesis was performed on the gene coding RRM3 WT to replace the S318 by an alanine (RRM3 S318A) or an aspartate (RRM3 S318D) so as to mimic the phosphorylation of HuR at S318.…”

A Non‐Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH‐Modulation of Protein–Nucleic Acid Interfaces

“…TIA-1 RRM23 and HuR RRM12 constructs used in the assay were prepared as previously described. [2,46,47] See the Supporting Information for further details.…”