Specific Protein Domains Mediate Cooperative Assembly of HuR Oligomers on AU-rich mRNA-destabilizing Sequences

Abstract

The RNA-binding factor HuR is a ubiquitously expressed member of the Hu protein family that binds and stabilizes mRNAs containing AU-rich elements (AREs). Hu proteins share a common domain organization of two tandemly arrayed RNA recognition motifs (RRMs) near the N terminus, followed by a basic hinge domain and a third RRM near the C terminus. In this study, we engineered recombinant wild-type and mutant HuR proteins lacking affinity tags to characterize their ARE-binding properties. Using combinations of electrophoretic mobility shift and fluorescence anisotropy-based binding assays, we show that HuR can bind ARE substrates as small as 13 nucleotides with low nanomolar affinity, but forms cooperative oligomeric protein complexes on ARE substrates of at least 18 nucleotides in length. Analyses of deletion mutant proteins indicated that RRM3 does not contribute to high affinity recognition of ARE substrates, but is required for cooperative assembly of HuR oligomers on RNA. Finally, the hinge domain between RRM2 and RRM3 contributes significant binding energy to HuR⅐ARE complex formation in an ARE length-dependent manner. The hinge does not enhance RNA-binding activity by increased ion pair formation despite extensive positive charge within this region, and it does not thermodynamically stabilize protein folding. Together, the results define distinct roles for the HuR hinge and RRM3 domains in formation of cooperative HuR⅐ARE complexes in solution.

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

“…12 The NMR titration data herein reported and earlier findings with HuB, 59 demonstrate that isolated RRM3 can bind to AREs, although previous studies suggested a negligible role of HuR RRM3 ¡ as part of HuR FL ¡ in AREs recognition. 34,[44][45][46] Isolated RRM3 is in a monomer/dimer exchange on a time scale unfavorable for NMR observation of the residues at the dimerization site, notably those in the W261-T271 region at the C-end of helix a 1 . Indeed, this region shows large fluctuations in MD simulations, which affect the H-bonding pattern at a 1 and the dynamics of W261 ring.…”

“…Interestingly, isolated HuR RRM3 preferably binds U-rich versus AU-rich RNA stretches, despite previous studies suggesting that RRM3 has a negligible contribution to ARE-binding events in comparison with the other 2 HuR RRM motifs. 34,[44][45][46] Although the HuR linker between RRM2 and RRM3 could contribute to the ARE stabilization, 34,35 it was unfeasible to obtain a linker-bearing RRM3 construct in a soluble manner. Moreover, HuR RRM3 phosphorylation, which was mimicked by a S318-to-D318 substitution, causes no significant structural changes of RRM3, in contrast to a phosphorylation-mediated unfolding described in other types of RNA domains.…”

“…28 HNS is mainly responsible for nuclear/cytoplasmic shuttling upon binding adaptor proteins for nuclear export such as pp32/PHAP-I and APRIL 29,30 and with import factors transportin-1, -2 and importin a: [31][32][33] Previous Electrophoretic Mobility Shift Assay (EMSA)-based report 34 suggests that the RRM23 linker, together with the RRM3 domain, could also have an additional role in stabilizing HuR-AREs complexes. This enhancement of ARE-binding activity was greater than that observed in Surface Plasmon Resonance (SPR) experiments with HuD, a highly homolog protein to HuR, 35 where the RRM23 linker showed a negligible effect in RNA binding.…”

“…43 Cytoplasmic binding of HuR to single-stranded ARE-containing mRNAs is mainly driven by RRM1 and RRM2 domains. 34,[44][45][46] RRM3 is involved in binding long poly-A tails of mRNAs [47][48][49] and in catalyzing the 3 0 -terminal adenosyl modification of non-polyadenylated RNA substrates. 50 Besides its role in RNA recognition, RRM3 is also responsible for the formation of HuR multimers, 34 as occurs with the homologous Drosophila ELAV protein.…”

“…34,[44][45][46] RRM3 is involved in binding long poly-A tails of mRNAs [47][48][49] and in catalyzing the 3 0 -terminal adenosyl modification of non-polyadenylated RNA substrates. 50 Besides its role in RNA recognition, RRM3 is also responsible for the formation of HuR multimers, 34 as occurs with the homologous Drosophila ELAV protein. 51 In fact, mutations in the RRM3 domain of Drosophila ELAV protein lead to a temperature-sensitive phenotype by impairing HuR oligomerization, highlighting the crucial role of this RRM module.…”