Abstract
HuR/ELAVL1 is an RNA-binding protein involved in differentiation and stress response that acts primarily by stabilizing messenger RNA (mRNA) targets. HuR comprises three RNA recognition motifs (RRMs) where the structure and RNA binding of RRM3 and of full-length HuR remain poorly understood. Here, we report crystal structures of RRM3 free and bound to cognate RNAs. Our structural, NMR and biochemical data show that RRM3 mediates canonical RNA interactions and reveal molecular details of a dimerization interface localized on the α-helical face of RRM3. NMR and SAXS analyses indicate that the three RRMs in full-length HuR are flexibly connected in the absence of RNA, while they adopt a more compact arrangement when bound to RNA. Based on these data and crystal structures of tandem RRM1,2-RNA and our RRM3-RNA complexes, we present a structural model of RNA recognition involving all three RRM domains of full-length HuR. Mutational analysis demonstrates that RRM3 dimerization and RNA binding is required for functional activity of full-length HuR in vitro and to regulate target mRNAs levels in human cells, thus providing a fine-tuning for HuR activity in vivo.
Highlights
HuR/ELAVL1 (Human antigen R/Embryonic Lethal Abnormal Vision-Like Protein 1) is a ubiquitously expressedRNA-binding protein implicated in several vital processes such as cell proliferation, differentiation or responses to stress and immune stimuli
In order to investigate the role of RRM3 in HuR function, we set out to optimize its expression and purification conditions to obtain the domain in amounts and purity suitable for biochemical and structural studies
We produced RRM3 fused to an N-terminal thioredoxin 1 (Trx) tag via a short uncleavable Gly-Ser-Ala-Met linker (Figure 1B) to improve solubility and suitability for crystallization as described previously [42]
Summary
HuR/ELAVL1 (Human antigen R/Embryonic Lethal Abnormal Vision-Like Protein 1) is a ubiquitously expressedRNA-binding protein implicated in several vital processes such as cell proliferation, differentiation or responses to stress and immune stimuli. HuR/ELAVL1 (Human antigen R/Embryonic Lethal Abnormal Vision-Like Protein 1) is a ubiquitously expressed. HuR is enriched in the nucleus under physiological conditions, its main function, mRNA stabilization, takes place in the cytoplasm where it can translocate, for example in response to cellular stress [2]. The function of HuR is regulated at several levels. The amount of HuR in cells is controlled and adjusted at the level of transcription, polyadenylation and mRNA stability [3,4]. Posttranslational modifications, such as phosphorylation, ubiquitinylation, neddylation and cleavage by caspases further regulate the cellular levels and localization of HuR protein. HuR binding to target mRNAs is controlled by its phosphorylation, methylation and ubiquitination [5,6,7,8,9,10]
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