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 HuR.ARE 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 HuR.ARE complexes in solution.
Highlights
Several mRNAs that are rapidly degraded, often with halflives of less than 1 h, include those encoding oncoproteins and signaling proteins such as cytokines, chemokines, and inflammatory mediators [3]
electrophoretic mobility shift assays (EMSAs) using glutathione S-transferase (GST)-HuR [40], we cannot rule out the possibility that the size of the GST tag may occlude a third binding site on the RNA or that additional GST-HuR1⁄7ARE
The HuR basic hinge domain contains the nuclear localization sequence element required for nucleocytoplasmic shuttling by this protein [25]
Summary
RNA Substrates—Synthesis, 2Ј-hydroxyl deprotection, and purification of the RNA substrates used in this study were performed by Dharmacon Research or Integrated DNA Technologies. The RNA substrate ARE38 includes the core ARE sequence from TNF␣ mRNA (Table 1). Other substrates designated ARExx are subsections of this TNF␣ mRNA sequence. The RNA substrate R encodes a fragment of the rabbit -globin mRNA coding sequence. Fluorescein groups were linked to the 5Ј-ends of some RNA substrates during solid-phase synthesis and are designated by the prefix “Fl” where applicable. Quantification of RNA yields and fluorophore labeling efficiency was performed by absorbance spectroscopy as described previously [28, 29]. For preparation of 5Ј-32P-radiolabeled RNA substrates, 5Ј-hydroxyl RNA oligonucleotides were incubated with [␥-32P]ATP (PerkinElmer Life Sciences) and T4 polynucleotide kinase (Promega) as described [30], yielding specific activities of 3–5 ϫ 103 cpm/fmol
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