Fine mapping of interactions between eEF1α protein and 3′UTR of metallothionein-1 mRNA |
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Authors: | Kunbo Fan John E Hesketh |
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Institution: | a Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK b Institute for Ageing and Health, Newcastle University, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK |
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Abstract: | The localization of metallothionein-1 (MT-1) mRNA to the perinuclear cytoskeleton is determined by a signal in the 3′untranslated region (3′UTR) and trans-acting binding proteins. The present study carried out detailed mapping of this signal and further characterized the binding to elongation factor 1 alpha (eEF1α) and other interacting proteins. Electrophoresis mobility shift assays demonstrated that shortening of a stem region proximal to nucleotides 66-76 abrogated binding. Full length recombinant rat eEF1α, and independently domains I and III, formed complexes with the mRNA. Proteins binding to biotinylated MT-1 3′UTR sequences were isolated using RNA-affinity techniques, and mass spectrometry identified histidine-tRNA ligase as one of the major MT-1 3′UTR binding proteins. We conclude that a 5-bp internal stem in the MT-1 3′UTR is critical for binding of eEF1α and histidine-tRNA ligase, and that binding of eEF1α is facilitated through domains I and III. |
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Keywords: | Metallothionein-1 Perinuclear mRNA localisation 3&prime Untranslated region Elongation factor 1α RNA binding protein RNA secondary structure Histidine-tRNA ligase |
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