Fine mapping of interactions between eEF1alpha protein and 3'UTR of metallothionein-1 mRNA.
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 (eEF1alpha) 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 eEF1alpha, 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 eEF1alpha and histidine-tRNA ligase, and that binding of eEF1alpha is facilitated through domains I and III.
Fan
Kunbo
K
Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Chrzanowska-Lightowlers
Zofia M A
ZM
Hesketh
John E
JE
D020413:3' Untranslated Regions / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D024202:Electrophoretic Mobility Shift Assay; D008668:Metallothionein / Q000235:genetics*; D009690:Nucleic Acid Conformation; D020648:Peptide Elongation Factor 1 / Q000378:metabolism*; D051381:Rats; D014158:Transcription, Genetic
D020413:3' Untranslated Regions; C491055:Mt2A protein, rat; D020648:Peptide Elongation Factor 1; D008668:Metallothionein