Elongation factor 1alpha binds to the region of the metallothionein-1 mRNA implicated in perinuclear localization--importance of an internal stem-loop.
In eukaryotic cells, mRNA localization can provide local protein synthesis. Metallothionein-1 (MT-1) mRNA is associated with the perinuclear cytoskeleton, and this is essential for subsequent nuclear import of the protein. The present study defines the cis-acting localization signal and a trans-acting binding protein. Gel retardation and UV cross-linking assays using MT-1 3'UTR transcripts and CHO cell extracts revealed formation of a complex containing a approximately 50-kDa protein. Only localization-positive mutant transcripts competed for binding of this protein. Using an RNA affinity technique, Western blotting, mass spectrometry, and a supershift assay, the protein was identified as Elongation factor 1alpha (eEF1alpha). Mutation and deletion analysis showed that two regions, nucleotides 21-36 and 66-76, were required for both binding and localization. RNA-folding prediction combined with chemical and enzymatic probing experiments suggest that these regions are in juxtaposition within a stem/internal loop structure. Mutations that are predicted to alter this structure abrogate protein binding. Our hypothesis is that the cis-acting signal in MT-1 3'UTR is formed by this stem/internal loop, that it binds eEF1alpha, and that eEF1alpha-cytoskeleton interactions play a role in perinuclear mRNA localization.
Mickleburgh
Ian
I
Institute for Cell and Molecular Biosciences, University of New Castle upon Tyne, UK.
Chabanon
Hervé
H
Nury
David
D
Fan
Kunbo
K
Burtle
Brian
B
Chrzanowska-Lightowlers
Zofia
Z
Hesketh
John
J
D020413:3' Untranslated Regions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D017931:DNA Primers; D008668:Metallothionein / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D020648:Peptide Elongation Factor 1 / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D014158:Transcription, Genetic
D020413:3' Untranslated Regions; D017931:DNA Primers; D020648:Peptide Elongation Factor 1; D012333:RNA, Messenger; D008668:Metallothionein