Format

Send to

Choose Destination
RNA. 2006 Jul;12(7):1397-407. Epub 2006 May 24.

Elongation factor 1alpha binds to the region of the metallothionein-1 mRNA implicated in perinuclear localization--importance of an internal stem-loop.

Author information

1
Institute for Cell and Molecular Biosciences, University of New Castle upon Tyne, UK.

Abstract

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.

PMID:
16723660
PMCID:
PMC1484432
DOI:
10.1261/rna.2730106
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center