The 3′ untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1γ and HAX-1
| Autor: | Zendra E. Zehner, May Al‐Maghrebi, Marina Padkina, Carrie Allen, Hervé Brulé, W. Michael Holmes |
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| Jazyk: | angličtina |
| Rok vydání: | 2002 |
| Předmět: |
Untranslated region
Cell Extracts Macromolecular Substances Vimentin RNA-binding protein Peptide Elongation Factor 1 Protein Interaction Mapping Genetics Humans Binding site 3' Untranslated Regions Adaptor Proteins Signal Transducing Messenger RNA Binding Sites biology Three prime untranslated region RNA Signal transducing adaptor protein Proteins RNA-Binding Proteins Articles Molecular biology Nuclear Pore Complex Proteins Mutation biology.protein HeLa Cells |
| Popis: | Previously, we have shown that the vimentin 3' untranslated region (3'UTR) contains a highly conserved region, which is sufficient for the perinuclear localization of a reporter mRNA. This region was shown to specifically bind protein(s) by band shift analyses. UV-cross-linking studies suggest these proteins are 46- and 35-kDa in mass. Here, we have used this sequence as 'bait' to isolate RNA binding proteins using the yeast three-hybrid method. This technique relies on a functional assay detecting bona fide RNA-protein interaction in vivo. Three cDNA isolates, HAX-1, eEF-1gamma and hRIP, code for proteins of a size consistent with in vitro cross- linking studies. In all cases, recombinant proteins were capable of binding RNA in vitro. Although hRIP is thought to be a general mRNA binding protein, this represents an unreported activity for eEF-1gamma and HAX-1. Moreover, HAX-1 binding appears to be specific to vimentin's 3'UTR. Both in vivo synthesized eEF-1gamma and HAX-1 proteins were 'pulled out' of HeLa whole cell extracts by binding to a RNA affinity column comprised of vimentin's 3'UTR. Moreover, size-fractionation of extracts results in the separation of large complexes containing either eEF-1gamma or HAX-1. Thus, in addition to their known functions, both eEF-1gamma and HAX-1 are RNA binding proteins, which suggests new roles in mRNA translation and/or perinuclear localization. |
| Databáze: | OpenAIRE |
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