Nucleic Acids Research, 2002, Vol. 30, No. 23 5017-5028
© 2002 Oxford University Press
The 3' untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1
and HAX-1
Department of Biochemistry and Molecular Biophysics and the Massey Cancer Center, 1 Institute for Structural Biology and Drug Discovery and 2 Department of Microbiology and Immunology, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, VA, USA and 3 Laboratory of Biochemical Genetics, Biological Institute, St Petersburg State University, Russia
*To whom correspondence should be addressed at Box 980614, Department of Biochemistry and Molecular Biophysics, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0614, USA. Tel: +1 804 828 8753; Fax: +1 804 828 1473; Email: zezehner{at}vcu.edu
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-1
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-1 and HAX-1. Moreover, HAX-1 binding appears to be specific to vimentin’s 3'UTR. Both in vivo synthesized eEF-1 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-1 or HAX-1. Thus, in addition to their known functions, both eEF-1 and HAX-1 are RNA binding proteins, which suggests new roles in mRNA translation and/or perinuclear localization.
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