The 3' untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1gamma and HAX-1.
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.
Al-Maghrebi
May
M
Department of Biochemistry and Molecular Biophysics and the Massey Cancer Center, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, VA 23298-0614, USA.
Brulé
Hervé
H
Padkina
Marina
M
Allen
Carrie
C
Holmes
W Michael
WM
Zehner
Zendra E
ZE
D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites; D002457:Cell Extracts / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D009154:Mutation; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000378:metabolism*; D025941:Protein Interaction Mapping; D011506:Proteins / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014746:Vimentin / Q000235:genetics* / Q000378:metabolism
D020413:3' Untranslated Regions; C119724:AGFG1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002457:Cell Extracts; C104759:HAX1 protein, human; D046911:Macromolecular Substances; D028861:Nuclear Pore Complex Proteins; D020648:Peptide Elongation Factor 1; D011506:Proteins; D016601:RNA-Binding Proteins; D014746:Vimentin