The 3′ untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1γ and HAX-1

May Al-Maghrebi, Hervé Brulé, Marina Padkina, Carrie Allen, W. Michael Holmes, Zendra E. Zehner

Research output

67 Citations (Scopus)

Abstract

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-1c 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-Iγ and HAX-1. Moreover, HAX-1 binding appears to be specific to vimentin's 3′ UTR. Both in vivo synthesized eEF-1γ 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-1γ or HAX-1. Thus, in addition to their known functions, both eEF-1γ and HAX-1 are RNA binding proteins, which suggests new roles in mRNA translation and/or perinuclear localization.

Original languageEnglish
Pages (from-to)5017-5028
Number of pages12
JournalNucleic Acids Research
Volume30
Issue number23
DOIs
Publication statusPublished - 1 Dec 2002

Fingerprint

3' Untranslated Regions
Vimentin
Messenger RNA
RNA-Binding Proteins
RNA
Proteins
Protein Biosynthesis
Cell Extracts
HeLa Cells
Recombinant Proteins
Carrier Proteins
Complementary DNA
Yeasts
In Vitro Techniques

Scopus subject areas

  • Genetics

Cite this

Al-Maghrebi, May ; Brulé, Hervé ; Padkina, Marina ; Allen, Carrie ; Holmes, W. Michael ; Zehner, Zendra E. / The 3′ untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1γ and HAX-1. In: Nucleic Acids Research. 2002 ; Vol. 30, No. 23. pp. 5017-5028.
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The 3′ untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1γ and HAX-1. / Al-Maghrebi, May; Brulé, Hervé; Padkina, Marina; Allen, Carrie; Holmes, W. Michael; Zehner, Zendra E.

In: Nucleic Acids Research, Vol. 30, No. 23, 01.12.2002, p. 5017-5028.

Research output

TY - JOUR

T1 - The 3′ untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1γ and HAX-1

AU - Al-Maghrebi, May

AU - Brulé, Hervé

AU - Padkina, Marina

AU - Allen, Carrie

AU - Holmes, W. Michael

AU - Zehner, Zendra E.

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AB - 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-1c 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-Iγ and HAX-1. Moreover, HAX-1 binding appears to be specific to vimentin's 3′ UTR. Both in vivo synthesized eEF-1γ 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-1γ or HAX-1. Thus, in addition to their known functions, both eEF-1γ and HAX-1 are RNA binding proteins, which suggests new roles in mRNA translation and/or perinuclear localization.

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