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.

PY - 2002/12/1

Y1 - 2002/12/1

N2 - 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.

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.

KW - INTERMEDIATE-FILAMENT PROTEIN

KW - INTRACELLULAR-LOCALIZATION

KW - 3'-UNTRANSLATED REGION

KW - NUCLEOTIDE-SEQUENCE

KW - 3-HYBRID SYSTEM

KW - ACTIN

KW - PHOSPHORYLATION

KW - SUBSTRATE

KW - DETECT

KW - MUSCLE

UR - http://www.scopus.com/inward/record.url?scp=0036884863&partnerID=8YFLogxK

U2 - 10.1093/nar/gkf656

DO - 10.1093/nar/gkf656

M3 - Review article

C2 - 12466525

AN - SCOPUS:0036884863

VL - 30

SP - 5017

EP - 5028

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 23

ER -