title: Interactions of elongation factor 1alpha with F-actin and beta-actin mRNA: implications for anchoring mRNA in cell protrusions.
abstract: The targeting of mRNA and local protein synthesis is important for the generation and maintenance of cell polarity. As part of the translational machinery as well as an actin/microtubule-binding protein, elongation factor 1alpha (EF1alpha) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1alpha colocalizes with beta-actin mRNA and F-actin in protrusions of chicken embryo fibroblasts and binds directly to F-actin and beta-actin mRNA simultaneously in vitro in actin cosedimentation and enzyme-linked immunosorbent assays. To investigate the role of EF1alpha in mRNA targeting, we mapped the two actin-binding sites on EF1alpha at high resolution and defined one site at the N-terminal 49 residues of domain I and the other at the C-terminal 54 residues of domain III. In vitro actin-binding assays and localization in vivo of recombinant full-length EF1alpha and its various truncates demonstrated that the C terminus of domain III was the dominant actin-binding site both in vitro and in vivo. We propose that the EF1alpha-F-actin complex is the scaffold that is important for beta-actin mRNA anchoring. Disruption of this complex would lead to delocalization of the mRNA. This hypothesis was tested by using two dominant negative polypeptides: the actin-binding domain III of EF1alpha and the EF1alpha-binding site of yeast Bni1p, a protein that inhibits EF1alpha binding to F-actin and also is required for yeast mRNA localization. We demonstrate that either domain III of EF1alpha or the EF1alpha-binding site of Bni1p inhibits EF1alpha binding to beta-actin mRNA in vitro and causes delocalization of beta-actin mRNA in chicken embryo fibroblasts. Taken together, these results implicate EF1alpha in the anchoring of beta-actin mRNA to the protrusion in crawling cells.
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, New York 10461, USA. liug@mail.amc.edu
authors.forename: Gang
authors.initials: G
authors.lastname: Liu
authors.forename: Wayne M
authors.initials: WM
authors.lastname: Grant
authors.forename: Daniel
authors.initials: D
authors.lastname: Persky
authors.forename: Vaughan M
authors.initials: VM
authors.lastname: Latham
authors.forename: Robert H
authors.initials: RH
authors.lastname: Singer
authors.forename: John
authors.initials: J
authors.lastname: Condeelis
mesh_terms: D000199:Actins / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats
chemical_list: D000199:Actins; D020648:Peptide Elongation Factor 1; D012333:RNA, Messenger