Two ZBP1 KH Domains Facilitate β-Actin mRNA Localization, Granule Formation, and Cytoskeletal Attachment

• Published in: The Journal of cell biology Vol. 160; no. 1; pp. 77 - 87
• Main Authors: Farina, Kim L., Hüttelmaier, Stefan, Musunuru, Kiran, Darnell, Robert, Singer, Robert H.
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 06.01.2003; The Rockefeller University Press
• Subjects: Actins; Actins - genetics; Actins - metabolism; Amino Acid Sequence; Animals; Avian Proteins; Cell motility; Cell Movement; Cells; Chick Embryo; Cytoskeleton; Cytoskeleton - metabolism; Dose-Response Relationship, Drug; Fibroblasts; Gene Deletion; Glutathione Transferase - metabolism; In Situ Hybridization; In Situ Hybridization, Fluorescence; Messenger RNA; Microfilaments; Microscopy, Fluorescence; Molecular Sequence Data; Neurons; Neurotransmitters; Perceptual localization; Phenotype; Precipitin Tests; Protein Structure, Tertiary; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic acid; RNA; RNA - metabolism; RNA, Messenger - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - metabolism; Sequence Homology, Amino Acid; Skeletal system; Transcription, Genetic; Transfection
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.200206003

Chicken embryo fibroblasts (CEFs) localize β-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of β-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of β-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized β-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both β-actin mRNA localization and cell motility.