Two ZBP1 KH domains facilitate beta-actin mRNA localization, granule formation, and cytoskeletal attachment.
Chicken embryo fibroblasts (CEFs) localize beta-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of beta-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 beta-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.
Farina
Kim L
KL
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Huttelmaier
Stefan
S
Musunuru
Kiran
K
Darnell
Robert
R
Singer
Robert H
RH
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins; D002465:Cell Movement; D002642:Chick Embryo; D003599:Cytoskeleton / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D017403:In Situ Hybridization; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010641:Phenotype; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection
D000199:Actins; D030161:Avian Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C498636:ZBP1 protein, Gallus gallus; D012313:RNA; D005982:Glutathione Transferase