Characterization of ZBP-1 and its function in beta-actin mRNA localization

Abstract

Chicken embryo fibroblasts (CEF's) localize beta-actin mRNA to their lamellae. This process requires a cis-localization element (zipcode), and involves, Zipcode Binding Protein-1 (ZBP1), a protein that specifically binds to the zipcode and localizes to the lamellae of polarized CEF's. beta-actin mRNA and ZBP-1 assemble into ribonucleoprotein (RNP) granules that are transported and anchored on the cytoskeleton. These granules are believed to contain machinery required for assembly, transport, localization, anchoring and translation of the transcript. This study details ZBP1 regions responsible for granule assembly, cytoskeletal interaction and RNA binding. Previous data linking ZBP1 function to beta-actin mRNA localization was correlative. This study provides the first evidence that ZBP1 plays a direct role in localizing the mRNA. Furthermore, a physiological role for the function of ZBP1 has been established.;ZBP1 truncations fused to green fluorescent protein (GFP) revealed that the KH domains of ZBP1 mediate granule formation, cytoskeleton retention and RNA binding while peripheral localization of ZBP1 containing granules depends on the RRM domains. Using recombinant protein fragments of ZBP1 in RNA binding assays, the two C-terminal KH domains were identified as the zipcode binding region of ZBP1. Overexpression of the GFP fusion constructs in conjunction with in situ hybridization analysis was employed to evaluate the role of ZBP1 in beta-actin mRNA localization. Overexpression of the two C-terminal KH domains, delocalizes beta-actin mRNA in CEF's, strongly supporting the direct involvement of ZBP1 in the localization of beta-actin mRNA.;beta-actin mRNA localization is required for maintenance of polarized cell shape and directed motility in. It seems likely, that the function of ZBP1 might also influence cellular motility given its association with beta-actin mRNA. To address this possibility, motion analysis studies were performed. Full length ZBP1 and dominant negative ZBP1 (KH1--KH4) were overexpressed in CEF's to assess the protein's role in cell motility. Cell motility was altered by overexpression of either construct, indicating that ZBP1 is indeed playing a role in the production of sustained directed cell movements. Furthermore, the development of an animal model that can be used to evaluate the role of ZBP1 in cell motility in vivo was developed.