ZBP-1 novel highly conserved RNA -binding protein and its role in beta-actin mRNA localization

Abstract

This dissertation focuses on mechanisms of mRNA transport and, specifically, a novel protein involved in the transport of beta-actin mRNA. Localization of beta-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3' UTR of the mRNA, including a 54-nucleotide zipcode (Kislauskis, Li 1993). We have purified, cloned and sequenced a novel 63 kDa RNA-binding protein that binds the zipcode with high affinity and specificity. Zipcode mutagenesis demonstrates that the binding of this protein correlates with the zipcode's ability to localize (Ross, Oleynikov 1997). The protein, termed ZBP-1, contains a classical RRM RNA-binding domain, four KH RNA-binding domains, a leucine-rich REV-like NES and putative NLS indicating its high RNA-binding capability and potential involvement in nuclear import-export. Epitope-tagged and endogenous ZBP-1 is mostly cytoplasmic and colocalizes with beta-actin mRNA at the leading edge of fibroblasts. Triton-extracted cells retain most ZBP-1 attached to cytoskeleton at the leading edge. Cytoskeletal drug treatments affect ZBP-1's ability to localize and myosin inhibitor BDM delocalizes ZBP-1 along with beta-actin mRNA. Nuclear export drug Leptomycin B increases amount of nuclear ZBP-1 suggesting it is involved in nuclear transport. In addition, upon beta-actin mRNA transcription activation by serum, ZBP-1 accumulates at the transcription site suggesting its involvement in nuclear export of the RNA. Antisense to the zipcode delocalizes ZBP-1 and beta-actin mRNA. The particles formed by ZBP-1 can be seen associating with microtubules and thin actin filaments but not actin stress fibers. GFP-ZBP-1 fusion protein localizes to the leading edge of CEFs and can be observed forming particles that move bi-directionally along filamentous structures in living fibroblasts as well as in neuronal cells. The data strongly suggests direct involvement of ZBP-1 in localization of beta-actin mRNA on actin cytoskeleton and microtubules, from the RNA transcription site to the leading edge.;ZBP-1 and its subsequently isolated homologs form a family of RNA-binding proteins implicated in mRNA localization, mRNA stability and translation regulation, and carcinogenesis. This indicates that ZBP-1 and its family are fundamentally important for cytoplasmic gene expression control utilizing various molecular mechanisms.*.;*Includes CD-ROM which requires Media Player (G2) or Real Player.