Mechanism and function of ZBP1 facilitated localization of beta-actin mRNA in neurons

Abstract

The molecular mechanism of mRNA localization in neurons is not well understood. The objective of my thesis was to characterize the molecular mechanism of beta-actin mRNA localization in neurons and perturb it to assess possible defects in actin morphology and structure. The localization of beta-actin mRNA in fibroblasts and neurons was shown to be dependent on a 54nt zipcode sequence in the 3 'UTR. In my thesis, I showed that cZBP1 is expressed in chick forebrain neurons and forms a complex with the beta-actin zipcode in vitro. In order to determine if the interaction between the zipcode and cZBP1 was necessary for beta-actin mRNA localization to growth cones of developing neurites, I showed that antisense oligonucleotides to the zipcode could disrupt the formation on an RNP complex in vitro which correlated with impaired localization of beta-actin mRNA in cultured neurons.;Further work was to investigate the role of rZBP1 in localization of beta-actin mRNA in dendrites using a cultured hippocampal neurons from rat embryos as a model system. I have cloned and sequenced the rat orthologue of chick ZBP1. rZBP1 was shown to bind the zipcode in vitro and in vivo. A yeast-three-hybrid method was used to identify domains on rZBP1 responsible for zipcode binding. The carboxyl terminal KH3 and KH4 domains plus some flanking sequences were shown to be the minimal regions necessary for zipcode binding. Transfections of truncated rZBP1 constructs fused to EGFP showed that all four KH domains were needed for granule formation and dendritic transport.;rZBP1 was necessary for beta-actin mRNA localization in dendrites. Knockdown of rZBP1 in cultured hippocampal neurons using antisense oligonucleotides to rZBP1 decreased beta-actin mRNA localization and impaired filopodial growth, whereas knockdown of rZBP1 did not impact other dendritically localized mRNA. Overexpression of EGFP/beta-actin resulted in growth of spine synapses in a zipcode dependent manner. These results suggest a novel mechanism underlying the regulation of actin dynamics and structure that involves the transport of a ZBP1-zipcode of beta-actin mRNP complex.