Characterization of ZBP1‐βactin mRNA complex

Abstract

Zipcode binding protein 1 (ZBP1) belongs to a family of highly conserved RNA‐binding proteins that have been linked to several steps in the post‐transcriptional regulation of gene expression including mRNA stability, localization and translation. This family of proteins functions during embryogenesis and is often found over‐expressed in a variety of cancers. ZBP1 was first identified by its ability to promote the localization of beta‐actin mRNA to the leading edge of chicken embryo fibroblasts through its interaction with cis‐acting elements contained within the beta‐actin mRNA.ZBP1 contains two RNA recognition motifs (RRM) followed by four hnRNP K homology (KH) domains. We have identified a fragment of ZBP1, which contains both KH3 and KH4, that binds to a 28 nucleotide region of the beta‐actin mRNA with high affinity. KH3 and KH4 function together as one domain and bind RNA as a monomer. Mutation of the amino acids that link KH3 and KH4 reduces binding affinity by greater than 100‐fold suggesting that the orientation of the RNA binding surfaces is important for RNA recognition.While ZBP1 KH34 has remained recalcitrant towards crystallization, we have recently obtained crystals of IMP2 KH34, a paralog of ZBP1 with 82% sequence identity, that diffract to 2Å resolution. A molecular replacement solution has not yet been identified, so experimental phases are being determined.