Identification of ZBP2 and characterization of its role in beta-actin RNA localization

Abstract

mRNA localization is an evolutionally conserved way to establish cell asymmetry by spatially restricting translation. The localization of beta-actin mRNA to the leading lamellae of chicken fibroblasts and growth cones of developing neurons requires a 54-nt localization signal (zipcode) within the 3 ' untranslated region. This process is important to maintain cell polarity and neurite outgrowth. Zipcode binding protein 1 (ZBP1) was previously identified in our lab which binds to the zipcode and regulates beta-actin mRNA localization. Four other proteins, in addition to ZBP1, were isolated by binding to the zipcode using RNA affinity chromatography. One of them, a 92-kD protein, was abundant in extracts from embryonic brain. We identified this protein as ZBP2 and showed that it is a homologue of the human hnRNP protein, KSRP, which is proposed to mediate pre-mRNA splicing. ZBP2 contains four hnRNP K homology domains (KH domains) as well as a C terminal protein-protein interaction domain. The subcellular localization of various portions of ZBP2 fused to GFP as well as heterokaryon assays of ZBP2 indicated that the protein was a predominantly nuclear protein but shuttles between the nucleus and the cytoplasm very rapidly. Expression of a truncated ZBP2 fragment including the four KH domains inhibited the localization of beta-actin mRNA in both fibroblasts and neurons. Although ZBP1 also colocalized with beta-actin mRNA in the nucleus, it was predominantly a cytoplasmic protein. ZBP2, on the contrary, was predominantly nuclear. A question was whether ZBP1 and ZBP2 cooperated to localize beta-actin mRNA and if they did, where and how that happened. We demonstrated that ZBP2 facilitated the binding of ZBP1 to nascent beta-actin transcripts. This occurred presumably by the stabilization of the beta-actin RNA secondary structure. ZBP2 was the first protein to recognize the transcripts, followed by ZBP1, which appeared to replace ZBP2 and continued to be bound to the RNA through out nuclear export and localization. ZBP2 family proteins are involved in various steps of RNA dynamics including transcription, splicing, editing and stability control. ZBP2 interacts with cytoplasmic and nuclear proteins including mDia1, the mammalian homologue of the yeast protein Bni1p, hnRNP D and SMN. These data suggest that ZBP2 may regulate mRNA by facilitating the formation of a mRNP in the nucleus that moves the RNA for localization in the cytoplasm.