Figure 1.

Over-expression of reporter mRNA containing the 38bp COXIV zipcode inhibits the axonal localization and mitochondrial targeting of nuclear-encoded mitochondrial mRNAs. (A) Schematic diagram showing the compartmented Campenot cell culture chambers (upper panel). Lower panel shows a single track in the bottom of the culture dish indicating the location of cell-bodies and proximal axons in the center compartment and distal axons in the side compartments. (B) Overview of constructs used in this study. Schematic representation of reporter gene plasmids carrying the dGFP cDNA followed by either the final 38bp or 22bp COXIV 3′UTR (dGFP+3′UTR [38bp] and dGFP+3′UTR [22bp]) or mCherry cDNA followed by final 38bp of COXIV 3′UTR(mCherry+3′UTR [38bp]). (C) Representative DIC and fluorescence images showing expression of dGFP constructs in the transfected cell-bodies and distal axons. The arrow shows reporter gene expression in axons of cell-bodies transfected with the chimeric construct that contained the 38bp zipcode. No reporter gene expression was observed in the distal axons of neurons transfected with either a plasmid containing the GFP alone or the dGFP+3′UTR[22bp] constructs. (D) Levels of γ-actin mRNA was used to assess the purity of the RNA prepared from the distal axons located in the side compartments of the Campenot cell culture chambers. (E) Quantitation of dGFP mRNA levels in distal axons of SCG neurons were determined by qRT-PCR 24 h after transfection with plasmid DNA. The dGFP mRNA levels were expressed relative to βactin transcript. (F) SCG neurons (6DIV) were transfected with dGFP constructs and total RNA was prepared from a mitochondrial fraction isolated from distal axons using a mitochondrial isolation kit (Pierce). Quantitation of dGFP mRNA levels from total mitochondrial RNA was determined by qRT-PCR 24 h after DNA transfection. The dGFP mRNA levels were expressed relative to COXII mRNA. (G) Quantitation of COXIV mRNA levels in transfected cell-bodies and distal axons of SCG neurons was determined by qRT-PCR 24 h after DNA transfection. COXIV mRNA levels are expressed relative to β-actin mRNA. (H) Quantitation of COXIV mRNA levels from total mitochondrial mRNA was determined by qRT-PCR 24 h after DNA transfection. COXIV mRNA levels are expressed relative to COXII mRNA. (I) mCherry constructs containing the 38bp COXIV zipcode or 22bp COXIV zipcode or mCherry alone were transfected into 6DIV SCG neurons. After transfection (24 h) COXIV mRNA levels were assessed by qRT-PCR. COXIV mRNA levels were expressed relative to β-actin mRNAs. Error bars represent SEM for n=3 samples. Two-way ANOVA, Posthoc Tukey’s test *,p<0.05, **, p<0.01.