Examination of the function of two kelch proteins generated by stop codon suppression

Abstract

The Drosophila kelch gene produces a single transcript with a UGA stop codon separating two open reading frames (ORF1 and ORF2). From the transcript, 76 kDa ORF1 and 160 kDa full-length (ORF1 + ORF2) proteins are made. The expression of these two proteins is regulated in a tissue-specific manner causing the ratio of full-length to ORF1 protein to vary in different tissues. The only detected defect for kelch mutants is female sterility, and kelch protein is localized to the ovarian ring canals. kelch mutant ring canals are disorganized and have partly occluded lumens, causing a failure to transport cytoplasm. ORF1 and full-length kelch proteins co-sediment with ring canals suggesting that both proteins are found in the ring canals. Transgenetic analysis reveals that ORF1 kelch protein is sufficient to rescue ring canal morphology and fertility. In addition, we have mutated the UGA stop codon to a UAA stop codon and to three sense codons that allow constitutive readthrough. Analysis of these mutants reveals that a full-length kelch protein can partially compensate for the loss of endogenous kelch, but the residue included at the stop codon is critical for function. Finally, these studies suggest that the mechanism of stop codon suppression of kelch is by tRNA suppression.