Isolation of intragenic suppressor of tsp-15-splicing mutant in Caenorhabditis elegans

TSP-15 is one of the 21 tetraspanins in Caenorhabditis elegans and is essential for exoskeletal (cuticle) development. A reduction in the function of TSP-15 results in a dumpy (Dpy) and/or blistered (Bli) phenotype (Moribe et al. 2004), and TSP-15 null mutants exhibit embryonic lethality (Moribe et al. 2012). sv15 is a hypomorphic allele of tsp-15, featuring a splicing error mutation in tsp-15’s fourth intron. The mutation results in the immediate production of a stop codon (Fig. 1A), and the resulting truncated form of TSP-15 protein has no function. sv15 mutants are viable despite severe cuticle deficiencies since some correctly spliced transcripts are produced (Moribe et al. 2004). To further understand the functions of TSP-15 in vivo, we screened genetic suppressor mutants of tsp-15(sv15). tsp-15(sv15) worms were mutagenized with 50 mM ethyl methanesulfonate (EMS) for 4 h and F2 progeny were screened for wild-type appearance. Nine suppressor alleles (im1 to im9) were isolated from a nonclonal screen from 15,000 haploid genomes. One dominant and eight recessive suppressor alleles were included among them. The alleles were mapped by single-nucleotide polymorphism (SNP) mapping using Hawaiian variant CB4856 and characterized by DNA sequence analysis. Mutations were confirmed by complementation test, rescue by DNA transformation, and RNAi analysis. We identified one dominant allele as described here and four of eight recessive alleles, which will be described elsewhere. The dominant allele im9 was revealed to feature an intragenic mutation in intron 4 of tsp-15 (Fig. 1A). sv15/sv15im9 showed the wild-type appearance with a very mild Dpy phenotype, and the appearance of sv15im9/sv15im9 homozygotes almost matched of the wild-type. im9 mutation abolished a cryptic splice donor site, which is used in sv15, suggesting that the original splice site was possibly reused in sv15im9 mutants. To test this hypothesis, we performed quantitative RT-PCR (qRT-PCR). To detect the correct tsp-15 transcript, we designed a TaqMan MGB probe specific for the border between exons 4 and 5 (Fig. 1B). The tsp-15 primer set and TaqMan probe were designed by Primer Express software (Applied Biosystems). Total RNA and first-strand cDNA were prepared as described previously (Moribe et al. 2004). TaqMan real-time PCR was performed and analyzed using the ABI 7500 system (Applied Biosystems). RNA polymerase II subunit ama-1 was used as an internal control to normalize the tsp-15 relative