Isolation of a Hypomorphic skn-1 Allele That Does Not Require a Balancer for Maintenance

Abstract

In Caenorhabditis elegans, the transcription factor SKN-1 has emerged as a central coordinator of stress responses and longevity, increasing the need for genetic tools to study its regulation and function. However, current loss-of-function alleles cause fully penetrant maternal effect embryonic lethality, and must be maintained with genetic balancers that require careful monitoring and labor intensive strategies to obtain large populations. In this study, we identified a strong, but viable skn-1 hypomorphic allele skn-1(zj15) from a genetic screen for suppressors of wdr-23, a direct regulator of the transcription factor. skn-1(zj15) is a point mutation in an intron that causes mis-splicing of a fraction of mRNA, and strongly reduces wildtype mRNA levels of the two long skn-1a/c variants. The skn-1(zj15) allele reduces detoxification gene expression and stress resistance to levels comparable to skn-1 RNAi, but, unlike RNAi, it is not restricted from some tissues. We also show that skn-1(zj15) is epistatic to canonical upstream regulators, demonstrating its utility for genetic analysis of skn-1 function and regulation in cases where large numbers of worms are needed, a balancer is problematic, diet is varied, or RNAi cannot be used.

Keywords: genetic screen; mutant; resource.

Figure 1

sow-1(zj15) maps to an intron of skn-1. (A) Schematic diagram of three skn-1 splice variant gene models; the mutated base is shown relative to exon 4 of skn-1a. (B) Direct RT-PCR product sequence results (top) and predicted proteins (bottom). sow-1(zj15) is an AT–GC mutation in an intron that is specific to skn-1a and c. Sequencing of cDNA reveals that a fraction of skn-1a/c transcripts have a splicing error that introduces an extra 5 bp (GCATG) from the beginning of this intron. The consequence of this spicing error is the addition of two amino acids and a stop codon that removes the entire c-terminus and DNA binding domain. (C) mRNA levels of skn-1b and skn-1a/c transcripts (mean ± SE, n = 5 replicates of worms, ***P < 0.001 relative to wildtype). (D) Rescuing effects of skn-1 gDNA on Pgst-4::GFP expression during exposure to 2.8 mM acrylamide [n = 29–78 total worms, ***P < 0.001 relative to sow-1(zj15)]; given full rescue, we conclude that sow-1 is skn-1, and refer to the allele as skn-1(zj15) from this point forward.

Figure 2

skn-1(zj15) worms are viable. (A) Developmental stages of wildtype and skn-1(zj15) worms grown from synchronized L1 for 2 d (n = 193–303 total worms, ***P < 0.001 relative to wildtype). (B) Body length of worms at the young adult stage (n = 30–37 total worms, ***P < 0.001 relative to wildtype). (C) Numbers of total eggs and hatched eggs produced from individual hermaphrodites [mean ± SE, n = 6–12 total worms, ***P < 0.001 relative to wildtype, ^###P < 0.001 relative to skn-1(zj15)].

Figure 3

skn-1(zj15) is functionally comparable to skn-1 RNAi. (A) Relative mRNA levels under basal conditions (mean ± SE, n = 5 replicates of worms, ***P < 0.001, **P < 0.01, *P < 0.05 relative to control RNAi). (B) Relative mRNA levels after exposure to 5 mM As for 1 hr [mean ± SE, n = 4–5 replicates of worms, ***P < 0.001 relative to control RNAi, and ^#P < 0.05 relative to skn-1(zj15)]. (C) Survival of 5 mM arsenite [n = 242–295 total worms from three trials, P < 0.0001 for control RNAi relative to skn-1 RNAi or skn-1(zj15), P = 0.7716 for skn-1 RNAi vs. skn-1(zj15), P = 0.0005 for skn-1(zj15) vs. skn-1(zj15); skn-1 RNAi, P < 0.0125 was taken to indicate statistical significance]. (D) Survival of 175 µM juglone [n = 252–322 total worms from three trials, P < 0.0001 for control RNAi relative to skn-1 RNAi or skn-1(zj15), P = 0.4792 for skn-1 RNAi vs. skn-1(zj15), P = 0.2967 for skn-1(zj15) vs. skn-1(zj15); skn-1 RNAi, P < 0.0125 was taken to indicate statistical significance]. (E) Lifespan analysis [n = 290–487 total worms from three trials, P < 0.0001 for control RNAi relative to skn-1 RNAi or skn-1(zj15), P < 0.0001 for skn-1 RNAi vs. skn-1(zj15), P = 0.0001 for skn-1(zj15) vs. skn-1(zj15); skn-1 RNAi, P < 0.0125 was taken to indicate statistical significance]. Note that the survival trials in Figure 3, C, D, and E were run with those in Figure 5, C, D, and E, respectively, and that the control curves are the same.

Figure 4

skn-1(zj15) suppressed gst-4 transcription in tissues that are resistant to RNAi. All panels are micrographs of Pgst-4::GFP fluorescence in at least seven worms. (A–D) Worms of the indicated genotypes were constitutively grown on plates containing 2.8 mM acrylamide for 48 hr. (E–H) Worms of the indicated genotypes were imaged.

Figure 5

skn-1(zj15) is a tool for epistasis. (A) skn-1(zj15) suppressed Pgst-4::GFP induction by loss of known inhibitory signals upstream of SKN-1. ***P < 0.001 relative to wildtype. (B) RNAi phenotype scoring for sterility (wdr-46 and plc-3) and dumpy (dpy-5 and dpy-7) in skn-1(zj15) worms. (C) Survival of 5 mM arsenite [n = 214–251 total worms from three trials, P < 0.001 for control RNAi relative to wdr-23 RNAi or skn-1(zj15), P < 0.001 for wdr-23 RNAi vs. skn-1(zj15); wdr-23 RNAi, P = 0.0654 for skn-1(zj15) vs. skn-1(zj15); wdr-23 RNAi, P < 0.0125 was taken to indicate statistical significance]. (D) Survival of 175 µM juglone [n = 252–353 total worms from three trials, P < 0.001 for control RNAi relative to wdr-23 RNAi or skn-1(zj15), P < 0.001 for wdr-23 RNAi vs. skn-1(zj15); wdr-23 RNAi, P = 0.490 for skn-1(zj15) vs. skn-1(zj15); wdr-23 RNAi, P < 0.0125 was taken to indicate statistical significance]. (E) Lifespan analysis [n = 278–462 total worms from three trials, P < 0.001 for control RNAi relative to wdr-23 RNAi or skn-1(zj15), P < 0.001 for wdr-23 RNAi vs. skn-1(zj15); wdr-23 RNAi, P = 0.7337 for skn-1(zj15) vs. skn-1(zj15); wdr-23 RNAi, P < 0.0125 was taken to indicate statistical significance]. Note that the survival trials in Figure 3, C, D, and E were run with those in Figure 5, C, D, and E, respectively, and that the control curves are the same.