Molecular Identification of daf-25 To identify the daf-25 genetic locus, we first used three-factor genetic crosses to map the m362 allele to the left arm of Chromosome I. Then, we employed a modified SNP mapping procedure [30], in which we selected for recombinants in the unc- 11-daf-25 interval to map daf-25 to the left-most 1 Mbp of Chromosome I. Finally, we used a custom-made high-density array for the left-most ,2.5 Mbp for comparative genomic hybridization (CGH). Two molecular lesions in daf-25(m362) were identified in exon 4 of Y48G1A.3 (Figure 2), including a 31 bp deletion and a G.A change 72 bp to the right of the deletion. Subsequent sequencing of PCR products from mutant genomic DNA uncovered the lesions in the remaining alleles. The m98 mutant has a 996 bp deletion that removes the first two exons, m137 has an ochre stop in the fourth exon, and m488 has a Tc1 transposon insertion in the third exon (Figure 2). Y48G1A.3 encodes the C. elegans ortholog of mammalian Ankmy2 (by reciprocal BLAST), a protein with three ankyrin repeats and a MYND-type zinc finger domain. The C. elegans protein shares throughout its length (388 residues) 52% similarity and 32% identity with mouse Ankmy2 (440 residues). The C. elegans ankyrin repeat domain is 40% identical and the MYND domain is 55% identical to the murine ortholog. Ankmy2 is very well conserved among chordates, with identity percentages compared to human Ankmy2 of 99% for macaque, 93% for cow, 88% for mouse, and 76% for zebrafish (Figure S4). Although the protein is highly conserved, there is no reported functional data for this gene from any organism. The MYND domain is thought to function in protein-protein interactions, although only a small number of MYND domain-containing proteins have been characterized, including the AML1/ETO protein, which binds SMRT/N-CoR through its MYND domain [31]. To analyze the transcript(s) generated by the daf-25 gene, we employed a PCR-based approach. Using primers for the SL1 transplice sequence or poly-T in combination with gene-specific DAF-25/Ankmy2 Functions in Ciliary cGMP Signaling primers, we were able to amplify only one isoform (Figure S5). This result is consistent with the RNA-Seq and trans-splice data found on Wormbase, which shows a daf-25 transcript sequence identical to that presented in Figure S5, including the 59 and 39 UTRs [32–34]. We were unable to amplify an SL2 trans-spliced product using multiple gene specific primers and an SL2 primer under any conditions tested. DAF-25 Functions in Cilia To determine the sub-cellular localization of DAF-25, a GFP- tagged protein was constructed. The daf-25 upstream promoter (approx. 2.0 kb 59 of the ATG) was fused to the daf-25 cDNA in frame into the pPD95.77 vector (gift from Dr. Andrew Fire) containing GFP (without a nuclear localization signal) and the unc- 54 39UTR. This construct was found to be expressed in many ciliated sensory neurons, including the following pairs of anterior neurons: AFD, ASK, ASI, ASH, ASJ, ASG, ASE, ADF, AWA, AWB, AWC and IL2 (Figure 3). It is also expressed in the PQR ciliated neuron and one ventral interneuron. We also show expression of the DAF-25::GFP construct in the 7A ciliated neuron in the male tail though we did not fully examine male expression due to the limited number examined and the mosaic expression associated with extra-chromosomal arrays. Most importantly, the fluorescence of the GFP-tagged protein was localized to the cilia of all these cells. The GFP-fusion construct was judged to be functional because it fully rescued the Daf-c phenotype of daf-25(m362) at 25uC while non-transgenic siblings arrested as dauers (n.200). To investigate whether the ciliary localization of DAF-25 might depend on the intraflagellar transport (IFT) machinery, the DAF- 25::GFP construct was crossed into che-11, which is required for retrograde transport in the cilia. In che-11 mutants, IFT-associated proteins accumulate in the cilia [35]. The DAF-25::GFP translational fusion protein accumulated within the cilia and basal body (base of cilia) despite a reduction in total GFP fluorescence Figure 2. daf-25 alleles encode the ortholog of mammalian Ankmy2. We identified the daf-25 gene using three-factor genetic crosses and SNP mapping followed by ArrayCGH. The four alleles of daf-25 include two EMS-induced deletions m98 (996 bp deletion at I:332481-333477) and m362 (31 bp deletion at I:335814-335844 which results in a premature stop 14 codons downstream), a transposon (Tc1) insertion at I:330927 (m488) and an EMS-induced ochre nonsense mutation m137 (at I:336013). DAF-25 is well conserved and has been named Ankmy2 in mammals for its three ankyrin repeats and MYND-type zinc finger domain. doi:10.1371/journal.pgen.1001199.g002 PLoS Genetics | www.plosgenetics.org 4 November 2010 | Volume 6 | Issue 11 | e1001199