Mutational analysis of the Caenorhabditis elegans ankyrin gene unc-44 demonstrates that the large spliceoform is critical for neural development

In Caenorhabditis elegans, unc-44 mutations affect axonal outgrowth and guidance, leading to locomotory defects. The wild-type unc-44 gene encodes a family of ankyrin proteins, which, in addition to the conventional ankyrins, includes a novel ankyrin isoform with an extended C-terminal domain, referred to AO13 ankyrin. Six spontaneous unc-44 mutations and their reversions were analyzed in order to localize regions critical for gene function. The q331::Tc1 and rh1013::Tc1 mutations were mapped to the portion of the gene encoding the conventional ankyrins, mn339 had an uncharacterized 2-kb insertion in the serine/threonine/glutamic acid/proline-rich (STEP) repeat block 5, st200::Tc5(variant) and rh1042::Tc1 were localized near the C-terminus, and mn259 resulted from two Tc1 insertions, one in STEP block 6 and the other near the C-terminus. Tc1 excisions in several revertants resulted either in the restoration of the wild-type sequence, or were associated with small in-frame deletions or insertions. Reversion of mn339 resulted in the net excision of 2463 bp of genomic DNA, including the region encoding parts of STEP blocks 5 and 6 and the intervening hydrophobic region. Interestingly, additional Tc1 insertions at a 5′ exon/intron boundary were found in revertants of st200 and rh1042. Reversion of the st200::Tc5 mutation resulted in excision of the Tc5 element, and the insertion of two copies of Tc1 at different sites. The wild-type unc-44 gene produces multiple transcripts – shorter RNAs determined to be approximately 1, 3.2, 5, 6, and 7 kb long, and two large transcripts estimated to be 22 and 26 kb in length. The largest transcripts were affected by all unc-44 mutations and are proposed to be essential for axonal outgrowth and guidance.