Fig. 2. The panm313 mutation causes mis-splicing of spt6 mRNA and truncation of Spt6 protein. (A) spt6 cDNA from pan mutants at 30 hpf contains a 92 nucleotide insertion. PCR primers that flank an 808 bp fragment of wild-type spt6 cDNA amplify a 900 bp fragment from pan mutant cDNA. This larger fragment represents the major splice isoform of spt6 in pan mutants, although we have infrequently detected trace amounts of normally spliced cDNA that could represent a low level of maintained maternal mRNA or a low level of normal splicing of zygotic mRNA. (B) Comparison of wild-type and pan mutant spt6 cDNA. Labeled regions in the wild-type ORF are proposed to encode discrete protein domains (see D below). The 92 nucleotide insertion in pan mutant cDNA is indicated by a loop, and the location of the in-frame stop codon is marked with an asterisk. (C) Genomic sequence of exon-intron boundaries flanking the mutated intron. The insertion in pan mutant cDNA corresponds to a 92 bp intron. pan genomic DNA contains a point mutation (boxed) in the second nucleotide of this intron. This mutation prevents correct splicing at this junction in pan mutants. The in-frame stop codon is underlined and marked with an asterisk. (D) Predicted protein structure for wild-type Spt6 protein (1726 amino acids) and truncated pan mutant Spt6 protein (829 amino acids). The acidic region, S1 RNA binding domain and SH2 domain are indicated. The insertion of intronic sequence in pan mutant cDNA is predicted to result in the addition of nine mis-sense amino acids before reaching a premature in-frame stop codon. Therefore, the pan mutant Spt6 protein would lack both the S1 RNA binding domain and SH2 domain.
