Characterization of the Splicing Defect in <i>hif-1(nu469)</i>

<p>(A) Diagram of <i>hif-1</i> gene structure and the two mutations in <i>hif-1(nu469),</i> a previously uncharacterized lesion in the background of the KP3365 strain. This lesion consists of two closely linked mutations: (1) C→A at position 2315 of the coding sequence, resulting in a P771Q mutation, and (2) an insertion of TTATCA after position 2373. Sequencing cDNA from KP3365 revealed that these two mutations result in the inappropriate splicing of the <i>hif-1</i> transcript (indicated by a dashed line), removing 135 base pairs of the last exon. Location of primers for PCR are indicated by half-arrows above exons 9 and 11.</p> <p>(B) RT-PCR confirmed the altered splicing of the <i>hif-1(nu469)</i> mRNA.</p

Supplemental Material for Zhao, Hao, and Kaplan, 2018

Supplemental Figure

Expression of TOM-1, the <i>C. elegans</i> Ortholog of Tomosyn

<p>(A) Schematic of worm tomosyn indicating the location of the premature stop found in <i>nu468</i> and deletion in <i>ok285</i>.</p> <p>(B–D) Expression pattern of <i>tom-1</i> characterized with 4.2 kb of sequence upstream of the start codon driving expression of green fluorescent protein. Expression is seen in ventral cord motor neurons, with cell bodies indicated by arrowheads (B) and a number of neurons in the head (C) and the tail (D). Scale bars = 10 μm.</p

Analysis of mRNA Abundance in <i>mec-3(e1338)</i> Animals

<p>Fold-change (<i>x</i>-axis) is plotted against the statistical significance (<i>y</i>-axis) for each probeset. Fold-changes are shown on log₂ scale. <i>p-</i>Values are shown on a negative log₁₀ scale. The symbol × indicates genes with reduced expression in <i>mec-3(e1338)</i> animals (fold-change < −1, <i>p</i> < 0.01). Light blue circles indicate genes with reduced expression that are also on Chromosome 4. Dark blue circles indicate genes with reduced expression that are within 1 cM to the left or right of <i>mec-3</i>. The open red circle indicates the <i>mec-3</i> gene.</p

Analysis of mRNA Abundance in the KP3365 <i>unc-43(n1186)</i> CaMKII Strain

<p>Expression data are illustrated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010002#pgen-0010002-g002" target="_blank">Figure 2</a>. The symbol × indicates probesets with reduced expression in KP3365 animals (fold-change < −0.5, <i>p</i> < 0.01). Filled blue circles indicate probesets with reduced expression that are also on Chromosome 4. Open red circles indicate probesets corresponding to the <i>unc-43</i> CaMKII gene. The black square indicates the probeset corresponding to the <i>hif-1</i> gene<i>.</i></p

Aldicarb Sensitivity and Rescue of KP3293 <i>nu468</i>

<p>Levels of ACh secretion were assayed by following the time course of paralysis of animals on 1 mM aldicarb. Filled circle, wild-type; filled square, <i>nu468,</i> mutant recovered in our screen; filled triangle, <i>ok285</i> deletion allele of <i>tom-1;</i> open circle, <i>nu468</i> with a transgene expressing <i>tom-1</i> cDNA under the <i>unc-17</i> promoter. Data shown are averages from seven trials, except for <i>ok285,</i> which is an average of five trials. Error bars represent standard error.</p

Analysis of Nonsense Mutants in <i>Arabidopsis</i> and Mouse

<p>(A) PMR4 mutant; (B) MDX mutant. Expression data are illustrated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010002#pgen-0010002-g002" target="_blank">Figure 2</a>. The symbol × indicates probesets with reduced expression in the nonsense mutant (fold-change < −1.0, <i>p</i> < 0.01) for PMR4 and (fold-change < −1.5, <i>p</i> < 0.0001) for MDX. Numbers of genes with significantly reduced expression are indicated for both mutants. Filled blue circles indicate probesets with reduced expression that are on same chromosome as the mutant gene. The open red circle indicates the probeset corresponding to the mutant gene.</p

Fraction of <i>C. elegans</i> Alleles That Are Nonsense Mutations

<p>Molecular information about alleles was obtained from WormBase and literature searches. Graph includes 117 genes for which molecular characterization of three or more alleles was available (770 alleles total). Of these 117 alleles, 22 (19%) have no known nonsense mutations. Many of these no-nonsense alleles are in genes that are required for viability.</p

Positional Cloning of <i>tom-1(nu468)</i>

<p>Expression data are illustrated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010002#pgen-0010002-g002" target="_blank">Figure 2</a>. The symbol × indicates probesets with reduced expression in KP3293 <i>nu468</i> (fold-change < −0.5, <i>p</i> < 0.01). Filled blue circles indicate probesets with reduced expression in KP3293 <i>nu468</i> that are also on Chromosome 1. The open red circle indicates the probeset corresponding to <i>tom-1.</i> Sequencing of the <i>tom-1</i> gene in KP3293 <i>nu468</i> revealed a W212Stop mutation in the <i>tom-1</i> gene (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010002#pgen-0010002-g007" target="_blank">Figure 7</a>A).</p