Fig. 1.

Genome editing generates likely null alleles of zebrafish larp6a and larp6b. (A) In situ RNA hybridisation for larp6a and larp6b at the indicated stages. (B) Schematic of larp6a and larp6b genes and proteins showing the position of kg139 and kg153 mutant alleles. The larp6a^kg139 frameshift mutation produces a truncated protein with the first 29 amino acids of Larp6a followed by a 72 amino acid tail lacking both La motif and RRM domains. The larp6b^kg153 frameshift allele truncates Larp6b at amino acid 17 with an eight amino acid tail terminating in coding exon 1. There is a lack of in-frame ATG codons near the termination site. (C) In situ RNA hybridization for larp6a mRNA on genotyped larp6a^kg139 mutant and wild-type siblings from a larp6a^kg139/+ incross reveals nonsense-mediated mRNA decay (NMD) of mutant larp6a^kg139 mRNA at 24 hpf. Eleven out of 47 low expressors were shown to be mutant and 10/47 normal expressors were wild type upon sequence genotyping. As larp6b mRNA is primarily maternally expressed, NMD was analysed at the 256-cell stage by larp6b mRNA in situ RNA hybridization on lays from incrosses of larp6b^kg153 F3 mutants or their wild-type siblings (see Table S1). The results shown were observed in 33/33 embryos from a wild-type female and 30/30 from a mutant female. Genotypes of mutant embryos shown were confirmed by sequencing. (D) QRT-PCR on RNA from 1k stage embryos from wild-type or larp6a^kg139;larp6b^kg153 double mutant incrosses confirmed reduction of each mutant mRNA. Symbols indicate results from three individual RNA preparations from three separate pairs of lays. (E) Lack of larp6b mRNA upregulation in larp6a^kg139 mutants. All images are lateral views with anterior to the left and dorsal upwards, except 128 cell and 50% epiboly, which are animal upward. Scale bars: 200 µm.