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TMEM16 and TMEM63/OSCA proteins share a conserved potential to permeate ions and phospholipids

• Authors : Lowry, Augustus J.; Liang, Pengfei; Serena Wan, Y.C.

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Author response: TMEM16 and TMEM63/OSCA proteins share a conserved potential to permeate ions and phospholipids

• Authors : Lowry, Augustus J.; Liang, Pengfei; Serena Wan, Y.C.

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TMEM16 and TMEM63/OSCA proteins share a conserved potential to permeate ions and phospholipids

• Authors : Lowry, Augustus J.; Liang, Pengfei; Serena Wan, Y.C.

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Figure 1—figure supplement 2. Alignment of the 5’ portions of the cw9 gene of CBS5557 and the wtf9 gene of the reference genome. ; The sequences were aligned using MAFFT via Jalview (Katoh et al., 2002; Waterhouse et al., 2009). Identical bases are indicated by blue background. Red and green boxes denote the first and second predicted protein coding exons, respectively. Predicted start codons are highlighted with red letters. Gene structure of cw9 was predicted using the AUGUSTUS web server (Stanke et al., 2008).

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Figure 2—figure supplement 1. Knockdown efficiency after RNAi with Pt-Ets4. ; In comparison to the control embryo (A–A’’) the expression of Pt-Ets4 is strongly silenced after Pt-Ets4 RNAi (B–B’’). Nascent Pt-Ets4 transcripts are still detectable in the nuclei of the primary thickening. The boxed region in A and B is magnified in A’’ and B’’. (C) Schematic representation of the AUGUSTUS prediction for Pt-Ets4 including the location of the untranslated regions (5’ and 3’ UTR), the coding sequence (CDS) and the primers (T7-Pt-CDS-Ets4-Fw (Fw1), T7-Pt-CDS-Ets4-Rev (Rev1), T7-Pt-3’Ets4-Fw (Fw2), T7-Pt-3’Ets4-Rev (Rev2), see Material and methods) that were used to generate two non-overlapping fragments of Pt-Ets4 that were used in the RNAi experiments (see Material and methods). (D–F) Statistical analysis of the knockdown efficiency after pRNAi with the CDS fragment and the 3’UTR fragment of Pt-Ets4. As many embryos were able to completely recover from the Pt-Ets4 knockdown at later stages of development (see embryo shown in G) the embryos were analyzed for their phenotypes at embryonic stages 6 and 7. (G) Stills from a time-lapse imaging experiment showing a strongly affected Pt-Ets4 RNAi embryo at developmental stages 6 and 7. The tube-like structure elongates from the posterior, folds back onto the yolk and re-establishes a DV axis during stages 8 and 9. The embryo has fully recovered from the early DV phenotype at stage 9 of development. This demonstrates the regulatory capacities of spider embryos.

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Figure 1. The identification of cw9 and cw27 genes as spore killers. ; (A) NGS-assisted bulk segregant analysis of viable progenies from a cross between DY9974, a heterothallic derivative of CBS5557, and DY8531, a laboratory strain harboring the 2.23 Mb chromosome I inversion (Hu et al., 2015). Reference allele frequencies at SNP positions are plotted as dots. Red trend lines are based on a rolling median calculation. Two vertical dashed lines denote the positions of cw9 and cw27 genes on chromosome III. Black triangles mark the positions of centromeres. (B and C) Bulk segregant analysis of viable progenies from crosses between a laboratory strain and the backcrossed-1 strain, and between a laboratory strain and the backcrossed-2 strain, respectively. Blue and orange colored segments in the diagrams and on the X-axes of the plots denote chromosome III regions with reference genome sequence and those with CBS5557 genome sequence, respectively, in the two backcrossed strains. (D and E) Schematics of the regions surrounding cw9 and cw27 in the CBS5557 genome and the corresponding regions in the reference genome. Gene structures of cw9 and cw27 were predicted using the AUGUSTUS web server (Stanke et al., 2008). Solo LTRs were annotated based on BLAST analysis.

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Figure 4. Workflows of assembly, annotation, and proteome generation. ; (A) Flowchart of the genome assembly. Two shotgun libraries and four mate-pair libraries with the indicated average sizes were prepared from a single male animal and sequenced to a predicted depth of 115x coverage after read filtering, based on a predicted size of 3.6 Gbp. Contigs were assembled at two different k-lengths with Abyss and the two assemblies were merged with GAM-NGS. Filtered contigs were scaffolded with SSPACE. (B) The final scaffolded assembly was annotated with a combination of Evidence Modeler to generate 847 high quality gene models and Augustus for the final set of 28,155 predictions. These protein-coding gene models were generated based on a Parhyale transcriptome consolidated from multiple developmental stages and conditions, their homology to the species indicated, and ab initio predictions with GeneMark and SNAP. (C) The Parhyale proteome contains 28,666 entries based on the consolidated transcriptome and gene predictions. The transcriptome contains 292,924 coding and non-coding RNAs, 96% of which could be mapped to the assembled genome.

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