Fig. 3: Altered SCNN1D genes in Antilopinae.

A Mutation of the splice donor following exon 11 to ‘AT’ in Ovis aries (representative of the Antilopinae) causes fusion of exons 11 and 12 to a ‘super exon’. B Reverse transcriptase (RT)-PCR confirmed the exon fusion in cDNA derived from mRNA isolated from O. aries gastrointestinal tissues. Om. Omasum, Oes. Oesophagus, Ru. Rumen. Arrows highlight expected amplicon sizes based on absence or presence of the exon fusion (e.f.) or contamination with genomic DNA (gDNA). bp base pairs. M = DNA size marker. C Reverse transcriptase (RT)-PCR confirmed expression of SCNN1D in sheep tongue tissue. bp base pairs. M = DNA size marker. D Presence of the SCNN1D exon fusion in Bovidae. SCNN1D with exon fusion (turquoise squares) is exclusively found in representative species of the Antilopinae, but not in the Bovinae. MYA million years ago. E Partial δ-ENaC amino acid sequence alignment of representative species of the Bovidae. The sequence highlighted in blue indicates amino acids encoded by exons 11 and 12, the sequence highlighted in turquoise indicates additional amino acids due to incorporation of the former intron. F O. aries δ-ENaC structures predicted by ColabFold suggest location of the additional amino acids in the ‘knuckle’ domain. For clarity, predictions of the intracellular N-/C-termini are not displayed. Left: The predicted structures follow the canonical ENaC subunit architecture. Dark blue parts are derived from former exons 11 and 12, turquoise parts highlight additional ‘intron-derived’ amino acids. Right: Alignment of the O. aries δ-ENaC structure containing the exon fusion (turquoise) to a hypothetical O. aries δ-ENaC lacking the exon fusion (magenta). The exon fusion does not impair the general channel architectures but might alter the position of loops (highlighted by asterisks) between the ‘knuckle’ and ‘finger’ domains.