The genome of D. melanogaster encodes three homologs of the Na+/K+ ATPase alpha subunit, as described in Roy et al., 2013 (FBrf0220434): ATPalpha (FBgn0002921), JYalpha (FBgn0040037) and CG3701 (FBgn0034732). In this FlyBase analysis, evidence for the CG3701 annotation is reviewed. We conclude that within the Sophophora subgenus, the ATPalpha homolog CG3701 has been split into two distinct genes, designated as CG45062 and CG45063.

A comparison of the genomic sequences for CG3701 among various Drosophila species reveals the presence of a genomic insertion exclusively within the Sophophora subgenus (e.g., D. melanogaster), but not in the Drosophila subgenus (e.g., D. virilis). This genomic insertion falls within the predicted E1-E2 ATPase domain encoded by the CG3701 annotation. This genomic insertion is spanned by the second intron in the Dmel\CG3701 annotation and orthologous annotations in other species of the Sophophora subgenus. However, no independent support of this junction could be found from a review of extensive RNA-Seq coverage data (FBrf0213330, FBrf0213503), two independent RNA-Seq exon junction datasets (FBrf0212682, FBrf0213064), EST or cDNA data. All other junctions annotated for CG3701 have extensive support corroborated by independent sources of evidence.

In the absence of support for a junction bridging the Sophophora-specific genomic insertion, alternative gene models were considered. Analysis of RNA-Seq coverage data, transcription start site data (FBrf0220331), EST and cDNA data supported the existence of an upstream transcriptional stop and nearby downstream transcriptional start in the Sophophora-specific genomic insertion of D. melanogaster. This suggests that the ATPalpha homolog (represented by the CG3701 annotation) actually exists as two partial, independent transcriptional units, designated here as CG45062 and CG45063.

The CG45063 gene (FBgn0266433) corresponds to the first two upstream exons of the now obsolete CG3701 annotation. The 5' transcription start site, exon structure and 3' polyadenylation site of the transcript are supported by RNA-Seq coverage, exon junction, EST and cDNA data. In further support of the CG45063 annotation, the novel C-terminus (EYIKD), which is specific to the new annotation and extends past the ATPalpha homologous sequence, is conserved in many species of the Sophophora subgenus (FBrf0202906 and BLAST analysis).

The CG45062 gene (FBgn0266432) corresponds to the seven downstream exons of the now obsolete CG3701 annotation. Importantly, the proposed 5' transcription start site is supported by CAGE data (FBrf0220331). Furthermore, the novel N-terminus of the new CG45062 polypeptide (MSSKTLSKWSSSN), which extends beyond the region of ATPalpha homology, is conserved in all species of the Sophophora subgenus (BLAST analysis). RNA-Seq coverage, exon junction and EST data support all other aspects of the CG45062 transcript structure.

To summarize, there is a genomic insertion within an ATPalpha homolog (CG3701) that is specific to the Sophophora subgenus. In D. melanogaster, there is no evidence in support of a junction that would bridge this insertion to preserve the nearby ATPalpha homologous sequences in a single transcriptional unit. Instead, various types of evidence, including transcriptome data and protein conservation analysis, support the existence of two distinct genes representing different portions of the ATPalpha homologous sequences. As such, the CG3701 annotation has been split into CG45062 and CG45063.