Turnover and lineage-specific broadening of the transcription start site in a testis-specific retrogene

Proteasomes are large multisubunit complexes responsible for regulated protein degradation. Made of a core particle (20S) and regulatory caps (19S), proteasomal proteins are encoded by at least 33 genes, of which 12 have been shown to have testis-specific isoforms in Drosophila melanogaster. Pros28.1A (also known as Prosalpha4T1), a young retroduplicate copy of Pros28.1 (also known as Prosalpha4), is one of these isoforms. It is present in the D. melanogaster subgroup and was previously shown to be testis-specific in D. melanogaster. Here, we show its testis-specific transcription in all D. melanogaster subgroup species. Due to this conserved pattern of expression in the species harboring this insertion, we initially expected that a regulatory region common to these species evolved prior to the speciation event. We determined that the region driving testis expression in D. melanogaster is not far from the coding region (within 272 bp upstream of the ATG). However, different Transcription Start Sites (TSSs) are used in D. melanogaster and D. simulans, and a "broad" transcription start site is used in D. yakuba. These results suggest one of the following scenarios: (1) there is a conserved motif in the 5' region of the gene that can be used as an upstream or downstream element or at different distance depending on the species; (2) different species evolved diverse regulatory sequences for the same pattern of expression (i.e., "TSS turnover"); or (3) the transcription start site can be broad or narrow depending on the species. This work reveals the difficulties of studying gene regulation in one species and extrapolating those findings to close relatives.