Evolution of the highly repetitive PEVK region of titin across mammals

The protein titin plays a key role in vertebrate muscle where it acts like a giant molecular spring. Despite its importance and conservation over vertebrate evolution, a lack of high quality annotations in non-model species makes comparative evolutionary studies of titin challenging. The PEVK region of titin—named for its high proportion of Pro-Glu-Val-Lys amino acids—is particularly difficult to annotate due to its abundance of alternatively spliced isoforms and short, highly repetitive exons. To understand PEVK evolution across mammals, we first developed a bioinformatics tool, PEVK_Finder, to annotate PEVK exons from genomic sequences of titin and then applied it to a diverse set of mammals. PEVK_Finder consistently outperforms standard annotation tools across a broad range of conditions and improves annotations of the PEVK region in non-model mammalian species. We find that the PEVK region can be divided into two subregions (PEVK-N, PEVK-C) with distinct patterns of evolutionary constraint and divergence. The bipartite nature of the PEVK region has implications for titin diversification. In the PEVK-N region, certain exons are conserved and may be essential, but natural selection also acts on particular codons. This region is also rich in glutamate and may contribute to actin binding. In the PEVK-C, exons are more homogenous and length variation of the PEVK region may provide the raw material for evolutionary adaptation in titin function. Taken together, we find that the very complexity that makes titin a challenge for annotation tools may also promote evolutionary adaptation.