This robustness reduces fitness losses due to mutation and mistranslation, which is believed to be a major force in coding sequence evolution [ 14].
Two of the five loci with major changes in protein coding sequence due to alternative splicing are predicted to have entirely different protein coding sequences; thus, these might actually represent misannotation of separate genes.
Sequence identity in major coding genes between our C. hongkongensis sequences and that of C. gigas is shown in Table 2. Considerable differentiation has occurred between the two sister-species at some genes (i.e., gene identity of 75.1% for nad2) despite the identical gene order.
As reported in that study, there are two major splice variants for the coding sequence of zebra finch FoxP2.
Previously evaluated was Gria4, encoding the ionotropic glutamate receptor GluR4 (also known as GLUR4 and GluR-D, the official protein symbol is now GRIA4), but neither coding sequence nor major gene expression differences were observed between HeJ and wild-type strains, provisionally excluding its candidacy (32).
In contrast, CAGE tags towards the 3'-end of a transcript exclude most or all of the coding sequence or the major part of a lncRNA, hence strongly suggesting a distinct transcript.
This is because the CAG repeat, which is transcribed into two different CACNA1A mRNA isoforms by an alternative splicing at the intron46/exon47 splice junction, is placed within a 3′-extended coding sequence of the major mRNA isoform, resulting in the CAG repeat being translated into a polyQ tract [ 1, 15, 65].
To confirm this, we directly sequenced PCR amplicons covering the major coding regions of ATPalpha from each mutant and two control strains.
The major fusion products are out-of-frame for mCherry coding sequence, which may explain why mCherry reporter expression is barely detected in dhx37 mutants and siblings.
