Functionality of unspliced XBP1 is required to explain evolution of overlapping reading frames.

Eukaryotic genes with overlapping reading frames exemplify some of the most striking biological phenomena. Transcript of one such gene, the gene encoding X-box protein (XBP1), has evolved a mechanism for "on-demand" switching of translation between two overlapping reading frames. Despite the existence of this elaborate system, only one reading frame was believed to be functional. Here, we show that XBP1 evolves in a fashion that is only consistent with functionality of both reading frames. Our study provides a novel evolutionary framework for the analysis of loci with overlapping reading frames, which can be used for identification and analyses of novel dual-coding genes.