Moonlighting genes harbor antisense ORFs that encode potential membrane proteins.

Moonlighting genes encode for single polypeptide molecules that perform multiple and often unrelated functions. These genes occur across all domains of life. Their ubiquity and functional diversity raise many questions as to their origins, evolution, and role in the cell cycle. In this study, we present a simple bioinformatics probe that allows us to rank genes by antisense translation potential, and we show that this probe enriches, reliably, for moonlighting genes across a variety of organisms. We find that moonlighting genes harbor putative antisense open reading frames (ORFs) rich in codons for non-polar amino acids. We also find that moonlighting genes tend to co-locate with genes involved in cell wall, cell membrane, or cell envelope production. On the basis of this and other findings, we offer a model in which we propose that moonlighting gene products are likely to escape the cell through gaps in the cell wall and membrane, at wall/membrane construction sites; and we propose that antisense ORFs produce "membrane-sticky" protein products, effectively binding moonlighting-gene DNA to the cell membrane in porous areas where intensive cell-wall/cell-membrane construction is underway. This leads to high potential for escape of moonlighting proteins to the cell surface. Evolutionary and other implications of these findings are discussed.