# Oscillating Evolution of a Mammalian Locus with Overlapping Reading Frames: An XLαs/ALEX Relay

## TL;DR

This paper explores how two proteins from overlapping DNA sequences in mammals evolve together in a balanced way to maintain their function.

## Contribution

The study reveals the first example of oscillating evolution in a locus with overlapping reading frames encoding interacting proteins.

## Key findings

- The overlapping region of the XLαs/ALEX locus has high GC content and evolves rapidly.
- The proteins evolve in an oscillating pattern to maintain functional interactions despite high mutation rates.
- This mechanism may contribute to species-specific neurological differences.

## Abstract

XLαs and ALEX are structurally unrelated mammalian proteins translated from alternative overlapping reading frames of a single transcript. Not only are they encoded by the same locus, but a specific XLαs/ALEX interaction is essential for G-protein signaling in neuroendocrine cells. A disruption of this interaction leads to abnormal human phenotypes, including mental retardation and growth deficiency. The region of overlap between the two reading frames evolves at a remarkable speed: the divergence between human and mouse ALEX polypeptides makes them virtually unalignable. To trace the evolution of this puzzling locus, we sequenced it in apes, Old World monkeys, and a New World monkey. We show that the overlap between the two reading frames and the physical interaction between the two proteins force the locus to evolve in an unprecedented way. Namely, to maintain two overlapping protein-coding regions the locus is forced to have high GC content, which significantly elevates its intrinsic evolutionary rate. However, the two encoded proteins cannot afford to change too quickly relative to each other as this may impair their interaction and lead to severe physiological consequences. As a result XLαs and ALEX evolve in an oscillating fashion constantly balancing the rates of amino acid replacements. This is the first example of a rapidly evolving locus encoding interacting proteins via overlapping reading frames, with a possible link to the origin of species-specific neurological differences.

One of the possible ways to achieve tight co-expression of two proteins is to encode them within a single mRNA. The GNAS1 gene in mammals does just that: it encodes two interacting signaling polypeptides within a single transcript using nested reading frames shifted one nucleotide relative to each other. The exceptionally high GC content of the region where the two reading frames overlap diminishes the probability of encountering stop codons but makes the locus highly mutable. To preserve their ability to interact functionally with each other despite the high mutation rate, the two polypeptides appear to evolve in an oscillating fashion, trying to maintain approximately equal rates of amino acid substitutions. This unexpected observation provides new insights into the evolution of mostly overlooked overlapping coding regions in eukaryotic genomes.