Wilburn, D. B. | Search bioRxiv

bioRxiv2018

An annual cycle of gene regulation in the red-legged salamander mental gland: from hypertrophy to expression of rapidly evolving pheromones

Cell differentiation is mediated by synchronized waves of coordinated expression for hundreds to thousands of genes, and must be an exquisitely regulated process to produce complex tissues and phenotypes. For many animal species, sexual selection has driven the development of elaborate male ornaments, requiring sex-specific differentiation pathways. One such male ornament is the pheromone-producing mental gland of the red-legged salamander (Plethodon shermani). Mental gland development follows an annual cycle of extreme hypertrophy, production of pheromones for the ~2 month mating season, and then complete resorption before repeating the process in the following year. At the peak of the mating season, the transcriptional and translational machinery of the mental gland are almost exclusively redirected to synthesis of many rapidly evolving pheromones. Of these pheromones, Plethodontid Modulating Factor (PMF) has experienced an unusual history of disjunctive evolution: following gene duplication, positive sexual selection has diversified the protein coding region while the untranslated regions have been conserved by purifying selection. However, the molecular underpinnings that bridge the processes of gland hypertrophy, pheromone synthesis, and disjunctive evolution remain to be determined and are the focus of the present investigation. Using Illumina sequencing, we prepared a de novo transcriptome of the mental gland at six stages of development. Differential expression analysis and immunohistochemistry revealed that the mental gland initially adopts a highly proliferative, almost tumor-like phenotype, followed by a rapid increase in pheromone mRNA and protein levels. One likely player in this transition is Cold Inducible RNA Binding Protein (CIRBP), which selectively and cooperatively binds the highly conserved PMF 3' UTR. CIRBP, along with other stress response proteins, have seemingly been co-opted to aid in mental gland development by helping to regulate pheromone synthesis. The P. shermani mental gland utilizes a complex system of transcriptional and post-transcriptional gene regulation to facilitate its hypertrophication and pheromone synthesis. The data support the evolutionary interplay of both coding and noncoding segments in rapid gene evolution, and necessitate study of the co-evolution of pheromone gene products along with their transcriptional and translational regulators. Additionally, the mental gland could be a powerful emerging model of regulated proliferation and subsequent resorption of a tissue, within the dermis, thus having potential links to skin cancer biology.

molecular biology↗

bioRxiv2017

Solution structure of sperm lysin yields novel insights into molecular dynamics of rapid protein evolution

Protein evolution is driven by the sum of different physiochemical and genetic processes that usually results in strong purifying selection to maintain biochemical functions. However, proteins that are part of systems under arms race dynamics often evolve at unparalleled rates that can produce atypical biochemical properties. In the marine mollusk abalone, lysin and VERL are a pair of rapidly coevolving proteins that are essential for species-specific interactions between sperm and egg. Despite extensive biochemical characterization of lysin, including crystal structures of multiple orthologs, it was unclear how sites under positive selection may facilitate recognition of VERL. Using a combination of targeted mutagenesis and multidimensional NMR, we present a high-definition solution structure of sperm lysin from red abalone (Haliotis rufescens). Unapparent from the crystallography data, multiple NMR-based analyses conducted in solution reveal clustering of the N-and C-termini to form a nexus of 13 positively selected sites that constitute a VERL binding interface. Evolutionary rate was found to be a significant predictor of backbone flexibility, which may be critical for lysin bioactivity and / or accelerated evolution. These flexible, rapidly evolving segments that constitute the VERL binding interface were also the most distorted regions of the crystal structure relative to what was observed in solution. While lysin has been the subject of extensive biochemical and evolutionary analyses for more than 30 years, this study highlights the enhanced insights gained from applying NMR approaches to rapidly evolving proteins.\n\nSignificanceThe fertilization of eggs by sperm is a critical biological process for nearly all sexually reproducing organisms to propagate their genetic information. Despite the importance of fertilization, the molecules that mediate egg-sperm interactions have been characterized for only a few species, and the biochemical mechanisms underlying these interactions are even less well understood. In the marine mollusk abalone, sperm lysin interacts with egg VERL in a species-specific manner to facilitate fertilization. In this report, we characterized the solution structure and molecular evolution of sperm lysin from red abalone (Haliotis rufescens), and identified the VERL binding interface as well as important lysin dimer interactions that have emerged as part of an incessant sexual arms race.

evolutionary biology↗