# Characterization of PRDM9 Multifunctionality in Yak Testes Through Protein Interaction Mapping

## TL;DR

This study identifies proteins that interact with PRDM9 in yak testes, revealing new functions related to spermatogenesis and hybrid male sterility.

## Contribution

The study establishes a complete PRDM9 interaction network in yaks and identifies novel functions related to meiosis and hybrid sterility.

## Key findings

- 267 PRDM9-interacting proteins were identified, with GO terms linked to spermatogenesis and DNA repair.
- MKX and PDCD5 were confirmed as strong PRDM9 interactors expressed in meiotic prophase I.
- 55 out of 58 interacting genes were downregulated in sterile cattleyak hybrids.

## Abstract

Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks during spermatogenesis. PRDM9 determines the localization of recombination hotspots by interacting with several protein complexes in mammals. The function of PRDM9 is not well understood during spermatogenesis in mice or yaks. In this study, we applied yeast two-hybrid assays combined with next-generation sequencing techniques to screen the complete set of PRDM9-interacting proteins and explore its novel functions in yak spermatogenesis. Our results showed that 267 PRDM9-interacting proteins were identified. The gene ontology (GO) analysis of the interacting proteins revealed that the GO terms were primarily associated with spermatogenesis, positive regulation of double-strand break repair via homologous recombination, RNA splicing, the ubiquitin-dependent ERAD pathway, and other biological processes. MKX and PDCD5 were verified to be strongly interacting with PRDM9 and expressed in prophase I of meiosis in both mouse and yak testes. The localizations of RNA splicing genes including THOC5, DDX5, and XRCC6 were expressed in spermatocytes. Cattleyak is the hybrid offspring of a yak and a domestic cow, and the male offspring are sterile. The gene expression of the interacting proteins was also examined in the sterile male hybrid of yak and cattle. Among the 58 detected genes, 55 were downregulated in cattleyak. In conclusion, we established a complete PRDM9 interaction network, and a novel function of PRDM9 was identified, which will further promote our understanding of spermatogenesis. It also provides new insights for the study of hybrid male sterility.