Primate piRNA cluster evolution suggests limited relevance of pseudogenes in piRNA-mediated gene regulation

Abstract

PIWI proteins and their guiding Piwi-interacting (pi-) RNAs direct the silencingof target nucleic acids in the animal germline andsoma. Although inmammal testes fetal piRNAs are involved in extensive silencing of transposons, pachytene piRNAs have additionally been shown to act in post-transcriptional gene regulation. The bulk of pachytene piRNAs is produced from large genomic loci, named piRNA clusters. Recently, the presence of reversed pseudogeneswithin piRNA clusters prompted the idea that piRNAs derived from such sequencesmight direct regulation of their parent genes.Here,we examine primatepiRNAclusters and integrated pseudogenes in a comparative approach to gain a deeper understanding about mammalian piRNA cluster evolution and the presumed generegulatory role of pseudogene-derived piRNAs. Initially, we provide a broad analysis of the evolutionary relationships of piRNA clusters and their differential activity among six primate species. Subsequently, we show that pseudogenes in reserve orientation relative to piRNA cluster transcription direction generally do not exhibit signs of selection pressure and cause weakly conserved targeting of homologous genes among species, suggesting a lack of functional constraints and thus only a minor significance for gene regulation in most cases. Finally, we report that piRNA-producing loci generally tend to be located in active genomic regions with elevated gene and pseudogene density. Thus,we conclude that the presence ofmost pseudogenes in piRNA clustersmight be regardedas abyproductofpiRNAclustergeneration,whereas thisdoes not exclude thatsomepseudogenesneverthelessplay critical roles in individual cases.