PIWI proteins and their guiding Piwi-interacting (pi-) RNAs direct the silencing of target nucleic acids in the animal germline, acting on transcriptional and post-transcriptional levels. While in primordial male germ cells of mammals so-called pre-pachytene piRNAs are involved in extensive silencing of transposons, pachytene piRNAs that are active from the pachytene meiotic phase of spermatogenesis have additionally been shown to act in post-transcriptional regulation of protein-coding genes. The bulk of pachytene piRNAs is produced from large genomic loci, named piRNA clusters, that harbor many different transposon fragments, which serve as the source for transposon-targeting piRNAs. Recently, the presence of reversed pseudogene copies within piRNA clusters lead to the idea that piRNAs derived from such pseudogenes might direct regulation of their parent genes. Here, we examine primate piRNA clusters and therein contained pseudogenes in a comparative approach in order to gain a deeper understanding about the evolution of mammalian piRNA clusters in general and the putative gene regulatory 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 cluster transcription do not show signs of selection pressure, compared to pseudogenes in parallel orientation. Further, the fact that only a minority of reversed pseudogenes produces piRNAs which target gene transcripts that are in turn processed within the PIWI/piRNA pathway and a weak conservation of targeting of homologous genes among species, suggest only a minor impact on gene regulation. Finally, possibly serving as an alternative explanation for the general enrichment of pseudogenes in piRNA clusters, we report that piRNA producing loci themselves tend to be located in genomic regions with elevated gene density, indicating open and active chromatin, but also correlating with pseudogene abundance. Hence, the occurrence of pseudogenes in piRNA clusters might be regarded as a by-product of cluster generation.
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