(222) Three Classes of piRNA Clusters in Human Spermatogenesis

Ahrend F^1,2; Konstantinidou P<sup>1; Loubalova Z¹; Haase AD¹

1. National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

2. Department of Biochemistry I, Faculty of Biology and Pre-Clinical Medicine, University of Regensburg, Regensburg, Germany

Abstract Text:

The PIWI-piRNA pathways in germ cells initiate silencing of transposable elements. PIWI-interacting RNAs (piRNAs) originate from long single-stranded precursors encoded by genomic piRNA clusters. Our recently developed toolkit, the ‘piRNA Cluster Builder’ (PICB), identifies, prioritizes and characterizes piRNA clusters, laying the foundation for systematic comparison between species. Here, we systematically characterize piRNA clusters in mammals and identify unique features of human piRNA pathways. Through the integration of bulk- and single-cell RNA-sequencing data and piRNA sequencing data, we disentangled functionally distinct piRNA pathways. First, we performed a comprehensive gene expression analysis of the four human PIWI genes, PIWIL1, 2, 3, and 4 during germ cell development. We observed that PIWIL4 expression was not restricted to juvenile (pre-puberty) gonads but was also present in adult testes, alongside PIWIL1. This pattern diverges from that in other mammals, where these individual PIWI genes are not expressed simultaneously. Single-cell RNA-seq data revealed that PIWIL4 and PIWIL1 signals originate from different cell types: early stages of Spermatogonial Stem Cells (SSCs) and meiotic and post-meiotic spermatocytes, respectively. Finally, considering the specific length profiles of piRNAs associated with different PIWI proteins, we could attribute the activity of individual piRNA clusters to specific cell types. We observed three classes of piRNA clusters: The first class produced piRNAs specifically in SSCs in juvenile and adult testes, the second class produced piRNAs specifically in meiotic and post-meiotic spermatocytes and was unique to adult testes. Finally, a third class produced piRNAs in SSCs and meiotic spermatocytes in adult testes. Our current work aims to understand the function of these different piRNA clusters for transposon silencing and gene regulation.