Long first exon and broad H3K27ac are two defining features of Eutherian conserved pachytene piRNA loci
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In the germ cells of animals, PIWI-interacting RNAs (piRNAs) guide silencing transposons and regulating genes to ensure normal fertility. In adult mammalian testes, most piRNAs derive from ~100 discrete intergenic loci called pachytene piRNA genes that produce canonical RNA polymerase II transcripts. Little is known about the conservation of pachytene piRNA genes. Several studies suggested pachytene piRNA genes evolve much faster than protein-coding genes and miRNA genes, although more than 20 of them are conserved in eutherian mammals. Whereas, due to the lack of detailed annotation, previous studies regarded pachytene piRNA-producing loci as “clusters” rather than “genes” and could not investigate the evolution of gene elements like promoters and splice junctions in mammals.
Taking advantage of published RNA-seq, small RNA-seq, and H3K4me3 ChIP-seq data, we annotated pachytene piRNA genes in eight mammals (rhesus, marmoset, rat, rabbit, cow, pig, dog, and opossum), along with manually curated TSS, TES, and splice junctions. We then performed a thorough analysis for the evolution of pachytene piRNA genes across 11 mammals by combining our annotation with the mouse, human, and koala pachytene piRNA gene annotation. We showed the two marsupials (koala and opossum) share more pachytene piRNA genes than laurasiatherias and glires in spite of their similar divergent times (~80 Myrs), suggesting variant rates of pachytene piRNA gene gain and loss in marsupial and eutherian mammals. Primates and glires diverged after separating from laurasiatheria, but there are less conserved pachytene piRNA genes in primates–glires than in glires–laurasiatheria and primates–laurasiatheria. Further analysis suggested that the sequence of pachytene piRNA genes is under neutral selection, and the conservation of sequence and splice junctions gradually decrease from 5’ end to 3’ end of the genes.
Together, we accurately annotated pachytene piRNA genes in eight marsupial and eutherian mammals. Our evolutionary analysis for these genes further indicated different gain and loss rates for marsupials, laurasiatherias, glires, and primates and implicated the importance of the promoters and the first splice junctions for piRNA biogenesis.