On the origin of the mammalian piRNA pathway
The piRNA pathway protects animal germlines form active transposons. Mammals employ a cytoplasmic pathway to post-transcriptionally destroy transposon transcripts and a nuclear pathway that mediates DNA methylation to epigenetically silence transposon expression. When the modern version of these pathways evolved remains unknown. We found that the mammalian version of the cytoplasmic pathway evolved in lobe-finned fish, as defined by the use of the 3' exonuclease Pnldc1 for precursor piRNA trimming. The nuclear pathway evolved during early in tetrapod evolution and is already found in its current configuration in the salamander, Ambystoma mexicanum. Interestingly, the nuclear pathway was lost during bird evolution. We found evidence that both the cytoplasmic and nuclear piRNA pathways are active in both the male and female axolotl germlines. Analysis of axolotl testes and oocytes revealed diverse repertoires of piRNAs and persuasive post-transcriptional targeting of young transposons. We found high levels of genome methylation in axolotl spermatozoa with full-length transposons being highly methylated. Furthermore, CpG island methylation in axolotl is associated with transposon silencing. In summary, we find that the mammalian version of the piRNA pathway has ancient vertebrate origins, and it has been safeguarding the germline across most of tetrapod evolution. Overall design: Small RNA-seq was performed on axolotl testis and oocyte samples, each with two biological replicates. EM-seq was performed on axolotl spermatozoa with three biological replicates. Samples were collected from the d/d white axolotl strain with hCG (human chorionic gonadotropin) injection to induce gamete maturation in adult axolotl.