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.

piRNA通路（piRNA pathway）可保护动物生殖系免受活性转座子的侵袭。哺乳动物拥有两条通路：一条为细胞质通路，通过转录后降解的方式清除转座子转录本；另一条为细胞核通路，介导DNA甲基化以表观遗传机制沉默转座子的表达。目前尚不清楚这些通路的现代版本究竟于何时演化形成。我们发现，以3'核酸外切酶Pnldc1（3' exonuclease Pnldc1）对前体piRNA进行修剪为判定标准，哺乳动物的细胞质piRNA通路在肉鳍鱼中便已演化形成。细胞核通路则在四足动物早期演化过程中出现，并在墨西哥钝口螈（Ambystoma mexicanum）中已具备当前的完整功能构型。值得注意的是，该细胞核通路在鸟类演化历程中发生了丢失。我们证实，细胞质与细胞核piRNA通路在雄性和雌性墨西哥钝口螈的生殖系中均处于激活状态。对墨西哥钝口螈的睾丸与卵母细胞的分析显示，其piRNA库具有丰富的多样性，且对年轻转座子存在显著的转录后靶向调控作用。我们还观察到，墨西哥钝口螈的精子中存在高水平的基因组甲基化，且全长转座子的甲基化程度极高。此外，墨西哥钝口螈中的CpG岛甲基化与转座子沉默存在显著关联。综上，我们证实哺乳动物的piRNA通路拥有古老的脊椎动物起源，并在四足动物演化的绝大多数阶段中守护着生殖系基因组的稳态。整体实验设计：对墨西哥钝口螈的睾丸与卵母细胞样本开展小RNA测序（Small RNA-seq），每组设置2个生物学重复；对墨西哥钝口螈的精子样本开展EM-seq（EM-seq），每组设置3个生物学重复。所有样本均取自经人绒毛膜促性腺激素（human chorionic gonadotropin，hCG）注射以诱导成年个体配子成熟的d/d白色钝口螈品系。