Exonuclease domain-containing 1 enhances MIWI2 piRNA biogenesis via its interaction with TDRD12

PIWI proteins and their associated small RNAs called PIWI-interacting RNAs (piRNAs) restrict transposon activity in animal gonads to ensure fertility. Distinct biogenesis pathways load piRNAs into the PIWI proteins MILI and MIWI2 in the mouse male embryonic germline. While most of MILI piRNAs derive via a slicer-independent pathway, a MILI slicer endonuclease-initiated pathway loads nuclear MIWI2 with a series of phased piRNAs. Tudor domain-containing 12 (TDRD12) and its interaction partner Exonuclease domain-containing 1 (EXD1) are required for loading MIWI2, but only Tdrd12 is essential for fertility, leaving us with no explanation for the physiological role of Exd1. Using an artificial piRNA precursor, we demonstrate that MILI-triggered piRNA biogenesis is greatly reduced in the Exd1 mutant. The situation deteriorates in the sensitized Exd1 mutant (Exd1-/-; Tdrd12+/-), where diminished MIWI2 piRNA levels de-repress LINE1 retrotransposons, causing infertility. Thus, EXD1 enhances slicing-triggered MIWI2 piRNA biogenesis via a functional interaction with TDRD12. Small RNA sequencing was used to identify differences in piRNA levels between various Exd1 and Tdrd12 mouse mutants. Total small RNAs were sequenced together with immunopurified MILI and MIWI2 associated piRNAs.PIWI蛋白（PIWI proteins）及其结合的PIWI互作RNA（PIWI-interacting RNAs，简称piRNAs）可通过抑制动物性腺中的转座子（transposon）活性，保障生物体生育能力。在小鼠雄性胚胎生殖系中，存在多条不同的生物发生通路，将piRNAs装载至PIWI家族蛋白MILI与MIWI2内。其中，绝大多数MILI结合的piRNAs通过不依赖切片酶（slicer）的通路生成，而由MILI的切片酶内切核酸酶启动的通路，则可为细胞核内的MIWI2装载一系列阶段性piRNAs（phased piRNAs）。含Tudor结构域蛋白12（Tudor domain-containing 12，TDRD12）及其互作伴侣含外切核酸酶结构域蛋白1（Exonuclease domain-containing 1，EXD1）均为MIWI2装载过程的必需因子，但仅有Tdrd12对小鼠生育能力不可或缺，这使得此前的研究无法阐释Exd1的生理功能。本研究借助人工piRNA前体开展实验，证实Exd1突变体中MILI触发的piRNA生物发生过程显著减弱。在致敏型Exd1突变体（Exd1-/-; Tdrd12+/-）中，表型进一步恶化：MIWI2结合的piRNA水平大幅下降，导致LINE1逆转座子（LINE1 retrotransposons）去抑制，最终引发不育。综上，EXD1可通过与TDRD12的功能性互作，增强由切片酶触发的MIWI2 piRNA生物发生通路。本研究采用小型RNA测序（small RNA sequencing）技术，鉴定了不同Exd1与Tdrd12小鼠突变体之间的piRNA水平差异，并对总小型RNA以及免疫纯化得到的与MILI、MIWI2结合的piRNAs进行了测序。