Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan.
Department of Agrobiology and Bioresources, School of Agriculture, Utsunomiya University, Utsunomiya, Japan.
Nature. 2020 Feb;578(7794):311-316. doi: 10.1038/s41586-020-1966-9. Epub 2020 Jan 29.
PIWI-interacting RNAs (piRNAs) of between approximately 24 and 31 nucleotides in length guide PIWI proteins to silence transposons in animal gonads, thereby ensuring fertility. In the biogenesis of piRNAs, PIWI proteins are first loaded with 5'-monophosphorylated RNA fragments called pre-pre-piRNAs, which then undergo endonucleolytic cleavage to produce pre-piRNAs. Subsequently, the 3'-ends of pre-piRNAs are trimmed by the exonuclease Trimmer (PNLDC1 in mouse) and 2'-O-methylated by the methyltransferase Hen1 (HENMT1 in mouse), generating mature piRNAs. It is assumed that the endonuclease Zucchini (MitoPLD in mouse) is a major enzyme catalysing the cleavage of pre-pre-piRNAs into pre-piRNAs. However, direct evidence for this model is lacking, and how pre-piRNAs are generated remains unclear. Here, to analyse pre-piRNA production, we established a Trimmer-knockout silkworm cell line and derived a cell-free system that faithfully recapitulates Zucchini-mediated cleavage of PIWI-loaded pre-pre-piRNAs. We found that pre-piRNAs are generated by parallel Zucchini-dependent and -independent mechanisms. Cleavage by Zucchini occurs at previously unrecognized consensus motifs on pre-pre-piRNAs, requires the RNA helicase Armitage, and is accompanied by 2'-O-methylation of pre-piRNAs. By contrast, slicing of pre-pre-piRNAs with weak Zucchini motifs is achieved by downstream complementary piRNAs, producing pre-piRNAs without 2'-O-methylation. Regardless of the endonucleolytic mechanism, pre-piRNAs are matured by Trimmer and Hen1. Our findings highlight multiplexed processing of piRNA precursors that supports robust and flexible piRNA biogenesis.
PIWI 相互作用 RNA（piRNA）的长度约为 24 到 31 个核苷酸，在动物性腺中指导 PIWI 蛋白沉默转座子，从而确保生育能力。在 piRNA 的生物发生过程中，PIWI 蛋白首先与称为 pre-pre-piRNA 的 5'-单磷酸化 RNA 片段结合，然后经历内切核酸酶切割产生 pre-piRNA。随后，pre-piRNA 的 3'末端被核酸外切酶 Trimmer（小鼠中的 PNLDC1）修剪，2'-O 甲基化由甲基转移酶 Hen1（小鼠中的 HENMT1）完成，生成成熟的 piRNA。假定内切核酸酶 Zucchini（小鼠中的 MitoPLD）是主要的酶，可将 pre-pre-piRNA 切割成 pre-piRNA。然而，该模型缺乏直接证据，pre-piRNA 的产生机制仍不清楚。在这里，为了分析 pre-piRNA 的产生，我们建立了 Trimmer 敲除的家蚕细胞系，并衍生出一个细胞游离系统，该系统忠实地再现了 Zucchini 介导的 PIWI 加载的 pre-pre-piRNA 的切割。我们发现 pre-piRNA 的产生是由平行的 Zucchini 依赖和非依赖机制实现的。Zucchini 的切割发生在 pre-pre-piRNA 上以前未被识别的共有基序上，需要 RNA 解旋酶 Armitage，并伴随着 pre-piRNA 的 2'-O 甲基化。相比之下，具有弱 Zucchini 基序的 pre-pre-piRNA 的切割是由下游互补的 piRNA 完成的，产生没有 2'-O 甲基化的 pre-piRNA。无论内切核酸酶机制如何，pre-piRNA 都由 Trimmer 和 Hen1 成熟。我们的研究结果突出了 piRNA 前体的多路处理，这支持了强大和灵活的 piRNA 生物发生。
