Topoisomerase 3b Facilitates piRNA Biogenesis to Promote Transposon Silencing and Germ Cell Development

Topoisomerases typically function in nucleus to relieve topological stress in DNA. Here we show that a dual-activity topoisomerase Top3b and its partner TDRD3 largely localize in cytoplasm, and interact biochemically and genetically with piRNA processing enzymes to promote piRNA biogenesis, post-transcriptional gene silencing (PTGS) of transposons, and Drosophila germ cell development. Top3b requires its topoisomerase activity to promote PTGS of a transposon reporter, and preferentially silences long and highly-expressed transposons, suggesting that RNAs with these features may produce more topological stress for topoisomerases to solve. The double mutants between Top3b and piRNA processing enzymes exhibit stronger disruption of the signatures and levels of germline piRNAs, more de-silenced transposons, and larger defects in germ cells, than either single mutant. Our data suggest that Top3b can act in an RNA-based process—piRNA biogenesis and PTGS of transposons; and this function is required for Top3b to promote normal germ cell function. Test if Top3b mutant exhibits de-silencing of transposons by genetic interaction with piRNA pathway mutants - aub, ago3, vas, armi, zuc. We performed RNA-seq and small RNA-seq to profile transposon level as compared to control.

拓扑异构酶（Topoisomerase）通常在细胞核内发挥功能，以缓解DNA中的拓扑张力。本研究发现，一种兼具双重活性的拓扑异构酶Top3b及其结合蛋白TDRD3主要定位于细胞质，且可与piRNA加工酶发生生化与遗传相互作用，进而促进piRNA生物发生、转座子的转录后基因沉默（post-transcriptional gene silencing, PTGS）以及果蝇生殖细胞发育。Top3b需依赖其拓扑异构酶活性来促进转座子报告基因的转录后基因沉默，且优先沉默长度较长、表达水平较高的转座子，这提示具备此类特征的RNA可能会产生更多拓扑张力，需要拓扑异构酶进行解除。相较于单突变体，Top3b与piRNA加工酶的双突变体对生殖系piRNA的特征与表达水平的破坏更为显著，转座子去沉默程度更高，生殖细胞缺陷也更为严重。本研究数据表明，Top3b可参与基于RNA的过程——piRNA生物发生与转座子的转录后基因沉默；且该功能是Top3b促进正常生殖细胞功能所必需的。为验证Top3b突变体是否会通过与piRNA通路突变体（aub、ago3、vas、armi、zuc）的遗传互作引发转座子去沉默，我们开展了RNA测序（RNA-seq）与小RNA测序（small RNA-seq），以相较于对照组表征转座子的表达水平。