Novel piRNA Pathway Components Identified Among the Class of TUDOR Domain Containing Proteins. Drosophila melanogaster

PIWI proteins and their bound piRNAs form the core of a gonad specific small RNA silencing pathway in animals that protects the genome against the deleterious activity of transposable elements. Recent studies linked the piRNA pathway to TUDOR biology, where TUDOR domains of various proteins recognize and bind symmetrically methylated Arginine residues in PIWI proteins. We systematically analyzed the Drosophila TUDOR protein family and identified three previously not characterized TUDOR domain-containing genes (CG4771, CG14303 and CG11133) as essential piRNA pathway members. We characterized CG4771 (Avocado) in detail and demonstrate a critical role for this protein during primary piRNA biogenesis in somatic and germline cells of the ovary. Avocado physically and/or genetically interacts with the primary pathway components Piwi, Armitage, Yb and Zucchini. Avocado also interacts with the Tdrd12 orthologs CG11133 and CG31755, which are essential for primary piRNA biogenesis in the germline and probably functionally replace the related and soma specific factor Yb. Overall design: small RNA libraries were prepared from total RNA isolation of 8 different genotypes

PIWI蛋白与其结合的piRNA（Piwi-interacting RNA）构成了动物体内性腺特异性小RNA沉默通路的核心，该通路可保护基因组免受转座因子的有害活性侵扰。近期研究将piRNA通路与TUDOR生物学联系起来，各类蛋白的TUDOR结构域（TUDOR domain）可识别并结合PIWI蛋白中对称甲基化的精氨酸残基。我们对果蝇TUDOR蛋白家族开展系统性分析，鉴定出3个此前未被表征的含TUDOR结构域的基因（CG4771、CG14303与CG11133），它们均为piRNA通路的必需成员。我们对CG4771（又名Avocado）进行了详细表征，并证实该蛋白在卵巢体细胞与生殖系细胞的初级piRNA生物发生过程中发挥关键作用。Avocado可与初级piRNA通路核心组分Piwi、Armitage、Yb及Zucchini发生物理或遗传互作。Avocado还可与Tdrd12的直系同源基因CG11133和CG31755发生互作，这两个基因在生殖系细胞的初级piRNA生物发生过程中不可或缺，且可能在功能上替代体细胞特异性相关因子Yb。实验整体设计：从8种不同基因型的总RNA提取物中构建小RNA文库。