Patil Anandrao Ashok, Tatsuke Tsuneyuki, Mon Hiroaki, Lee Jae Man, Morokuma Daisuke, Hino Masato, Kusakabe Takahiro
Laboratory of Insect Genome Science, Department of Bioresource Science, Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Laboratory of Insect Genome Science, Department of Bioresource Science, Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Biochem Biophys Res Commun. 2017 Nov 18;493(2):971-978. doi: 10.1016/j.bbrc.2017.09.107. Epub 2017 Sep 20.
Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with PIWI subfamily proteins, which play an important role in transposon silencing in animal germ cell. The piRNAs biogenesis is divided into two major pathways: primary and secondary, and both pathways are independent of double-stranded RNA-processing enzyme Dicer, which processes the single-stranded RNA transcripts in microRNA (miRNA) and siRNA (small interfering RNA) pathway. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters. Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily protein is conserved among the animals and involved in piRNA biogenesis. Recent studies showed that the Zucchini is an endoribonuclease essential for primary piRNA maturation and production of phased piRNA in secondary piRNA biogenesis of drosophila germ cell. Based on these reports, here we identified and studied the silkworm Zucchini (BmZuc) at subcellular level in ovary-derived BmN4 cell. The silkworm Zuc specifically expressed in germ-related tissues and localized on mitochondria and partially co-localized with perinuclear nuage-piRNA pathway components and nuage marker protein BmVasa. Molecular dissection analyses revealed that the conserved mitochondrial localization sequence, RGV motif, PLDc 2 domain and HKD motif are important for the BmZuc mitochondrial localization. Moreover, the knockdown analyses showed that the piRNA pathway components are independent on BmZuc for their nuage localization, whereas BmZuc depend on piRNA pathway components for the proper localization. Our data provides vital information on mitochondrial BmZuc and its relationship to "nuage" in ovary-derived BmN4 cell.
Piwi相互作用RNA（piRNA）是一类小的非编码RNA，它们与PIWI亚家族蛋白相关联，在动物生殖细胞的转座子沉默中发挥重要作用。piRNA的生物合成分为两条主要途径：初级途径和次级途径，这两条途径都独立于双链RNA加工酶Dicer，Dicer在微小RNA（miRNA）和小干扰RNA（siRNA）途径中加工单链RNA转录本。初级piRNA由从piRNA簇转录的长链非编码RNA前体加工而来。西葫芦（Zuc）是一种线粒体磷脂酶D（PLD）超家族蛋白，在动物中保守，并参与piRNA的生物合成。最近的研究表明，西葫芦是果蝇生殖细胞次级piRNA生物合成中初级piRNA成熟和阶段性piRNA产生所必需的一种核糖核酸内切酶。基于这些报道，我们在此在卵巢来源的BmN4细胞的亚细胞水平上鉴定并研究了家蚕西葫芦（BmZuc）。家蚕Zuc在生殖相关组织中特异性表达，定位于线粒体，并与核周生殖质-piRNA途径成分和生殖质标记蛋白BmVasa部分共定位。分子剖析分析表明，保守的线粒体定位序列、RGV基序、PLDc 2结构域和HKD基序对BmZuc的线粒体定位很重要。此外，敲低分析表明，piRNA途径成分的生殖质定位不依赖于BmZuc，而BmZuc的正确定位依赖于piRNA途径成分。我们的数据提供了关于卵巢来源的BmN4细胞中线粒体BmZuc及其与“生殖质”关系的重要信息。
