Ribosomal protein S5b is essential for oogenesis and is required to maintain mitochondrial integrity and function in Drosophila melanogaster

Increasing evidence indicates that ribosomes are heterogeneous and perhaps dynamic, in contrast to the classical view of them as constitutive machinery for protein synthesis [1-11]. In Drosophila melanogaster, nine ribosomal protein genes are each present in two paralogs, and in many of these cases one of the paralogs is primarily expressed in germline tissues [12-14]. Among these are two genes encoding ribosomal protein S5, called RpS5a and RpS5b. We demonstrate that RpS5b is specifically required for oogenesis. Females lacking RpS5b produce ovaries with polarity defects, fused egg chambers, and over-proliferation of posterior follicle cells. Oogenesis ceases at stage 8-9, and widespread apoptosis follows. Females lacking germline RpS5a are fully fertile, but germline expression of interfering RNA targeting RpS5a in an RpS5b mutant background worsens the RpS5b phenotype and causes complete germline lethality. A broad spectrum of mRNAs co-purified in immunoprecipitations with RpS5a, while RpS5b-associated mRNAs were specifically enriched for GO terms related to mitochondrial electron transport and cellular metabolic processes. Consistent with this, proteome comparison of wildtype and RpS5b ovaries revealed that RpS5b mitochondria were depleted for proteins linked to oxidative phosphorylation and mitochondrial respiration. As observed by immunohistochemical staining and transmission electron microscopy, RpS5b nurse cell mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls. We also observed that RpS5b ovaries have elevated ROS, consistent with mitochondrial dysfunction. We conclude that RpS5b-containing ribosomes preferentially associate with mRNAs encoding mitochondrial components and serve an essential function in oogenesis. Overall design: The mRNAs associated to either RpS5b and RpS5a were identified by co-IP followed by deep sequencing. The mRNAs enriched to either of the two parologs of RpS5, when applicable, were analyzed in three genetic backgrounds, wildtype, RpS5b-null and the ovary with germline RpS5a knocked down by RNAi.越来越多的证据表明，核糖体（ribosomes）具有异质性且可能处于动态变化之中，这与将核糖体视作蛋白质合成组成型机器的经典观点相悖[1-11]。 在黑腹果蝇（Drosophila melanogaster）中，9个核糖体蛋白基因各自拥有两个旁系同源基因（paralogs），其中多数情况下其中一个旁系同源基因主要在生殖系组织中表达[12-14]。此类基因包含两个编码核糖体蛋白S5的基因，分别命名为RpS5a与RpS5b。 本研究证实，RpS5b是卵子发生（oogenesis）过程中特异性必需的基因。缺失RpS5b的雌性个体所产生的卵巢存在极性缺陷、卵室融合以及后卵泡细胞过度增殖的表型。卵子发生会在第8-9期停滞，随后出现广泛的细胞凋亡。生殖系RpS5a缺失的雌性个体育性完全正常，但在RpS5b突变背景下，通过干扰RNA靶向抑制生殖系RpS5a的表达，会加剧RpS5b的表型，并导致完全的生殖系致死。 免疫沉淀（immunoprecipitation）实验显示，与RpS5a共纯化的mRNA种类广泛，而与RpS5b结合的mRNA则显著富集于与线粒体电子传递及细胞代谢过程相关的基因本体（Gene Ontology, GO）术语条目。与此一致的是，对野生型与RpS5b突变卵巢的蛋白质组比较分析显示，RpS5b突变体的线粒体中，与氧化磷酸化及线粒体呼吸相关的蛋白质含量显著降低。 通过免疫组织化学染色与透射电子显微镜（transmission electron microscopy）观察发现，RpS5b突变体的滋养细胞（nurse cell）线粒体倾向于形成大型簇状结构，且其形态异质性相较于对照组更为显著。本研究同时观察到，RpS5b突变卵巢中的活性氧（reactive oxygen species, ROS）水平升高，这与线粒体功能异常的表型相符。综上，包含RpS5b的核糖体可优先与编码线粒体组分的mRNA结合，并在卵子发生过程中发挥不可或缺的关键功能。 整体实验设计：通过免疫共沉淀（co-IP）结合深度测序（deep sequencing），鉴定与RpS5a或RpS5b结合的mRNA。针对两种RpS5旁系同源基因富集的mRNA，在三种遗传背景下进行分析，分别为野生型、RpS5b纯合缺失型，以及通过RNA干扰（RNA interference, RNAi）敲低生殖系RpS5a表达的卵巢样本。