Original research RSBP15 interacts with and stabilizes dRSPH3 during sperm axoneme assembly in Drosophila Ya Wang a, b , Rui Xu c, d , Yiwei Cheng a, b , Haowei Cao c , Zibin Wang a , Tianyu Zhu a, b , Jiayin Jiang a, b , Hao Zhang a, b , Chang Wang a, b , Lin Qi a, b , Mingxi Liu a, b , Xuejiang Guo a, b, *** , Juan Huang c, d, ** , Jiahao Sha a, b, * a State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, 211166, China b Department of Histology and Embryology, Nanjing Medical University, Nanjing, 211166, China c Department of Medical Genetics, Nanjing Medical University, Nanjing, 211166, China d Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, 211166, China article info Article history: Received 14 March 2019 Received in revised form 28 May 2019 Accepted 29 May 2019 Available online xxx Keywords: RSBP15 RSPH3 Flagellum Radial spoke Drosophila abstract Flagellum in sperm is composed of over 200 different proteins and is essential for sperm motility. In particular, defects in the assembly of the radial spoke in the flagellum result in male infertility due to loss of sperm motility. However, mechanisms regulating radial spoke assembly remain unclear in metazoans. Here, we identified a novel Drosophila protein radial spoke binding protein 15 (RSBP15) which plays an important role in regulating radial spoke assembly. Loss of RSBP15 results in complete lack of mature sperms in seminal vesicles (SVs), asynchronous individualization complex (IC) and defective “9 þ 2” structure in flagella. RSBP15 is colocalized with dRSPH3 in sperm flagella, and interacts with dRSPH3 through its DD_R_PKA superfamily domain which is important for the stabilization of dRSPH3. Moreover, loss of dRSPH3, as well as dRSPH1, dRSPH4a and dRSPH9, showed similar phenotypes to rsbp15 KO mutant. Together, our results suggest that RSBP15 acts in stabilizing the radial spoke protein complex to anchor and strengthen the radial spoke structures in sperm flagella. Copyright © 2019, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. 1. Introduction During fertilization a sperm must use its flagellum to propel itself to the oocyte, and defects in sperm flagella are a main cause of male-related infertility in humans (Lee et al., 2011). During sper- matogenesis, mature sperms with long flagella develop from round sperms through a series of ultrastructural changes including nu- clear condensation, flagellum elongation and microtubule assem- bly (Tokuyasu et al., 1972; Tokuyasu, 1975; White-Cooper, 2009). Sperm axoneme, a highly ordered microtubule-based “9 þ 2” structure, is composed of nine outer microtubule doublets and a central pair of microtubules (Inaba, 2007; Fabian and Brill, 2012; Gottardo et al., 2013; Soulavie et al., 2014; Vieillard et al., 2016). Inside axoneme, radial spokes function as linkers and mechano- chemical transducers which transmit signals from the central pair apparatus to the outer microtubule doublets for local control of dynein activity (Diener et al., 1993; Lin et al., 2012; Pigino and Ishikawa, 2012). Radial spokes were originally described as slender threads be- tween the central paired microtubules and the nine outer micro- tubule doublets in the axoneme of sea urchin sperm flagella (Afzelius, 1959). Each individual radial spoke is a T-shaped structure composed of a stalk, which is anchored on the A microtubule of an outer doublet, and a head, which is thought to have transient contacts with the inner sheath and the central paired microtubules (Warner and Satir, 1974; Goodenough and Heuser, 1985). The radial spoke head was first characterized in the flagella of Sarcophaga bullata and Chlamydomonas reinhardtii (Warner, 1970). Later, the T- shaped radial spoke structure was also observed in the cilia and * Corresponding author. State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, 211166, China. ** Corresponding author. Department of Medical Genetics, Nanjing Medical Uni- versity, Nanjing, 211166, China. *** Corresponding author. State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, 211166, China. E-mail addresses: guo_xuejiang@njmu.edu.cn (X. Guo), huangjuan@njmu.edu. cn (J. Huang), shajh@njmu.edu.cn (J. Sha). Contents lists available at ScienceDirect Journal of Genetics and Genomics Journal homepage: www.journals.elsevier.com/journal-of-genetics- and-genomics/ https://doi.org/10.1016/j.jgg.2019.05.001 1673-8527/Copyright © 2019, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. Journal of Genetics and Genomics xxx (xxxx) xxx Please cite this article as: Wang, Y et al., RSBP15 interacts with and stabilizes dRSPH3 during sperm axoneme assembly in Drosophila, Journal of Genetics and Genomics, https://doi.org/10.1016/j.jgg.2019.05.001