<i>HEATR2</i> Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.纤毛（Cilia）是高度保守的基于微管的结构，在发育过程与成体稳态中执行多种感知与运动功能。在人类中，特异性影响运动纤毛的缺陷会导致慢性气道感染、不孕症，以及遗传异质性疾病原发性纤毛运动障碍（Primary Ciliary Dyskinesia, PCD）中的左右发育缺陷。我们利用相对简易的果蝇（Drosophila）系统开展研究，该系统的机械感觉神经元拥有修饰后的运动纤毛；我们采用新近阐明的纤毛转录RFX-FOX调控代码，以筛选新型PCD候选基因。本文报道了CG31320/HEATR2的特征解析，该基因在果蝇与人类中均对形成纤毛运动所需的轴丝动力蛋白臂发挥保守的关键作用。CG31320突变果蝇的轴丝以及携带新型剪接受体型HEATR2突变的PCD患者体内，均缺失内臂与外臂动力蛋白。HEATR2同源基因上游紧密排布的RFX-FOX结合位点的功能保守性，可能促使早期运动纤毛发生过程中HEATR2的胞质表达水平升高。免疫沉淀（immunoprecipitation）实验显示，HEATR2可与DNAI2相互作用，但无法结合HSP70或HSP90，这使其区别于其他参与动力蛋白臂组装的胞质蛋白（如DNAAF1-4）所报道的底物/分子伴侣功能。上述数据表明，CG31320/HEATR2参与了日益拓展的细胞内预组装与转运网络，该网络对于将功能性动力蛋白机器递送至纤毛区室并整合入运动性轴丝是必需的。