Hwang Hye Jin, Sheard Kelsey M, Cox Rachel T
Department of Biochemistry and Molecular Biology, Uniformed Services University, Bethesda, MD 20814.
Henry M. Jackson Foundation, Rockville, MD.
bioRxiv. 2024 Aug 22:2024.08.21.609023. doi: 10.1101/2024.08.21.609023.
The cytoplasm is populated with many ribonucleoprotein (RNP) particles that post-transcriptionally regulate mRNAs. These membraneless organelles assemble and disassemble in response to stress, performing functions such as sequestering stalled translation pre-initiation complexes or mRNA storage, repression and decay. Clueless (Clu) is a conserved multi-domain ribonucleoprotein essential for mitochondrial function that forms dynamic particles within the cytoplasm. Unlike well-known RNP particles, stress granules and Processing bodies, Clu particles completely disassemble under nutritional or oxidative stress. However, it is poorly understood how disrupting protein synthesis affects Clu particle dynamics, especially since Clu binds mRNA and ribosomes. Here, we capitalize on and imaging of female germ cells to determine what domains of Clu are necessary for Clu particle assembly, how manipulating translation using translation inhibitors affects particle dynamics, and how Clu particle movement relates to mitochondrial association. Using Clu deletion analysis and live and fixed imaging, we identified three protein domains in Clu, which are essential for particle assembly. In addition, we demonstrated that overexpressing functional Clu disassembled particles, while overexpression of deletion constructs did not. To examine how decreasing translation affects particle dynamics, we inhibited translation in germ cells using cycloheximide and puromycin. In contrast to stress granules and Processing bodies, cycloheximide treatment did not disassemble Clu particles yet puromycin treatment did. Surprisingly, cycloheximide stabilized particles in the presence of oxidative and nutritional stress. These findings demonstrate that Clu particles have novel dynamics in response to altered ribosome activity compared to stress granules and Processing bodies and support a model where they function as hubs of translation whose assembly heavily depends on the dynamic availability of polysomes.
细胞质中存在许多核糖核蛋白（RNP）颗粒，它们在转录后对mRNA进行调控。这些无膜细胞器会根据应激反应进行组装和拆卸，执行诸如隔离停滞的翻译起始前复合物或mRNA储存、抑制和降解等功能。无头绪蛋白（Clu）是一种对线粒体功能至关重要的保守多结构域核糖核蛋白，它在细胞质中形成动态颗粒。与众所周知的RNP颗粒、应激颗粒和加工小体不同，Clu颗粒在营养或氧化应激下会完全解体。然而，目前对于破坏蛋白质合成如何影响Clu颗粒动态变化了解甚少，尤其是因为Clu会结合mRNA和核糖体。在这里，我们利用秀丽隐杆线虫雌性生殖细胞的荧光标记和成像技术，来确定Clu的哪些结构域对于Clu颗粒组装是必需的，使用翻译抑制剂操纵翻译如何影响颗粒动态变化，以及Clu颗粒运动与线粒体关联之间的关系。通过Clu缺失分析以及活细胞和固定细胞成像，我们在Clu中鉴定出三个对颗粒组装至关重要的蛋白质结构域。此外，我们证明过表达功能性Clu会使颗粒解体，而过表达缺失构建体则不会。为了研究降低翻译水平如何影响颗粒动态变化，我们使用环己酰亚胺和嘌呤霉素抑制秀丽隐杆线虫生殖细胞中的翻译。与应激颗粒和加工小体不同，环己酰亚胺处理不会使Clu颗粒解体，而嘌呤霉素处理则会。令人惊讶的是，在存在氧化和营养应激的情况下，环己酰亚胺会使颗粒稳定。这些发现表明，与应激颗粒和加工小体相比，Clu颗粒在核糖体活性改变时具有新的动态变化，并支持了一个模型，即它们作为翻译中心发挥作用，其组装在很大程度上依赖于多核糖体的动态可用性。
