Sheu-Gruttadauria Jessica, Yan Xiaowei, Stuurman Nico, Vale Ronald D, Floor Stephen N
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.
Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA.
bioRxiv. 2024 Oct 15:2023.09.26.559432. doi: 10.1101/2023.09.26.559432.
Ribosome biogenesis occurs in the nucleolus, a nuclear biomolecular condensate that exhibits dynamic biophysical properties thought to be important for function. However, the relationship between ribosome assembly and nucleolar dynamics is incompletely understood. Here, we present a platform for high-throughput fluorescence recovery after photobleaching (HiT-FRAP), which we use to screen hundreds of genes for their impact on dynamics of the nucleolar scaffold nucleophosmin (NPM1). We find that scaffold dynamics and nucleolar morphology respond to disruptions in key stages of ribosome biogenesis. Accumulation of early ribosomal intermediates leads to nucleolar rigidification while late intermediates lead to increased fluidity. We map these biophysical changes to specific ribosomal intermediates and their affinity for NPM1. We also discover that disrupting mRNA processing impacts nucleolar dynamics and ribosome biogenesis. This work mechanistically ties ribosome assembly to the biophysical features of the nucleolus and enables study of how dynamics relate to function across other biomolecular condensates.
核糖体生物发生在核仁中进行，核仁是一种核生物分子凝聚物，具有被认为对其功能很重要的动态生物物理特性。然而，核糖体组装与核仁动态之间的关系尚未完全了解。在这里，我们展示了一个用于高通量光漂白后荧光恢复（HiT-FRAP）的平台，我们用它来筛选数百个基因对核仁支架蛋白核磷蛋白（NPM1）动态的影响。我们发现支架动态和核仁形态对核糖体生物发生关键阶段的破坏有反应。早期核糖体中间体的积累导致核仁僵化，而晚期中间体则导致流动性增加。我们将这些生物物理变化映射到特定的核糖体中间体及其对NPM1的亲和力上。我们还发现，破坏mRNA加工会影响核仁动态和核糖体生物发生。这项工作从机制上将核糖体组装与核仁的生物物理特征联系起来，并能够研究动态如何与其他生物分子凝聚物的功能相关。
