Matsumori Haruka, Watanabe Kenji, Tachiwana Hiroaki, Fujita Tomoko, Ito Yuma, Tokunaga Makio, Sakata-Sogawa Kumiko, Osakada Hiroko, Haraguchi Tokuko, Awazu Akinori, Ochiai Hiroshi, Sakata Yuka, Ochiai Koji, Toki Tsutomu, Ito Etsuro, Goldberg Ilya G, Tokunaga Kazuaki, Nakao Mitsuyoshi, Saitoh Noriko
Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan.
Cancer Institute of Japanese Foundation for Cancer Research, Tokyo, Japan.
Life Sci Alliance. 2022 Mar 23;5(7). doi: 10.26508/lsa.202101045. Print 2022 Jul.
The nucleolus is the site of ribosome assembly and formed through liquid-liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm. We revealed that cells lacking a specific 60S ribosomal protein set exhibited common nucleolar disintegration. The depletion of RPL5 (also known as uL18), the liquid-liquid phase separation facilitator, was most effective, and resulted in an enlarged and un-separated sub-nucleolar compartment. Single-molecule tracking analysis revealed less-constrained mobility of its components. rDNA arrays were also unbundled. These results were recapitulated by a coarse-grained molecular dynamics model. Transcription and processing of ribosomal RNA were repressed in these aberrant nucleoli. Consistently, the nucleoli were disordered in peripheral blood cells from a Diamond-Blackfan anemia patient harboring a heterozygous, large deletion in Our combinatorial analyses newly define the role of RPL5 in rDNA array bundling and the biophysical properties of the nucleolus, which may contribute to the etiology of ribosomopathy.
核仁是核糖体组装的场所，通过液-液相分离形成。多个核糖体DNA（rDNA）阵列在核仁中聚集，但其潜在机制和意义尚不清楚。在本研究中，我们进行了高内涵筛选，随后使用wndchrm机器学习算法进行图像分析。我们发现，缺乏特定60S核糖体蛋白组的细胞表现出常见的核仁解体。液-液相分离促进因子RPL5（也称为uL18）的缺失最为有效，并导致一个扩大且未分离的核仁亚区室。单分子追踪分析显示其组分的流动性受限较小。rDNA阵列也解聚。这些结果通过粗粒度分子动力学模型得到了重现。在这些异常核仁中，核糖体RNA的转录和加工受到抑制。同样，在一名患有钻石-黑范贫血的患者的外周血细胞中，核仁也出现紊乱，该患者的rDNA存在杂合性大片段缺失。我们的组合分析新定义了RPL5在rDNA阵列聚集和核仁生物物理特性中的作用，这可能有助于核糖体病的病因学研究。
