Pooled CRISPR/Cas9 screen for genes that contribute to ribosome biogenesis and turnover. Pooled CRISPR/Cas9 screen for genes that contribute to ribosome biogenesis and turnover

While features of ribosome assembly are shared between species, our understanding of the diversity, complexity, dynamics, and regulation of ribosome production in multicellular organisms remains incomplete. To gain insights into ribosome biogenesis in human cells, we performed a genome wide loss of function screen combined with differential labeling of pre-existing and newly assembled ribosomes. These efforts identified two functionally uncharacterized genes, C1ORF09 and SPATA5. We provide evidence that these factors, together with CINP and SPATA5L1, control a late step of human pre-60S maturation in the cytoplasm. Loss of either C1ORF109 or SPATA5 impairs global protein synthesis. These results link ribosome assembly with neurodevelopmental disorders associated with recessive SPATA5 mutations. Based on these findings, we propose the expanded repertoire of ribosome biogenesis factors likely enables multicellular organisms to coordinate multiple steps of ribosome production in response to different developmental and environmental stimuli. Overall design: A genome-scale CRISPR/Cas9-mediated loss of function screen for ribosome biogenesis/turnover factors was performed in HEK293T cells with endogenous RPL28-SNAP tagging. The pre-existing and newly produced ribosomes were differentially labeled with red and green fluorescence for FACS enrichment.

尽管不同物种间核糖体组装的特征具有保守性，但我们对多细胞生物核糖体生成的多样性、复杂性、动态性及调控机制仍缺乏完整认知。为深入解析人类细胞中的核糖体生物发生过程，我们开展了全基因组功能丧失筛选，并结合已存在与新组装核糖体的差异标记策略。本研究通过上述工作鉴定出两个功能未被注释的基因：C1ORF09与SPATA5。我们证实，这些因子与CINP、SPATA5L1协同作用，调控人类细胞质内60S前体成熟过程（pre-60S）的晚期步骤。敲除C1ORF109或SPATA5均会损害细胞的全局蛋白质合成过程。本研究结果将核糖体组装与携带隐性SPATA5突变的神经发育障碍疾病建立了关联。基于上述发现，我们提出：核糖体生物发生因子的扩展库使得多细胞生物能够协调核糖体生成的多个步骤，以响应不同发育与环境刺激。
实验整体设计：在携带内源性RPL28-SNAP标签标记的HEK293T细胞中，针对核糖体生物发生/周转因子开展了全基因组规模的CRISPR/Cas9介导的功能丧失筛选。以红色与绿色荧光对已存在与新生成的核糖体进行差异标记，通过荧光激活细胞分选（FACS）富集目标细胞群。