Small molecule RBI2 disrupts ribosome biogenesis through pre-rRNA depletion

In all domains of life, the rate of protein synthesis is directly linked to the rates of cell growth and proliferation. Consequently, highly proliferative cancer cells are especially sensitive to perturbations in ribosome biogenesis. While ribosome synthesis and cancer have a well-established relationship, only recently has ribosome biogenesis drawn interest as a cancer therapeutic target. Here, we have exploited the relationship between ribosome biogenesis and cancer cell proliferation by using a potent ribosome biogenesis inhibitor: RBI2 (Ribosome Biogenesis Inhibitor 2) to perturb cancer cell growth and viability. We demonstrate that RBI2 significantly decreases cell viability in malignant melanoma cells and breast cancer cell lines. Treatment with RBI2 dramatically and rapidly decreases ribosomal RNA (rRNA) synthesis, without affecting the occupancy of RNA polymerase I (Pol I) on the ribosomal DNA template. Next-generation RNA sequencing (RNA-seq) reveals that RBI2 and previously described ribosome biogenesis inhibitor CX-5461 induce distinct changes in the transcriptome. Investigation of the content of the pre-rRNAs through RT-qPCR reveals an increase in polyadenylation of cellular rRNA after treatment with RBI2, a known pathway by which rRNA degradation occurs. Northern blotting revealed that RBI2 does not appear to impair or alter rRNA processing. Collectively, these data suggest that RBI2 inhibits rRNA synthesis distinctly from other previously described ribosome biogenesis inhibitors, potentially through a novel pathway that upregulates turnover of premature rRNAs. Overall design: MDA-MB-231 cells treated with 5 µM of RBI2 or CX-5461 as a function of time (10 minutes, 30 minutes, and 120 minutes). Two replicates were included for each time point. Another two samples were not treated and served as untreated controls

在所有生命域中，蛋白质合成速率与细胞生长及增殖速率直接相关。因此，高增殖性癌细胞对核糖体生物发生（ribosome biogenesis）的扰动尤为敏感。尽管核糖体合成与癌症之间的关联已被充分证实，但直至近年，核糖体生物发生才作为癌症治疗靶点受到关注。本研究利用核糖体生物发生与癌细胞增殖之间的关联，采用强效核糖体生物发生抑制剂RBI2（Ribosome Biogenesis Inhibitor 2）扰动癌细胞的生长与存活能力。研究表明，RBI2可显著降低恶性黑色素瘤细胞及乳腺癌细胞系的细胞存活率。RBI2处理可快速且显著抑制核糖体RNA（ribosomal RNA, rRNA）的合成，且不影响RNA聚合酶I（RNA polymerase I, Pol I）在核糖体DNA模板上的结合占有率。下一代RNA测序（RNA-seq）结果显示，RBI2与此前报道的核糖体生物发生抑制剂CX-5461可诱导转录组产生截然不同的变化。通过RT-qPCR（逆转录定量聚合酶链反应，Reverse Transcription Quantitative Polymerase Chain Reaction）对前体核糖体RNA（pre-rRNA）含量的检测发现，经RBI2处理后，细胞内rRNA的多腺苷酸化水平升高——这是已知的rRNA降解途径之一。Northern印迹（Northern blotting）实验结果显示，RBI2似乎并未损伤或改变rRNA的加工过程。综上，上述数据表明，RBI2与其他已报道的核糖体生物发生抑制剂的作用机制不同，它可通过一条上调前体rRNA周转的新型途径来抑制rRNA合成。实验整体设计：将MDA-MB-231细胞分别用5 μM的RBI2或CX-5461处理，设置不同时间梯度（10分钟、30分钟、120分钟）；每个时间点设置两个生物学重复；另外设置两组未处理的样本作为空白对照。