An ultraconserved snoRNA-like element in long noncoding RNA CRNDE promotes ribosome biogenesis and cell proliferation (CRISPRi screen) [ACHN]

Cancer cells frequently upregulate ribosome production to support tumorigenesis. While small nucleolar RNAs (snoRNAs) are critical for ribosome biogenesis, the roles of other classes of noncoding RNAs in this process remain largely unknown. Here we performed CRISPR screens to identify essential long noncoding RNAs (lncRNAs) in renal cell carcinoma (RCC) cells. This revealed that an alternatively-spliced isoform of lncRNA Colorectal Neoplasia Differentially Expressed containing an ultraconserved element (UCE), referred to as CRNDEUCE, is required for RCC cell proliferation. CRNDEUCE localizes to the nucleolus and promotes 60S ribosomal subunit biogenesis. The UCE of CRNDE functions as an unprocessed C/D box snoRNA that directly interacts with ribosomal RNA precursors. This facilitates delivery of eIF6, a key 60S biogenesis factor, which binds to CRNDEUCE through a sequence element adjacent to the UCE. These findings highlight the functional versatility of snoRNA sequences and expand the known mechanisms through which noncoding RNAs orchestrate ribosome biogenesis. Overall design: A genome-wide CRISPRi screen was performed in ACHN cells expressing dCas9. Two independent clones were used for the screen. Samples were collected before (D0, control samples) and 12 days after prologned growth (D12).

癌细胞通常会上调核糖体生成以支持肿瘤发生。尽管小核仁RNA（small nucleolar RNAs, snoRNAs）对核糖体生物发生至关重要，但其他类别非编码RNA在该过程中的作用仍知之甚少。本研究通过CRISPR筛选，在肾细胞癌（renal cell carcinoma, RCC）细胞中鉴定必需性长非编码RNA（long noncoding RNAs, lncRNAs）。研究发现，携带超保守元件（ultraconserved element, UCE）的结直肠癌差异表达lncRNA（Colorectal Neoplasia Differentially Expressed, CRNDE）的可变剪接异构体（命名为CRNDEUCE）是肾细胞癌细胞增殖所必需的。CRNDEUCE定位于核仁，并可促进60S核糖体亚基的生物发生。CRNDE的超保守元件可作为未加工的C/D盒小核仁RNA，直接与核糖体RNA前体结合。这一过程可促进关键60S生物发生因子真核翻译起始因子6（eIF6）的递送，该因子通过UCE附近的序列元件与CRNDEUCE结合。上述研究结果凸显了小核仁RNA序列的功能多样性，并拓展了我们对非编码RNA调控核糖体生物发生的已知机制的认知。实验整体设计：在表达失活型Cas9（dCas9）的ACHN细胞中开展全基因组CRISPR干扰（CRISPRi）筛选，本次筛选使用两株独立的细胞克隆，分别在持续培养前（D0，对照样本）和持续培养12天后（D12）收集样本。