A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting cellular host-factors that can modify prion propagation. We exposed prion-infected cells in high-density microplates to 35,364 ternary pools of 52,746 siRNAs targeting 17,582 genes representing the majority of the mouse protein-coding transcriptome. We identified 1,191 modulators of prion propagation. While 1,151 modified the expression of both the pathological prion protein, PrP^Sc, and its cellular counterpart, PrP^C, 40 genes selectively affected PrP^Sc. Of the latter 40 genes, 20 augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrP^C. Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Further, the unexpected identification of a prion-controlling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.

中文翻译：

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排列的全基因组扰动屏幕将核糖核蛋白 Hnrnpk 识别为朊病毒传播的限速器

哺乳动物朊病毒的一个决定性特征是它们自我维持繁殖的能力。理论考虑和实验证据表明，朊病毒的传播受细胞自主和非自主调节剂的调节。使用新型定量磷脂酶保护测定 (QUIPPER) 进行高通量朊病毒测量，我们进行了阵列式全基因组 RNA 干扰 (RNAi) 筛选，旨在检测可以修改朊病毒传播的细胞宿主因子。我们将高密度微孔板中的朊病毒感染细胞暴露于 52,746 种 siRNA 的 35,364 个三元池中，这些 siRNA 靶向代表小鼠蛋白质编码转录组的大部分的 17,582 个基因。我们确定了 1,191 种朊病毒传播调节剂。虽然 1,151 修饰了病理性朊病毒蛋白 PrP ^Sc的表达, 及其细胞对应物 PrP ^C , 40 个基因选择性地影响 PrP ^Sc。在后 40 个基因中，有 20 个基因在受到抑制时会增加朊病毒的产生。朊病毒传播的一个突出限制因素是异质核核糖核蛋白 Hnrnpk。Psammaplysene A (PSA) 结合 Hnrnpk，降低培养细胞中的朊病毒水平并保护它们免受细胞毒性。PSA 还降低了受感染的小脑器官切片中的朊病毒水平，并减轻了表达绵羊 PrP ^{C的受朊病毒感染的}黑腹果蝇的运动缺陷. 因此，基于全基因组 QUIPPER 的扰动可以发现参与朊病毒传播的可操作细胞通路。此外，控制朊病毒的核糖核蛋白的意外鉴定表明 RNA 在传染性朊病毒的产生中发挥作用。