Arrayed Genome-Wide Perturbation Screen Identifies Hnrnpk As Rate-Limiting for Prion Propagation

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 1191 modulators of prion propagation. While 1151 modified the expression of both the PrPSc pathological prion protein and its cellular counterpart, PrPC , 40 genes selectively affected PrPSc. 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 PrPC . 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.

哺乳动物朊病毒（prions）的标志性特征，在于其具备自我持续增殖的能力。理论推导与实验证据均表明，朊病毒的增殖过程受到细胞自主性与非自主性调控因子的调节。本研究采用一种全新的、用于高通量朊病毒检测的定量磷脂酶保护测定法（QUIPPER），开展了全基因组阵列式RNA干扰（RNAi）筛选，旨在识别可调控朊病毒增殖的宿主细胞因子。我们将高密度微孔板中的朊病毒感染细胞，暴露于由52746条小干扰RNA（siRNA）组成的35364个三联体混合池，这些siRNA靶向17582个基因，覆盖了小鼠蛋白质编码转录组的绝大多数。最终共筛选得到1191个朊病毒增殖调控因子。其中1151个调控因子可同时改变致病性朊蛋白（PrPSc）及其细胞型朊蛋白（PrPC）的表达水平，另有40个基因仅对PrPSc产生选择性调控作用。在这40个选择性调控基因中，有20个在被敲低后会促进朊病毒的生成。其中一个显著的朊病毒增殖抑制因子是异质性细胞核核糖蛋白（Hnrnpk）。可结合Hnrnpk的Psammaplysene A（PSA）可降低培养细胞中的朊病毒载量，并减轻其细胞毒性。此外，PSA还可降低感染朊病毒的小脑器官型脑片的朊病毒载量，并缓解表达绵羊源PrPC（ovine PrPC）的朊病毒感染黑腹果蝇（Drosophila melanogaster）的运动功能缺陷。综上，基于QUIPPER的全基因组扰动筛选可用于发现参与朊病毒增殖的可靶向细胞通路。此外，本次研究意外发现了一类可调控朊病毒的核糖蛋白，这表明RNA在感染性朊病毒的生成过程中发挥了一定作用。