Reconstitution of the RNAi response in human cells using drosophila gene products. Homo sapiens

While mammalian somatic cells are incapable of mounting an effective RNA interference (RNAi) response to viral infections, plants and invertebrates are able to generate high levels of functional short interfering RNAs (siRNAs) of viral origin that can effectively control many infections. In Drosophila, the RNAi response is mediated by the Dicer 2 enzyme (dDcr2) acting in concert with two co-factors called Loqs-PD and R2D2. To examine whether a functional RNAi response could be reconstituted in human somatic cells by expression of these insect proteins, we expressed dDcr2, in the presence or absence of Loqs-PD and/or R2D2, in a previously described human cell line, NoDice/ΔPKR, that lacks functional forms of both the human Dicer (dcr) and EIF2AK2 (pkr) gene. Upon expression of dDcr2, Loqs-PD and R2D2 in these human cells, we observed the production of ~21-nt long siRNAs, derived from a co-transfected double stranded RNA (dsRNA) expression vector, that were loaded into the human RNA-induced silencing complex (RISC) and were able to significantly reduce the expression of a cognate indicator gene. We conclude that it is possible to at least partly rescue the ability of mammalian somatic cells to express functional siRNAs by using gene products of invertebrate origin. Overall design: Combinations of Drosophila Dicer2 and cofactors were transfected into 425-PKR cells to determine whether they were sufficient for genesis of siRNAs in mammalian cells.

尽管哺乳动物体细胞无法对病毒感染产生有效的RNA干扰（RNA interference, RNAi）应答，但植物与无脊椎动物能够生成大量源自病毒的功能性小干扰RNA（short interfering RNAs, siRNAs），可有效抑制多种病毒感染。在果蝇中，RNAi应答由Dicer 2酶（dDcr2）介导，该酶需与两种辅因子Loqs-PD及R2D2协同发挥作用。为探究能否通过表达这些昆虫蛋白在人类体细胞中重建功能性RNAi应答，我们在既往已报道的、同时缺失功能性人类Dicer（dcr）与EIF2AK2（pkr）基因的人类细胞系NoDice/ΔPKR中，分别或联合表达dDcr2、Loqs-PD及R2D2。在该细胞系中表达dDcr2、Loqs-PD及R2D2后，我们观察到由共转染的双链RNA（double stranded RNA, dsRNA）表达载体产生的约21 nt长度的siRNA，这些siRNA可加载至人类RNA诱导沉默复合物（RNA-induced silencing complex, RISC）中，并能显著降低同源报告基因的表达水平。综上，我们证实可通过引入无脊椎动物来源的基因产物，至少部分恢复哺乳动物体细胞表达功能性siRNA的能力。整体实验设计：将果蝇Dicer2及其辅因子的不同组合转染至425-PKR细胞中，以验证其是否足以在哺乳动物细胞中生成siRNA。