Ferguson Chantal M, Echeverria Dimas, Hassler Matthew, Ly Socheata, Khvorova Anastasia
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Mol Ther Nucleic Acids. 2020 Sep 4;21:384-393. doi: 10.1016/j.omtn.2020.06.006. Epub 2020 Jun 12.
RNA interference (RNAi) is a potent mechanism that silences mRNA and protein expression in all cells and tissue types. RNAi is known to exert many of its functional effects in the cytoplasm, and thus, the cellular localization of target mRNA may impact observed potency. Here, we demonstrate that cell identity has a profound impact on accessibility of apolipoprotein E (ApoE) mRNA to RNAi. We show that, whereas both neuronal and glial cell lines express detectable ApoE mRNA, in neuronal cells, ApoE mRNA is not targetable by RNAi. Screening of a panel of thirty-five chemically modified small interfering RNAs (siRNAs) did not produce a single hit in a neuronal cell line, whereas up to fifteen compounds showed strong efficacy in glial cells. Further investigation of the cellular localization of ApoE mRNA demonstrates that ApoE mRNA is partially spliced and preferentially localized to the nucleus (∼80%) in neuronal cells, whereas more than 90% of ApoE mRNA is cytoplasmic in glial cells. Such an inconsistency in intracellular localization and splicing might provide an explanation for functional differences in RNAi compounds. Thus, cellular origin might have an impact on accessibility of mRNA to RNAi and should be taken into account during the screening process.
RNA干扰（RNAi）是一种强大的机制，可使所有细胞和组织类型中的mRNA和蛋白质表达沉默。已知RNAi在细胞质中发挥其许多功能作用，因此，靶mRNA的细胞定位可能会影响观察到的效力。在这里，我们证明细胞身份对载脂蛋白E（ApoE）mRNA对RNAi的可及性有深远影响。我们表明，虽然神经元和神经胶质细胞系都表达可检测到的ApoE mRNA，但在神经元细胞中，ApoE mRNA不能被RNAi靶向。对一组35种化学修饰的小干扰RNA（siRNA）进行筛选，在神经元细胞系中未产生任何有效结果，而多达15种化合物在神经胶质细胞中显示出强大的效力。对ApoE mRNA细胞定位的进一步研究表明，ApoE mRNA在神经元细胞中部分剪接并优先定位于细胞核（约80%），而在神经胶质细胞中超过90%的ApoE mRNA位于细胞质中。细胞内定位和剪接的这种不一致可能为RNAi化合物的功能差异提供解释。因此，细胞来源可能会影响mRNA对RNAi的可及性，在筛选过程中应予以考虑。
