Messenger RNA (mRNA) turnover is a crucial and highly regulated step of gene expression in mammalian cells. This includes mRNA surveillance pathways such as nonsense-mediated mRNA decay (NMD), which assesses the fidelity of transcripts and eliminates mRNAs containing a premature translation termination codon (PTC). When studying mRNA degradation pathways, reporter mRNAs are commonly expressed in cultivated cells. Traditionally, the molecular mechanism of NMD has been characterized using pairs of reporter constructs that express the same mRNA with ("PTC-containing mRNA") or without ("wild-type mRNA") a PTC. Cell lines stably expressing an NMD reporter have been reported to yield very robust and highly reproducible results, but establishing the cell lines can be very time-consuming. Therefore, transient transfection of such reporter constructs is frequently used and allows analysis of many samples within a short period of time. However, the behavior of transiently and stably transfected NMD constructs has not been systematically compared so far. Here, we report that not all commonly used human cell lines degrade NMD targets following transient transfection. Furthermore, the degradation efficiency of NMD substrates can depend on the manner of transfection within the same cell line. This has substantial implications for the interpretation of NMD assays based on transient transfections.

中文翻译：

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质粒转染影响人类细胞中无意义介导的mRNA衰变报告基因测定的读数。

信使RNA（mRNA）转换是哺乳动物细胞中基因表达的关键且高度受调控的步骤。这包括mRNA监测途径，例如无义介导的mRNA衰变（NMD），它可以评估转录物的保真度并消除含有过早翻译终止密码子（PTC）的mRNA。在研究mRNA降解途径时，报告子mRNA通常在培养的细胞中表达。传统上，NMD的分子机制已通过使用成对的报告基因构建体来表征，这些构建体表达具有（带有“ PTC的mRNA”）或不带有（“野生型mRNA”）PTC的相同mRNA。据报道稳定表达NMD报告基因的细胞系可产生非常稳健和高度可重复的结果，但是建立细胞系可能非常耗时。所以，这种报告基因构建体的瞬时转染是经常使用的，并允许在短时间内分析许多样品。然而，到目前为止，尚未系统地比较瞬时和稳定转染的NMD构建体的行为。在这里，我们报道并不是所有常用的人类细胞系在瞬时转染后都会降解NMD靶标​​。此外，NMD底物的降解效率可能取决于同一细胞系内的转染方式。这对于基于瞬时转染的NMD分析的解释具有重大意义。我们报道并非所有常用的人类细胞系在瞬时转染后都能降解NMD靶标​​。此外，NMD底物的降解效率可能取决于同一细胞系内的转染方式。这对于基于瞬时转染的NMD分析的解释具有重大意义。我们报道并非所有常用的人类细胞系在瞬时转染后都能降解NMD靶标​​。此外，NMD底物的降解效率可能取决于同一细胞系内的转染方式。这对于基于瞬时转染的NMD分析的解释具有重大意义。