Abstract
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.
Highlights
The transfer of correct genetic information from the DNA to the translated protein is essential for the survival of a cell
We established inducible HeLa and HEK-293 Flp-In T-REx stable cell lines expressing triosephosphate isomerase (TPI) or globin reporter Messenger RNA (mRNA) with or without a premature translation termination codon (PTC). In both types of cells, the levels of PTCcontaining full-length reporter mRNA were markedly reduced (Fig. 1b,c), indicating that these transcripts are efficiently degraded by nonsense-mediated mRNA decay (NMD)
When we transiently transfected the TPI and globin NMD reporter constructs in HeLa Flp-In T-REx (HeLa FT) cells, the PTC-containing reporter mRNA was strongly reduced (Fig. 1d)
Summary
The transfer of correct genetic information from the DNA to the translated protein is essential for the survival of a cell. Aberrant translation termination at the PTC causes stalling of the ribosome and the assembly of a surveillance complex, which recruits RNA degradation enzymes[3]. This prevents the production of truncated proteins with a potentially dominant-negative effect[4]. In the study of NMD, reporter mRNAs with and without a PTC are utilized to monitor the degradation of transcripts in standard human cell lines such as HeLa and HEK-293 cells. Generation of stable cell lines can be laborious and especially when a high number of reporter constructs are systematically compared, transient transfections yield faster results. Human cells effectively degrade NMD targets when reporter mRNAs are expressed from extrachromosomal plasmid DNA. For the widely used HEK-293 cells we observed differential NMD efficiency depending on whether stable or transient transfections have been performed
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