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.

Nonsense-mediated mRNA decay (NMD) is a cellular process that eliminates messenger RNA (mRNA) substrates with premature translation termination codons (PTCs). In addition, NMD regulates the expression of a number of physiological mRNAs, for example transcripts containing long 3' UTRs. Current models implicate the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and translation termination in NMD. Accordingly, PABPC1 present within close proximity of a termination codon antagonizes NMD. Here, we use reporter mRNAs with different NMD-inducing 3' UTRs to establish a general NMD-inhibiting property of PABPC1. NMD-inhibition is not limited to PABPC1, but can also be achieved by peptides consisting of the PABP-interacting motif 2 (PAM2) of different proteins when recruited to an NMD-inhibiting position of NMD reporter transcripts. The short PAM2 peptides efficiently suppress NMD activated by a long 3' UTR, an exon-junction complex (EJC) and individual EJC components, and stabilize a PTC-containing β-globin mRNA. In conclusion, our results establish short PABPC1-recruiting peptides as potent but position-dependent inhibitors of mammalian NMD.