Figure 1.

Transiently transfected 293 FT cells show a strongly reduced NMD efficiency in comparison to stably transfected cells. (a) Schematic representation of the experimental setup. The reporter constructs are generated by cloning the gene of interest into the pcDNA5/FRT/TO vector. The blue bar represents the coding sequence of triosephosphate isomerase (TPI) or β-globin with or without a premature termination codon (PTC) and with a normal stop codon (stop). Downstream, a viral XRN1-resistant sequence (xrRNA) is present that allows the detection of decay intermediates resulting from 5′–3′ exonucleolytic decay. The grey boxes correspond to four repeats of a northern blot probe binding site. Cells are transfected with these constructs using either a stable or transient transfection system. Flp-In T-REx (FT) cells constitutively express a Tet repressor (TetR) that blocks transcription of the reporter by binding to Tet operators (TetO₂) present downstream of the cytomegalovirus (CMV) promoter in the reporter construct. Therefore, FT cells are induced with tetracycline(Tet)/doxycycline. In non-FT cells, the reporter is always expressed. Unstable reporter mRNAs are degraded with participation of the cellular 5′–3′ exonuclease XRN1. Full-length reporter levels and decay fragment (xrFrag) caused by stalling of XRN1 at the xrRNA are detected via northern blotting. p(A): poly(A) tail. (b,c) Expression of reporter mRNAs in HeLa FT (b) and 293 FT (c) stable cell lines was induced with doxycycline for 24 h and the reporter levels detected by northern blotting. The lower band (xrFrag) corresponds to a 5′–3′ exonucleolytic decay intermediate. Endogenous 7SL RNA levels were detected and quantified on the same blot and are shown as control. The mean values ± SD (n = 3) for relative transcript levels were quantified and the PTC values normalized to the wild-type control. The xrRNA/reporter ratio is indicated below the graph. (d,e) HeLa FT (d) and 293 FT (e) cells were transiently transfected with 3 µg plasmid DNA per 6-well and transcription was induced with doxycycline for 24 h. Reporter mRNA levels were analyzed by northern blotting. LacZ was co-expressed and serves as a transfection control. The mean values ± SD (n = 3) were quantified and the PTC values normalized to the wild-type control. The xrRNA/reporter ratio is shown below the graph.