Effect of the premature stop codon (PTC) on mRNA stability and the synthesis rate. (A) Scheme of the two plasmidic gene fusions used for these experiments. The GAL1 promoter directs the synthesis of an mRNA that contains the yeast PHO5 and E. coli lacZ open reading frames fused in-frame until the natural lacZ stop (bottom), or containing a PTC between the two ORFs (top). The CYC1 terminator is placed at the end of constructs. The three probes (1–3) used for transcription run-on (TRO) and RNA polymerase II chromatin immunoprecipitation (ChIP) with the anti-Rbp3 antibody are shown. (B) The mRNA half-life (HL) was determined by the transcription shut-off of the GAL1 promoter by changing cells from galactose to glucose-containing media, and a northern blot of the extracted RNAs, which was successively tested with probes PHO5, GAL1, and 18S rRNA. Note that the presence of a PTC (pink bars) provokes a one-third reduction in the HL in the fusion transcript, but no decrease in the endogenous GAL1 mRNA in the wild-type cells. Conversely, the PTC-containing fusion transcript was significantly stabilized in an upf1 mutant. The greater stability of the PTC-containing transcript versus the noncontaining one in upf1Δ could be related to the much shorter translated region of the former. The shown HL values correspond to the average of at least three independent experiments. (C) The TRO experiment with the wild-type strain shows no significant difference in elongating RNA pol II (SR) in the fusion genes containing (pink bars) a PTC, or not (blue bars). Values were presented after normalizing to the plasmid copy number measured by Q-PCR. The results were similar using probes 1–3. (D) ChIP using anti-Rpb3 shows similar results as the TRO experiment. (E) The TRO experiment in the upf1 mutant shows that there is no significant difference in elongating RNA pol II (SR) on fusion genes containing (pink bars), or not (blue bars), a PTC. In fact the SR is slightly lower in the PTC-containing mRNA, but not statistically significant. The results were similar using probes 1–3. (F) ChIP using anti-Rpb3 in the upf1 strain gave similar results to the TRO experiment. Bars represent the average and SD of three independent replicates of the experiment. The statistical significance of the differences between the averages of the indicated samples was estimated using a two-tailed Student's t-test ([*] indicates P < 0.01; [***] indicates P < 0.0001).
