The EST2 À1 RF signal at nucleotide 1653 is primarily destabilized by À1 RF induced NMD Figure 2 suggests that À1 RF induced NMD is the major cause of decreased mRNA steady-state abundance by the EST2 À1 RF signal beginning at nucleotide 1653. To confirm this, a series of time course mRNA decay assays were performed employing the PGK1-EST2 À1 RF reporter, the readthrough control, and the PTC containing construct in cells harboring the temperature sensitive rpb1-1 allele of RNA polymerase II. At the zero time point, cells were shifted to the non-permissive temperature (42 C) to arrest transcription of mRNAs, total cellular mRNAs were extracted at 0, 1, 2, 4, 8 and 16 min. subsequent to the temperature shift, and RNA blots were hybridized with the firefly luciferase and U3 snoRNA probes. While the readthrough control was stable in wild-type cells ( Figure 3A and D) , both the PTC containing control and the reporter containing the EST2 À1 RF signal promoted rapid exponential decay of the reporter mRNA, thus demonstrating that this À1 RF signal can operate as an mRNA destabilizing element ( Figure 3B-D) . In a parallel experiment using rpb1-1 upf1D cells, all of the reporter mRNAs remained stable ( Figure 3E-H) . The rapid decay kinetic profile of the EST2 À1 RF containing reporter, and its stabilization in NMD-deficient cells are consistent with NMD being the major decay pathway triggered by this element (19) . To independently test of this, the A AAA AAT slippery site was partially inactivated by mutating it to G AAG AAC. This silent mutation stabilized the reporter mRNA $19-fold compared to the wild-type slippery site in wild-type, i.e. DOM34 cells ( Figure 3I ). Interestingly, this is less than the 35-fold stabilization in upf1D cells. One would expect that, since NMD is dependent of À1 RF, then inactivation of À1 RF should be quantitatively the same as inactivation of NMD. To address this, the steady state abundance of the G AAG AAC slippery site containing PGK1 reporter was assayed in an isogenic dom34D strain ( Figure 3I, dom34D lane) . This combination increased the steady-state abundance of the reporter mRNA to near wild-type levels.