Full length EST2 expression vectors (pJD641), EST2 mutant vectors (pJD796) and null plasmids (pJD315) were transformed into WT (JD1281), EST2 deletion (JD1287), UPF2 (JD1288) and EST2/UPF2 (JD1276) deletion strains. Total RNA was extracted with acid phenol/chloroform (pH = 4.5) from mid-logarithmic cell cultures. In parallel, total RNAs were extracted from isogenic rpl3D strains expressing wild-type RPL3 (JD1228), the down-frameshifting rpl3-R247A allele (AM-L3R247A), or the up-frameshifting rpl3-W255C/ P257S allele (JD1229). To prevent amplification from contaminating cellular DNA, RNA was treated with DNase I before reverse transcription using Turbo DNase (Ambion). cDNA was generated using the Bio-Rad iScript cDNA synthesis kit and used in the LightCycler real-time PCR system. PCR reactions were performed with 2 ml of cDNA in 20-ml reaction mixtures containing $10 nM each sense and antisense primer, and 1x LightCycler 480 SYBR Green I Master Mix (Roche). PCR cycles were run as follows: 1 cycle of 95 C for 10 min; 40 cycles of 95 C for 10 s, 54 C for 20 s and 72 C for 20 s. U3 snoRNA was chosen as a reference gene.