The EST family of yeast genes is named after their 'Ever Shortening Telomere' phenotype (30) . EST2 encodes the catalytic subunit of telomerase and the other three EST genes either encode protein subunits of telomerase (EST1 and EST3) or a telomere-associated regulator of telomerase (CDC13/EST4) (31) . Telomere elongation occurs in late S phase, although Est2p is associated to varying extents with telomeric chromatin throughout the cell cycle, and telomerase defects result in chromosome instability and rapid senescence (32) . The very low abundance EST2 mRNA is stabilized in NMD-deficient cells (33, 34) . Computational analyses revealed that EST2 contains four additional high confidence À1 RF signals beginning at positions 72, 1215, 1326 and 1995 (Supplementary Figure S1 ). The positions of the five predicted À1 RF signals in the EST2 ORF are shown in Figure 4A . Silent protein coding changes were introduced into the slippery sites of all 5 of the À1 RF signals in a full-length EST2 clone expressed from a low copy vector (pEST2ssÁ, Figure 4A ). Clones expressing either wild-type EST2 (pEST2) or pEST2ssÁ were introduced into isogenic est2D or est2D upf1D cells, and qRT-PCR analyses were performed. These silent mutations resulted $8.5-fold increase in the abundance of the full-length EST2ssÁ mRNA relative to wild-type EST2 mRNA ( Figure 4B) . Similarly, abrogation of NMD increased the abundance of the wild-type EST2 and EST2ssÁ mRNAs $5.8-fold and $7.0-fold, respectively. To independently monitor the influence of À1 RF on mRNA abundance, the steady-state abundance of the EST2 mRNA was monitored in isogenic cells expressing up-and down-frameshift promoting alleles of RPL3 (which encodes ribosomal protein L3) by qRT PCR. EST2 mRNA abundances were normalized to U3 snoRNA in cells expressing wild-type RPL3, the rpl3-R247A allele which decreases À1 RF from the L-A frameshift signal to $55% of wild-type levels (35) , and the rpl3-W255C/P247S allele which increases À1 RF by $1.6-fold (36) . Relative to wild-type cells, steady-state abundance of the EST2 mRNA was increased by 1.29 ± 0.04 fold in cells expressing rpl3-R247A, and decreased to 0.55 ± 0.02 in cells expressing rpl3-W255C/P247S ( Figure 4C ). Taken together, these experiments demonstrate that À1 RF induced NMD plays a significant role in destabilizing EST2 mRNA.