Yeast telomere maintenance is globally controlled by programmed ribosomal frameshifting and the nonsense-mediated mRNA decay pathway

Abstract

We have previously shown that ~10% of all eukaryotic mRNAs contain potential programmed -1 ribosomal frameshifting (-1 PRF) signals and that some function as mRNA destabilizing elements through the Nonsense-Mediated mRNA Decay (NMD) pathway by directing translating ribosomes to premature termination codons. Here, the connection between -1 PRF, NMD and telomere end maintenance are explored. Functional -1 PRF signals were identified in the mRNAs encoding two components of yeast telomerase, EST1 and EST2, and in mRNAs encoding proteins involved in recruiting telomerase to chromosome ends, STN1 and CDC13. All of these elements responded to mutants and drugs previously known to stimulate or inhibit -1 PRF, further supporting the hypothesis that they promote -1 PRF through the canonical mechanism. All affected the steady-state abundance of a reporter mRNA and the wide range of -1 PRF efficiencies promoted by these elements enabled the determination of an inverse logarithmic relationship between -1 PRF efficiency and mRNA accumulation. Steady-state abundances of the endogenous EST1, EST2, STN1 and CDC13 mRNAs were similarly inversely proportional to changes in -1 PRF efficiency promoted by mutants and drugs, supporting the hypothesis that expression of these genes is post-transcriptionally controlled by -1 PRF under native conditions. Overexpression of EST2 by ablation of -1 PRF signals or inhibition of NMD promoted formation of shorter telomeres and accumulation of large budded cells at the G2/M boundary. A model is presented describing how limitation and maintenance of correct stoichiometries of telomerase components by -1 PRF is used to maintain yeast telomere length.

Keywords: Nonsense-mediated decay; cell cycle; frameshifting; mRNA; ribosome; telomerase.

Figure 1. -1 PRF signals in the yeast EST1, EST2, STN1 and CDC13 mRNAs respond to cellular mutants (panel A) and a drug (panel B) that were previously found to promote changes in -1 PRF promoted by viral -1 PRF signals. -1 PRF was monitored using dual luciferase reporters in isogenic cells expressing RPL3, rpl3-mak8 or rpl3-R247A as the sole forms of ribosomal protein L3,^, or the CBF5 or cbf5-D95A allele of the yeast homolog of Dyskerin. All assays were performed enough times to generate meaningful statistical data. Error bars represent standard error. Note that the following the gene name denotes the specific mRNA and first nucleotide of the -1 PRF signal. This nomenclature is used throughout.

Figure 2. Delineation mathematical relationships between -1 PRF and mRNA abundance in wild-type (Panel A), and NMD-deficient cells (Panel B). The -1 PRF signals from the yeast EST1, EST2, STN1 and CDC13 mRNAs were cloned into yeast PGK1 reporters in their native orientations so that frameshift events would direct elongating ribosomes to premature termination codons (PTC). A PGK1 vector without inserted sequences was used as a control. These reporters were transformed and expressed in isogenic wild-type (A), or upf1Δ cells (B), and mRNA steady-state abundances were determined by qRT-PCR using the endogenous yeast G3PD mRNA as an internal standard. (A) Steady-state abundances of -1 PRF signal containing PGK1 reporter mRNAs/PGK1 control mRNA (i.e., no -1 PRF) in wild-type cells, fit to a logarithmic function. (B) Steady-state abundances of -1 PRF signal containing PGK1 reporter mRNAs in isogenic upf1Δ vs. UPF1 cells fit to a logarithmic function. Error bars denote standard deviation.

Figure 3. Steady-state abundances of native EST1, EST2, STN1 and CDC13 mRNAs in mutant and drug treated cells. qRT PCR was used to determine steady-state abundances of the native mRNAs using the G3PD mRNA as an internal standard. (A) mRNA abundances were monitored in isogenic UPF1 and upf1Δ upf2Δ upf3Δ cells. (B) mRNA abundances in isogenic wild-type and rpl3-mak8-1, rpl3-R247A (compared with wild-type RPL3), and cbf5-D95A (compared with CBF5) cells. (C) mRNA abundances were monitored in wild-type cells treated with the indicated concentrations of anisomycin. Error bars denote standard error.

Figure 4. Ablation of -1 PRF in EST2, or of NMD affects telomere length and promotes G2/M cell cycle arrest. (A) Southern blot of PstI digested DNAs isolated from isogenic wild-type, upf1Δ, est2Δ, or upf1Δ est2Δ cells were transformed with either an empty CEN6 low copy vector (vector), the same vector expressing the wild-type EST2 gene (pEST2), or one in which the slippery sites of all 5 tested -1 PRF signals had been silently mutated (see Table 1 and²⁴). The blot was probed using DNA oligonucleotedes complementary to telomeric repeat sequences as described. Letters along the right hand side indicate (L)ong, (I)ntermediate, and (S)hort telomeres respectively. (B) Phase contrast microscopy (100x) of cells used in panel A harvested from logarithmically growing cultures.

Figure 5. Model: telomerase recruitment to uncapped telomeres is controlled by the relative stoichiometries of telomerase components. Yeast telomeres exist in a range of states, from fully capped and full length to uncapped and short. As telomeres age, they progressively shorten, and at some point reach an intermediate, uncapped status, recruiting the MRX+Tel1p complex. Tel1p phosphorylates Cdc13p (part of the Cdc13, Stn1, Ten1 comples shown as TSC), promoting telomerase recruitment through Est1p, and inducing checkpoint arrest at the G2/M boundary. Telomerase recruitment stimulates telomere repair (up arrow on left), and releases cells from checkpoint arrest. Failure to recruit telomerase leads to further telomere shortening (down arrow on right), where they eventually resemble double-stranded breaks (DSB-like). These short telomeres also promote checkpoint arrest, but since they cannot recruit telomerase they enter crisis. Recruitment of the DSB repair machinery promotes chromosome end joining in some cells, where homologous recombination is used to amplify telomere sequences enabling checkpoint bypass. We propose that maintaining the correct stoichiometric ratios of telomerase components is critical for telomerase recruitment and telomere length homeostasis. Alteration in the expression of one of these factors, e.g., overexpression of Est2p by ablation of -1 PRF, weakly inhibits telomerase recruitment resulting in accumulation of shorter, intermediate length telomeres and accumulation of cells arrested at G2/M. Overexpression of all of these factors by inactivation of NMD has strong dominant-negative effects on telomerase recruitment, increasing the proportion of cells with short telomeres with similarly strong cell cycle effects.