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. 1997 Oct 14;94(21):11190-5.
doi: 10.1073/pnas.94.21.11190.

Three Ever Shorter Telomere (EST) genes are dispensable for in vitro yeast telomerase activity

J Lingner  1 , T R Cech, T R Hughes, V Lundblad
Affiliations

Three Ever Shorter Telomere (EST) genes are dispensable for in vitro yeast telomerase activity

J Lingner et al. Proc Natl Acad Sci U S A. .

Abstract

Telomerase is a specialized reverse transcriptase consisting of both RNA and protein components. Previous characterization of yeast telomerase function in vivo identified four EST (for ever shorter telomeres) genes that, when mutated, result in the phenotypes expected for a defect in telomerase. Consistent with this genetic prediction, the EST2 gene has recently been shown to encode the catalytic component of telomerase. Using an in vitro assay, we show here that telomerase activity is present in extracts prepared from yeast strains carrying est1-Delta, est3-Delta, and cdc13-2(est) mutations. Therefore, while these three genes are necessary for telomerase function in vivo, they do not encode components essential for core catalytic activity. When Est2p, the one EST gene product found to be essential for catalytic activity, was immunoprecipitated from extracts, the telomerase RNA subunit was also specifically precipitated, supporting the conclusion that these two components are in a stable complex.

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Figures

Figure 1
Figure 1
A quadruple-mutant est1-Δ est2-Δ est3-Δ cdc13–2est strain has the same phenotype as single-mutant est strains. Haploid quadruple- and single-mutant strains were generated by dissection of DVL176, and freshly germinated haploid spores of the indicated genotype were streaked for single colonies on YEPD (yeast extract/peptone/dextrose) rich medium plates. Two successive streak-outs, differing from each other by ≈25 generations (g.), were assembled on YEPD plates and incubated for 3 days at 30°C prior to photography. A total of four to five strains for each genotype were analyzed in parallel, with similar results observed for each genotype; a representative example is shown for each.
Figure 2
Figure 2
Sedimentation of the yeast telomerase RNP from est mutant strains. Yeast extracts were fractionated by glycerol gradient sedimentation, and the yeast TLC1 RNA (Upper) and U1 snRNA (Lower, shown for the EST+ strain) were quantitated by PhosphorImager analysis following Northern blotting. The U1 snRNA distribution was consistently bimodal, which may represent the free snRNP and higher-order complexes. Marker proteins were run in a parallel gradient (alcohol dehydrogenase, 7.6S; catalase, 11.3S; apoferritin, 17.3S; and thyroglobulin, 19.3S).
Figure 3
Figure 3
Telomerase activity is present in est1-Δ, est3-Δ, and cdc13–2est strains. (A) Telomerase activity as measured in the standard reaction, in the presence of [α-32P]dTTP and dGTP. Pooled glycerol gradient fractions were concentrated, to equalize the amount of telomerase RNA in the various samples. The +1 marker was generated by terminal deoxynucleotidyltransferase extension of the same primer used in the telomerase assays with [α-33P]ddTTP. (B) Enzyme activity monitored in the presence of [α-32P]dTTP and the chain-terminator ddGTP, using the same pooled fractions as in A.
Figure 4
Figure 4
Est2p is in a complex with the yeast telomerase RNA. Immunoprecipitation of Est2p and TLC1 RNA from extracts of a HA3-tagged Est2 strain and of a control untagged Est2 strain by incubation with the anti-HA antibody. (A) Western blot, using 12CA5 antibody; lanes 1 and 2, 5 μl (35 μg) of extracts from strains carrying pVL782 (HA3-tagged Est2) or pVL369 (untagged Est2), respectively; lanes 3 and 4, immunoprecipitates derived from 550 μl of the same extracts used in lanes 1 and 2, respectively. (B) Western blot as in A; lanes 1 and 2, 10 μl (60 μg) of extracts from strains carrying pVL782 (HA3-tagged Est2) or pVL406 (untagged Est2), respectively; lanes 3 and 4, immunoprecipitates derived from 250 μl of the same extracts used in lanes 1 and 2, respectively. A longer exposure is shown for lanes 1 and 2 than for lanes 3 and 4; the ≈50-kDa band in lanes 1 and 2 is a protein that crossreacts with the 12CA5 antibody, detected on long exposures. (C) Northern blot, using TLC1 and U1 probes; lane 1, molecular mass markers; lanes 2 and 3, RNA extracted from the same extracts and extract volume as in lanes 1 and 2, part A; lanes 4 and 5, RNA extracted from the same immunoprecipitates (and same volume) as shown in lanes 3 and 4, part A. (D) Northern blot, using TLC1 and U1 probes; lanes 1 and 2, RNA extracted from the same extracts and extract volume as in lanes 1 and 2, part B; lanes 3 and 4, RNA extracted from the same immunoprecipitates (and same volume) as shown in lanes 3 and 4, part B.
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