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. 1998 Oct 15;12(20):3286-300.
doi: 10.1101/gad.12.20.3286.

Specific Telomerase RNA Residues Distant From the Template Are Essential for Telomerase Function

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Free PMC article

Specific Telomerase RNA Residues Distant From the Template Are Essential for Telomerase Function

J Roy et al. Genes Dev. .
Free PMC article

Abstract

The reverse transcriptase telomerase is a ribonucleoprotein complex that adds telomeric repeats to chromosome ends, using a sequence within its endogenous RNA component as a template. Although templating domains of telomerase RNA have been studied in detail, little is known about the roles of the remaining residues, particularly in yeast. We examined the functions of nontemplate telomerase residues in the telomerase RNA of budding yeast Kluyveromyces lactis. Although approximately half of the RNA residues were dispensable for function, four specific regions were essential for telomerase action in vivo. We analyzed the effects of mutating these regions on in vivo function, in vitro telomerase activity, and telomerase RNP assembly. Deletion of two regions resulted in synthesis of stable RNAs that appeared unable to assemble into a stable RNP. Mutating a region near the 5' end of the RNA allowed RNP assembly but abolished enzymatic activity. Mutations in another specific small region of the RNA led to an inactive telomerase RNP with an altered RNA conformation.

Figures

Figure 1
Figure 1
Deletion of TER1 results in telomere shortening, which is complemented by TER1–BclI. Southern blots of yeast genomic DNA showing telomeric profiles from wild-type strain 7B520, ΔTER1, or derivatives thereof. DNAs are digested with EcoRI (A) or in pairs of digestions with EcoRI (R) and EcoRI plus BclI (R/B) (B,C) and probed with radiolabeled oligonucleotide probes for telomeric repeats as indicated. The wild-type (WT) probe (oligonucleotide Klac 1–25) hybridizes to wild-type and mutant repeats. The BclI probe (oligonucleotide KTelBcl) hybridizes only to BclI-marked repeats. The same filter was probed first with the BclI probe (C) and subsequently with the wild-type probe (B). DNA markers in kilobases are indicated to the left of each panel. All 12 telomeres are visualized. The band around 1 kb contains seven telomeres and the band just above 1.6 kb contains two telomeres. The telomeric fragment marked with an asterisk in B contains a BclI restriction site between the subtelomeric EcoRI site and the telomere end. The arrowheads demonstrate examples of telomeric fragment shortening with BclI digestion. Each passage represents ∼25 cell doublings.
Figure 2
Figure 2
A schematic diagram of the TER1 RNA. The template region is contained within residues 435 and 464. The boxed areas represent regions removed or substituted in mutant alleles.
Figure 3
Figure 3
Telomeric profile of genomic DNA from the ΔTER1 strain transformed with control pHIS3 and pTERBclI plasmids (A,B) or fully functional TER1 deletion mutants (C,D). Each pair of lanes contains DNA digested with EcoRI (R) and EcoRI plus BclI (R/B) enzymes, respectively. Each passage represents ∼25 doublings after the loss of the pTERWT plasmid. The DNAs in C and D were prepared from cells after five passages. The same filters were probed first with the BclI probe (B,D) and subsequently with the wild-type probe (A,C). The asterisk in B is a nonspecific band.
Figure 3
Figure 3
Telomeric profile of genomic DNA from the ΔTER1 strain transformed with control pHIS3 and pTERBclI plasmids (A,B) or fully functional TER1 deletion mutants (C,D). Each pair of lanes contains DNA digested with EcoRI (R) and EcoRI plus BclI (R/B) enzymes, respectively. Each passage represents ∼25 doublings after the loss of the pTERWT plasmid. The DNAs in C and D were prepared from cells after five passages. The same filters were probed first with the BclI probe (B,D) and subsequently with the wild-type probe (A,C). The asterisk in B is a nonspecific band.
Figure 4
Figure 4
Steady-state levels of wild-type and mutant TER1 RNAs. Northern blot of RNA extracted from the ΔTER1 strain expressing pHIS3, TER1–BclI or deletion mutants of TER1 and probed with a TER1 DNA probe (top) or a probe for RP59 mRNA (bottom).
Figure 5
Figure 5
BclI repeat incorporation and telomere shortening in partially functional TER1 mutants. Telomeric profile of DNAs prepared from the fifth passage of the ΔTER1 strain expressing TER1–BclI or partially functional TER1 alleles alone (A,B) or together with the pTERWT plasmid (C). Each pair of lanes contains DNA digested with EcoRI (R) and EcoRI plus BclI (R/B) enzymes, respectively. The same filter was probed first with the BclI probe (B) and subsequently with the WT probe (A). The asterisk in B and C is a nonspecific band.
Figure 5
Figure 5
BclI repeat incorporation and telomere shortening in partially functional TER1 mutants. Telomeric profile of DNAs prepared from the fifth passage of the ΔTER1 strain expressing TER1–BclI or partially functional TER1 alleles alone (A,B) or together with the pTERWT plasmid (C). Each pair of lanes contains DNA digested with EcoRI (R) and EcoRI plus BclI (R/B) enzymes, respectively. The same filter was probed first with the BclI probe (B) and subsequently with the WT probe (A). The asterisk in B and C is a nonspecific band.
Figure 6
Figure 6
Overexpression of a partially functional TER1 mutant restores telomere length. (A,B) Telomeric profile of DNAs prepared from the fifth passage of the ΔTER1 strain expressing TER1 alleles on plasmids as indicated. The cells were maintained in medium containing galactose as the carbon source. Each pair of lanes contains DNA digested with EcoRI (R) and EcoRI plus BclI (R/B) enzymes, respectively. The blots are probed with the BclI probe. (C) Northern blot of RNA prepared from the ΔTER1 strain expressing TER1 alleles on plasmids. The cells were grown in galactose-containing medium. The blot is probed with radiolabeled TER1 DNA. Equal amounts of RNA were loaded in each lane.
Figure 7
Figure 7
BclI repeats are not incorporated in telomeres of nonfunctional TER1 mutants. Telomeric profiles of DNAs prepared from the ΔTER1 strain expressing TER1–BclI or TER1 deletion alleles, as indicated. Each pair of lanes contains DNA digested with EcoRI (R) and EcoRI plus BclI (R/B) enzymes, respectively. Each passage represents ∼25 doublings after the loss of the pTERWT plasmid. The same filter was probed first with the BclI probe (A) and subsequently with the wild-type (WT) probe (B).
Figure 8
Figure 8
Mutations in a small region of TER1 abolish telomerase function in vivo. (A) Predicted secondary structure of the TER1 RNA between residues 901 and 1054. (B,C) Telomeric profiles of DNAs prepared from the 10th passage of the ΔTER1 strain expressing TER1 deletion alleles. Each pair of lanes contains DNA digested with EcoRI (R) and EcoRI plus BclI (R/B) enzymes, respectively. The same filter was probed first with the BclI probe (C) and subsequently with the wild-type (WT) probe (B).
Figure 9
Figure 9
(A,B) Nonfunctional TER1 mutants contain undetectable telomerase activity in vitro. DEAE fractions of extracts from strains expressing TER1 alleles were assayed for K. lactis telomerase activity in vitro with primer KL13(12). The sequence of the TER1 templating domain and the predicted alignment of the primer are shown. The boxed template residue corresponds to the site of the A → G mutation in the TER1–BclI strains examined. Terminal transferase-labeled KL13(12) primer is shown in M lanes, and the positions of the +1 products are marked correspondingly. (A) Telomerase reactions with DEAE fractionated extracts were carried out with all four dNTPs. RNase pretreatment (lanes 2,4,6) consisted of incubation of extracts with 10 μg/ml RNase A at 25°C for 5 min. Mid-template products are denoted with brackets, and near-terminal products are marked with arrowheads. A background ladder of RNase A insensitive bands was detected in lanes 5 and 6 and is most likely caused by contaminating polymerases in the fractions assayed. The asterisk marks a nontelomerase generated background band described previously (Fulton and Blackburn 1998). (B) Reactions with DEAE fractionated extracts from TER1 (lane 1), TER1–Bcl (lane 2), ter1–sb980–987loop (lane 3), ter1-Δ630–730 (lane 4), ter1-Δ20–60 (lane 5), and ter1-Δ493–580 (lane 6) strains were carried out as in A but with ddTTP substituted for dTTP. (C,D) Profiles of TER1 RNA-containing complexes in wild-type and mutant cell extracts. DEAE fractions of extracts from strains expressing TER1 alleles were fractionated on nondenaturing gels and probed with a radio-labeled TER1 DNA probe. The gel in C was run for 13 hr, whereas the gel in D was run for 10 hr.
Figure 9
Figure 9
(A,B) Nonfunctional TER1 mutants contain undetectable telomerase activity in vitro. DEAE fractions of extracts from strains expressing TER1 alleles were assayed for K. lactis telomerase activity in vitro with primer KL13(12). The sequence of the TER1 templating domain and the predicted alignment of the primer are shown. The boxed template residue corresponds to the site of the A → G mutation in the TER1–BclI strains examined. Terminal transferase-labeled KL13(12) primer is shown in M lanes, and the positions of the +1 products are marked correspondingly. (A) Telomerase reactions with DEAE fractionated extracts were carried out with all four dNTPs. RNase pretreatment (lanes 2,4,6) consisted of incubation of extracts with 10 μg/ml RNase A at 25°C for 5 min. Mid-template products are denoted with brackets, and near-terminal products are marked with arrowheads. A background ladder of RNase A insensitive bands was detected in lanes 5 and 6 and is most likely caused by contaminating polymerases in the fractions assayed. The asterisk marks a nontelomerase generated background band described previously (Fulton and Blackburn 1998). (B) Reactions with DEAE fractionated extracts from TER1 (lane 1), TER1–Bcl (lane 2), ter1–sb980–987loop (lane 3), ter1-Δ630–730 (lane 4), ter1-Δ20–60 (lane 5), and ter1-Δ493–580 (lane 6) strains were carried out as in A but with ddTTP substituted for dTTP. (C,D) Profiles of TER1 RNA-containing complexes in wild-type and mutant cell extracts. DEAE fractions of extracts from strains expressing TER1 alleles were fractionated on nondenaturing gels and probed with a radio-labeled TER1 DNA probe. The gel in C was run for 13 hr, whereas the gel in D was run for 10 hr.
Figure 9
Figure 9
(A,B) Nonfunctional TER1 mutants contain undetectable telomerase activity in vitro. DEAE fractions of extracts from strains expressing TER1 alleles were assayed for K. lactis telomerase activity in vitro with primer KL13(12). The sequence of the TER1 templating domain and the predicted alignment of the primer are shown. The boxed template residue corresponds to the site of the A → G mutation in the TER1–BclI strains examined. Terminal transferase-labeled KL13(12) primer is shown in M lanes, and the positions of the +1 products are marked correspondingly. (A) Telomerase reactions with DEAE fractionated extracts were carried out with all four dNTPs. RNase pretreatment (lanes 2,4,6) consisted of incubation of extracts with 10 μg/ml RNase A at 25°C for 5 min. Mid-template products are denoted with brackets, and near-terminal products are marked with arrowheads. A background ladder of RNase A insensitive bands was detected in lanes 5 and 6 and is most likely caused by contaminating polymerases in the fractions assayed. The asterisk marks a nontelomerase generated background band described previously (Fulton and Blackburn 1998). (B) Reactions with DEAE fractionated extracts from TER1 (lane 1), TER1–Bcl (lane 2), ter1–sb980–987loop (lane 3), ter1-Δ630–730 (lane 4), ter1-Δ20–60 (lane 5), and ter1-Δ493–580 (lane 6) strains were carried out as in A but with ddTTP substituted for dTTP. (C,D) Profiles of TER1 RNA-containing complexes in wild-type and mutant cell extracts. DEAE fractions of extracts from strains expressing TER1 alleles were fractionated on nondenaturing gels and probed with a radio-labeled TER1 DNA probe. The gel in C was run for 13 hr, whereas the gel in D was run for 10 hr.
Figure 9
Figure 9
(A,B) Nonfunctional TER1 mutants contain undetectable telomerase activity in vitro. DEAE fractions of extracts from strains expressing TER1 alleles were assayed for K. lactis telomerase activity in vitro with primer KL13(12). The sequence of the TER1 templating domain and the predicted alignment of the primer are shown. The boxed template residue corresponds to the site of the A → G mutation in the TER1–BclI strains examined. Terminal transferase-labeled KL13(12) primer is shown in M lanes, and the positions of the +1 products are marked correspondingly. (A) Telomerase reactions with DEAE fractionated extracts were carried out with all four dNTPs. RNase pretreatment (lanes 2,4,6) consisted of incubation of extracts with 10 μg/ml RNase A at 25°C for 5 min. Mid-template products are denoted with brackets, and near-terminal products are marked with arrowheads. A background ladder of RNase A insensitive bands was detected in lanes 5 and 6 and is most likely caused by contaminating polymerases in the fractions assayed. The asterisk marks a nontelomerase generated background band described previously (Fulton and Blackburn 1998). (B) Reactions with DEAE fractionated extracts from TER1 (lane 1), TER1–Bcl (lane 2), ter1–sb980–987loop (lane 3), ter1-Δ630–730 (lane 4), ter1-Δ20–60 (lane 5), and ter1-Δ493–580 (lane 6) strains were carried out as in A but with ddTTP substituted for dTTP. (C,D) Profiles of TER1 RNA-containing complexes in wild-type and mutant cell extracts. DEAE fractions of extracts from strains expressing TER1 alleles were fractionated on nondenaturing gels and probed with a radio-labeled TER1 DNA probe. The gel in C was run for 13 hr, whereas the gel in D was run for 10 hr.

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