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First published online 7 October 2008
doi: 10.1242/jcs.033308

Journal of Cell Science 121, 3553-3560 (2008)
Published by The Company of Biologists 2008
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Hypermethylation of yeast telomerase RNA by the snRNA and snoRNA methyltransferase Tgs1

Jacqueline Franke*, Jessica Gehlen and Ann E. Ehrenhofer-Murray{ddagger}

Zentrum für Medizinische Biotechnologie, Universität Duisburg-Essen, Universitätsstr. 5, 45117 Essen, Germany


Figure 1
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  		Fig. 1. m3G cap formation of TLC1 is dependent on Tgs1. (A) Whole cell extracts were prepared from wild-type and tgs1{Delta} cells. After precipitation using an antibody against the m3G structure, RNA was extracted and analyzed by northern blotting. For hybridization, the random primed [{alpha} 32P]dCTP-labeled TLC1 DNA was used as a probe. Input (10%), anti-m3G immunoprecipitates (RNA immunoprecipitates of the m3G antibody; 90%) and supernatant (unbound RNA; 25%) are shown. Duplicates of immunoprecipitates and supernatant were loaded. (B) Telomeres are elongated in the absence of Tgs1. Genomic DNA was prepared from wild-type and tgs1{Delta} cells and digested with XhoI. DNA fragments were analyzed by Southern blotting. As a probe, a CA-rich fragment of telomere VII was labeled with [{alpha} 32P]dCTP. Two wild-type and three tgs1{Delta} genomic DNA preparations are shown. Black and white arrows indicate the average telomere length in the WT and tgs1 mutant, respectively. (C) Telomeres are elongated in the catalytically inactive tgs1-W178A mutant. Genomic DNA was analyzed as in B. Black and white arrows indicate the average telomere length in the WT and tgs1 mutant, respectively.

 

Figure 2
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  		Fig. 2. Loss of Tgs1 affects the telomere length of several mutants involved in telomere length regulation. Genomic DNA from wild-type, rap1-12 (AEY567) and rap1-12 tgs1{Delta} cells (AEY3593) (A), rif2{Delta} (AEY3578), rif2{Delta} tgs1{Delta} (AEY3611) and wild-type cells (B), ku70{Delta} (AEY1507), wild-type and ku70{Delta} tgs1{Delta} (AEY3573) cells (C) and wild-type, sir4{Delta} (AEY21) and sir4{Delta} tgs1{Delta} (AEY3567) cells (D). Cells were grown at 30°C. DNA was probed with a CA-rich fragment of telomere VII labeled with [{alpha} 32P]dCTP as in Fig. 1B.

 

Figure 3
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  		Fig. 3. The absence of Tgs1 causes increased silencing at telomeres. (A) Wild-type (AEY3595), tgs1{Delta} (AEY3591), tgs1-W178A (AEY3793), rap1-12 (AEY3590) and rap1-12 tgs1{Delta} (AEY3594) were grown on rich medium for 3 days. (B) Quantification of white and red-sectoring colonies from the strains used in A. Less sectoring is indicative of increased silencing of the telomeric ADE2 reporter gene.

 

Figure 4
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  		Fig. 4. Single-stranded telomeric DNA accumulates in tgs1{Delta} cells. Genomic DNA was prepared in triplicate from wild-type and tgs1{Delta} cells, digested with XhoI and analyzed by non-denaturing (A) and denaturing (B) Southern blot analysis. WTd, DNA boiled for 10 minutes after digestion; positive control for single-stranded DNA hybridization.

 

Figure 5
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  		Fig. 5. tgs1{Delta} affects the coupling of telomerase to the DNA replication machinery. (A) tgs1{Delta} exacerbates the effect of cdc13-5 and stn1-13. (B) tgs1{Delta} partially suppresses the effect of pol1-17 and pol12-216. Cells were grown at 30°C. Analysis of telomere length was performed as in Fig. 1B.

 

Figure 6
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  		Fig. 6. tgs1{Delta} and tgs1-W178A stabilize artificially induced telomeric recombination. AEY3575, AEY3576, AEY3616 and AEY3791 (top to bottom) were grown overnight at 30°C in YPD. Diluted cells were plated on YPD and incubated at 30°C for 3 days.

 

Figure 7
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  		Fig. 7. tgs1{Delta}, tgs1-W178A and pol12-216 had a shortened replicative lifespan. Forty virgin mother cells of wild-type and tgs1{Delta} strains (A) and wild-type, tgs1-W178A and pol12-216 (B) were plated on YPD at 30°C. Daughter cells were separated by micromanipulation. The percentage of survival of the mother cells after each generation was scored.

 

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© The Company of Biologists Ltd 2008