Low abundance of telomerase in yeast imp ... r telomerase haploinsufficiency

Telomerase is an RNA-dependent reverse transcriptase that maintains telomeric DNA at a species-specific equilibrium length. To determine an upper limit for the number of telomerase molecules in a Saccharomyces cerevisiae cell, we have established real-time RT-PCR assays to quantify the noncoding telomerase RNA, TLC1. We find that the number of TLC1 molecules in a haploid yeast cell is approximately 29, less than the number of chromosome ends (64) in late S-phase. Wild-type diploid cells contain approximately 37 telomerase RNAs, while diploids heterozygous for a null tlc1 allele have half the wild-type amount, approximately 19 TLC1 molecules. For comparison, there are approximately 480 molecules of the U2 snRNA per haploid cell. We show that a biological consequence of this low level of telomerase is haploinsufficiency: A TLC1/tlc1Delta heterozygote maintains shorter telomeres. A dominant-negative telomerase RNA, with a deletion of the template for telomeric DNA synthesis, further demonstrates that yeast telomere length is sensitive to telomerase dosage. Sixfold overexpression of tlc1Deltatemplate establishes a new telomere length set point, approximately 160 bp shorter than wild type. Removing telomerase protein-interaction sites from the tlc1Deltatemplate RNA mitigates the dominant-negative effect, suggesting that the tlc1Deltatemplate RNA competes with wild-type TLC1 for a limited supply of telomerase proteins or for telomeres. Because yeast telomerase is tethered at chromosome ends, the finding that it may be outnumbered by its telomeric DNA substrates provides a new perspective for interpreting the results of telomere maintenance studies.