Low abundance of telomerase in yeast: Implications for telomerase haploinsufficiency
Low abundance of telomerase in yeast: Implications for telomerase haploinsufficiency
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DOI：
10.1261/rna.134706
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发表时间：
2006-09-01
期刊：
影响因子：
4.5
通讯作者：
Cech, Thomas R.
中科院分区：
文献类型：
--
作者：
Mozdy, Amy D.;Cech, Thomas R.
端粒酶是一种RNA依赖的逆转录酶，其维持端粒DNA在物种特异性平衡长度。为了确定酿酒酵母细胞中端粒酶分子数量的上限，我们建立了实时RT-PCR测定来定量非编码端粒酶RNA，TLC 1。我们发现，在一个单倍体酵母细胞中的TLC 1分子的数目为29，小于染色体末端的数目（64）在晚期S期。野生型二倍体细胞含有37个端粒酶RNA，而对于无效tlc 1等位基因杂合的二倍体具有野生型量的一半，19个TLC 1分子。为了比较，每个单倍体细胞有480个U2 snRNA分子。我们发现，这种低水平的端粒酶的生物学后果是单倍不足：TLC 1/TLC 1 Delta杂合子保持较短的端粒。缺失端粒DNA合成模板的显性负性端粒酶RNA进一步证明酵母端粒长度对端粒酶剂量敏感。tlc 1 δ模板的六倍过表达建立了一个新的端粒长度设定点，比野生型短160 bp。从tlc 1D模板RNA中去除端粒酶蛋白相互作用位点减轻了显性负效应，表明tlc 1D模板RNA与野生型TLC 1竞争有限的端粒酶蛋白或端粒。由于酵母端粒酶是拴在染色体末端，它可能被其端粒DNA底物的数量超过的发现提供了一个新的视角来解释端粒维持研究的结果。
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; 29, less than the number of chromosome ends ( 64) in late S-phase. Wild-type diploid cells contain; 37 telomerase RNAs, while diploids heterozygous for a null tlc1 allele have half the wild-type amount,; 19 TLC1 molecules. For comparison, there are; 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/tlc1 Delta 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 tlc1 Delta template establishes a new telomere length set point,; 160 bp shorter than wild type. Removing telomerase protein-interaction sites from the tlc1Dtemplate RNA mitigates the dominant-negative effect, suggesting that the tlc1Dtemplate 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.