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. 2004 Aug;24(16):7024-31.
doi: 10.1128/MCB.24.16.7024-7031.2004.

Expression of telomerase RNA template, but not telomerase reverse transcriptase, is limiting for telomere length maintenance in vivo

Affiliations

Expression of telomerase RNA template, but not telomerase reverse transcriptase, is limiting for telomere length maintenance in vivo

Y Jeffrey Chiang et al. Mol Cell Biol. 2004 Aug.

Abstract

Telomerase consists of two essential components, the telomerase RNA template (TR) and telomerase reverse transcriptase (TERT). The haplo-insufficiency of TR was recently shown to cause one form of human dyskeratosis congenita, an inherited disease marked by abnormal telomere shortening. Consistent with this finding, we recently reported that mice heterozygous for inactivation of mouse TR exhibit a similar haplo-insufficiency and are deficient in the ability to elongate telomeres in vivo. To further assess the genetic regulation of telomerase activity, we have compared the abilities of TR-deficient and TERT-deficient mice to maintain or elongate telomeres in interspecies crosses. Homozygous TERT knockout mice had no telomerase activity and failed to maintain telomere length. In contrast, TERT(+/-) heterozygotes had no detectable defect in telomere elongation compared to wild-type controls, whereas TR(+/-) heterozygotes were deficient in telomere elongation. Levels of TERT mRNA in heterozygous mice were one-third to one-half the levels expressed in wild-type mice, similar to the reductions in telomerase RNA observed in TR heterozygotes. These findings indicate that both TR and TERT are essential for telomere maintenance and elongation but that gene copy number and transcriptional regulation of TR, but not TERT, are limiting for telomerase activity under the in vivo conditions analyzed.

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Figures

FIG. 1.
FIG. 1.
Generation of mTERT−/− mice by gene targeting. +/+, mTERT+/+ mice; +/−, mTERT+/− mice; −/−, mTERT−/− mice. (A) Gene targeting strategy and restriction map of the mTERT gene. The black boxes represent the exons of the mTERT gene. GFP and neomycin genes were inserted into exon 1 5′ of the translation initiation codon ATG. E, EcoRI; X, XbaI; EX1, exon 1; EX2, exon 2; B, BamHI; H, HindIII; TK, thymidine kinase. (B) Southern blot analysis of mouse tail DNA. The 7- and 9.3-kb bands represent the germ line and target allele, respectively. (C) PCR genotype of mTERT knockout mice. The wild-type band is 150 bp; the knockout band of an mTERT knockout mouse is 420 bp. (D) Reverse transcription-PCR analysis of mTERT gene expression. 5′mTERT represents the reverse transcription-PCR product from the mTERT658F and mTERT950R primers. 3′mTERT represents the reverse transcription-PCR product from the mTERT2646F and mTERT2952R primers. (E) TRAP assay for telomerase activity. Cell lysates were prepared from testes of mTERT+/+ (wild-type [WT]), mTERT+/−, and mTERT−/− (knockout [KO]) mice. Telomerase activity is shown for each type of mouse for (from left to right) 90, 30, 10, and 3.3 ng of lysate. ▵, serial dilutions of cell lysates made before TRAP analysis; ○, protein samples heated to 85°C for 10 min before TRAP assay; IS, internal standard.
FIG. 2.
FIG. 2.
SPRET/Ei telomeres undergo elongation in (B6 × SPRET/Ei)F1 mice. Telomeric repeat fragment distribution was analyzed by pulsed-field gel electrophoresis and in-gel hybridization of telomeric DNA from B6, SPRET/Ei (Sp), and (B6 × SPRET/Ei)F1 (F1) mice. The boxed area represents elongated telomeres of parental SPRET/Ei origin. MK, molecular size marker.
FIG. 3.
FIG. 3.
Telomere maintenance and elongation in [(B6 × SPRET/Ei) × B6] mice is dependent upon the expression of both mTR and mTERT. Q-FISH frequency distributions of telomere signals in SPRET/Ei mice (A), mTERT+/+ mice [(B6 × SPRET/Ei) × B6] (B), mTERT−/− mice [(B6 × SPRET/Ei) × B6] (C), mTR+/+ mice [(B6 × SPRET/Ei) × B6] (D), and mTR−/− mice [(B6 × SPRET/Ei) × B6] (E) are shown. All Q-FISH data represent the analysis of at least 10 metaphases and 800 telomeres for mTR mutant mice and 20 metaphases and 1,600 telomeres for mTERT mutant mice.
FIG. 4.
FIG. 4.
Telomere elongation is deficient in mTR+/− but not in mTERT+/− (B6 × SPRET/Ei)F1 mice. Q-FISH frequency distributions of telomere signals from mTR+/+ mice (B6 × SPRET/Ei)F1 (A), mTR+/− mice (B6 × SPRET/Ei)F1 (B), mTERT+/+ mice (B6 × SPRET/Ei)F1 (C), and mTERT+/− mice (B6 × SPRET/Ei)F1 (D) are shown. All Q-FISH data represent the analysis of at least 10 metaphases and 800 telomeres for mTR mutant mice and 20 metaphases and 1,600 telomeres for mTERT mutant mice.
FIG. 5.
FIG. 5.
mTERT mRNA expression is reduced in mTERT+/− mice. Relative mRNA units were calculated from a standard curve. (A) Open circles represent mTERT mRNA from mTERT+/+ (B6 × SPRET/Ei)F1 mice, and filled circles represent mTERT mRNA from mTERT+/− (B6 × SPRET/Ei)F1 mice. (B) Open squares represent mGAPDH mRNA from mTERT+/+ (B6 × SPRET/Ei)F1 mice, and filled squares represent mGAPDH mRNA from mTERT+/− (B6 × SPRET/Ei)F1 mice.
FIG. 6.
FIG. 6.
SPRET/Ei telomeres undergo the same elongation in wild-type and mTERT-transgenic B6 or (B6 × SPRET/Ei)F1 mice. Telomeric repeat fragment distribution was analyzed by pulsed-field gel electrophoresis and in-gel hybridization of telomeric DNA from SPRET/Ei (Sp), B6, and (B6 × SPRET/Ei)F1 mice with a wild-type genotype (WT) and/or the mTERT-transgene (+). The results shown are representative of those from six mTERT-transgenic (B6 × SPRET/Ei)F1 mice from two independent transgenic lines. The boxed area represents elongated telomeres of parental SPRET/Ei origin. MK, molecular size marker; Tg, transgene.

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