Telomerase is a ribonucleoprotein complex that elongates telomeres, allowing the stable maintenance of chromosomes during multiple cell divisions. Here, we describe the isolation and characterization of the catalytic subunit of mouse telomerase, mTERT (mouse telomerase reverse transcriptase), an essential protein component of the telomerase complex. During embryonic development, mTERT mRNA is abundantly expressed in the whole embryo, especially in regions of intense proliferation. We found that the mTERT mRNA expression in both embryonic and adult tissues is independent of the essential RNA component of telomerase, mTR, and therefore, of the formation of active telomerase complexes. mTERT protein is present exclusively in tissues with telomerase activity, such as testis, spleen, and thymus. mTERT protein is barely detectable in the thymus of mTR ؊/؊ mice, suggesting that mTERT protein stability in this tissue may depend on the actual assembly of active telomerase complexes. Finally, we found that mouse and human telomerase catalytic subunit is located in the cell nucleus, and its localization is not regulated during cell cycle progression.

Telomere length must be tightly regulated in highly proliferative tissues, such as the lymphohematopoietic system. Under steady-state conditions, the levels and functionality of hematopoietic-committed or multipotent progenitors were not affected in late-generation telomerase-deficient mice (mTerc ؊/؊ ) with critically short telomeres. Evaluation of self-renewal potential of mTerc ؊/؊ day-12 spleen colonyforming units demonstrated no alteration as compared with wildtype progenitors. However, the replating ability of mTerc ؊/؊ granulocyte-macrophage CFUs (CFU-GMs) was greatly reduced as compared with wildtype CFU-GMs, indicating a diminished capacity of late-generation mTerc ؊ IntroductionEukaryotic chromosomes are capped by a special structure, the telomere, that in all vertebrates consists of tandem repeats of the DNA sequence TTAGGG and of associated proteins. Telomeres guarantee chromosome integrity by preventing illegitimate recombination, degradation, and end fusions. 1,2 Telomere shortening occurs in each replication cycle and is proposed to mediate replicative senescence in human cells in culture, as well as the aging process. 3,4 Telomere maintenance involves a ribonucleoprotein with reverse-transcriptase activity, called telomerase. 5,6 Telomerase is active during human embryonic development and is downregulated immediately after birth. 7,8 In adults, most normal somatic cells lack detectable telomerase activity, whereas cells from germline tissues and most tumors express high levels of telomerase activity. 9 Telomerase activity is also detected in normal human somatic tissues containing cells with self-renewal capacity, such as those of the lymphohematopoietic system 10 and the skin epithelium. 11 Hematopoiesis requires self-renewal of stem cells, as well as proliferation and differentiation of the committed progenitors. This process demands an extraordinary replicative capacity in certain cell types, especially those of the immune system. Telomeres in blood cells from bone marrow (BM) transplant recipients are shorter than those in cells from the BM donor, 12,13 suggesting that the additional cell divisions in the stem cell compartment required for BM regeneration result in a measurable decline in telomere length. Analysis of human BM cells showed that, in vitro, telomerase activity is repressed in quiescent stem cells, expressed at low levels in cycling stem cells, and up-regulated following cytokine stimulation. 10,14,15 Moreover, cytokine-induced differentiation of CD34 ϩ cells results in a decrease in telomerase activity. 16 In murine fetal liver and adult BM, results based on single-cell analysis 17 showed that the majority of long-term reconstituting BM hematopoietic stem cells (HSCs) and transiently self-renewing multipotent progenitors exhibit telomerase activity.Mice genetically deficient for the mouse telomerase RNA (mTerc) gene lack telomerase activity and show telomere shortening at a rate of 4 to 5 kilobases (kb) per mouse generation. 7,18 This shortening is accompanied by an increase in...

The telomerase holoenzyme consists of two essential components, a reverse transcriptase, TERT (telomerase reverse transcriptase), and an RNA molecule, TR (telomerase RNA, also known as TERC), that contains the template for the synthesis of new telomeric repeats. Telomerase RNA has been isolated from 32 different vertebrates, and a common secondary structure has been proposed (Chen, J.-L., Blasco, M. A., and Greider, C. W. (2000) Cell 100, 503-514). We have generated 25 mutants in the four conserved structural domains of the mouse telomerase RNA molecule, mTR, and assayed their ability to reconstitute telomerase activity in mTR ؊/؊ cells in vivo. We found that the pseudoknot and the CR4/CR5 domains are required for telomerase activity but are not essential for mTR stability in the cell, whereas mutations in the BoxH/ACA and the CR7 domains affect mTR accumulation in the cell. We have also identified mTR mutants that are able to inhibit wild type telomerase in vivo.