Telomerase RNA levels limit the telomere length equilibrium.

Small functional RNAs play essential roles in many biological processes. Regulating the level of these small RNAs can be as important as maintaining their function in cells. The telomerase RNA is maintained in cells at a steady-state level where small changes in concentration can have a profound impact on function. Cells that have half the level of the telomerase RNA cannot maintain telomeres through many cell divisions. People who are heterozygous for telomerase RNA mutations have the diseases dyskeratosis congenita and aplastic anemia, caused by short telomeres that result in loss of tissue renewal capacity. Mice heterozygous for telomerase RNA show haploinsufficiency in telomere length maintenance and also show loss of tissue renewal capacity. It is remarkable that small changes in the level of this functional RNA can have such profound effects in cells. This tight regulation highlights the importance of controlling the action of telomerase in cells.

Telomerase and cancer stem cells.

Telomerase is critical for the integrity of stem cell compartments. Mutations in telomerase components lead to telomere shortening and hematopoietic stem cell failure in autosomal dominant dyskeratosis congenita and aplastic anemia. Telomerase activity is readily detected in most cancers but not in adult somatic cells. The telomere hypothesis for cancer states that telomerase is reactivated in late stages of carcinogenesis. However, recent evidence has suggested a stem cell origin for certain cancers, implying that the genetic alterations that lead to cancer accumulate in tissue-specific stem cells and not in adult somatic cells. In these cancers, stem cells would already have telomerase and it would not need to be reactivated. Here, we reconsider the telomere hypothesis in view of this evidence and propose that, rather than telomerase reactivation, enzyme activity may increase in later stages of carcinogenesis due to increased expression or efficient assembly of telomerase components. Understanding these mechanisms will refine approaches to telomerase inhibition in cancer.

Tetrahymena proteins p80 and p95 are not core telomerase components.

Telomeres provide stability to eukaryotic chromosomes and consist of tandem DNA repeat sequences. Telomeric repeats are synthesized and maintained by a specialized reverse transcriptase, termed telomerase. Tetrahymena thermophila telomerase contains two essential components: Tetrahymena telomerase reverse transcriptase (tTERT), the catalytic protein component, and telomerase RNA that provides the template for telomere repeat synthesis. In addition to these two components, two proteins, p80 and p95, were previously found to copurify with telomerase activity and to interact with tTERT and telomerase RNA. To investigate the role of p80 and p95 in the telomerase enzyme, we tested the interaction of p80, p95, and tTERT in several different recombinant expression systems and in Tetrahymena extracts. Immunoprecipitation of recombinant proteins showed that although p80 and p95 associated with each other, they did not associate with tTERT. In in vitro transcription and translation lysates, tTERT was associated with telomerase activity, but p80 and p95 were not. p80 bound telomerase RNA as well as several other unrelated RNAs, suggesting p80 has a general affinity for RNA. Immunoprecipitations from Tetrahymena extracts also showed no evidence for an interaction between the core tTERT/telomerase RNA complex and the p80 and p95 proteins. These data suggest that p80 and p95 are not associated with the bulk of active telomerase in Tetrahymena.

The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability.

Loss of telomere function can induce cell cycle arrest and apoptosis. To investigate the processes that trigger cellular responses to telomere dysfunction, we crossed mTR-/- G6 mice that have short telomeres with mice heterozygous for telomerase (mTR+/-) that have long telomeres. The phenotype of the telomerase null offspring was similar to that of the late generation parent, although only half of the chromosomes were short. Strikingly, spectral karyotyping (SKY) analysis revealed that loss of telomere function occurred preferentially on chromosomes with critically short telomeres. Our data indicate that, while average telomere length is measured in most studies, it is not the average but rather the shortest telomeres that constitute telomere dysfunction and limit cellular survival in the absence of telomerase.

Telomere dysfunction increases mutation rate and genomic instability.

The increased tumor incidence in telomerase null mice suggests that telomere dysfunction induces genetic instability. To test this directly, we examined mutation rate in the absence of telomerase in S. cerevisiae. The mutation rate in the CAN1 gene increased 10- to 100-fold in est1Delta strains as telomeres became dysfunctional. This increased mutation rate resulted from an increased frequency of terminal deletions. Chromosome fusions were recovered from est1Delta strains, suggesting that the terminal deletions may occur by a breakage-fusion-bridge type mechanism. At one locus, chromosomes with terminal deletions gained a new telomere through a Rad52p-dependent, Rad51p-independent process consistent with break-induced replication. At a second locus, more complicated rearrangements involving multiple chromosomes were seen. These data suggest that telomerase can inhibit chromosomal instability.

Telomere dysfunction triggers developmentally regulated germ cell apoptosis.

Telomere dysfunction results in fertility defects in a number of organisms. Although data from fission yeast and Caenorhabditis elegans suggests that telomere dysfunction manifests itself primarily as defects in proper meiotic chromosome segregation, it is unclear how mammalian telomere dysfunction results in germ cell death. To investigate the specific effects of telomere dysfunction on mammalian germ cell development, we examined the meiotic progression and germ cell apoptosis in late generation telomerase null mice. Our results indicate that chromosome asynapsis and missegregation are not the cause of infertility in mice with shortened telomeres. Rather, telomere dysfunction is recognized at the onset of meiosis, and cells with telomeric defects are removed from the germ cell precursor pool. This germ cell telomere surveillance may be an important mechanism to protect against the transmission of dysfunctional telomeres and chromosomal abnormalities.

Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events.

Yeast cells can survive in the absence of telomerase RNA, TLC1, by recombination-mediated telomere elongation. Two types of survivors, type I and type II, can be distinguished by their characteristic telomere patterns. RAD52 is essential for the generation of both types of survivors. Deletion of both RAD50 and RAD51 produces a phenotype similar to that produced by deletion of RAD52. Here we examined the effects of the RAD50 and the RAD51 epistasis groups as well as the RAD52 homologue, RAD59, on the types of survivors generated in the absence of telomerase. rad59 mutations completely abolished the ability to generate type II survivors, while rad50 mutations decreased the growth viability of type II survivors but did not completely eliminate their appearance. Mutations in RAD51, RAD54, and RAD57 had the converse affect: they eliminated the ability of cells to generate type I survivors in a tlc1 strain. The triple mutant, tlc1 rad51 rad59, was not able to generate survivors. Thus either type I or type II recombination pathways can allow cells to survive in the absence of telomerase; however, elimination of both pathways in a telomerase mutant leads to the inability to elongate telomeres and ultimately cell death.

Wild-derived inbred mouse strains have short telomeres.

Telomere length and telomerase activity directly affect the replicative capacity of primary human cells. Some have suggested that telomere length influences organismal lifespan. We compared telomere length distributions in a number of inbred and outbred established mouse strains with those of strains recently derived from wild mice. Telomere length was considerably shorter in wild-derived strains than in the established strains. We found no correlation of telomere length with lifespan, even among closely related inbred mouse strains. Thus, while telomere length plays a role in cellular lifespan in cultured human cells, it is not a major factor in determining organismal lifespan.

Recombination in telomere-length maintenance.

Although telomerase is the major mechanism for telomere elongation in most cells, telomerase-independent mechanisms of telomere maintenance can allow cell survival. Yeast cells that lack telomerase maintain telomere length through a form of recombination known as gene conversion. Understanding the role that telomeric recombination might play in mammalian cells has important implications for cancer therapeutics.

Identification of two RNA-binding proteins associated with human telomerase RNA.

Telomerase plays a crucial role in telomere maintenance in vivo. To understand telomerase regulation, we have been characterizing components of the enzyme. To date several components of the mammalian telomerase holoenzyme have been identified: the essential RNA component (human telomerase RNA [hTR]), the catalytic subunit human telomerase reverse transcriptase (hTERT), and telomerase-associated protein 1. Here we describe the identification of two new proteins that interact with hTR: hStau and L22. Antisera against both proteins immunoprecipitated hTR, hTERT, and telomerase activity from cell extracts, suggesting that the proteins are associated with telomerase. Both proteins localized to the nucleolus and cytoplasm. Although these proteins are associated with telomerase, we found no evidence of their association with each other or with telomerase-associated protein 1. Both hStau and L22 are more abundant than TERT. This, together with their localization, suggests that they may be associated with other ribonucleoprotein complexes in cells. We propose that these two hTR-associated proteins may play a role in hTR processing, telomerase assembly, or localization in vivo.

Secondary structure of vertebrate telomerase RNA.

Telomerase is a ribonucleoprotein enzyme that maintains telomere length by adding telomeric sequence repeats onto chromosome ends. The essential RNA component of telomerase provides the template for telomeric repeat synthesis. To determine the secondary structure of vertebrate telomerase RNA, 32 new telomerase RNA genes were cloned and sequenced from a variety of vertebrate species including 18 mammals, 2 birds, 1 reptile, 7 amphibians, and 4 fishes. Using phylogenetic comparative analysis, we propose a secondary structure that contains four structural domains conserved in all vertebrates. Ten helical regions of the RNA are universally conserved while other regions vary significantly in length and sequence between different classes of vertebrates. The proposed vertebrate telomerase RNA structure displays a strikingly similar topology to the previously determined ciliate telomerase RNA structure, implying an evolutionary conservation of the global architecture of telomerase RNA.

G-strand overhangs on telomeres in telomerase-deficient mouse cells.

Telomeres of eukaryotic chromosomes contain 3' overhangs which are thought to be essential for the maintenance of proper chromosome end structure and function. We examined the requirement for telomerase activity for the generation of these G-strand overhangs in mammalian cells. Using non-denaturing in-gel hybridization to both tissue and cultured cells from mice deficient for the telomerase RNA component, we found that G-strand overhangs exist in the absence of telomerase activity. Quantitation of overhang signal intensity showed no significant reduction in telomerase-deficient cells relative to wild-type. These results support a telomerase-independent mechanism for generating G-strand overhangs.

Short dysfunctional telomeres impair tumorigenesis in the INK4a(delta2/3) cancer-prone mouse.

Maintenance of telomere length is predicted to be essential for bypass of senescence and crisis checkpoints in cancer cells. The impact of telomere dysfunction on tumorigenesis was assessed in successive generations of mice doubly null for the telomerase RNA (mTR) and the INK4a tumor suppressor genes. Significant reductions in tumor formation in vivo and oncogenic potential in vitro were observed in late generations of telomerase deficiency, coincident with severe telomere shortening and associated dysfunction. Reintroduction of mTR into cells significantly restored the oncogenic potential, indicating telomerase activation is a cooperating event in the malignant transformation of cells containing critically short telomeres. The results described here demonstrate that loss of telomere function in a cancer-prone mouse model possessing intact DNA damage responses impairs, but does not prevent, tumor formation.

p53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis.

Maintenance of telomere length and function is critical for the efficient proliferation of eukaryotic cells. Here, we examine the interactions between telomere dysfunction and p53 in cells and organs of telomerase-deficient mice. Coincident with severe telomere shortening and associated genomic instability, p53 is activated, leading to growth arrest and/or apoptosis. Deletion of p53 significantly attenuated the adverse cellular and organismal effects of telomere dysfunction, but only during the earliest stages of genetic crisis. Correspondingly, the loss of telomere function and p53 deficiency cooperated to initiate the transformation process. Together, these studies establish a key role for p53 in the cellular response to telomere dysfunction in both normal and neoplastic cells, question the significance of crisis as a tumor suppressor mechanism, and identify a biologically relevant stage of advanced crisis, termed genetic catastrophe.

RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase.

Telomere length is maintained by the de novo addition of telomere repeats by telomerase, yet recombination can elongate telomeres in the absence of telomerase. When the yeast telomerase RNA component, TLC1, is deleted, telomeres shorten and most cells die. However, gene conversion mediated by the RAD52 pathway allows telomere lengthening in rare survivor cells. To further investigate the role of recombination in telomere maintenance, we assayed telomere length and the ability to generate survivors in several isogenic DNA recombination mutants, including rad50, rad51, rad52, rad54, rad57, xrs2, and mre11. The rad51, rad52, rad54, and rad57 mutations increased the rate of cell death in the absence of TLC1. In contrast, although the rad50, xrs2, and mre11 strains initially had short telomeres, double mutants with tlc1 did not affect the rate of cell death, and survivors were generated at later times than tlc1 alone. While none of the double mutants of recombination genes and tlc1 (except rad52 tlc1) blocked the ability to generate survivors, a rad50 rad51 tlc1 triple mutant did not allow the generation of survivors. Thus RAD50 and RAD51 define two separate pathways that collaborate to allow cells to survive in the absence of telomerase.

Telomerase activation. One step on the road to cancer?

Ever since the discovery that telomeres are short in cancer cells and telomerase is activated in immortal cells, telomerase has been an oncogene wannabe. Oncogenes have been the glamour genes of molecular biology for 20 years, garnering flashy headlines and name recognition. More recently, tumor-suppressor genes have joined oncogenes on center stage. Recent evidence has shown that MYC upregulates the catalytic subunit of telomerase, TERT, and that TERT cooperates with HPV E7 in cell immortalization. This evidence now supports the placement of telomerase among the cancer gene elite.

Longevity, stress response, and cancer in aging telomerase-deficient mice.

Telomere maintenance is thought to play a role in signaling cellular senescence; however, a link with organismal aging processes has not been established. The telomerase null mouse provides an opportunity to understand the effects associated with critical telomere shortening at the organismal level. We studied a variety of physiological processes in an aging cohort of mTR-/- mice. Loss of telomere function did not elicit a full spectrum of classical pathophysiological symptoms of aging. However, age-dependent telomere shortening and accompanying genetic instability were associated with shortened life span as well as a reduced capacity to respond to stresses such as wound healing and hematopoietic ablation. In addition, we found an increased incidence of spontaneous malignancies. These findings demonstrate a critical role for telomere length in the overall fitness, reserve, and well being of the aging organism.