Cells that lack telomerase components undergo replicative senescence resulting in a reduction of proliferative potential. The NMD pathway accelerates senescence in otherwise wild-type cells that lack either telomerase holoenzyme components (TLC1 and Est2p) or accessory factors (Est1p and Est3p). Loss of the NMD pathway causes a delay in the senescence process such that telomerase-deficient upfΔ strains live for ~10 to 25 PDs longer than UPF+ strains. As in wild-type telomerase-deficient cells, senescence in upfΔ strains is accompanied by gradual erosion of telomeric DNA and culminates in crisis.
The extension of proliferative potential in upfΔ strains involves neither accelerated survivor formation nor RAD52. Yet, as with wild-type strains, survivors arise eventually in later passages of upfΔ telomerase-deficient strains. This supports the idea that survivor formation is not due to a stochastic process that occurs continuously. Rather, the RAD52-dependent activities that give rise to survivors are induced at a critical point in the senescence process. Interestingly, in upfΔ strains, type I survivors are more prevalent, while type II survivors appear with less frequency (data not shown). Furthermore, the delay of senescence in the telomerase-deficient upfΔ mutants is not due to the MEC3-dependent telomere checkpoint.
STN1 and TEN1 mRNA levels are regulated by NMD through their 5′-UTR sequences; upf
Δ strains have elevated levels of STN1 and TEN1 mRNAs (28
). Increased levels of STN1 and TEN1 expression contribute to the short-telomere phenotype of upf
Δ in telomerase-proficient cells (30
). Increased levels of STN1
also phenocopy most of the senescence delay seen in upf
Δ strains (Table ), indicating that upf
Δ-mediated senescence delay is primarily due to changes in the telomere cap structure. We propose that the extended proliferative potential of telomerase-deficient upf
Δ strains is caused by a reinforcement of the telomere cap structure when extra Stn1p is available (Fig. ). We propose that excess Stn1p strengthens the telomere cap and thus protects the chromosome ends from degradation in telomerase-deficient cells. In cells with active telomerase, extra copies of STN1
may result in shorter telomeres (9
), because the reinforced telomere cap reduces the access of telomerase to the telomeres (Fig. ).
FIG. 4. Model for the role of the telomere cap in senescence in telomerase-deficient cells. The three columns represent three states: wild-type, increased Stn1p levels, and cdc13-2 strains. The top row in each case indicates the situation in the presence of telomerase. (more ...)
Increased expression of TEN1
alone had no effect on senescence rates (Table ). Similarly, extra TEN1
had little effect on telomere length or telomeric silencing phenotypes associated with upf
Δ mutations (9
). This is consistent with previous reports that found, in contrast to excess STN1
, excess TEN1
had no effect on the suppression of cdc13
temperature sensitivity or on the negative regulation of telomere length (9
). However, as in other situations (9
), increased TEN1 expression enhanced the effect of extra STN1 expression.
Cdc13p is a component of the telomere cap that recruits telomerase to the telomere (12
was isolated as an est
). However, cdc13-2
strains senesce more slowly than tlc1
Δ, and est3
Δ strains, and in cdc13-2
strains, the rate of senescence is not affected by the NMD pathway. This suggests that the telomere cap structure in cdc13-2
mutants is different from that in the other telomerase-deficient (est
Δ and tlc1
Δ) mutants. The presence of similar rates of senescence in cdc13-2
Δ strains is consistent with the idea that these mutations delay senescence by altering the telomere cap structure. For upf
Δ mutants, the cap is altered by increasing levels of Stn1p, which, we propose, also limits access of telomerase and senescence activities to the telomere (Fig. ). cdc13-2
alters the cap in a different way that makes it insensitive to upf
mutations and to levels of Stn1p. Alternatively, because Cdc13p has multiple roles at the telomere, it could affect senescence independent of its role as a telomere cap component.
One function of the telomere cap is to preserve the chromosome ends from degradation. The rate of senescence is influenced by the initial telomere length at the time that cells lose telomerase: strains with shorter telomeres progress through senescence more rapidly than those with initially longer telomeres. Furthermore, telomerase-deficient upf
Δ strains with shorter telomeres, due to upf
Δ parents or to high levels of Stn1p in the parent strains, senesce more rapidly than telomerase-deficient upf
Δ strains with wild-type telomere length. Our results support the assumption that telomere length is a critical factor in determining a cell's proliferative potential. However, it is important to note that the NMD pathway does not affect the rate of senescence by affecting telomere length: telomerase-deficient upf
Δ cells have average telomere lengths that, at the time of crisis, appear to be shorter than the average telomere lengths in otherwise wild-type telomerase-deficient cells. Because the telomeric cap components bind telomeric G-overhang structures (3
), we cannot rule out the possibility that strains with altered cap proteins may also have altered G-overhang structures. Nonetheless, telomere cap structure, rather than telomere length alone, is an important contributor to the rate of senescence.
Based on our results, it is appealing to consider the idea that increasing telomere end protection may facilitate the modulation of life span in other organisms. This is consistent with the observation that overexpression of mammalian TRF2
protects shortened telomeres and delays senescence (25
). In most human cells, telomerase is not active. Cultured cells exhibit replicative senescence partially due to limited telomerase activity (reviewed in reference 54
). Much remains unknown, however, about life span and replicative senescence in whole organisms.
An intriguing connection between telomeres and the NMD pathway was recently revealed in humans. KIAA0732 (hEST1A)was identified by two groups as being a human homologue of Est1p (40
). Overexpression of KIAA0732 (hEST1A) caused end-to-end chromosome fusions (40
) and altered telomere length (46
). At the same time, KIAA0732 was also shown to be similar to SMG5/7a, Caenorhabditis elegans
genes involved in the NMD pathway. KIAA0732 (hSmg5/7a) copurifies with Upf1p, Upf2p, and Upf3p and may target a protein phosphatase 2A to Upf1p (7
). The mechanisms by which NMD affects telomere functions and the role of KIAA0732 in human cell life span remain to determined.
One approach to extending life span or combating the ill effects of replicative senescence is to reactivate telomerase. A reasonable criticism of this approach is that telomerase activation may be a barrier to cellular immortalization, which would be lost and therefore may lead to an increased risk of carcinogenesis (19
), although several experiments have suggested that telomerase activation alone does not increase the occurrence of cancers in mice (5
). Our results suggest an alternative approach to increasing life span, without generating immortal cells, by reinforcing the telomere cap structure through increased expression of a cap binding protein. Reinforcing the telomere end structure does not activate telomerase, and at least in yeast, upf
Δ cells appear normal for ~20 additional PDs and then undergo crisis in an apparently normal manner that produces rare survivor cells. It has been assumed that in the absence of new telomere synthesis, telomeres would erode during each cell cycle, and thus cell life span would be determined by telomere length. An alternative mechanism to extend cellular replicative potential may be to alter the chromosome end protection complex stoichiometry or structure.