- Email: [email protected]
Structure and function of telomerase
374
Structure and function of telomerase Kathleen Collins The study of eukaryotic telomeres
at the molecular
level
began with the discovery of short, tandem repeats at Tetrahymena decades,
chromosome
ends. In the following two
major insights about telomere structure and function
have come from investigations polymerase
that synthesizes
three areas of telomerase
telomere
the DNA
these repeats. In the past year,
research have been particularly
intense: assays of telomerase components,
of telomerase,
ditional background concerning telomerase or telomere structure and function, including details of the unique telomeres of Drosophila which are not addressed in this review, readers are referred to several recent publications [3-71.
activity, isolation of telomerase
and studies of the regulation of telomerase
and
length in Go.
Address Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204, USA; e-mail: [email protected] Current Opinion in Cell Biology 1996, 8:374-380 0 Current Biology Ltd ISSN 0955-0674 Abbreviation RNP ribonucleoprotein
Introduction The complete replication of eukaryotic chromosomes involves the participation of an RNA-dependent DNA polymerase known as telomerase ([1,2]; for reviews, see [3,4]). Telomerase recognizes chromosome ends and extends 3’ termini by addition of tandem simple sequence repeats. This de no210 DNA addition compensates for the sequence loss that results from incomplete terminal replication. Telomerase adds a specific repeat sequence to telomeres, using as a template a short region within the RNA component of the ribonucleoprotein (RNP) telomerase enzyme. Although cells can maintain a constant number of telomeric simple sequence repeats by balancing telomere loss during replication and addition by telomerase, not all cells do so (for reviews, see [3,5,6]). Human germline cells maintain a constant number of telomeric simple sequence repeats, but human somatic cells lose telomeric repeats with each cycle of cell division. Previous studies have suggested a correlation between a lack of telomerase activity, failure to maintain a stable telomere length, and limited proliferative capacity [3,5,6]. Cells with critically shortened telomeres appear to stop proliferating, as if halted by a checkpoint sensing dangerously small protective caps at chromosome ends. The past year has been an exciting one in the study of telomerase and telomere-length regulation. The first three sections of this review summarize new findings about telomerase activity and telomerase components. The fourth section relates these studies to an evolving understanding of telomere-length regulation, which must be more complex than previously anticipated. For ad-
Telomerase activity: more assays, more uncertainties An assay of telomerase activity in vitro requires only a few reaction components, namely a single-stranded DNA primer (which mimics a chromosome end), nucleotides, and telomerase enzyme [2]. As a result of these simple substrate requirements and the RNase-sensitivity of the reaction (which is a consequence of the essential RNA component of the enzyme), telomerase can often be detected specifically even in crude cell lysates. However, current assay protocols are not entirely reliable for telomerase obtained from some species or for quantitative analysis. The finicky nature of the in vitro telomerase assays is highlighted by three recent reports of Sadaromyces cerevisiae telomerase activity; the use of different protocols and different extracts led to entirely different descriptions of enzyme properties [8*,9,10]. The lack of qualitative and quantitative reliability derives partly from the following circumstances: first, the presence of contaminating RNA-dependent primer extension activities (such as reverse transcriptases of retro-elements); second, non-specific inhibition of telomerase in crude extracts; third, non-linear dependence of product synthesis on enzyme, primer, and nucleotide concentrations; and fourth, assay-dependent alteration of the processivity of elongation (i.e. alteration of the number of telomeric repeats added to each substrate). Hopefully, a consensus concerning the in vitro properties of yeast telomerase will develop from additional characterization of assay specificities and the use of telomerase preparations of greater purity. Another issue in the interpretation of telomerase activity assays is the relation of activity assayed in vitro (with high concentrations of single-stranded oligonucleotides as substrates) with activity assayed in vivo (on native chromosome ends). Many sequences will serve as primers for telomerase in vitro that do not promote telomere formation in vivo. In fact, the sequence specificity of telomere formation in human cells [ll] appears to reflect the sequence-specificity of DNA binding by a human telomere protein [ 12,13’] rather than the primer specificity of telomerase itself. In vitro, telomerase from the ciliate Oxytticha can elongate a telomeric primer DNA bound to telomere proteins [14]. These results suggest that telomerase recognizes a chromosome substrate composed of both DNA and protein. The interaction of telomerase with
Structure and function of telomerese Collins
telomeric repeat binding proteins topic for future exploration.
Telomerase
remains
an interesting
components
RNA
The RNA component of telomerase, originally identified in Tetrahymena [15], has been subsequently identified in a number of other ciliates [16X0]. In the past year, telomerase RNAs have also been cloned from nonciliate species. A genetic screen led to the cloning of the S. cerevisiae telomerase RNA: when overexpressed in incomplete form, the telomerase RNA reduced telomeric silencing [Zl”]. Singer and Gottschling [Zl”] noted in their clone a 16 nucleotide region of exact complementarity to the repeat sequence initially added to heal broken yeast chromosomes [ZZ]. Changes in the sequence of this RNA region in vivo resulted in telomeric incorporation of the predicted mutant repeats [21**]. Disruption of the gene encoding the 1.3 kb telomerase RNA led to telomere shortening and a delayed-growth defect of the culture, as first described for S. cewvisiae estl strains deficient in telomere maintenance [23]. The Khyveromyces lactis telomerase RNA was identified using a different approach [24”]. A genomic DNA fragment which hybridized to the long K. lactis telomeric repeats, but was not itself telomeric, was cloned. When the region of the clone complementary to the telomeric repeat was altered in vivo, mutant repeats were incorporated into telomeric DNA. Disruption of the gene encoding the (also) 1.3 kb telomerase RNA resulted in telomere shortening and a delayed-growth defect of the culture. For both S. cerevisiae and K. /actis strains that lack telomerase-dependent telomere elongation, a small percentage of cells eventually overcome their growth defect and acquire longer telomeres [21**,24**,25]. This suggests that a telomerase-independent telomere elongation mechanism exists (see below). Telomerase RNAs have also been cloned from two mammalian sources. The human RNA was isolated by employing molecular techniques to enrich for small RNAs containing a short region of telomere-complementary sequence [26”]. The gene encoding the human RNA was used as a hybridization probe to clone the mouse RNA [27**]. Sequence alterations in the predicted human or mouse template region resulted in the synthesis of mutant repeats in in vitro experiments using extracts of cells transfected with plasmids encoding mutant RNAs. Both mammalian telomerase RNAs are approximately 500 nucleotides in length. In several tissues, levels of the mouse telomerase RNA decline during development [27**]. Different human tissues, and primary versus immortalized cell lines, also show variation in telomerase RNA levels, although not as much as might be expected from relative differences in telomerase activity and telomere maintenance [26.*]. The yeast and mammalian telomerase RNAs show a high divergence of primary sequence, as previously observed for the ciliate RNAs [ 1S-201.
375
Analysis of telomerase RNA function has continued as in previous years [28-301, using Tetraymena telomerase assembled from mutant RNAs and wild-type proteins either in &JO or in vitro. Studies of mutant RNA function have focused on the RNA template region for technical reasons. In vivo assembly requires competition of overexpressed mutant RNAs with the wild-type RNA for limiting proteins; thus, mutations which decrease the efficiency of RNP assembly cannot be analyzed. In vitro assembly is performed in the presence of fragments of partially digested native RNA, thus, mutant RNAs may need to displace native RNA fragments or may not require essential domains of the RNA due to residual function of the native fragments. Continued analysis of the template region has yielded several interesting results. When reconstituted in vitro, mutation of a region of the RNA immediately 5’ of the template, a region conserved in ciliate RNAs, alters selection of the template residues that are most efficiently copied into product DNA [31*]. Using in vivo reconstitution, mutations in the template region were discovered that alter elongation efficiency and also, unexpectedly, affect enzyme fidelity [32*,33*]. The latter result suggests that the telomerase RNA plays a role in catalysis in addition to its template function. Finally, the properties of wild-type ciliate telomerase RNAs have been examined. The structure of i’hrahymena telomerase RNA was analyzed in vivo [34], as was the structure of the same RNA transcribed in vitro [35]. A comparison of these two structures suggests that the overall folding of the RNA is similar whether or not proteins are bound, and is similar to the structure predicted from phylogenetic comparison. In another study, the nuclear localization of Oxytticha telomerase RNA was examined by in situ hybridization [36]. Chromosomes in an Oxytrida macronucleus are replicated by a concerted progression of DNA synthesis machinery, which forms a replication ‘band’. Telomerase RNA was shown to be localized at the trailing end of the replication band (consistent with its function in telomere synthesis) but also at large structures that are likely to be storage sites. Thus, assembly or localization of the RNP could regulate telomerase activity, allowing the enzyme to be active only when appropriate substrates are present. Proteins, and the ribonucleoprotein
in action
Ten years after the identification of telomerase activity in vitro [2], protein components of Tetralzymena telomerase have been identified using a biochemical approach [37**]. Two polypeptides of 80 kDa and 95 kDa copurified with telomerase activity and the telomerase RNA, and by several criteria were shown to form a stable, threecomponent RNP complex with the telomerase RNA. Nucleic acid cross-linking and binding assays demonstrated that p80 specifically interacts with the telomerase RNA and p95 specifically interacts with telomeric primers. Cloning of the cDNAs encoding p80 and p95 revealed novel proteins, with limited homology to any sequence in
376
Nucleus and gene expression
the available databases [37”]. Although no polypeptides other than p80 and ~95 were associated in stoichiometric quantities with the telomerase RNA, it remains to be shown that one or both proteins are sufficient, in combination with the telomerase RNA, to catalyze enzyme activity.
Fiaure 1
A weak homology has been observed between the carboxy-terminal region of ~95 and conserved active-site residues in a family of viral RNA-dependent RNA polymerases [37”]. Whether this minimal structural homology reflects a conservation of function is unknown, although biochemical evidence also suggests that telomerase shares the properties of an RNA-dependent RNA polymerase: i’&ra/?vmena telomerase shows greater affinity for RNA primers than for DNA primers of the same sequence and can incorporate ribonucleotides [38*]. These studies extend previous indications of the remarkable lack of specificity of telomerase in oitro. They also suggest the possibility that telomerase evolved from an RNA-dependent RNA polymerase expected to have catalyzed genome replication in the primordial RNA world [39]. A hypothetical model for Tetrahymena telomerase structure is shown in Figure 1. For illustrative purposes, telomerase is drawn to resemble models for the reverse transcriptase of HIV-l [40]. This reverse transcriptase contains two subunits with homologous sequences but distinct structures. The larger subunit harbors the active site; the smaller subunit forms part of the template-primer-binding site and may position tRNA primers such that the tRNA 3’ end forms base pairs with the template in the active site. In Figure 1, telomerase p95 contains the active site whereas p80 brings the telomerase RNA to ~95 such that the template is held in the active site. Primers typically bind with their 3’ end at the RNA template and with a more 5’ region at an RNA-independent binding site which is, at least partially, composed of ~95 [37”,41,42]. How product binding is regulated at these two sites to accomplish processivity and to moderate the nucleolytic cleavage reaction catalyzed by telomerase [41] is not known. However, telomerase and the DNA-dependent RNA polymerases are likely to share a similar mechanism, judging from biochemical similarities between the two enzymes [41-43].
Telomerase and telomere-length in vivo
regulation
Telomeres have several confirmed roles in the cell, and several more are suspected. They are required for chromosome stability and complete linear genome replication, and they appear to regulate gene expression, chromosome segregation, and nuclear architecture (for reviews, see [3,7]). However, much of the emphasis of the past year has been placed on a single question: is telomere length regulated by telomerase to determine a cell’s proliferative capacity? Numerous studies have shown a correlation between replication-dependent loss of telomeric
Q 1996 Current Opinion m Cell Biology
A model of the Tetrahymena telomerase RNP. The telomerase RNA is drawn with regions of single-stranded or double-stranded structure as derived from phylogenetic and direct physical evidence [16-20,34,35]. A primer is bound at its 3’ end to the RNA (binding is shown as three base pairs or parallel lines) and also to an RNA-independent site (drawn as a cleft) on the p95 protein subunit [37’*]. The p60 subunit interacts with telomerase RNA [37”1; the interaction is shown here between p60 and a long, bent double helix (which represents combined stems I and IV; 1161). Protein-RNA interactions which fasten the template in the active site area are suggested
by dark gray shaded boxes.
simple sequence repeats and limited proliferative capacity (for reviews, see [3,5,6]). Cells appear to stop dividing when they sense that telomeres are too short to safely allow additional rounds of cell division. In general, cells that exhibit finite proliferative capacity and shortening telomeres lack telomerase activity, whereas cells with indeterminate proliferative capacity and stable telomere length possess active telomerase enzyme (for reviews, see [3,5,6]). In the past year, however, several interesting exceptions have been reported (see below). These results suggest that we are missing unanticipated pieces of the telomere length regulation puzzle. At least three lines of research have converged to indicate the need for a new, more complex model of telomere-length regulation; these lines of research are additional investigations of telomerase activity and telomere length in mammalian cells, genetic manipulation of telomeres in model systems, and analysis of telomere replication intermediates in ciliates. In a large number of recent reports (only a selection of which can be cited here), some immortal cell lines that maintain telomere length have been shown to possess
Structure and function of telomerase
telomerase activity [44*,45] whereas other immortal cell lines have been shown to lack it [46,47’,48]. In addition, not all cells possessing telomerase activity do maintain telomere length. For example, some normal human blood cell populations were found to be telomerase-positive [49*-51.1 although telomere shortening also occurs in some of these cell types ([52*]; see chapter 9 in 131). Telomerase activity has been detected in somatic tissues of mice that experience telomere loss [53,54], although the telomerase-positive cell populations are not known. We conclude from these studies that not all immortal cells possess detectable telomerase activity and that not all cells with active telomerase maintain a stable number of telomeric simple sequence repeats.
In addition, genetic manipulations have revealed that activities other than telomerase are important for telomere maintenance. S. cemisiae and K. /act& strains lacking in viva telomerase activity exhibit growth defects after telomere attrition, but they then recover, yielding ‘survivor’ populations with extended telomeres [21”,24**,25]. these results suggest that a telomeraseAlthough independent telomere maintenance mechanism can ‘kick in’ if required, the uneven growth of the survivor strains suggests that telomerase-dependent telomere maintenance may be the healthier alternative. Experiments in yeast also suggest that telomere-length regulation in the presence of active telomerase is influenced by additional factors. In a revealing set of experiments, mutations in the K. /a& telomerase RNA were employed to alter telomeric repeat sequences ([24.*]; see above). Previously, when Tetrahymena telomeric repeats were altered using a similar method, some mutations caused immediate loss of telomere-length regulation [28,29,32’,33*]. In K. lacks, telomeric incorporation of some mutant repeats also resulted in immediate loss of length regulation. Other mutant repeats, however, affected telomere length only after many generations, when almost all wild-type repeats had been replaced with the mutant sequence. Thus, even in the presence of telomerase, proper control of telomere length appears to depend on factor(s) which recognize centromere-proximal and centromere-distal telomere repeat sequences.
It is striking that even very centromere-proximal telomeric repeats could be replaced in K. /ads strains with altered telomerase specificity. According to current dogma, telomerase should have replaced only sequences at the extreme termini, repeatedly adding back the same few terminal repeats lost during each round of replication. The substitution of centromere-proximal telomeric repeats in K. lads could proceed either by non-homologous and unequal recombination or by access of templatereprogrammed telomerase to ‘internal’ locations. The latter possibility would suggest extensive telomere trimming, balanced with compensatory elongation, in a single S phase (see Fig. 2). Perhaps telomerase itself recruits the
trimming machinery to prevent absence of elongation.
excess
Collins
trimming
377
in the
Figure 2 I
I
0
1996Current
O&on
in Cell B~oloav
Multiple processing events in telomere synthesis. Various activities that contribute to the maintenance of telomere length by telomerase are suggested. Each processing step could occur multiple times on the same telomere in a single S phase, or a step could be missed completely. (a) Elongation of one strand of telomeric DNA is mediated by telomerase (the newly synthesized, G-rich telomeric DNA strand is represented by a wavy black arrow) [281.(b-d) Cleavage of the single-stranded telomeric repeat DNA could occur both (b) before and (d) after (c) completion of second-strand synthesis. Cleavage before second-strand synthesis could promote replacement of internal telomeric repeats I24”l whereas cleavage after second-strand synthesis would provide a mechanism for polishing ends to produce a precise overhang 155’1. (e) Cleavage or deletion of double-stranded telomeric repeats is also likely to occur; this is particularly noticeable when telomeres
become extremely long (see [24”,5g]
several examples). How these processing coordinated remains to be determined.
for two of
activities are regulated
and
Some evidence for regulated, replication-independent telomere trimming derives from the investigation of telomere replication intermediates. Following Ize n~uo telomere synthesis in the ciliate Euplotes crassus, telomeric repeat tracts are longer and more heterogeneous than those of mature macronuclei i55.1. This suggests that telomere elongation itself need not be precisely controlled. Trimming of overly long telomeres to obtain a precise terminal structure occurs independently of DNA replication (see Fig. 2) [56]. In S. cemisiae, long G-rich regions of single-stranded DNA were observed transiently in late S phase, possibly reflecting a similar excess of telomeric repeat addition [57]. An accumulation of single-stranded telomeric DNA can also be observed in cell division cycle mutants of yeast [58]. If balancing the number of telomeric repeats involves not only loss of repeats by incomplete replication and elongation by telomerase but also active trimming, subtle alterations in this balance of multiple components could readily establish a different ‘normal’ telomere length.
378
Nucleus and gene expression
Conclusions The past year has seen enormous progress in the identification of telomerase components. Telomerase RNAs cloned from species other than ciliates reveal even less conservation of length or primary sequence than previously observed among ciliate RNAs. The first telomerase proteins have been identified from Tetrahymenaand share very little, if any, primary sequence homology with other known proteins. With these reagents in hand, the future holds promise for a more detailed molecular investigation of this unique polymerase. Although limited by the lack of molecular reagents, studies of telomerase regulation using activity assays have revealed a greater complexity than originally envisioned. The past year’s research was full of exceptions: not all mammalian somatic tissues lack telomerase activity and not all immortalized cells possess it. In addition, exceptions have been identified to the correlations between telomerase activity and stable telomere length, and between telomerase absence and telomere shortening. Mechanisms for executing the various regulations of telomerase will be a prime area of interest for future studies. Experimentally induced ‘regulation’ of telomerase has provided a convenient starting point for addressing more general questions of telomere structure and function. When a telomere sequence was altered by mutation of the telomerase RNA template, sequence-specific effects on telomere-length regulation and cell viability were observed. Future use of this technique, combined with a biochemical analysis of altered telomeric chromatin, should prove extremely informative in the pursuit of the molecular basis of telomere functions, including the which limits cell putative ‘short telomere’ checkpoint proliferation.
5.
Greider CW: Mammalian teiomere dynamics: healing, fragmentation, shortening and stabilization. Curr Opin Gener Dev 1994,4:203-211.
6.
Hariev CB. Kim NW. Prowse KR. Weinrich SL. Hirsch KS. West.MD, ‘Bacchett/ S, Hirte Hh, Counter &I, Greider CW et al.: Teiomerase, ceil immortality, and cancer. Cold Spring Harb Symp &ant Biol1994, 59:307-315.
7.
Zakian VA: Teiomeres: beginning to understand Science 1995, 270:1601-l 607.
8.
Cohn M, Blackburn EH: Teiomerase in yeast Science 1995, 269:396-400. yhis paper describes the most convincing in vitro assay for S. cerevisiae telomerase. Fractionated whole call extracts were examined for RNasesensitive, primer sequence dependent incorporation of dGTP and lTP. Nucleotides were added in register with the 3’ end of sequence-permuted telomeric primers, and ddNTPs were incorporated at predicted positions in the oroduct DNA. Product DNA svnthesis raouired the teiomerase RNA but was’indistinguishable in the presence or absahce of EST1 p, a gene product required for telomere maintenance [231. Hence EST7 probably does not encode the catalytic subunit of telomerase. 9.
Lin J, Zakian VA: An in vitro assay for Sacchumyces telomerase requires ESTl. Cell 1995, 81 :1127-l 135.
10.
Lue NF, Wang JC: ATP-dependent processivity of a teiomerase activity from Sacchefomyces cefevisiae. J Biol Chem 1995, 270121453-21456.
11.
Hanish JP, Yanowitz JL, De Lange T: Stringent sequence requirements for the formation of human teiomeres. froc Nat/ Acad Sci USA 1994, 91:8881-8865.
12.
Zhong Z, Shiue L, Kaplan S, De Lange T: A mammalian factor that binds teiomeric TTAGGG repeats in vitro. MO/ Cell Biol 1992,12:4834-4843.
13. .
Chona L. Van Steensal B. Broccoli D. Erdiument-Bromaae H. Hanisi J; Tempst P, De Lange T: A himar; teiomeric p&ein. Science 1995, 270:1663-l 667. . This paper describes the punhcatlon and cloning of the gene encodmg a human protein that specifically binds to double-stranded teiomeric repeats. The protein, human teiomeric repeat binding factor, is shown to localize to teiomeres in vivo, and represents the first vertebrate telomeric repeat binding protein whose encoding gene has been cloned. 14.
Shippen DE, Blackburn EH, Price CM: DNA bound by the hyffiche teiomere protein is accessible to teiomerase and other DNA poiymerases. Proc Nat/ Acad Sci USA 1994, 91:405-409.
15.
Greider CW. Blackburn EH: A teiomeric seouence in the RNA of Tetrahynkne telomerase required for teiomere repeat synthesis. Nature 1989, 337:331-337.
16.
Shippan-Lentz D, Blackburn EH: Functional evidence for an RNA template in telomerase. Science 1990, 247:546-552.
1 7.
Romero DP, Blackburn EH: A conserved secondary teiomerase RNA Cell 1991, 67~343-353.
18.
Greider CW, Blackburn EH: The telomere terminal transferase of Tetrahymene is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell 1967, 513887-898.
Meiek M, Davis BT, Shippen DE: Oligonucieotides complementary to the Oxykiche nov8 teiomerase RNA delineate the template domain and uncover a novel mode of primer utilization. MO/ Cell Biol 1994, 14:7827-7838.
19.
Greider CW, Blackburn EH: identification of a specific telomere terminal transferase activity in Tetrabymena extracts. Cell 1985, 43:405-413.
Lingner J, Hendrick LL. Cech TR: Teiomerase RNAs of different ciiiates have a common secondary structure and a permuted template. Genes Dev 1994, 8:1984-l 998.
20.
McCormick-Graham structural features.
Acknowledgements I thank members of the Collins laboratory (namely, L Gandhi, R Mitchell, JM Andresen and J Mitchell), Paul Kaufman and Donald Rio for comments on this review.
References and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
. .. 1.
2.
3.
4.
the end.
of special interest of outstanding interest
Blackburn EH, Greider CW: Telomeres. Plainview, New York: Cold Spring Harbor Laboratory Press; 1995. Blackburn EH: Telomerase. In The RNA World. Edited by Gestiand RF, Atkins JF. Plainview, New York: Cold Spring Harbor Laboratory Press; 1993:557-576.
structure for
M, Rornero DP: Ciliate telomerase RNA Nucleic Acids Res 1995, 23:1091-l 097.
Singer MS, Gottschling DE: TLC1 : template RNA component of S8cc/t8rofnyces cerevisiae teiomerase. Science 1994, 266:404-409. After much hunting for teiomerase components in S. cerevisiae, the first sighting is described in this paper. Partial telomerase RNA clones were
21. ..
Structure and function of telomerese
identified as high-copy suppressors of silencing. Genetic disruption of the RNA resulted in telomere shortening and in senescence; mutations of the RNA template region were copied into telomeric DNA in viva. 22.
Kramer KM, Haber JE: New telomeres in yeast are initiated with a highly selected subset of TGI -3 repeats. Genes fJev 1993, 712345-2356.
23.
Lundblad V, Szostak JW: A mutant with a defect in telomere elongation leads to senescence In yeast Cell 1989, 57:633-643.
McEachem MJ, Blackburn EH: Runaway telomere elongation caused by telomerase RNA gene mutations. Nature 1995, 376:403-409. The K /a&s telomerase RNA was cloned by exploiting the long telomerit repeats found in this organism. Genetic disruption of the RNA caused senescence, and mutations of the RNA in the template region resulted in mutant telomeric repeat synthesis, loss of telomere-length regulation, and loss of viability. The use of telomerases with altered specificity, in combination with other techniques, will continue to provide insights into mechanisms for telomere-length regulation. 24. ..
25.
Lundblad V, Blackburn EH: An alternative pathway for yeast telomere maintenance rescues estl -senescence. Cell 1993, 731347-360.
Feng J, Funk WD, Wang S, Weinrich SL, Avilion AA, Chiu C, Adams RR, Chang E, Allsopp RC, Yu J et al: The RNA component of human telomerase. Science 1995, 269:1236-l 241. The human telomerase RNA was cloned and expression of the RNA was examined in cells and tissues. Many or all cells and tissues appear to contain telomerase RNA, but with up to 1 O-fold differences in levels. Antisense RNA expression caused crisis in some cultures, although significant telomere length and telomerase activity were maintained.
26. ..
Blasco MA, Funk W, Villeponteau B, Greider CW: Functional characterization and developmental regulation of mouse telomerase RNA Science 1995, 269:1267-l 270. The mouse telomerase RNA was cloned and expression of the RNA examined in cells and tissues. Like the human RNA, many or all mouse cells and tissues appear to contain telomerase RNA, with up to 1O-fold differences in the levels found. In several tissues, RNA levels decreased during development. 27. ..
26.
Yu G, Bradley JD, Attardi LD, Blackburn EH: In viva alteration of telomere sequences and senescence caused by mutated Tebabymena telomerase RNAs. Nature 1990, 344:126-l 32.
29.
Yu G, Blackburn EH: Developmentally programmed healing of chromosomes by telomerase in Tefrahymena. Cell 1991, 67:823-832.
30.
Autexier C, Greider CW: Functional reconstitution of wildtype and mutant Tetrahymena telomerase. Genes Dev 1994, 8:563-575.
31. .
Autexier C, Greider CW: Boundary elements of the Tetrahymena telomerase RNA template and alignment domains. Genes Dev 1995,9:2227-2239. Mutant telomerase RNAs were combined with wild-type proteins in vitro to assay the effect of template region changes on telomerase activity. Alteration of the template had complex effects. A region 5’ of the template sequence helped to define template boundaries. 32. .
Gilley D, Lee MS, Blackburn EH: Altering specific telomerase RNA template residues affects active site function. Genes Dev 1995,9:2214-2226. Mutant telomerase RNAs were assembled with wild-type proteins in viva then assayed in cell extracts. Mutation of the template region affected DNA synthesis in a complex manner, including alterations of enzyme fidelity. See also 133.1. 33. .
Gilley D, Blackburn EH: Specific RNA residue interactions required for enzymatic functions of Tetrehymene telomerase. MO/ Cell Biol 1996, 16:66-75. See annotation 132.1.
Collins
379
34.
Zaug Al, Cech TR: Analysis of the structure of Tetrahymena nuclear RNAs in viva: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA RNA 1995, 1:363-374.
35.
Bhattacharyya A, Blackburn EH: Architecture EM60 J 1994,13:5721-5731.
36.
Fang G, Cech TR: Telomerase RNA localized in the replication band and spherical subnuclear organelles in hypotrichous ciliates. J Ce// Biol 1995, 130:243-253.
of telomerase
RNA
37. ..
Collins K, Kobayashi R, Greider CW: Purification of Tetrahymene telomerase and cloning of genes encoding the two protein components of the enzyme. Cell 1995,81:677-666. This paper represents the first identification of protein components of telomerase in any species. Tefrahymena telomerase was puriiied and shown to contain two proteins in addition to the 159 nucleotide telomerasa RNA. Nucleic acid binding properties of the proteins were examined. Genes encoding the proteins were cloned using peptide sequence information and the genes were shown to encode novel proteins. 38. .
Collins K, Greider CW: Utilization of ribonucleotldes and RNA primers by Tetrahymena telomerase. EMBO J 1995, 14~5422-5432. Ribonucleotides and RNA primers were shown to serve as substrates for telomerase. Substitution of RNA at different positions in a primer had different consequences, suggesting that there are multiple distinct sites of enzyme-primer interaction. 39.
Joyce GF, Orgel LE: Prospects for understanding the origin of the RNA world. In The RNA World. Edited by Gesteland RF, Atkins JF. Plainview, New York: Cold Spring Harbor Laboratory Press; 1993:1-25.
40.
Kohlstaedt LA, yang J, Freidman JM, Rice PA, Steitz TA: Crystal structure at 3.5A resolution of HIV-l reverse transcriptase complexed with an inhibitor. Science 1992, 256:1783-l 790.
41.
Collins K, Greider CW: Nucleolytic cleavage and nonprocessive elongation catalyzed by Tetrahymena telomerase. Genes Dev 1993, 7:1364-l 376.
42.
Lee MS, Blackburn EH: Sequence-specific DNA primer effects on telomerase polymerization activity. MO/ Cell Biol 1993, 13:6586-6599.
43.
Chamberlin MJ: New models for the mechanism of transcription elongation and its regulation. In The Harvey Lectures. Wiley-Liss; 1993:1-21.
44. .
Kim NW, Piatyszek MA, Prowsa KR, Harley CB, West MD, Ho PLC, Coviello GM, Wright WE, Weinrich SL, Shay JW: Specific association of human telomerase activity with immortal cells and cancer. Science 1994, 266:201 l-201 4. A telomeraae assay wtth increased sensltiwty was developed (using the polymerase chain reaction to amplify telomerase product DNA) and employed to assay telomerase activity in a va:iety of human cells, tissues, and cancers. Most immortal cell lines and cancers showed higher telomerase activity than did primary cells or normal tissue. 45.
Hiyama E, Hiyama K, Yokoyama T, Matsuura Y, Piatyszek MA, Shay JW: Correlating telomerase activity levels with human neuroblastoma outcomes. Nat Med 1995,1:249-255.
46.
Murnane JP, Sabatier L, Marder BA, Morgan WF: Telomere dynamics in an immortal cell line. EM60 J 1994, 13:4953-4962.
47. .
Bryan TM, Englezw A, Gupta J, Bacchetti S, Reddel RR: Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J 1995, 1414240-4240. Telcmere length and telomerase activity were assayed in a number of im mortalized human cell lines. Not ail cell lines possessed active telomerase, but all immortalized cell lines that lacked telomerase had telcuneres which were longer and more heterogeneous than in telomerase-positive cell lines. In combination with other results, these findings suggest that a non-telomerase dependent mechanism for telomere elongation is likely to exist, characterized by more variable telomere length. 48.
Rogan EM, Bryan TM, Hukku B, MacLean K, Chang AC, Moy EL, Englezou A, Warnford SG, Dalla-Poua L, Reddel RR: ARerations
380
Nucleus
and gene expression
in p53 and pl6lNK4 expression and telomere length during spontaneous immortalization of Li-Fraumeni syndrome fibroblasts. MO/ Cell Biol 1995, 1514745-4753. 49. .
Counter CM, Gupta J, Harley CB, Leber B, Bacchetti S: Telomerase activity in normal leukocytes and in hematologic malignancies. Blood 1995, 65:2315-2320. Telomerase activity was detected in leukocytes, with some differences in activity level between normal and malignant cells. 50. .
Broccoli D, Young JW, De Lange T: Telomerase activity in normal and malignant hematopoietic cells. Proc Nat/ Acad Sci USA 1995,92:9082-9066. Telomeraae activity was detected in a variety of normal human blood cell populations and at similar levels in hematologic malignancies. Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Piatyazek MA, Shay JW, lshioka S, Yamakido M: Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. I Immunol1995, 155:371 l-371 5. Telomeraae activity was detected in normal human blood calls and was shown to increase with mitogenic stimulation of cells in vitro.
53.
Prowae KR, Greider CW: Developmental and tissue-specific regulation of mouse telomerase and telomere length. Proc Nat/ Acad Sci USA 1995,92:4818-4822.
54.
Chadeneau C, Siegel P, Harley CB, Muller WJ, Bacchetti S: Telomerase activity in normal and malignant murine tissues. Oncogene 1995, 11:893-898.
55. .
Vermeeach JR, Price CM: Telomeric DNA sequence and structure following de nova telomere synthesis in Euplotas crassus. MO/ Cell Biol 1994, 14:554-566. Exploiting the well defined timing of macronuclear development in Euplotes crassus, the structure of nascent telomeres was examined. Initially, telomeres were longer than in mature macronuclei, and the G-rich strand was more heterogeneous in length than the C-rich strand. 56.
Vermeesch JR, Williams D, Price CM: Telomere processing Euplotes. Nucleic Acids Res 1993, 21:5368-5371.
57.
Wellinger RJ, Wolf AJ, Zakian VA: Saccbaromyces telomeres acquire single-strand TGI -3 tails late in S phase. Cell 1993, 72:51-60.
58.
Gatvik B, Carson M, Hartwell L: Single-stranded DNA arising at telomeres in cdcl3 mutants may constitute a specific signal for the RAD9 checkpoint MO/ Cell Biol 1995, 15:6128-6138.
59.
Kyrion G, Boakye K, Lustig A: C-terminal truncation of RAP1 results in deregulation of telomere size, stability, and function in Saccharomyces cerevisiae. MO/ Cell Biol 1992, 12:5159-5173.
51. .
52. .
Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lanadorp PM: Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Nat/ Acad Sci USA 1994,91:9657-9860. Telomeres in a stem cell population from human blood were shown to be eroded with cell proliferation, raising the possibility that the proliferative lifespan of these cells is limited.
in
Structure and function of telomerase Kathleen Collins The study of eukaryotic telomeres
at the molecular
level
began with the discovery of short, tandem repeats at Tetrahymena decades,
chromosome
ends. In the following two
major insights about telomere structure and function
have come from investigations polymerase
that synthesizes
three areas of telomerase
telomere
the DNA
these repeats. In the past year,
research have been particularly
intense: assays of telomerase components,
of telomerase,
ditional background concerning telomerase or telomere structure and function, including details of the unique telomeres of Drosophila which are not addressed in this review, readers are referred to several recent publications [3-71.
activity, isolation of telomerase
and studies of the regulation of telomerase
and
length in Go.
Address Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204, USA; e-mail: [email protected] Current Opinion in Cell Biology 1996, 8:374-380 0 Current Biology Ltd ISSN 0955-0674 Abbreviation RNP ribonucleoprotein
Introduction The complete replication of eukaryotic chromosomes involves the participation of an RNA-dependent DNA polymerase known as telomerase ([1,2]; for reviews, see [3,4]). Telomerase recognizes chromosome ends and extends 3’ termini by addition of tandem simple sequence repeats. This de no210 DNA addition compensates for the sequence loss that results from incomplete terminal replication. Telomerase adds a specific repeat sequence to telomeres, using as a template a short region within the RNA component of the ribonucleoprotein (RNP) telomerase enzyme. Although cells can maintain a constant number of telomeric simple sequence repeats by balancing telomere loss during replication and addition by telomerase, not all cells do so (for reviews, see [3,5,6]). Human germline cells maintain a constant number of telomeric simple sequence repeats, but human somatic cells lose telomeric repeats with each cycle of cell division. Previous studies have suggested a correlation between a lack of telomerase activity, failure to maintain a stable telomere length, and limited proliferative capacity [3,5,6]. Cells with critically shortened telomeres appear to stop proliferating, as if halted by a checkpoint sensing dangerously small protective caps at chromosome ends. The past year has been an exciting one in the study of telomerase and telomere-length regulation. The first three sections of this review summarize new findings about telomerase activity and telomerase components. The fourth section relates these studies to an evolving understanding of telomere-length regulation, which must be more complex than previously anticipated. For ad-
Telomerase activity: more assays, more uncertainties An assay of telomerase activity in vitro requires only a few reaction components, namely a single-stranded DNA primer (which mimics a chromosome end), nucleotides, and telomerase enzyme [2]. As a result of these simple substrate requirements and the RNase-sensitivity of the reaction (which is a consequence of the essential RNA component of the enzyme), telomerase can often be detected specifically even in crude cell lysates. However, current assay protocols are not entirely reliable for telomerase obtained from some species or for quantitative analysis. The finicky nature of the in vitro telomerase assays is highlighted by three recent reports of Sadaromyces cerevisiae telomerase activity; the use of different protocols and different extracts led to entirely different descriptions of enzyme properties [8*,9,10]. The lack of qualitative and quantitative reliability derives partly from the following circumstances: first, the presence of contaminating RNA-dependent primer extension activities (such as reverse transcriptases of retro-elements); second, non-specific inhibition of telomerase in crude extracts; third, non-linear dependence of product synthesis on enzyme, primer, and nucleotide concentrations; and fourth, assay-dependent alteration of the processivity of elongation (i.e. alteration of the number of telomeric repeats added to each substrate). Hopefully, a consensus concerning the in vitro properties of yeast telomerase will develop from additional characterization of assay specificities and the use of telomerase preparations of greater purity. Another issue in the interpretation of telomerase activity assays is the relation of activity assayed in vitro (with high concentrations of single-stranded oligonucleotides as substrates) with activity assayed in vivo (on native chromosome ends). Many sequences will serve as primers for telomerase in vitro that do not promote telomere formation in vivo. In fact, the sequence specificity of telomere formation in human cells [ll] appears to reflect the sequence-specificity of DNA binding by a human telomere protein [ 12,13’] rather than the primer specificity of telomerase itself. In vitro, telomerase from the ciliate Oxytticha can elongate a telomeric primer DNA bound to telomere proteins [14]. These results suggest that telomerase recognizes a chromosome substrate composed of both DNA and protein. The interaction of telomerase with
Structure and function of telomerese Collins
telomeric repeat binding proteins topic for future exploration.
Telomerase
remains
an interesting
components
RNA
The RNA component of telomerase, originally identified in Tetrahymena [15], has been subsequently identified in a number of other ciliates [16X0]. In the past year, telomerase RNAs have also been cloned from nonciliate species. A genetic screen led to the cloning of the S. cerevisiae telomerase RNA: when overexpressed in incomplete form, the telomerase RNA reduced telomeric silencing [Zl”]. Singer and Gottschling [Zl”] noted in their clone a 16 nucleotide region of exact complementarity to the repeat sequence initially added to heal broken yeast chromosomes [ZZ]. Changes in the sequence of this RNA region in vivo resulted in telomeric incorporation of the predicted mutant repeats [21**]. Disruption of the gene encoding the 1.3 kb telomerase RNA led to telomere shortening and a delayed-growth defect of the culture, as first described for S. cewvisiae estl strains deficient in telomere maintenance [23]. The Khyveromyces lactis telomerase RNA was identified using a different approach [24”]. A genomic DNA fragment which hybridized to the long K. lactis telomeric repeats, but was not itself telomeric, was cloned. When the region of the clone complementary to the telomeric repeat was altered in vivo, mutant repeats were incorporated into telomeric DNA. Disruption of the gene encoding the (also) 1.3 kb telomerase RNA resulted in telomere shortening and a delayed-growth defect of the culture. For both S. cerevisiae and K. /actis strains that lack telomerase-dependent telomere elongation, a small percentage of cells eventually overcome their growth defect and acquire longer telomeres [21**,24**,25]. This suggests that a telomerase-independent telomere elongation mechanism exists (see below). Telomerase RNAs have also been cloned from two mammalian sources. The human RNA was isolated by employing molecular techniques to enrich for small RNAs containing a short region of telomere-complementary sequence [26”]. The gene encoding the human RNA was used as a hybridization probe to clone the mouse RNA [27**]. Sequence alterations in the predicted human or mouse template region resulted in the synthesis of mutant repeats in in vitro experiments using extracts of cells transfected with plasmids encoding mutant RNAs. Both mammalian telomerase RNAs are approximately 500 nucleotides in length. In several tissues, levels of the mouse telomerase RNA decline during development [27**]. Different human tissues, and primary versus immortalized cell lines, also show variation in telomerase RNA levels, although not as much as might be expected from relative differences in telomerase activity and telomere maintenance [26.*]. The yeast and mammalian telomerase RNAs show a high divergence of primary sequence, as previously observed for the ciliate RNAs [ 1S-201.
375
Analysis of telomerase RNA function has continued as in previous years [28-301, using Tetraymena telomerase assembled from mutant RNAs and wild-type proteins either in &JO or in vitro. Studies of mutant RNA function have focused on the RNA template region for technical reasons. In vivo assembly requires competition of overexpressed mutant RNAs with the wild-type RNA for limiting proteins; thus, mutations which decrease the efficiency of RNP assembly cannot be analyzed. In vitro assembly is performed in the presence of fragments of partially digested native RNA, thus, mutant RNAs may need to displace native RNA fragments or may not require essential domains of the RNA due to residual function of the native fragments. Continued analysis of the template region has yielded several interesting results. When reconstituted in vitro, mutation of a region of the RNA immediately 5’ of the template, a region conserved in ciliate RNAs, alters selection of the template residues that are most efficiently copied into product DNA [31*]. Using in vivo reconstitution, mutations in the template region were discovered that alter elongation efficiency and also, unexpectedly, affect enzyme fidelity [32*,33*]. The latter result suggests that the telomerase RNA plays a role in catalysis in addition to its template function. Finally, the properties of wild-type ciliate telomerase RNAs have been examined. The structure of i’hrahymena telomerase RNA was analyzed in vivo [34], as was the structure of the same RNA transcribed in vitro [35]. A comparison of these two structures suggests that the overall folding of the RNA is similar whether or not proteins are bound, and is similar to the structure predicted from phylogenetic comparison. In another study, the nuclear localization of Oxytticha telomerase RNA was examined by in situ hybridization [36]. Chromosomes in an Oxytrida macronucleus are replicated by a concerted progression of DNA synthesis machinery, which forms a replication ‘band’. Telomerase RNA was shown to be localized at the trailing end of the replication band (consistent with its function in telomere synthesis) but also at large structures that are likely to be storage sites. Thus, assembly or localization of the RNP could regulate telomerase activity, allowing the enzyme to be active only when appropriate substrates are present. Proteins, and the ribonucleoprotein
in action
Ten years after the identification of telomerase activity in vitro [2], protein components of Tetralzymena telomerase have been identified using a biochemical approach [37**]. Two polypeptides of 80 kDa and 95 kDa copurified with telomerase activity and the telomerase RNA, and by several criteria were shown to form a stable, threecomponent RNP complex with the telomerase RNA. Nucleic acid cross-linking and binding assays demonstrated that p80 specifically interacts with the telomerase RNA and p95 specifically interacts with telomeric primers. Cloning of the cDNAs encoding p80 and p95 revealed novel proteins, with limited homology to any sequence in
376
Nucleus and gene expression
the available databases [37”]. Although no polypeptides other than p80 and ~95 were associated in stoichiometric quantities with the telomerase RNA, it remains to be shown that one or both proteins are sufficient, in combination with the telomerase RNA, to catalyze enzyme activity.
Fiaure 1
A weak homology has been observed between the carboxy-terminal region of ~95 and conserved active-site residues in a family of viral RNA-dependent RNA polymerases [37”]. Whether this minimal structural homology reflects a conservation of function is unknown, although biochemical evidence also suggests that telomerase shares the properties of an RNA-dependent RNA polymerase: i’&ra/?vmena telomerase shows greater affinity for RNA primers than for DNA primers of the same sequence and can incorporate ribonucleotides [38*]. These studies extend previous indications of the remarkable lack of specificity of telomerase in oitro. They also suggest the possibility that telomerase evolved from an RNA-dependent RNA polymerase expected to have catalyzed genome replication in the primordial RNA world [39]. A hypothetical model for Tetrahymena telomerase structure is shown in Figure 1. For illustrative purposes, telomerase is drawn to resemble models for the reverse transcriptase of HIV-l [40]. This reverse transcriptase contains two subunits with homologous sequences but distinct structures. The larger subunit harbors the active site; the smaller subunit forms part of the template-primer-binding site and may position tRNA primers such that the tRNA 3’ end forms base pairs with the template in the active site. In Figure 1, telomerase p95 contains the active site whereas p80 brings the telomerase RNA to ~95 such that the template is held in the active site. Primers typically bind with their 3’ end at the RNA template and with a more 5’ region at an RNA-independent binding site which is, at least partially, composed of ~95 [37”,41,42]. How product binding is regulated at these two sites to accomplish processivity and to moderate the nucleolytic cleavage reaction catalyzed by telomerase [41] is not known. However, telomerase and the DNA-dependent RNA polymerases are likely to share a similar mechanism, judging from biochemical similarities between the two enzymes [41-43].
Telomerase and telomere-length in vivo
regulation
Telomeres have several confirmed roles in the cell, and several more are suspected. They are required for chromosome stability and complete linear genome replication, and they appear to regulate gene expression, chromosome segregation, and nuclear architecture (for reviews, see [3,7]). However, much of the emphasis of the past year has been placed on a single question: is telomere length regulated by telomerase to determine a cell’s proliferative capacity? Numerous studies have shown a correlation between replication-dependent loss of telomeric
Q 1996 Current Opinion m Cell Biology
A model of the Tetrahymena telomerase RNP. The telomerase RNA is drawn with regions of single-stranded or double-stranded structure as derived from phylogenetic and direct physical evidence [16-20,34,35]. A primer is bound at its 3’ end to the RNA (binding is shown as three base pairs or parallel lines) and also to an RNA-independent site (drawn as a cleft) on the p95 protein subunit [37’*]. The p60 subunit interacts with telomerase RNA [37”1; the interaction is shown here between p60 and a long, bent double helix (which represents combined stems I and IV; 1161). Protein-RNA interactions which fasten the template in the active site area are suggested
by dark gray shaded boxes.
simple sequence repeats and limited proliferative capacity (for reviews, see [3,5,6]). Cells appear to stop dividing when they sense that telomeres are too short to safely allow additional rounds of cell division. In general, cells that exhibit finite proliferative capacity and shortening telomeres lack telomerase activity, whereas cells with indeterminate proliferative capacity and stable telomere length possess active telomerase enzyme (for reviews, see [3,5,6]). In the past year, however, several interesting exceptions have been reported (see below). These results suggest that we are missing unanticipated pieces of the telomere length regulation puzzle. At least three lines of research have converged to indicate the need for a new, more complex model of telomere-length regulation; these lines of research are additional investigations of telomerase activity and telomere length in mammalian cells, genetic manipulation of telomeres in model systems, and analysis of telomere replication intermediates in ciliates. In a large number of recent reports (only a selection of which can be cited here), some immortal cell lines that maintain telomere length have been shown to possess
Structure and function of telomerase
telomerase activity [44*,45] whereas other immortal cell lines have been shown to lack it [46,47’,48]. In addition, not all cells possessing telomerase activity do maintain telomere length. For example, some normal human blood cell populations were found to be telomerase-positive [49*-51.1 although telomere shortening also occurs in some of these cell types ([52*]; see chapter 9 in 131). Telomerase activity has been detected in somatic tissues of mice that experience telomere loss [53,54], although the telomerase-positive cell populations are not known. We conclude from these studies that not all immortal cells possess detectable telomerase activity and that not all cells with active telomerase maintain a stable number of telomeric simple sequence repeats.
In addition, genetic manipulations have revealed that activities other than telomerase are important for telomere maintenance. S. cemisiae and K. /act& strains lacking in viva telomerase activity exhibit growth defects after telomere attrition, but they then recover, yielding ‘survivor’ populations with extended telomeres [21”,24**,25]. these results suggest that a telomeraseAlthough independent telomere maintenance mechanism can ‘kick in’ if required, the uneven growth of the survivor strains suggests that telomerase-dependent telomere maintenance may be the healthier alternative. Experiments in yeast also suggest that telomere-length regulation in the presence of active telomerase is influenced by additional factors. In a revealing set of experiments, mutations in the K. /a& telomerase RNA were employed to alter telomeric repeat sequences ([24.*]; see above). Previously, when Tetrahymena telomeric repeats were altered using a similar method, some mutations caused immediate loss of telomere-length regulation [28,29,32’,33*]. In K. lacks, telomeric incorporation of some mutant repeats also resulted in immediate loss of length regulation. Other mutant repeats, however, affected telomere length only after many generations, when almost all wild-type repeats had been replaced with the mutant sequence. Thus, even in the presence of telomerase, proper control of telomere length appears to depend on factor(s) which recognize centromere-proximal and centromere-distal telomere repeat sequences.
It is striking that even very centromere-proximal telomeric repeats could be replaced in K. /ads strains with altered telomerase specificity. According to current dogma, telomerase should have replaced only sequences at the extreme termini, repeatedly adding back the same few terminal repeats lost during each round of replication. The substitution of centromere-proximal telomeric repeats in K. lads could proceed either by non-homologous and unequal recombination or by access of templatereprogrammed telomerase to ‘internal’ locations. The latter possibility would suggest extensive telomere trimming, balanced with compensatory elongation, in a single S phase (see Fig. 2). Perhaps telomerase itself recruits the
trimming machinery to prevent absence of elongation.
excess
Collins
trimming
377
in the
Figure 2 I
I
0
1996Current
O&on
in Cell B~oloav
Multiple processing events in telomere synthesis. Various activities that contribute to the maintenance of telomere length by telomerase are suggested. Each processing step could occur multiple times on the same telomere in a single S phase, or a step could be missed completely. (a) Elongation of one strand of telomeric DNA is mediated by telomerase (the newly synthesized, G-rich telomeric DNA strand is represented by a wavy black arrow) [281.(b-d) Cleavage of the single-stranded telomeric repeat DNA could occur both (b) before and (d) after (c) completion of second-strand synthesis. Cleavage before second-strand synthesis could promote replacement of internal telomeric repeats I24”l whereas cleavage after second-strand synthesis would provide a mechanism for polishing ends to produce a precise overhang 155’1. (e) Cleavage or deletion of double-stranded telomeric repeats is also likely to occur; this is particularly noticeable when telomeres
become extremely long (see [24”,5g]
several examples). How these processing coordinated remains to be determined.
for two of
activities are regulated
and
Some evidence for regulated, replication-independent telomere trimming derives from the investigation of telomere replication intermediates. Following Ize n~uo telomere synthesis in the ciliate Euplotes crassus, telomeric repeat tracts are longer and more heterogeneous than those of mature macronuclei i55.1. This suggests that telomere elongation itself need not be precisely controlled. Trimming of overly long telomeres to obtain a precise terminal structure occurs independently of DNA replication (see Fig. 2) [56]. In S. cemisiae, long G-rich regions of single-stranded DNA were observed transiently in late S phase, possibly reflecting a similar excess of telomeric repeat addition [57]. An accumulation of single-stranded telomeric DNA can also be observed in cell division cycle mutants of yeast [58]. If balancing the number of telomeric repeats involves not only loss of repeats by incomplete replication and elongation by telomerase but also active trimming, subtle alterations in this balance of multiple components could readily establish a different ‘normal’ telomere length.
378
Nucleus and gene expression
Conclusions The past year has seen enormous progress in the identification of telomerase components. Telomerase RNAs cloned from species other than ciliates reveal even less conservation of length or primary sequence than previously observed among ciliate RNAs. The first telomerase proteins have been identified from Tetrahymenaand share very little, if any, primary sequence homology with other known proteins. With these reagents in hand, the future holds promise for a more detailed molecular investigation of this unique polymerase. Although limited by the lack of molecular reagents, studies of telomerase regulation using activity assays have revealed a greater complexity than originally envisioned. The past year’s research was full of exceptions: not all mammalian somatic tissues lack telomerase activity and not all immortalized cells possess it. In addition, exceptions have been identified to the correlations between telomerase activity and stable telomere length, and between telomerase absence and telomere shortening. Mechanisms for executing the various regulations of telomerase will be a prime area of interest for future studies. Experimentally induced ‘regulation’ of telomerase has provided a convenient starting point for addressing more general questions of telomere structure and function. When a telomere sequence was altered by mutation of the telomerase RNA template, sequence-specific effects on telomere-length regulation and cell viability were observed. Future use of this technique, combined with a biochemical analysis of altered telomeric chromatin, should prove extremely informative in the pursuit of the molecular basis of telomere functions, including the which limits cell putative ‘short telomere’ checkpoint proliferation.
5.
Greider CW: Mammalian teiomere dynamics: healing, fragmentation, shortening and stabilization. Curr Opin Gener Dev 1994,4:203-211.
6.
Hariev CB. Kim NW. Prowse KR. Weinrich SL. Hirsch KS. West.MD, ‘Bacchett/ S, Hirte Hh, Counter &I, Greider CW et al.: Teiomerase, ceil immortality, and cancer. Cold Spring Harb Symp &ant Biol1994, 59:307-315.
7.
Zakian VA: Teiomeres: beginning to understand Science 1995, 270:1601-l 607.
8.
Cohn M, Blackburn EH: Teiomerase in yeast Science 1995, 269:396-400. yhis paper describes the most convincing in vitro assay for S. cerevisiae telomerase. Fractionated whole call extracts were examined for RNasesensitive, primer sequence dependent incorporation of dGTP and lTP. Nucleotides were added in register with the 3’ end of sequence-permuted telomeric primers, and ddNTPs were incorporated at predicted positions in the oroduct DNA. Product DNA svnthesis raouired the teiomerase RNA but was’indistinguishable in the presence or absahce of EST1 p, a gene product required for telomere maintenance [231. Hence EST7 probably does not encode the catalytic subunit of telomerase. 9.
Lin J, Zakian VA: An in vitro assay for Sacchumyces telomerase requires ESTl. Cell 1995, 81 :1127-l 135.
10.
Lue NF, Wang JC: ATP-dependent processivity of a teiomerase activity from Sacchefomyces cefevisiae. J Biol Chem 1995, 270121453-21456.
11.
Hanish JP, Yanowitz JL, De Lange T: Stringent sequence requirements for the formation of human teiomeres. froc Nat/ Acad Sci USA 1994, 91:8881-8865.
12.
Zhong Z, Shiue L, Kaplan S, De Lange T: A mammalian factor that binds teiomeric TTAGGG repeats in vitro. MO/ Cell Biol 1992,12:4834-4843.
13. .
Chona L. Van Steensal B. Broccoli D. Erdiument-Bromaae H. Hanisi J; Tempst P, De Lange T: A himar; teiomeric p&ein. Science 1995, 270:1663-l 667. . This paper describes the punhcatlon and cloning of the gene encodmg a human protein that specifically binds to double-stranded teiomeric repeats. The protein, human teiomeric repeat binding factor, is shown to localize to teiomeres in vivo, and represents the first vertebrate telomeric repeat binding protein whose encoding gene has been cloned. 14.
Shippen DE, Blackburn EH, Price CM: DNA bound by the hyffiche teiomere protein is accessible to teiomerase and other DNA poiymerases. Proc Nat/ Acad Sci USA 1994, 91:405-409.
15.
Greider CW. Blackburn EH: A teiomeric seouence in the RNA of Tetrahynkne telomerase required for teiomere repeat synthesis. Nature 1989, 337:331-337.
16.
Shippan-Lentz D, Blackburn EH: Functional evidence for an RNA template in telomerase. Science 1990, 247:546-552.
1 7.
Romero DP, Blackburn EH: A conserved secondary teiomerase RNA Cell 1991, 67~343-353.
18.
Greider CW, Blackburn EH: The telomere terminal transferase of Tetrahymene is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell 1967, 513887-898.
Meiek M, Davis BT, Shippen DE: Oligonucieotides complementary to the Oxykiche nov8 teiomerase RNA delineate the template domain and uncover a novel mode of primer utilization. MO/ Cell Biol 1994, 14:7827-7838.
19.
Greider CW, Blackburn EH: identification of a specific telomere terminal transferase activity in Tetrabymena extracts. Cell 1985, 43:405-413.
Lingner J, Hendrick LL. Cech TR: Teiomerase RNAs of different ciiiates have a common secondary structure and a permuted template. Genes Dev 1994, 8:1984-l 998.
20.
McCormick-Graham structural features.
Acknowledgements I thank members of the Collins laboratory (namely, L Gandhi, R Mitchell, JM Andresen and J Mitchell), Paul Kaufman and Donald Rio for comments on this review.
References and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
. .. 1.
2.
3.
4.
the end.
of special interest of outstanding interest
Blackburn EH, Greider CW: Telomeres. Plainview, New York: Cold Spring Harbor Laboratory Press; 1995. Blackburn EH: Telomerase. In The RNA World. Edited by Gestiand RF, Atkins JF. Plainview, New York: Cold Spring Harbor Laboratory Press; 1993:557-576.
structure for
M, Rornero DP: Ciliate telomerase RNA Nucleic Acids Res 1995, 23:1091-l 097.
Singer MS, Gottschling DE: TLC1 : template RNA component of S8cc/t8rofnyces cerevisiae teiomerase. Science 1994, 266:404-409. After much hunting for teiomerase components in S. cerevisiae, the first sighting is described in this paper. Partial telomerase RNA clones were
21. ..
Structure and function of telomerese
identified as high-copy suppressors of silencing. Genetic disruption of the RNA resulted in telomere shortening and in senescence; mutations of the RNA template region were copied into telomeric DNA in viva. 22.
Kramer KM, Haber JE: New telomeres in yeast are initiated with a highly selected subset of TGI -3 repeats. Genes fJev 1993, 712345-2356.
23.
Lundblad V, Szostak JW: A mutant with a defect in telomere elongation leads to senescence In yeast Cell 1989, 57:633-643.
McEachem MJ, Blackburn EH: Runaway telomere elongation caused by telomerase RNA gene mutations. Nature 1995, 376:403-409. The K /a&s telomerase RNA was cloned by exploiting the long telomerit repeats found in this organism. Genetic disruption of the RNA caused senescence, and mutations of the RNA in the template region resulted in mutant telomeric repeat synthesis, loss of telomere-length regulation, and loss of viability. The use of telomerases with altered specificity, in combination with other techniques, will continue to provide insights into mechanisms for telomere-length regulation. 24. ..
25.
Lundblad V, Blackburn EH: An alternative pathway for yeast telomere maintenance rescues estl -senescence. Cell 1993, 731347-360.
Feng J, Funk WD, Wang S, Weinrich SL, Avilion AA, Chiu C, Adams RR, Chang E, Allsopp RC, Yu J et al: The RNA component of human telomerase. Science 1995, 269:1236-l 241. The human telomerase RNA was cloned and expression of the RNA was examined in cells and tissues. Many or all cells and tissues appear to contain telomerase RNA, but with up to 1 O-fold differences in levels. Antisense RNA expression caused crisis in some cultures, although significant telomere length and telomerase activity were maintained.
26. ..
Blasco MA, Funk W, Villeponteau B, Greider CW: Functional characterization and developmental regulation of mouse telomerase RNA Science 1995, 269:1267-l 270. The mouse telomerase RNA was cloned and expression of the RNA examined in cells and tissues. Like the human RNA, many or all mouse cells and tissues appear to contain telomerase RNA, with up to 1O-fold differences in the levels found. In several tissues, RNA levels decreased during development. 27. ..
26.
Yu G, Bradley JD, Attardi LD, Blackburn EH: In viva alteration of telomere sequences and senescence caused by mutated Tebabymena telomerase RNAs. Nature 1990, 344:126-l 32.
29.
Yu G, Blackburn EH: Developmentally programmed healing of chromosomes by telomerase in Tefrahymena. Cell 1991, 67:823-832.
30.
Autexier C, Greider CW: Functional reconstitution of wildtype and mutant Tetrahymena telomerase. Genes Dev 1994, 8:563-575.
31. .
Autexier C, Greider CW: Boundary elements of the Tetrahymena telomerase RNA template and alignment domains. Genes Dev 1995,9:2227-2239. Mutant telomerase RNAs were combined with wild-type proteins in vitro to assay the effect of template region changes on telomerase activity. Alteration of the template had complex effects. A region 5’ of the template sequence helped to define template boundaries. 32. .
Gilley D, Lee MS, Blackburn EH: Altering specific telomerase RNA template residues affects active site function. Genes Dev 1995,9:2214-2226. Mutant telomerase RNAs were assembled with wild-type proteins in viva then assayed in cell extracts. Mutation of the template region affected DNA synthesis in a complex manner, including alterations of enzyme fidelity. See also 133.1. 33. .
Gilley D, Blackburn EH: Specific RNA residue interactions required for enzymatic functions of Tetrehymene telomerase. MO/ Cell Biol 1996, 16:66-75. See annotation 132.1.
Collins
379
34.
Zaug Al, Cech TR: Analysis of the structure of Tetrahymena nuclear RNAs in viva: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA RNA 1995, 1:363-374.
35.
Bhattacharyya A, Blackburn EH: Architecture EM60 J 1994,13:5721-5731.
36.
Fang G, Cech TR: Telomerase RNA localized in the replication band and spherical subnuclear organelles in hypotrichous ciliates. J Ce// Biol 1995, 130:243-253.
of telomerase
RNA
37. ..
Collins K, Kobayashi R, Greider CW: Purification of Tetrahymene telomerase and cloning of genes encoding the two protein components of the enzyme. Cell 1995,81:677-666. This paper represents the first identification of protein components of telomerase in any species. Tefrahymena telomerase was puriiied and shown to contain two proteins in addition to the 159 nucleotide telomerasa RNA. Nucleic acid binding properties of the proteins were examined. Genes encoding the proteins were cloned using peptide sequence information and the genes were shown to encode novel proteins. 38. .
Collins K, Greider CW: Utilization of ribonucleotldes and RNA primers by Tetrahymena telomerase. EMBO J 1995, 14~5422-5432. Ribonucleotides and RNA primers were shown to serve as substrates for telomerase. Substitution of RNA at different positions in a primer had different consequences, suggesting that there are multiple distinct sites of enzyme-primer interaction. 39.
Joyce GF, Orgel LE: Prospects for understanding the origin of the RNA world. In The RNA World. Edited by Gesteland RF, Atkins JF. Plainview, New York: Cold Spring Harbor Laboratory Press; 1993:1-25.
40.
Kohlstaedt LA, yang J, Freidman JM, Rice PA, Steitz TA: Crystal structure at 3.5A resolution of HIV-l reverse transcriptase complexed with an inhibitor. Science 1992, 256:1783-l 790.
41.
Collins K, Greider CW: Nucleolytic cleavage and nonprocessive elongation catalyzed by Tetrahymena telomerase. Genes Dev 1993, 7:1364-l 376.
42.
Lee MS, Blackburn EH: Sequence-specific DNA primer effects on telomerase polymerization activity. MO/ Cell Biol 1993, 13:6586-6599.
43.
Chamberlin MJ: New models for the mechanism of transcription elongation and its regulation. In The Harvey Lectures. Wiley-Liss; 1993:1-21.
44. .
Kim NW, Piatyszek MA, Prowsa KR, Harley CB, West MD, Ho PLC, Coviello GM, Wright WE, Weinrich SL, Shay JW: Specific association of human telomerase activity with immortal cells and cancer. Science 1994, 266:201 l-201 4. A telomeraae assay wtth increased sensltiwty was developed (using the polymerase chain reaction to amplify telomerase product DNA) and employed to assay telomerase activity in a va:iety of human cells, tissues, and cancers. Most immortal cell lines and cancers showed higher telomerase activity than did primary cells or normal tissue. 45.
Hiyama E, Hiyama K, Yokoyama T, Matsuura Y, Piatyszek MA, Shay JW: Correlating telomerase activity levels with human neuroblastoma outcomes. Nat Med 1995,1:249-255.
46.
Murnane JP, Sabatier L, Marder BA, Morgan WF: Telomere dynamics in an immortal cell line. EM60 J 1994, 13:4953-4962.
47. .
Bryan TM, Englezw A, Gupta J, Bacchetti S, Reddel RR: Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J 1995, 1414240-4240. Telcmere length and telomerase activity were assayed in a number of im mortalized human cell lines. Not ail cell lines possessed active telomerase, but all immortalized cell lines that lacked telomerase had telcuneres which were longer and more heterogeneous than in telomerase-positive cell lines. In combination with other results, these findings suggest that a non-telomerase dependent mechanism for telomere elongation is likely to exist, characterized by more variable telomere length. 48.
Rogan EM, Bryan TM, Hukku B, MacLean K, Chang AC, Moy EL, Englezou A, Warnford SG, Dalla-Poua L, Reddel RR: ARerations
380
Nucleus
and gene expression
in p53 and pl6lNK4 expression and telomere length during spontaneous immortalization of Li-Fraumeni syndrome fibroblasts. MO/ Cell Biol 1995, 1514745-4753. 49. .
Counter CM, Gupta J, Harley CB, Leber B, Bacchetti S: Telomerase activity in normal leukocytes and in hematologic malignancies. Blood 1995, 65:2315-2320. Telomerase activity was detected in leukocytes, with some differences in activity level between normal and malignant cells. 50. .
Broccoli D, Young JW, De Lange T: Telomerase activity in normal and malignant hematopoietic cells. Proc Nat/ Acad Sci USA 1995,92:9082-9066. Telomeraae activity was detected in a variety of normal human blood cell populations and at similar levels in hematologic malignancies. Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Piatyazek MA, Shay JW, lshioka S, Yamakido M: Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. I Immunol1995, 155:371 l-371 5. Telomeraae activity was detected in normal human blood calls and was shown to increase with mitogenic stimulation of cells in vitro.
53.
Prowae KR, Greider CW: Developmental and tissue-specific regulation of mouse telomerase and telomere length. Proc Nat/ Acad Sci USA 1995,92:4818-4822.
54.
Chadeneau C, Siegel P, Harley CB, Muller WJ, Bacchetti S: Telomerase activity in normal and malignant murine tissues. Oncogene 1995, 11:893-898.
55. .
Vermeeach JR, Price CM: Telomeric DNA sequence and structure following de nova telomere synthesis in Euplotas crassus. MO/ Cell Biol 1994, 14:554-566. Exploiting the well defined timing of macronuclear development in Euplotes crassus, the structure of nascent telomeres was examined. Initially, telomeres were longer than in mature macronuclei, and the G-rich strand was more heterogeneous in length than the C-rich strand. 56.
Vermeesch JR, Williams D, Price CM: Telomere processing Euplotes. Nucleic Acids Res 1993, 21:5368-5371.
57.
Wellinger RJ, Wolf AJ, Zakian VA: Saccbaromyces telomeres acquire single-strand TGI -3 tails late in S phase. Cell 1993, 72:51-60.
58.
Gatvik B, Carson M, Hartwell L: Single-stranded DNA arising at telomeres in cdcl3 mutants may constitute a specific signal for the RAD9 checkpoint MO/ Cell Biol 1995, 15:6128-6138.
59.
Kyrion G, Boakye K, Lustig A: C-terminal truncation of RAP1 results in deregulation of telomere size, stability, and function in Saccharomyces cerevisiae. MO/ Cell Biol 1992, 12:5159-5173.
51. .
52. .
Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lanadorp PM: Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Nat/ Acad Sci USA 1994,91:9657-9860. Telomeres in a stem cell population from human blood were shown to be eroded with cell proliferation, raising the possibility that the proliferative lifespan of these cells is limited.
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