Telomerases chemistry biology and clinical applications

TELOMERASES


Cover art: Model of telomerase extending telomeric DNA (blue). This model is
rendered from available crystal and NMR structures of the telomerase RNA (green;
2K95, 2L3E, 1Z31, and 1OQ0), the telomerase reverse transcriptase (TEN, pink;
2B2A; TRBD, light red; RT, red; and CTE, dark red; 3KYL), the H/ACA snoRNP
complex (dyskerin, light blue; Gar1, blue; Nop10, sky blue; and Nhp2, dark blue;
2HVY), and the Pot1-Tpp1 complex (yellow, 1XJV and orange, 2I46; respectively).
Image provided by Josh D. Podlevsky and Julian J.-L. Chen (Arizona State
University).


TELOMERASES
Chemistry, Biology, and Clinical
Applications
Edited by
NEAL F. LUE
Weill Medical College of Cornell University, New York, NY, USA

CHANTAL AUTEXIER
Departments of Anatomy and Cell Biology, and Medicine, McGill University
Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research,
Jewish General Hospital, Montreal, Quebec, Canada


Copyright Ó 2012 by John Wiley & Sons, Inc. All rights reserved
Published by John Wiley & Sons, Inc., Hoboken, New Jersey
Published simultaneously in Canada
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Library of Congress Cataloging-in-Publication Data:
Telomerases : chemistry, biology, and clinical applications / edited by Neal F. Lue,
Chantal Autexier. – 1st. ed.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-0-470-59204-5 (hardback)
I. Lue, F. Neal, 1962- II. Autexier, Chantal, 1963[DNLM: 1. Telomerase. QU 56]
572.80 6–dc23
2011047556
Printed in the United States of America

ISBN: 9780470592045
10 9 8 7 6 5 4 3 2 1


CONTENTS

Preface

vii

Contributors

ix

1

The Telomerase Complex: An Overview

1

Johanna Mancini and Chantal Autexier

2

Telomerase RNA: Structure, Function, and Molecular
Mechanisms

23

Yehuda Tzfati and Julian J.-L. Chen

3

TERT Structure, Function, and Molecular Mechanisms

53

Emmanuel Skordalakes and Neal F. Lue

4

Telomerase Biogenesis: RNA Processing, Trafficking,
and Protein Interactions

79

Tara Beattie and Pascal Chartrand

5

Transcriptional Regulation of Human Telomerase

105

Antonella Farsetti and Yu-Sheng Cong

6

Telomerase Regulation and Telomere-Length Homeostasis

135

Joachim Lingner and David Shore

7

Telomere Structure in Telomerase Regulation

157

Momchil D. Vodenicharov and Raymund J. Wellinger
v


vi

8

CONTENTS

Off-Telomere Functions of Telomerase

201

Kenkichi Masutomi and William C. Hahn

9

Murine Models of Dysfunctional Telomeres and Telomerase

213

Yie Liu and Lea Harrington

10

Cellular Senescence, Telomerase, and Cancer in Human Cells

243

Phillip G. Smiraldo, Jun Tang, Jerry W. Shay, and Woodring E. Wright

11

Telomerase, Retrotransposons, and Evolution

265

Irina R. Arkhipova

Index

301


PREFACE

This year marks the 27th anniversary of the discovery of telomerase. In retrospect,
even though hints of a special activity needed to maintain linear chromosome ends
could be traced to earlier theoretical arguments and experimental observations, it was

the exposure of an autoradiogram on Christmas day, 1984 that finally brought the
activity into sharp focus and enabled it to be captured, dissected, and manipulated.
The fascinating story of the discovery of telomerase has been told elsewhere and will
not be repeated here. Our goal for this volume is instead to take stock of what has been
learned about this fascinating reverse transcriptase in the ensuing 27 years, in the hope
of providing more impetus for the investigation into its chemistry, biology, and
clinical applications. If the past 27 years can serve as a guide, than the payoff for the
next 27 years of telomerase research would be great indeed.
We have organized this compendium with a view toward offering integrated
discussions of the three aspects of telomerase covered by the subtitle. The collection
starts with an overview of the telomerase complex, followed by in-depth discussions
of the chemistry of its two critical components: TERTand TER. The next two chapters
highlight the biological regulatory mechanisms that control the synthesis and
assembly of the telomerase complex. Equally significant are the regulations imposed
by the nucleoprotein complex at chromosome ends, the topics of the two ensuing
chapters. Three more chapters accent studies that bring considerable spotlight to
telomerase as a promising target and a useful tool in medical interventions. The
collection then concludes with an essay that puts telomerase in evolutionary context
and illuminates its place in the extraordinarily diverse family of reverse transcriptases.
Although telomerase research is far from unique in the exploitation of model
organisms, it has perhaps uniquely benefited from this approach, as evidenced by the
initial discovery of the enzyme in ciliated protozoa, and the demonstration of its
vii


viii

PREFACE

importance in chromosome maintenance in budding yeast. The proliferation of model

system analysis, while arguably indispensable, also made it difficult even for
specialists to keep abreast of all the relevant developments, not to say students and
investigators newly attracted to a vibrant research field. A main objective for authors
of this volume, then, is not only to gather significant experimental observations, but
also to provide an integrated discussion of each significant topic across different
model systems. We thank all of the authors for their tremendous efforts in this
difficult but admirable endeavor.
This project would not have taken place without the initial suggestion and expert
guidance of Anita Lekwani at Wiley. Rebekah Amos and Catherine Odal’s help in
shepherding the initial drafts into the final texts is greatly appreciated. Finally, we
thank our coworkers and colleagues for making the study of telomerase an “endlessly”
stimulating and fascinating endeavor.
NEAL F. LUE
CHANTAL AUTEXIER


CONTRIBUTORS

Irina Arkhipova, Josephine Bay Paul Center for Comparative Molecular Biology
and Evolution, Marine Biological Laboratory, Woods Hole, MA, USA
Chantal Autexier, Departments of Anatomy and Cell Biology, and Medicine, McGill
University; Bloomfield Centre for Research in Aging, Lady Davis Institute for
Medical Research, Jewish General Hospital, Montreal, Quebec, Canada
Tara Beattie, Southern Alberta Cancer Research Institute and Departments of
Biochemistry and Molecular Biology and Oncology, University of Calgary,
Calgary, Alberta, Canada
Pascal Chartrand, Departement de Biochimie, Universite de Montreal, Montreal,
Quebec, Canada
Julian J.-L. Chen, Department of Chemistry and Biochemistry, and School of Life
Sciences, Arizona State University, Tempe, AZ, USA

Yu-Sheng Cong, Institute of Aging Research, Hangzhou Normal University School
of Medicine, Hangzhou, China
Antonella Farsetti, National Research Council (CNR) and Department of Experimental Oncology, Regina Elena Cancer Institute, Rome, Italy
William Hahn, Department of Medical Oncology, Dana-Farber Cancer Institute and
Departments of Medicine, Brigham and Women’s Hospital and Harvard Medical
School, Boston, MA, USA; Broad Institute of Harvard and MIT, Cambridge,
MA, USA

ix


x

CONTRIBUTORS

Lea Harrington, Wellcome Trust Centre for Cell Biology, University of Edinburgh,
Edinburgh, United Kingdom
Joachim Lingner, Swiss Institute for Experimental Cancer Research (ISREC),
School of Life Sciences, Frontiers in Genetics National Center of Competence
in Research, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne,
Switzerland
Yie Liu, Laboratory of Molecular Gerontology, National Institute on Aging, National
Institutes of Health Baltimore, MD, USA
Neal F. Lue, Department of Microbiology and Immunology, Weill Medical College
of Cornell University, New York, NY, USA
Johanna Mancini, Bloomfield Centre for Research in Aging, Lady Davis Institute
for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada
Kenkichi Masutomi, Cancer Stem Cell Project, National Cancer Center Research
Institute, Chuo-ku, Tokyo, Japan; PREST, Japan Science and Technology Agency,
Saitama, Japan

Jerry W. Shay, Department of Cell Biology, UT Southwestern Medical Center,
Dallas, TX, USA
David Shore, Department of Molecular Biology, University of Geneva, Frontiers in
Genetics National Center of Competence in Research, Geneva, Switzerland
Emmanuel Skordalakes, Gene Expression and Regulation Program, The Wistar
Institute, Philadelphia, PA, USA
Phillip G. Smiraldo, Department of Cell Biology, UT Southwestern Medical Center,
Dallas, TX, USA
Jun Tang, Department of Cell Biology, UT Southwestern Medical Center, Dallas,
TX, USA
Yehuda Tzfati, Department of Genetics, The Silberman Institute of Life Sciences,
The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem, Israel
Momchil Vodenicharov, Departement de biologie and Departement de microbiologie et infectiologie, Universite de Sherbrooke, Sherbrooke, Quebec, Canada
Raymund Wellinger, Departement de biologie and Departement de microbiologie et
infectiologie, Universite de Sherbrooke, Sherbrooke, Quebec, Canada
Woodring E. Wright, Department of Cell Biology, UT Southwestern Medical
Center, Dallas, TX, USA


FIGURE 2.1 Common secondary structure models for ciliates, vertebrates, and budding
yeast TERs. Indicated are the conserved regions/sequences (CR or CS), pairings/stems (P or S),
loops (L), template recognition element (TRE), and template boundary element (TBE).

Telomerases: Chemistry, Biology, and Clinical Applications, First Edition.
Edited by Neal F. Lue and Chantal Autexier.
Ó 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.


FIGURE 2.2 Template boundary elements in human, mouse, yeast, and ciliate TERs.
The secondary structure models of regions flanking the template are shown for human,

mouse, Kluyveromyces lactis, and Tetrahymena thermophila TERs. The sequence of the
template region is shown in a black box and the sequences essential for template boundary
definition are shown in a red box with a red line to indicate function in boundary definition.


FIGURE 2.3 The pseudoknot structures of human, K. lactis, and Tetrahymena thermophila
telomerase RNAs. (a) Ribbon representations of the three-dimensional solution structure of the
human (Kim et al., 2008) pseudoknot, and the computer models of the K. lactis (Shefer et al.,
2007) and T. thermophila (Ulyanov et al., 2007) pseudoknot, illustrated using the computer
program Chimera (Couch et al., 2006). Stem 1 is shown in gray, residues of stem 2 not
participating in base triples are shown in blue. Residues of stem 2 that are part of the triplex, are
shown in orange (purines) and yellow (pyrimidines). Bulged-out U residues are shown in red.
Residues of loop 1 that are part of the triplex, are shown in cyan. The rest of loop 1, as well as
loop 2 if present, are shown in green. Loop 3 is shown in magenta. (b) A schematic
representation of base pairing in the pseudoknot, including also the predicted scheme for the
S. cerevisiae pseudoknot (Gunisova et al., 2009; Qiao and Cech, 2008). Vertical lines represent
Watson–Crick interactions; tilted lines, Hoogsteen hydrogen bonds; and “.,” a G:U wobble pair.
Note that in the K. lactis pseudoknot, the region of the junction between stem 1 and 2 is
illustrated as unpaired, since the interactions among these nucleotides are unknown.


FIGURE 2.4 Conservation of the assembly/activation stem-loop elements in budding yeast,
vertebrates, and Tetrahymena species. (See text for full caption).

FIGURE 2.5 The 50 arm of Saccharomyces and Kluyveromyces telomerase RNAs.
Schemes illustrate the 50 arm, template, and template boundary of Saccharomyces sensu
stricto (a) and Kluyveromyces (b) TERs. Blue lines indicate sequence conservation.
Sequence alignments showing the conservation of the Ku80-binding site (48 nt stem-loop)
in Saccharomyces and a CGGA sequence motif in the Kluyveromyces Reg 2 element were
made by the computer program ClustalX (Chenna et al., 2003).

FIGURE 3.1 (a) The domain organizations of TERTs from different species are illustrated. (b) The structure of TcTERT (PDB ID: 3DU6): the TRBD, fingers, palm, and thumb
domains are colored in blue, orange, wheat, and red, respectively. (c) The structure of the
p66 subunit of HIV-1 RT without the nuclease domain (PDB ID: 1RTD) is shown.


FIGURE 3.2 (a) The RNA-binding domain of Tetrahymena thermophila TERT with
motif T in cyan and CP in yellow: conserved residues that comprise these motifs are shown
in the stick representation. (b) The fingers (orange) and palm (wheat) subdomains of
TcTERT: conserved motifs implicated in nucleotide and nucleic acid binding and catalysis
are displayed in the designated colors. (c) The thumb domain of TcTERT with the two
DNA-binding structural elements (the thumb loop and helix) highlighted in green. (d) The
TEN domain of T. thermophila TERT is displayed in a surface representation; the putative
DNA-binding groove and the residues implicated in DNA binding (Q168, F178, and W187)
are accented.


FIGURE 3.3 (a) A complex between TcTERT and an RNA–DNA hairpin (PDB ID: 3KYL):
the domain orientation and color scheme are similar to those shown in Figure 3.1B.
(b) A close view of the contacts between the RNA template and motifs 2 and B0 of TcTERT.
(c) A close view of the contacts between the RNA–DNA hybrid and the thumb helix (light
blue) and thumb loop (light blue) in the complex. (d) The primer grip region (motif E) is
juxtaposed to the 30 -end of the DNA primer at the active site of the enzyme.

FIGURE 3.4 (a) Same as Figure 3.3a. (b) The structure of the HIV-1 RT bound to an
RNA–DNA heteroduplex (PDB ID: 1RTD).


FIGURE 4.2 An integrated model of telomerase biogenesis in yeast. The Saccharomyces

cerevisiae telomerase RNA TLC1 is transcribed by the RNA polymerase II machinery (1) and
targeted to the nucleolus where its 50 mono-methylguanosine cap is hypermethylated by Tgs1
(2). Following its 50 cap hypermethylation, the TLC1 RNA is exported in the cytoplasm via the
Crm1p-dependent pathway (3). In the cytoplasm, the TLC1 RNA recruits the proteic components of the telomerase complex (4), assembles into a mature telomerase particle (5), and is
imported back in the nucleus via a Mtr10/Kap122 pathway (5). Once in the nucleus, it can be
recruited at the telomeres via the interaction between the TLC1 RNA and the yKu heterodimer
(6). (The telomerase holoenzyme at the telomere depicted in this figure would correspond to the
one in S phase. As Est1 is actively degraded or not depending on the phase of the cell cycle, so
the constitution of the telomerase recruited at the telomeres will vary accordingly). Taken from
Gallardo and Chartrand (2008). ÓLandes Bioscience.


FIGURE 4.4 Assembly of the human telomerase complex. After the transcription and
processing of the human telomerase RNA, the H/ACA proteins (dyskerin—blue, Nop10—
green, NHP2—orange, and NAF1—yellow) bind to the 30 end of the telomerase RNA.
Subsequently, NAF1 is exchanged for GAR1 (burgundy), and TCAB1 (purple) binds to the
hTR. After TERT (red) is localized to the nucleolus, mediated in part by interactions with 14-33 (gray) and nucleolin (pink) it is assembled with the ATPases pontin and reptin (shown in
green), to form a pretelomerase complex. During S-phase, pontin and reptin are released from
hTERT and the complex is remodeled or assembled with the help of additional factors (such as
the molecular chaperones Nat10, GNL3L, heat shock proteins, and SMN) with hTR to form an
active telomerase holoenzyme.


(a)
HIFs/O2 signaling

eNOS/NO signaling
eNOS
E2

HIFs

E2

ER ER
ER
ER

E2

+1

E2

+1

ER ER
ER
ER

ERE
E
RE
RE

ERE
E
RE
RE

(b)
?

HIFs

eNOS
E2

E2

ER
ER ER
ER

+1

ERE
E
RE
RE

FIGURE 5.1 Cartoons illustrating the functional cooperation between the ERs, eNOS and
HIFs pathways in the regulation of hTERT. (a) In primary cultures of human endothelium and in
prostate cancer cell lines, ligand-activated ER and eNOS form a combinatorial complex on the
estrogen response element (ERE) within the hTERT gene promoter (left panel). In prostate
cancer cell lines with a constitutive hypoxic phenotype, ER/HIF-1a or ER/HIF-2a complexes
are recruited upon estrogen treatment onto the hTERT–ERE (right panel). All these events lead
to increased hTERT gene transcription and telomerase activity. (b) Speculative model of
formation of a ER/eNOS/HIF trimeric complex. Since eNOS, ERs, and HIFs play a key role in
prostate cancer progression, it is conceivable that they may cooperate in the tumor microenviroment by coregulating their transcriptional targets. We propose that in the presence of

estrogen and of reduced O2 availability (hypoxia), these factors may form a trimeric complex
recruited by the ERE. This event may induce a local chromatin remodeling significantly
affecting the transcription of target genes.

telomerase inhibitors

telomerase

Est2
telomere repeat
addition by telomerase

cell division and telomere
repeat loss due to end
replication problem

activation/
recruitment
?

FIGURE 6.1 Schematic representation of the negative feedback “protein counting” model
for telomere length regulation. Details described in the text.


Tel1

telomerase

?
Est1

?
Rif1
Rif2
Rap1

Est3

?

Est2

MRX

Ku

3'

Exo

Pol1
Pol12

primase

FIGURE 6.2 Proteins and interactions implicated in telomere length regulation in the
budding yeast S. cerevisiae. Protein–protein interactions are indicated by double-headed
arrows. See text for details.

(a)

telomerase

POZ1TPZ1
POT1
RAP1

(b)
TPP1
TIN2
RAP1

Est2

RIF1
TAZ1

CCQ1

TRF2

RAP1
RIF1
TAZ1

Est2
POT1

Est2

CCQ1
TPZ1

TIN2
RAP1 T
TRF2
2

POT1

TRF1

TPP1

Est2

POT1
TRF1

POZ1

FIGURE 6.3 Schematic representations of models for telomerase activation at telomeres
in the fission yeast S. pombe (a) and in human cells (b). See text for details.


FIGURE 7.1 Telomere replication and the generation of a proper chromosome end
structure. At telomeres, the G-strand always serves as a template for lagging-strand synthesis
while the C-strand templates the telomere leading strand. Telomeric overhangs, the G-tails,
serve as a substrate for telomerase annealing and are formed through different mechanisms on
the leading and the lagging telomere sister chromatids. On the lagging strand, they may result

simply from incomplete replication at the chromosomal ends or form the removal or the
outmost RNA primer by the combined activity of specialized enzymes such as helicases, flap
endonucleases or RNases (orange sphere and yellow triangle). The initial 30 -overhang may be
further extended due to the activity of an exonuclease. On the leading-strand end, which is
predicted to be blunt ended after replication, resection of the C-strand by exonucleolytic
activities (magenta sphere) will generate the G-tail. Once overhangs of sufficient length are
generated, the binding of ss telomeric DNA binding proteins (the assembly of yellow, orange
ovals and green triangle) will obstruct more excessive nucleolytic degradation by blocking
access to telomeric ends. Concomitantly, the G-tail bound proteins may modulate the
cleavage sites for C- and G-strand specific endonucleases (small grey triangles) and dictate
the composition of terminal nt on telomeric DNA and the different length of G-tails on
leading versus lagging strand.


FIGURE 7.2 Regulation of telomerase by telomere-associated proteins. The current view
is that the telomeric proteins (grey ovals) bound to the ds telomere repeats (duplex zig-zag
line) negatively regulate telomerase. In several experimental systems, the activity of
telomerase is reversely correlated with the number of ds telomeric repeats and, respectively,
the number of telomeric proteins bound in cis, thereby establishing a negative feedback loop
or a counting mechanism. In higher eukaryotes, additional negative regulation may be
achieved by organization of sufficiently long telomeres into t-loops (illustrated on top).
The G-tail-binding proteins (the assembly of yellow, orange ovals and green triangle)
appear to facilitate telomerase access and positively regulate its activity at telomeres. Based
on data primarily from budding yeast, it has been proposed that at long telomeres, the
increased numbers of dsDNA-bound protein molecules inhibit telomerase access and the
recruitment of factors promoting the activity of telomerase, such as Mre11 complex.
Short telomeres, on the other hand, are permissive for Mre11 recruitment, which in turn
recruits checkpoint kinases, like Tel1/ATM (blue square). Together they signal the presence
of a short telomere by preparing telomere structure and modifying telomere proteins
(P; phosphorylation) to facilitate the recruitment and extension of telomeric DNA by

telomerase. The telomerase-mediated extension is tightly coordinated with the conventional
replication machinery, which limits the addition of new telomeric repeats by telomerase.


TERT
5’
3’

(a)
TIN2

TPP1
POT1

Telomerase

3’

5’

TERC

DNA damage response
Non-homologous recombination

5’-TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3’
3’-AATCCCAATCCCAAT CCC AATCCCAAT-5’
TRF1

GGTTAG 3’

CAAUCCCAAUC

Homologous recombination

TRF2/RAP1

Nucleus degradation

Loss of telomere DNA repeats or loss of shelterin protection

(b)
5’

3’

γ-H2AX, 53BP1, MRE11/RAD50/NBS1, phosphorylated ATM (TIF foci formation)

ATM/ATR kinase activation

p53/p21 dependent cell apoptosis or senescence

FIGURE 9.1 Telomere maintenance by telomerase and shelterin, and the consequences of
telomere dysfunction. (a) Telomere DNA, telomerase, and shelterin. Telomeres cap the
chromosome ends and protect against NHEJ, HR, DNA damage signaling, and nucleolytic
degradation. The access of telomerase to the telomere is limited by telomere-bound POT1 and
TRF1. (b) Dysfunctional telomeres arise via loss of telomere DNA repeats or loss of protection
of shelterin, resulting in the induction of DNA damage foci at telomeres (TIF) and activation of
ATM–ATR kinase pathways. These signaling cascades in turn can lead to p53/p21 dependent
cell apoptosis, cell cycle arrest, and cellular senescence.

FIGURE 11.6 Structural organization of Penelope-like elements (a) and their similarity to
telomerases (b). The EN( À ) (endonuclease-deficient) PLEs exhibit specificity for telomeres in
diverse eukaryotes. In panel (b), secondary structure predictions for representative TERT
(top 8) and PLE (bottom 8) sequences are compared in selected portions of the RT amino acid
alignment, showing the N-terminal T-motif region and the C-terminal motif 7 of the core RT
domain. Arrows designate characteristic beta-hairpins in the secondary structure. Sequences
were viewed with the aid of a structure-based sequence alignment program (STRAP) (http://
www.bioinformatics.org/strap/).