Telomerase Regulation by Alternative Splicing

Telomerase regulation by alternative splicing

Shanjadia Khondaker

Department of Medicine, Division of Experimental Medicine,

McGill University

Montreal, Quebec, Canada

Submitted June, 2012

A thesis submitted to McGill University in partial fulfillment of the requirements to the

degree of Master of Science

Preface

This M.Sc. thesis is written in accordance with the McGill University Faculty of Graduate and Postdoctoral Studies thesis preparation guidelines. I have included section 1 as an Introduction and literature review. Section 2 presents the Methods and Materials used in my studies. Section 3 presents the Results obtained. Finally, Section 4 and 5 present my Discussion and concluding remparks. The references appear at the end of the thesis.

Contribution of Authors

The candidate performed most of the work presented in this thesis with support from Dr. Chantal Autexier. The primers (Table 1) and optimal conditions (Table 2) necessary for mTERT isoform detection (Figure 1A) required for the detection assays (Figure 1B) were done by Shusen Zhu. Furthermore, the primers (Table 3) and optimal conditions (Table 4) necessary for the variant-specific PCRs (Figure 2B) and creation of the map of mTERT alternative splice sites (Figure 2A) were also done by Shusen Zhu. The initial pcDNA3.1-FLAG-Hygromycin-mTERT constructs were built by Dr. Johans Fakhoury (Figure 6A, Figure 7A, Figure 8A), as published in (Fakhoury, Marie- Egyptienne et al. 2010). The stable mTERT-overexpressing CB17 cell lines (Figure 10A, Figure 11A, Figure 12, Figure 13) were created by Dr. Delphine T. Marie-Egyptienne. All experiments were conducted under the supervision of Dr. Autexier, who also contributed to the design of the study. Contribution to Original Knowledge The work presented in this thesis is the first to report: 1. The identification of mouse telomerase reverse transcriptase (mTERT) alternatively-spliced variants, some species-specific, some conserved across species. 2. The presence of mTERT isoforms in different mouse cells and tissues. 3. The behaviour of alternatively-spliced variants in vitro (telomerase activity, telomeric DNA binding, telomerase RNA binding). 4. The dominant-negative characteristics of one of the alternatively-spliced variants (a deletion in the C-terminus). 5. The phenotype of telomerase-positive mouse cells when transfected with the alternatively-spliced isoforms.

Acknowledgements

This dissertation would not have been possible without the support and the help of my supervisor, Dr. Chantal Autexier. Thank you for accepting me into your laboratory and for guiding me through my journey as a new scientist. I am extremely appreciative of your contribution to my education with your easy accessibility, direction and patience while allowing me the space to learn and grow on my own. To past and present members of the Autexier Lab, Johans Fakhoury, Ricky Kwan, Catherine Lauzon, Marie-Eve Brault, May Shawi and Nahid Golabi, thank you for the continuous encouragement and for sharing ideas and stimulating conversations. Each of you have been abundantly helpful and have offered valuable help during my time here.

My deepest thanks go to my colleagues who, in one way or another, have contributed to the completion of this study. To Johanna Mancini, my first friend in the laboratory and in the institute. Thank you for always being within reach and for your kindness and constant support which have given me the strength to hold on at times when I wanted to give up. Thank you to Josephine Chu for your friendliness and generosity, both of which will remain with me for the rest of my life. The late nights spent with you brainstorming and troubleshooting have helped me grow as a scientist in more ways than you’ll ever know. Thank you to Mehdi Belgnaoui for sharing your experience and words of wisdom about both science and life. I am deeply grateful for your contributions in editing parts of my thesis.

To my mentors, Shusen Zhu and Yasmin D’Souza, you have provided me with knowledge about the telomere field and taught me the technical skills required for my Master’s degree. Shusen, thank you for sharing your expertise while teaching me lessons in molecular cloning and for your continued patience in training me as a new student in the lab. Yasmin, you are an exceptional scientist, teacher and friend. Thank you for selflessly spending hours teaching me about cell culture, radiation safety and the many assays required to complete my graduate studies.

Most importantly, to my family and friends, thank you from the bottom of my heart. I cherish each and every one of you and am very thankful of your contributions as I complete my journey. You have been my invaluable network of support with your forgiving, generous and loving ways. To my fiancé, Saem Ahmed, you encouraged me to pursue a Master’s degree and helped me see it to the end. Your strength, kind- heartedness and perception have lifted me time and time again from the shackles of my own uncertainty. There are few men in this world who can make their beloved feel like they are capable of accomplishing whatever their heart desires. You are definitely one of them.

I - Abstract

Telomerase, the main mechanism for telomere maintenance, is active in 85% of

cancer cells. Telomerase inhibition leads to telomere erosion, chromosome damage, and cell death, thereby validating the enzyme as a prime target in the search for anticancer therapies. hTERT genome analysis revealed the potential for complex splicing patterns that may reflect a specific aspect of telomerase regulation in proliferation, differentiation and apoptosis (Sykorova and Fajkus 2009).

A number of alternatively-spliced TERT mRNAs in vertebrates and plants have been identified, yet their role in telomere maintenance and cell survival is poorly characterized. We have identified several novel mTERT variants and are pursuing

characterization of two variants, one contains an in-frame insertion in the N-terminus

(Ins-i1[1-102]), a region important for binding to the telomerase RNA and telomeric

substrates, and the other a C-terminally located deletion (Del-e12[1-40] in the RT domain

that generates a premature stop codon and encodes a protein lacking the C-terminus. In-

vitro, the alternatively spliced mTERT variants display decreased telomerase activity,

most likely due to their defect in template RNA and telomeric DNA binding affinities.

For the deletion variant, this decrease in activity was consistent in telomerase positive

cells, which, by middle passage, appeared to have a noticeable growth defect. By

acquiring a detailed understanding of mouse TERT alternative splicing, we can advance

our knowledge on telomerase regulation and new mechanisms for inhibiting telomerase

in tumour cells.

II - Résumé

La télomérase est le mécanisme principal de maintien des télomères et est actif dans 85% des cellules cancéreuses. L’inhibition de la télomérase entraîne l'érosion des télomères, des lésions chromosomiques et la mort cellulaire, ce qui démontre que cette enzyme pourrait être une cible importante dans la recherche de thérapies anticancéreuses. L'analyse du génome de la télomérase a révélé le potentiel des motifs d'épissage complexes qui peuvent démontrer un aspect spécifique de la régulation de l’enzyme dans la prolifération, la différenciation et l'apoptose

(Sykorova and Fajkus 2009)

Un certain nombre d'ARNm TERT alternativement-épissés ont été identifiées chez les vertébrés et les plantes, mais leurs rôles dans le maintien des télomères et la survie cellulaire sont mal caractérisés. Nous avons identifié plusieurs nouvelles variantes de TERT murine et, en parallèle, nous poursuivons la caractérisation de deux autres variantes. La première variante, Ins- i1[1-102], contient une insertion en cadre de lecture, dans le domaine N-terminal qui une région importante pour la l’attachement aux ARN de la télomérase et aux substrats d’ADN télomérique.

La deuxième variante est une délétion localisée dans le domaine C-terminal. Celle-ci est plus spécifiquement située dans le domaine RT qui génère un codon stop prématuré et code pour une protéine manquant sa partie C-terminale, Del-e12 [1-40]. In-vitro, les activités télomériques des variantes mTERT sont diminuées, probablement dû à leur manque d’affinité pour l’ADN et l’ARN télomérique. Pour la variante Del-e12 [1-40], cette baisse d'activité a été confirmée dans des cellules télomérase-positives qui, après un certain nombre de passages, semblent développer des déficiences de croissance. La compréhension détaillée des mécanismes de l’épissage alternatif de la TERT murine pourrait contribuer à l’amélioration de nos connaissances sur les différentes méthodes de régulation de la télomérase. Ceci pourrait aussi aboutir à la découverte de nouveaux mécanismes d’inhibition de la télomérase dans les cellules tumorales.

Table of Contents

I - Abstract (English) 4 II - Abstract (French) 5 III - Tables and Figures 7

1. Introduction 1.1 Historical Background 8 1.2 The End Replication Problem and Telomeres 9 1.3 Telomere structure and function 12 1.4 Telomere Binding Proteins 12 1.5 Telomerase 17 1.6 Telomeres and Telomerase in Humans and Mice 21 1.7 Telomerase and Cancer 23 1.8 Telomere Theory of Aging 25 1.9 Telomerase Reverse Transcriptase 27 1.10 Telomerase Regulation 31 1.11 Alternative Splicing of the TERT gene 33 1.12 Alternative splicing of mouse TERT gene 38

2. Materials and Methods 2.1 Cell Line Preparation for RNA Isolation and Cell Culture 42 2.2 Mouse tissue preparation for total RNA isolation 43 2.3 RNA isolation, RT-PCR and Protein Extraction 43 2.4 Plasmid construction 55 2.5 In vitro transcription and translation, SDS-PAGE and TRAP Assay 56 2.6 In vitro DNA-binding assay and quantification 58 2.7 In vitro RNA-binding assay and quantification 58 2.8 Telomere Restriction Fragment Assay 59

3. Results & Figures 3.1 Identification of alternatively-spliced mTERT mRNA variants 61 3.2 mTERT isoforms exist in different mouse cells and tissues 63 3.3 Del-e12[1-40] variant behaves in a dominant-negative manner 64 3.4 Variants have decreased binding affinities for DNA and RNA substrates 66 3.5 Growth defects of mTERT deletion variant-expressing cells 68 3.6 Deletion variant-expressing cells have reduced telomerase activity 68 3.7 Deletion variant expression shows no change in telomere length 69

4. Discussion 104

5. Conclusion and Summary 116

IV - Bibliography 117 V - Appendix 124

List of Figures and Tables Figure I: The End-Replication problem 11 Figure II – General Features of the Telomeric Region 14 Figure III - Senescence/Crisis Cycle 20 Figure IV – Structure of TERT Subunit 30 Figure V – Full-Length and Alternative-Spliced Variants of Human TERT 35 Figure 1: Identification of alternatively-spliced mTERT mRNA variants 71 Figure 2 - Alternatively-spliced variants of mouse TERT 73 Figure 3: Specific PCR products of alternatively spliced mTERT variants 74 Figure 4A: Presence of alternatively-spliced variants in mouse cell lines 75 Figure 4B: Presence of alternatively-spliced variants in adult mouse tissues 76 Figure 5: Telomerase activity of WT TERT and variant reconstituted enzymes 78 Figure 6A: Telomerase activity of mixed WT and Insertion variant reconstituted enzymes80 Figure 6B: Telomerase activity of mixed WT and Deletion variant reconstituted enzymes 82 Figure 7A, B: In vitro DNA binding affinities of Alternatively-Spliced mTERT variants 84 Figure 7C, D: In vitro DNA binding affinities of Alternatively-Spliced mTERT variants 86 Figure 8: In vitro RNA binding affinities of alternatively-spliced mTERT variants 88 Figure 9: Screening of clones following CB17 cell transfection 90 Figure 10A: Growth Phenotype of CB17 cells transfected with TERT Variants 91 Figure 10B: Growth Phenotype of CB17 cells transfected with TERT Variants 92 Figure 11A: Telomerase Activity of CB17 Cells transfected with WT mTERT 94 Figure 11B: Telomerase Activity of CB17 Cells stably expressing Del-e12[1-40] Variant 96 Figure 11C: Quantification of Telomerase Activity of transfected CB17 Cells 98 Figure 12: Telomerase Activity of transfected CB17 cells (Early passage) 100 Figure 13: Telomere length of CB17 Cells as measured by TRF protocol 102

Table 1: Primers used to detect presence of alternatively-spliced mouse TERT variants 46 Table 2: PCR conditions used to detect alternatively-spliced mouse TERT variants 48 Table 3: PCR Reactions with TAQ DNA Polymerase 49 Table 4: PCR Reactions with High-Fidelity PfuTurbo DNA Polymerase 50 Table 5: Variant-Specific Primers for Isolation of mTERT Isoforms 51 Table 6: PCR conditions used to isolate alternatively-spliced mouse TERT variants 53

1. Introduction

1.1 Historical Background

In 1908, French Nobel prize-winning surgeon Alexis Carrel became interested in

the growth of cells in culture. In 1912, he established a culture of chick heart fibroblast

cells, which he claimed grew continuously in the laboratory for 34 years (Carrel and

Ebeling 1921). This led to the general idea that all vertebrate cells could divide indefinitely in cell culture. However, Carrel’s original observations could not be reproduced by other scientists, and may have been due to an experimental error

(Hayflick, Jacobs et al. 1964). However, it was scientists Leonard Hayflick and Paul

Moorhead who first began to report on difficulties in long-term cell culture. Performing

many of the same experiments, they disproved Carrel’s theory, demonstrating limited

replicative capacity of normal human fibroblasts and defining this process as cellular

aging.

The link of cellular aging to the compound structure at the ends of chromosomes was initiated a few decades later. Derived from the Greek words "telos," meaning end and "meros," meaning part, telomeres are protective structures at the terminal end of

linear chromosomes. They were first described in the 1930s by Barbara McClintock,

working with maize (McClintock 1931) and Hermann Muller, working with fruit flies

(Muller 1927). Collectively, they observed that when this structure is absent, end-to-end

fusion of the chromosome occurs leading to chromosomal instability and to cell death.

Both investigators proposed that chromosome ends have special protective structures required for chromosome stability and to ensure faithful segregation of genetic material into daughter cells upon cell division.

Returning to the Carrel-Hayflick debate, Hayflick’s senescence model eventually

became accepted. In the early 1960s, Leonard Hayflick described a biological view of

aging. He found that human cells proliferate a limited number of times in a cell culture

and then stop dividing by a process now known as replicative senescence (Hayflick and

Moorhead 1961). The "Hayflick limit" is the maximal number of divisions that a cell can achieve in vitro. When cells reach this limit, they undergo morphologic and biochemical changes that eventually lead to arrest of cell proliferation, a process called "cell

senescence" (Hayflick 1965). What was still unclear was the role of cellular senescence

in humans. Hayflick proposed that the cell culture phenomenon could be used as a model

to study human aging at a molecular and cellular level. However, the mechanism behind

why most primary human cells in culture stop dividing after a limited number of

divisions remained unclear long after the discovery of the “Hayflick limit”. The link

between cellular senescence and the replication of telomeres, however, wasn’t made until

the observations of scientist Alexei Olovnikov.

1.2 The End Replication Problem and Telomeres

The role and the importance of telomeres were not fully appreciated until the

interpretations by Alexei Olovnikov in 1971, of the DNA structure and the mechanisms

by which it is replicated. The conventional replication of DNA presents a few problems

mainly because the process only works in the 5' to 3' direction (Olovnikov 1973). During

lagging strand replication (Figure I), DNA polymerase requires binding of an RNA

primer. A primase creates RNA/DNA hybrids to which DNA polymerase α binds and

extends to form Okazaki fragments (Lundblad 1997). The RNA primers are then

removed and replaced with DNA by DNA polymerase δ. At the 5’- end, however, DNA

polymerase δ cannot bind to fill in the 3’ overhang. This leads to the loss of a small 5'

nucleotide segment as DNA synthesis takes place, with progressive replication-induced

telomere shortening. That the DNA replication process only functions in one direction

and leads to incomplete replication of chromosome ends was also recognized by James

D. Watson (Watson 1972). In 1972, Harvard scientist James D. Watson recognized that

the end-to-end joining of individual genomes could allow the complete replication of the

linear bacteriophage genome. Watson highlighted that DNA polymerases act in the 5‘ to

3‘ direction and require an RNA primer and that when the polymerase reaches the end of

a linear DNA molecule, there would be a problem in completing replication. Finally, he

also reported that phage genomes could escape this ‘end replication problem’ by linking multiple genomes before replication, leading to less damage due to incomplete replication. He proposed the need for “compensatory mechanism” which was needed to fill this terminal gap in the chromosome, without which the telomere would shortened with each successive cell division.

In 1978, Yale scientist Elizabeth Blackburn was studying the DNA sequence that allowed the Tetrahymena rDNA molecule to be maintained as a linear chromosome. Her work led to the finding that chromosome ends, or telomeres, are made of simple repeated

DNA sequences (Blackburn and Gall 1978). Soon after, experiments revealed that this motif is conserved throughout evolution and that this structure is of great physiological importance.

Figure I: The End-Replication problem

The replication of DNA presents two problems: the process only works in the 5' to 3'

direction and DNA polymerase requires binding of an RNA primer, leaving gaps at each interval in the absence of the primers. The gaps are the filled by DNA polymerase, except at the very end. This leads to the loss of a small 5' nucleotide segment as DNA synthesis

takes place, with progressive replication-induced telomere shortening.

1.3 Telomere structure and function

Telomeres are comprised of tracts of G-rich nucleotide repeats that serve to

protect the ends of eukaryotic chromosomes (Greider and Blackburn 1985). They are

composed of repeats of a short, G-rich DNA sequence (TTAGGG in humans). While the

sequence is highly conserved across many species (Shampay, Szostak et al. 1984), the

length of the repeats varies between chromosomes and between species. As such, the

lengths of the double-stranded TTAGGG tracks vary from human (10 - 15 kilobases,

depending on the type of tissue, the age of the donor, and the replicative history of the

cells) to mouse inbred strains (up to 40 kilobases) (Zijlmans et al., 1997) and to ciliate

Euplotes Aediculatus (less than 50 base pairs)(Klobutcher, Swanton et al.,1981).

Telomeres prevent the ends of linear chromosomes from being recognized as

DNA double-strand breaks, protect chromosome ends from degradation and fusion and

are dynamic in structure. Telomeric DNA in human cells, which is normally composed of

10-15 kb of G-rich repeats, terminates in a 50-300 nucleotide G-rich 3' overhang that is

important for telomere function (de Lange, 2002). The G-rich strand is replicated by the

lagging-strand machinery, whereas the C-rich strand is replicated by the leading-strand

machinery. Consequently, the parental G-rich strand is not completely duplicated, leading

to a G-rich 3' overhang (Gilson et al., 2009). The length of this G-tail can be different

depending on whether it corresponds to the length of the last primer (~10–12 nucleotides)

or to an inhibition in the synthesis of the last Okazaki fragment, leading to a longer

overhang of ~200 nucleotides (Gilson and Geli 2007).

1.4 Telomere Binding Proteins

Furthermore, telomeric regions are bound by a specialized six-protein complex

known as the Shelterin complex (Figure IB). These proteins are comprised of TRF1,

TRF2, TIN2, TPP1, Rap1, and POT1 and play fundamental roles in the regulation of

telomere length and telomere capping, as reviewed by (Martinez and Blasco 2010).

Without the protective activity of shelterin, telomeres are subject to the DNA damage

surveillance and chromosome ends are inappropriately processed by DNA repair pathways (de Lange 2005). To begin with, in order to avoid being recognized as a double-stranded break by DNA damage response proteins, the G-rich 3’-overhang can fold back and invade the double-stranded region of the telomere forming a T-loop (Figure

IA), an action mediated by the shelterin complex. This invasion separates a stretch of the

telomeric region, creating a second structure known as the displacement loop or D-loop

(Figure IA). Disruption of the T-loop or D-loop structures lead to telomere dysfunction.

This may occur though disruption of proteins necessary for their maintenance (de Lange

2005), by loss of the G-rich overhang itself, or by a critically short tract of telomeric

repeats. Furthermore, interference with the proper function of either the T-loop or the D-

loop can lead to the induction of the DNA damage response pathway (d'Adda di Fagagna,

Reaper et al. 2003).

A -

B-

Figure II – General Features of the Telomeric Region (A) The single-stranded overhang

(gray strand) invades the doubled-stranded DNA region of the telomere to form a

protective telomere T-loop with a displacement D-loop at the invasion site. (B) Telomeric

DNA is bound by Shelterin complex proteins TRF1, TRF2, RAP1, TPP1, TIN2 and

POT1. Six components of the shelterin and their DNA and protein binding abilities are

depicted. The telomerase is the enzyme that elongates telomeres. The specific function

associated with each shelterin and to the telomerase is highlighted in gray boxes.

Reprinted by permission from Blackwell Publishing Ltd: Aging Cell Volume 9, Issue

5, pages 653–666, © October 2010

As previously stated, telomeres are dynamic structures and may be associated to

different proteins under different conditions. For example, FRAP studies also showed

that POT1 and TRF2 bind to telomeric DNA in at least two different modes (Mattern,

Swiggers et al. 2004)one unstable (rapid exchange with unbound proteins) and one more

stable mode (slow exchange with unbound proteins). Some of these proteins, for example

POT1, bind to the single-stranded G-rich DNA whereas other proteins such as TRF1 and

TRF2 bind to the double-stranded telomeric repeats (Martinez and Blasco 2010). While

these three shelterin subunits directly recognize TTAGGG repeats, they are

interconnected by the three other subunits, TIN2, TPP1, and Rap1 (de Lange, Shiue et al.

1990). Together, they form a complex that allows cells to distinguish telomeres from sites

of DNA damage.

Overexpression of both TRF1 and TRF2 (van Steensel and de Lange 1997) have

negative regulatory effects on telomere length, suggesting that when bound by these

proteins, telomeres are stabilized and their elongation is prevented. When either TRF2 or

POT1 are inhibited, the overall amount of single-stranded TTAGGG repeats is

diminished by 30%–50% (van Steensel, Smogorzewska et al. 1998; Hockemeyer, Sfeir et

al. 2005). When TRF2 protein is inhibited alone, one of its effects is the defect in its ability to interact with the TTAGGG repeats. This induces rapid onset of apoptosis or senescence in an ATM/p53- or p16/Rb-dependent manner, end-to-end fusions and chromosomal instability, as well as erosion of the 3’-overhang (Karlseder, Broccoli et al.

1999; Smogorzewska, van Steensel et al. 2000). When POT1 is inhibited alone, 5’ ends often lose their homogeneity and end with AA, AT, TC, CC, CA, or AT (Hockemeyer,

Sfeir et al. 2005). TRF1-negative cells exhibit a growth defect with an extended

population doubling time, chromosomal instability in the form of end-to-end sister

chromatid fusions and decreased association of TRF2 and telomere binding protein TIN2

with the telomeres (Iwano, Tachibana et al. 2004).

Uncapping of telomeres may occur due to the disruption of the telomere binding

proteins causing telomere dysfunction in a length-independent fashion. Uncapped

telomeres have been shown to be associated to DNA damage response factors such as

53BP1, y-H2AX, Rad17, ATM and Mre11 (Takai, Smogorzewska et al. 2003). This is

important because the binding proteins cap the telomere to prevent genomic instability

and to regulate the activity of telomerase. Thus, either by critical shortening or inhibition

of binding proteins, uncapping of the telomeres induces a DNA damage response (de

Lange 2002).

Many proteins that are known to transiently associate with telomeric DNA have

roles unrelated to telomeres. However, the how these interactions are regulating their

physical traffic to the telomeres is not well understood. The most plausible explanation is

through posttranslational protein modifications including phosphorylation,

dephosphorylation, poly-ADP ribosylation, deribosylation, acetylation, ubiquitination and

sumoylation, as reviewed by (Aubert and Lansdorp 2008). For reasons yet to be

explained, many “telomeric” proteins can also be found at cytoplasmic and non-telomeric

nuclear sites, and some proteins appear to localize at telomeres. In general, it can be

stated that the “cross-talk” between the many proteins involved in telomere function and

various cellular signaling pathways is necessary yet poorly understood.

1.5 Telomerase

Olovnikov connected cell senescence with end-replication problems in his

"Theory of Marginotomy," in which telomere shortening was proposed as an internal

clock of aging that controls the number of cell divisions before replicative senescence

sets in (Olovnikov 1973). In 1990, this theory was supported by Carol Greider and

colleagues when they observed a progressive loss in telomere length in dividing cells

cultured in vitro (Harley, Futcher et al. 1990).

This problem is solved by the widely conserved enzyme telomerase, which is

capable of elongating the telomeres (Greider and Blackburn 1985). The telomeric

terminal 3’ overhang allows priming outside of “useful” DNA, permitting full synthesis

of the DNA end as it is now primed from the overhang. This led to the discovery of the

enzyme in Tetrahymena thermophila and to the observation that it was able to replace the

lost telomeric sequences, thereby maintaining chromosomal integrity (Greider and

Blackburn 1985). This enzyme, which was named telomerase, was shown to contain an

essential RNA component, a portion of which serves as the template for the synthesis of

the telomeric sequence, thus placing telomerase within the family of reverse

transcriptases. (Greider and Blackburn 1987; Greider and Blackburn 1989). Telomerase

is the main positive regulator of telomere length.

The human core enzyme consists of a reverse transcriptase protein (TERT) of

1,132 amino acids encoded by the hTERT gene (Harrington, McPhail et al. 1997),

located on chromosome 5p15.33 and telomerase RNA (TR) containing 451 nucleotides

(including the 5’-CAAUCCCAAUC-3’ telomere template) encoded by the telomerase

RNA gene hTERC (Feng, Funk et al. 1995), located on chromosome 3q21-q28. The

protein dyskerin (encoded by the DKC1 gene on the X chromosome) is required for proper folding and stability of telomerase RNA (Wong and Collins 2006) and was

recently added to the basic and necessary subunits of the human telomerase holoenzyme

(Cohen, Graham et al. 2007).

By reverse transcribing the RNA template region within TR, TERT can add the

telomeric repeat sequence, TTAGGG, to the 3' strand of linear chromosomes. Briefly, it

does so in the 3 following steps. First, there is recognition and binding between the

telomeric DNA substrate and the RNA template through complementary base pairing.

These and other RNA-protein interactions between TERT and TER are critical to

telomerase function. Also, DNA-protein interactions between TERT and the DNA

substrate are critical for this recognition step. The telomerase RNA is bound at high

affinity by conserved domains of TERT that lie outside its RT homology domain (Lin, Ly

et al. 2004). A short region of the telomerase RNA, the template domain, is then copied

during the elongation step leading to the addition of nucleotides onto the telomeric DNA.

Finally, in the translocation step, there is repositioning between the DNA substrate and

the telomerase enzyme, following which the entire process starts again for another round

of nucleotide addition.

As stated earlier, in the absence of telomerase, there is a loss of a small 5'

nucleotide segment and progressive telomere shortening with each replication cycle.

While stem cells and germline cells express telomerase to maintain telomere length

throughout their life cycle, most somatic cells stringently repress telomerase (Greider

1998). In culture, normal human cells grown in vitro replicate for a limited period of time

until their telomeres shorten to a critical length, beyond which they can no longer divide.

This state of replicative senescence is currently defined as the Hayflick limit, a point at

which cells undergo irreversible growth arrest (Figure III). However, cells can sometimes

bypass the senescence checkpoints. This is the case in which core cell cycle checkpoint

proteins, such as p53 or Rb, are mutated in the cells (Shay and Wright 2004). Cells

eventually reach a second proliferative block often referred to as crisis, which is

characterized by genomic instability and massive cell death. Rare variants, appearing at a

frequency of less than one cell in 107 emerge from this population of crisis cells and

acquire the ability to maintain their telomeres at a stable length. Such cells can continue

to grow despite the presence of dysfunctional telomeres. The loss of telomere function in

such cells results in chromosome fusions, broken chromosomes, break-fusion bridge

cycles, translocations, and aneuploidy. This large-scale genomic instability allows

selection of cells with abnormal growth characteristics and also facilitates rapid

acquisition of genetic alterations, such as telomerase reactivation, that provide further

growth advantages. The above is of utmost importance as 85-90% of cancer cells contain

short telomeres and high levels of telomerase activity.

Figure III - Senescence/Crisis Cycle. As normal human cells divide in culture, there is a decrease in telomere length, which ultimately leads to replicative senescence. The loss of cell-cycle checkpoint pathways, such as mutated p53 or Rb in the cell, leads to an extended lifespan but continued telomere losses. This eventually leads to the crisis stage of replicative senescence. To escape crisis, a rare human cell (about 1 in 10 million) can reactivate or up-regulate telomerase activity. Even more rarely, a cell may engage an alternative (ALT) to telomerase for maintaining telomeres that involves DNA

recombination between telomere sister chromatids.

1.6 Alternative Lengthening of Telomeres

In most human cancers telomerase activity is reactivated, but in 10–15% of

cancers, telomere length maintenance is achieved through a mechanism known as

alternative lengthening of telomeres or ALT. In this second class of cells, telomerase

activity remains undetectable (Murnane, Sabatier et al. 1994; Bryan, Englezou et al.

1997). In a telomerase-independent manner, alternative lengthening of telomeres in

human cells is believed to take place through a recombination-dependent process

(Lundblad and Blackburn 1993). ALT cells have characteristic heterogeneous telomere

lengths ranging from <2 to > 50 kb. Telomeres are normally maintained in the human

germline at lengths around 15kb (Allshire, Dempster et al. 1989; de Lange, Shiue et al.

1990). ALT has been detected in a variety of human tumors as well as tumor cell lines.

These include bone and soft tissue sarcomas, glioblastomas, and carcinomas of the lung, kidney, adrenal, breast, and ovary (Mehle, Piatyszek et al. 1996; Bryan, Englezou et al.

1997). The phenotypic characteristics associated with ALT are useful for determining

whether a cell line or tumour is likely to be ALT-positive, but it has been observed that

some of these characteristics can be induced in the absence of ALT activity.

1.6 Telomeres and Telomerase in Humans and Mice

As previously stated, telomere length in the mouse is significantly longer than in human cells (about 20 kilobases longer) (Kipling and Cooke 1990). This disparity in length may be the reason behind the differences in the response to telomere shortening in these two species (de Lange, Shiue et al. 1990). In humans, telomerase repression is necessary to protect against the generation of overly long telomeres and to suppress

tumor development (Collins and Mitchell 2002). This is balanced against telomerase

activation to extend tissue renewal capacity. In most cases, human telomerase is active

early in development and this is used to compensate for telomere loss in the upcoming

cycles of proliferation necessary for tissue growth and differentiation. After this, most

somatic human tissues and primary cells express low or undetectable telomerase activity.

This progressive telomere shortening has been shown to provide a signal for entry of cells

into a state of replicative senescence (Harley, Futcher et al. 1990; Kim, Piatyszek et al.

1994; Bodnar, Ouellette et al. 1998). In mouse somatic tissues, however, telomerase

activity is not stringently repressed. In most cases, there is moderate basal telomerase

activity as opposed to the lack of it in most human tissues. While this could provide a

basis for the greater ease of immortalization and higher cancer incidence in the mouse

(Prowse and Greider 1995), telomere erosion is unlikely to be a primary tumor suppressor

mechanism in rodents.

Mice overexpressing telomerase have a higher cancer incidence and hence a

shorter lifespan (Artandi, Alson et al. 2002), mice lacking telomerase are viable up to six

generations. In laboratory mice (Mus musculus), complete loss of telomerase is endurable for at least a few generations, principally due to their long telomeres. In contrast, only a

twofold reduction in telomerase levels has been shown to cause severe clinical symptoms

including aplastic anemia, immune deficiencies, and pulmonary fibrosis after one to three

generations (Blasco, Lee et al. 1997; Lee, Blasco et al. 1998; Johnson, Marciniak et al.

2001). At first, telomerase-knockout mice seem remarkably normal (Blasco, Lee et al.

1997). Telomere length decreases progressively in mTR-/- mice with increasing

generations in the absence of telomerase. Continued breeding reveal a decrease in

telomere length with successive generations accompanied by severe degeneration of

highly proliferative tissues. G5–G6 TERC mice become infertile, show high rates of

apoptosis in testis and germ cell depletion, exhibit impaired bone marrow function and

diminished proliferation of lymphocytes (Lee, Blasco et al. 1998). Furthermore, they

display shortened lifespan and suffer from multi-organ degenerative decline, with

evidence of ‘signal-free’ ends, chromosomal end-to-end fusions and anaphase bridging at

mitosis (Hande, Samper et al. 1999). These phenotypes are especially evident in highly

proliferative tissues where there are high rates of progenitor cell apoptosis in the

intestinal crypts with resultant villus atrophy (Rudolph, Chang et al. 1999; Wong, Kusdra

et al. 2002) as well as a diminished transplant potential of hematopoietic stem cells from

telomerase-deﬁcient mice.

1.7 Telomerase and Cancer

Telomerase, a ribonucleoprotein DNA polymerase, maintains telomere length by

balancing telomere shortening with net telomere elongation. The telomere shortening

observed in human somatic cells led to the hypothesis that telomerase is repressed in

human somatic cells, limiting their lifespan (Huschtscha, Thompson et al. 1986). In

contrast, it is expressed in cells that become immortalized in culture. As previously

stated, rare immortal cell clones can continue to divide beyond the first growth arrest

point and even escape crisis and survive by telomerase activation (Wright, Pereira-Smith

et al. 1989).

Following these observations, various studies have been done on telomere length

and telomerase activity in mortal and immortal human cultured cells. Analysis of

telomere lengths in human embryonic kidney cells transformed with simian virus 40 T

antigen shows a decline in telomere length until the crisis checkpoint, and then telomere

maintenance in the survivors. And so, in most cancer cells, telomeres are not so much

lengthened as they are stabilized by the presence of telomerase. Consistent with these

observations, the telomerase enzyme is inactive in the precrisis mortal cells and is

activated in the immortalized cell clones (Counter, Botelho et al. 1994).

The stabilized telomeres allow for cell immortalization (Zhang, Mar et al. 1999)

and these immortalized tumor cells are able to overcome the barrier of the senescence and

crisis phases and have the ability to proliferate indefinitely. It is important to note,

however, that immortalization is not sufficient for malignant transformation. Malignant

transformation is a multiple-step process, including the activation of oncogenes and the

inactivation of tumor suppressor genes, such as p53 and pRB (Hahn, Dessain et al. 2002).

Although telomerase expression is sufficient to immortalize some cell types, such as

fibroblasts, other cell types, such as keratinocytes, require the presence of at least an

oncogene for direct immortalization (Kiyono, Foster et al. 1998). Having stated the

above, exogenous expression of the telomerase catalytic subunit gene in certain

telomerase-negative cells is able to extend the lifespan of these cells, proving that, in

many cases, telomerase can overcome the barriers of human cell mortality.

In the 1990s, Shay and Harley detected telomerase in 90 of 101 human tumor cell

samples (from 12 different tumor types), but found no activity in 50 normal somatic cell

samples (from 4 different tissue types) (Kim, Piatyszek et al. 1994). Since then, more

than 2600 human tumor samples have been examined and telomerase activity detected in

about 90% of all tumor cells (Shay and Bacchetti 1997). In recent studies, hTERT

expression led to an extension in the lifespan of mortal fibroblasts and retinal epithelial

cells because they were able to bypass the growth arrest checkpoints (Bodnar, Ouellette

et al. 1998). hTERT-immortalized BJ foreskin fibroblasts have been found to divide for

an extra 200 population doublings compared to control cell clones which double at a

normal rate (Morales, Holt et al. 1999). Taken together, the above studies imply that telomerase may play a major role in the pathogenesis of cancer.

1.8 Telomere Theory of Aging

As previously stated, early studies in Drosophila (Muller 1927) and Maize

(McClintock 1931) showed that broken chromosomes lacking telomeres are highly

unstable, producing translocations, fusions, and other aberrations. Furthermore,

incomplete replication of DNA at the ends of linear chromosomes (Watson 1972) leads to

replicative senescence. However, it was only much later that the issue of organismal

aging as a consequence of short telomeres was raised. One of the earliest cases

demonstrating the above was Dolly the sheep, the first mammal to be cloned from an

adult somatic cell cloned. This was accomplished through the transfer of an adult

mammary gland nucleus into an enucleated egg. Experiments following the transfer

revealed that, because the nucleus carried genetic material from an “adult” gland into the

egg, it had short telomeres (Shiels, Kind et al. 1999). On the other hand, nuclear transfer

experiments using nuclei from senescent bovine fibroblasts yielded offspring with longer

than expected telomeres and a “youthful” phenotype (Lanza, Cibelli et al. 2000). Finally,

the “immortal” growth properties of germline or stem cells derived from pre-implantation

embryos of many species suggest that telomere length can be maintained or telomere loss

attenuated in early development.

The telomere hypothesis for cellular aging provides a basis for the limited replicative

capacity of normal somatic cells. In brief, because telomerase, the enzyme responsible for

maintaining telomere length, is expressed during gametogenesis, allowing long-term

viability of the germline, but is repressed during somatic cell differentiation, there is a

loss of telomeric DNA associated with finite replicative capacity of these cells (Harley,

Vaziri et al. 1992). As previously stated, when telomere length reaches a certain size,

cells stop dividing. The telomere hypothesis for cellular aging can be easily extended to

account for cell immortalization, including characteristics of stem cells. It can also be

studied in the context of disease. For example, telomere shortening is often accelerated in

human diseases associated with mutations in telomerase, such as some cases of

dyskeratosis congenita and aplastic anemia (Martinez and Blasco 2010). People with

these diseases, as well as TR-deficient mice, in which telomere length cannot be

maintained, show decreased lifespan coincidental with a premature loss of tissue renewal,

which suggests that telomerase is rate-limiting for tissue homeostasis and organismal

survival.

Human fibroblasts from most young persons have long telomeres, whereas older

individuals have shorter telomeres (Harley, Futcher et al. 1990). Similarly, patients with

premature aging syndrome (eg, Werner's syndrome and Progeria syndrome) have shorter

telomeres as compared with healthy individuals of the same age (Johnson, Marciniak et

al. 2001). Critically short telomeres induce cellular and tissue defects through checkpoint

signaling pathways that converge on the p53 tumor suppressor protein. In late-generation

TR -/- mice, p53 protein is stabilized and massive cell death occurs (Chin, Artandi et al.

1999). Loss of p53 signiﬁcantly attenuates the cell cycle arrest and cell death phenotypes

in TR -/- tissues. Furthermore, telomere uncapping through disruption of TRF2 also strongly activates p53 in cell culture (Karlseder, Broccoli et al. 1999). The genes downstream of p53 that are responsible for executing the responses to dysfunctional telomeres have been less well studied. However, the cyclin-dependent kinase inhibitor p21 has been shown to participate in the p53-dependent telomere checkpoint response in both human ﬁbroblasts and in TR -/- mice (Brown, Wei et al. 1997; Choudhury, Ju et al.

2007). The introduction of the catalytic subunit of hTERT into normal human cells can partially reverse this effect leading to indefinite and unchecked proliferation. Finally, late-generation telomerase-knockout mice exhibit a premature aging syndrome characterized by hair graying, shortened survival and impaired responses to acute and chronic stress (Herrera, Samper et al. 1999; Rudolph, Chang et al. 1999). All the above data support the theory that telomere shortening detected in aging humans may contribute to many aging phenotypes. The fact that loss of telomere function has consequences both for aging and carcinogenesis explains much of the interest in telomeres today.

1.9 Telomerase Reverse Transcriptase

Human telomerase reverse transcriptase, hTERT, is divided into four regions, the telomerase essential N-terminal (TEN) domain, the telomerase RNA binding domain

(TRBD), the enzymatic RT domain, and C-terminal extension (CTE). The reverse transcriptase domain contains seven conserved motifs shared with conventional RTs: motifs 1, 2 and A, B, C, D, and E (Figure IV).

Most TERTs have a long N-terminal extension that is lacking in viral reverse transcriptases (Kelleher, Teixeira et al. 2002). What also makes TERT unique is the

presence of the first telomerase-specific motif (T-motif), which was identified as a

characteristic feature distinguishing telomerase from viral RTs (Nakamura, Morin et al.

1997). Further analyses revealed more telomerase-specific motifs located in the N-

terminus of TERT (Bryan, Sperger et al. 1998). The function of the CTE region is not

fully understood but it has been observed that there is a higher similarity in the CTE

region among vertebrate TERTs than among yeasts. The CTE motif has also been split

into motifs considered as vertebrate- specific (Kuramoto, Ohsumi et al. 2001) or

chordate-specific (Li, Yates et al. 2007). However, there is in fact a lower similarity

between vertebrate and urochordate CTE regions than between vertebrate motifs and

those found in plants (Sykorova, Leitch et al. 2006).

Briefly, the N-terminal domain is essential for enzymatic activity, but its role in

telomerase function remains less well defined and might have diverged between TERTs form distantly related organisms. Interestingly, TERT has DNA- and RNA-binding domains localized in the TEN domain (Jacobs, Podell et al. 2006)and in the TRBD domain (Rouda and Skordalakes 2007). Unlike the RT and CTE domains, which are highly conserved between TERT and conventional RTs, the TEN and TRBD domains are telomerase-specific and unique to the TERT protein (Kelleher, Teixeira et al.

2002; Delany and Daniels 2004).

Recently, portions of TERT proteins have been identified in terms of crystal

structure. The ﬁrst high-resolution structure of TERT was the TEN domain from

Tetrahymena thermophila (Jacobs, Podell et al. 2006). Its identification revealed

phylogenetically-conserved amino acid residues in a groove on its surface which are

crucial telomerase catalytic activity and for sequence-speciﬁc binding of a single-

stranded telomeric DNA primer. In Tribolium castaneum, the crystal structure of the active subunit TERT subunit bound to an RNA-DNA hairpin was unveiled a few years later (Mitchell, Gillis et al. 2010). Here, the RNA-DNA duplex assumes a helical structure, positioned in the inner cavity of the TERT ring. These TERT–RNA template and TERT–telomeric DNA interactions are similar to those in retroviral reverse transcriptases, supporting the notion that there exists many common features between the two families of enzymes.

The TRBD, or the RNA binding domain, functions in binding the RNA templates to the DNA of the telomeres. The catalytic RT domain, as in other HIV reverse transcriptase domains, carries the enzymatic activity of telomerase. It permits the reverse transcription of the telomerase RNA templates into DNA to continue the elongation of telomeres. Finally, the C-terminal extension, like the N-terminal domain, is essential for enzymatic activity but it also has a role in nucleotide addition processivity (Sykorova and

Fajkus 2009)

These domains have not been well-characterized in other species, such as mouse

(Middleman, Choi et al. 2006; Fakhoury, Marie-Egyptienne et al. 2010); however, what is known is that across most species, the expression of telomerase activity is regulated at different levels, including transcription and mRNA splicing of the TERT gene.

Figure IV – Structure of TERT Subunit. The highly conserved motifs are RT motifs (1, 2,

A–E; red boxes) and telomerase-specific motifs (T2, T, CP, QFP). The TERT subunit can be split into the N-terminal portion and the catalytic domains with the RT motifs and the

CTE. Some of the above domains have been functionally identified as shown: RID1 and

RID2 (Moriarty, Marie-Egyptienne et al. 2004), N-DAT and C-DAT domains

(Armbruster, Banik et al. 2001; Banik, Guo et al. 2002). At the bottom is a structural description including the TEN domain(Jacobs, Podell et al. 2006), RNA-binding domain

[TRBD, (Rouda and Skordalakes 2007)], and RT domain and CTE domain (Gillis,

Schuller et al. 2008). Reprinted by permission from Portland Press Ltd: Biology of the

1.10 Telomerase Regulation

Telomerase activity is stringently repressed in many human tissues.

Transcriptional regulation of TERT mRNA seems to be the most prevalent and rate-

limiting step for telomerase activation (Meyerson, Counter et al. 1997). To begin with, in

humans, the correlation between hTERT mRNA and telomerase activity indicates

transcriptional regulation of the hTERT gene (Cong, Wen et al. 1999). The hTERT

promoter consists of three main regions important for the control of its regulation

(Horikawa, Cable et al. 1999). The first region, consisting of a 258 base pair sequence

corresponds to the core promoter and contains a fragment essential for transcriptional

activation of the hTERT gene. The second one, an activating region, is located between

positions -1397 and -798 on the hTERT gene. The third one is an inhibitory region

located between positions -798 and -400. Overall, the hTERT promoter is GC rich and

contains mainly two CpG islands, a larger CpG island from -845 to exon 2 and a small

one between -4245 and -4545. Several transcription factors are implicated in hTERT

expression, with some of them being activators (e.g., c-Myc, Max, Sp1, Ets1) and others

inhibitors (e.g., Mad1, WT1, p53, MZF-2) (Kyo, Takakura et al. 2008). While

transcription seems to be the most widespread means of telomerase repression in cells,

other levels of regulation exist as well.

Another mechanism for telomerase regulation is the post-translational TERT

modification. For example, recent studies have shown that telomerase activity is

regulated by protein phosphorylation in human breast cancer cells (Li, Zhao et al. 1997).

Inhibition of telomerase activity by protein phosphatase PP2A was both concentration-

and time-dependent and was prevented by the protein phosphatase inhibitor okadaic acid.

Phosphorylation has also been shown to be a regulator of telomerase expression (Li, Zhao

et al. 1998). For example, again in breast cancer cells, human telomerase- associated

protein 1 (hTEP1) and the telomerase catalytic subunit hTERT were used as

phosphoproteins to show that their phosphorylation is a prerequisite for the activation of

telomerase (Li, Zhao et al. 1998). This phosphorylation was mediated by kinase C-alpha,

inducing a significant increase in telomerase activity. This represented an essential step in

the generation of a functional telomerase complex in the initiation and maintenance of

telomerase activity in human cancer. Along the same lines, the Akt protein kinase was

also shown to enhance human telomerase activity through phosphorylation of TERT

subunit (Kang, Kwon et al. 1999). The hTERT subunit contains two putative Akt kinase

phosphorylation sites (220GARRRGGSAS229) and (817AVRIRGKSYV826).

Phosphorylation of the hTERT peptide was observed by a human melanoma cell lysate or

an activated recombinant Akt kinase protein in vitro. Collectively, the above data suggest

that protein phosphorylation regulates the function of various human cancer cells and that

this process is often reversible.

Telomerase has been shown to be regulated by sub-cellular TERT localization as

well (Wong, Kusdra et al. 2002). Catalytically active human telomerase has a regulated

intranuclear localization that is dependent on the cell-cycle stage, transformation state

and DNA damage. Confocal microscopy shows that the release of telomerase to the

nucleoplasm from sequestration at nucleolar sites is enhanced at the expected time of

telomere replication. Finally, telomerase is also regulated by the alternative splicing of

the TERT gene.

1.11 Alternative Splicing of the TERT gene

Alternative splicing of the hTERT transcript plays a role in telomerase regulation

(Kilian, Bowtell et al. 1997). In humans, a total of 13 alternate splicing sites have been

identified within the TERT mRNA (Figure V). Of interest is the α-deletion isoform,

corresponding to an in-frame deletion in the RT motif, which appears to be a dominant

negative inhibitor of telomerase activity (Colgin, Wilkinson et al. 2000; Yi, White et al.

2000). When overexpressed, the alpha-deletion inhibits telomerase activity in telomerase-

positive cells, causing telomere shortening and eventually cell death in a dominant-

negative manner. The remaining alternatively-spliced variants represent exonic deletions

and/or insertion of intronic sequences that cause frame shift and premature termination of

the open reading frame (ORF). Two of these variants, the α-deletion and γ-deletion, result from in-frame deletions of exonic sequences in exon 6 and 11, respectively. Interestingly, telomerase is down-regulated in many tissues by a shift to the abundant β-deletion splicing form. In the β deletion, exons 7 and 8 are deleted resulting in a truncated enzymatically inactive version of hTERT β. Both deletions can combine into a hTERTα

+ β variant, and there are also several additional splicing variants in humans (Hisatomi,

Ohyashiki et al. 2003; Saeboe-Larssen, Fossberg et al. 2006).

While the function of alternately spliced variants is largely unknown, several of the splice sites remove critical reverse transcriptase motifs. It is safe to say, in this case, that splicing may play a role in the regulation of telomerase activity. In particular, telomerase is down-regulated in many tissues by a shift to β-deletion splicing mode, in which exons 7 and 8 are deleted (Saeboe-Larssen, Fossberg et al. 2006). Alternative splicing resolves many of the discrepancies observed between hTERT mRNA levels and

lack of telomerase activity, but telomerase-negative cells frequently contain hTERT

mRNA of which a fraction apparently is normal full-length (Ulaner, Hu et al. 2000).

Figure V – Full-Length and Alternative-Spliced Variants of Human TERT. (A)

Exon/intron organization of the hTERT gene. Exons are shown as black boxes with numbering above. Start/Stop indicates the beginning and end of the opening reading frame (ORF). (B) Schematic drawing of hTERT mRNA alternatively-spliced sites

(ASPSs). Exons are shown as open boxes with numbering inside and black boxes represent intronic sequences. The ORF is indicated below each ASPS by arrows. The hTERT ASPSs have been assigned descriptive names; for example, Ins-i would be an insertion of intronic sequence and Del-e, deletion of exonic sequence. Reprinted by

“Open Access” permission from BioMed Central Ltd: BMC Molecular

Biology 2006, 7:26

Of great interest is the presence of the alternate splicing forms in cancer cells. In

general, splicing has been shown to play a very important role in disease progression.

For example, a reduction in the level of full-length cyclic AMP–dependent chloride

channel protein, often as a result of differential splicing, results in atypical, adult onset or

monosymptomatic forms of Cystic Fibrosis (Noone and Knowles 2001). Along the same

lines, when exon 10 of microtubule-associated protein, Tau, is included or skipped during

alternative splicing, it gives rise to either the three-microtubule repeat (3R) or the four-

microtubule repeat (4R) alternatively-spliced variant of the protein. Often, this leads to disruptions in various important functions of the brain. Normally the ratio of 4R to 3R is roughly one, and this stable isoform ratio seems to be critical for proper neuronal function (Goedert and Jakes 1990; Hong, Zhukareva et al. 1998; Hutton, Lendon et al.

1998; Spillantini, Murrell et al. 1998). When it comes to tumor progression, cancer cells

often take advantage of alternatively-spliced variants of different genes and their ability to give rise proteins that may support growth and survival. Because tumor cells behave in a manner which would usually induce apoptosis, they must constantly find an alternative

means to inhibit this process (Letai 2008). As such, transcripts from a significant number of genes involved in apoptosis, such as Bcl-x, caspase-2 and Fas, are alternatively-spliced and are used to promote tumor progression, as reviewed by (David and Manley 2010).

When it comes the TERT gene, alternative splicing of the alpha/beta double deletion hTERT transcripts was present in 51 melanomas and absent in the eight normal skin samples (Villa, Porta et al. 2001). Oligomer-mediated modulation of hTERT alternative splicing induces telomerase inhibition and cell growth decline in human prostate cancer cells (Brambilla, Folini et al. 2004). These findings could indicate the involvement of

alternative splicing of hTERT in the control of telomerase activity in melanomas or in

development. Such data support the concept that down-regulation of hTERT expression

can cause short-term effects on tumour cell growth.

The majority of data about alternatively spliced variants yielded from studies of

human TERT in different cell lines, cellular compartments, carcinoma and adjacent

tissues (Sykorova and Fajkus 2009). Four TERT splicing variants have been described in

rats with major expression of inactive rTERTb variant in adult brain (Kaneko, Esumi et

al. 2006). One splicing variant was reported for hamster TERT (Guo, Okamoto et al.

2001) five splicing variants were identified for canine TERT (Angelopoulou, Zavlaris et

al. 2008). The pattern of spliced variants is rather complex in chicken, where 19 isoforms

have been reported (Chang and Delany 2006; Hrdlickova, Nehyba et al. 2006). Finally, in

plants while fewer genes exhibit alternative splicing than in animals, they play roles in

many important biological processes such as photosynthesis, defense response and

ﬂowering quality (Reddy 2007; Barbazuk, Fu et al. 2008).

1.12 Alternative splicing of mouse telomerase reverse transcriptase (mTERT)

Searches for alternatively-spliced TERT variants in mice have failed but there are

two candidates in public databases, both coding for a partial deletion within exon 2.

These include deletion of exons 7 and 8 (supported by accession FJ154427; Kunicka et

al. 2002) and retention of partial intron 5 (supported by FJ210936) and a deletion inside

of exon 2, out-of-frame, (supported by ESTs CF531121, CF531069, BY783093,

BY784804, BY775178 and cDNA BC082327; Strausberg et al., 2002), two partial

deletions of exon 2, out-of-frame (supported by cDNA BC127068; Strausberg et al.,

2002).

The importance of hTERT mRNA spliced variants should not be underestimated

since products of other alternatively-spliced mRNAs are associated with a variety of

human diseases, including cancer (Faustino and Cooper 2003; Blencowe 2006; Venables

2006). Interestingly, two dyskerin mutations that affect splicing are found in patients afflicted by the X-linked form of the telomere maintenance syndrome dyskeratosis congenita (Knight, Heiss et al. 1999; Knight, Vulliamy et al. 2001; Marrone, Walne et al.

2005). Moreover, as indicated above, the potential for modulating hTERT alternative

splicing in anti-cancer therapy is supported by a recent study (Brambilla, Folini et al.

2004). Understanding the role of alternatively spliced hTERT variants in telomerase

regulation may provide a molecular framework for the pathogenesis of hTERT-associated

diseases. The functional relevance of spliced hTERT variants is strengthened by the

identification of spliced TERT variants in rice (Heller-Uszynska, Schnippenkoetter et al.

2002), chicken (Chang and Delaney 2006) and rat (Kaneko, Esumi et al. 2006). The

pharmacological assessment of anti-telomerase therapies can be accelerated using rodent

models and the conservation of the TERT variants among these closely related species

would be an indication of their importance.

The objective of the current thesis was to thus to identify potential alternative

splice sites in mouse TERT. We identified several novel mTERT variants and we

pursued the characterization of two variants, one containing an in-frame insertion in the

N-terminus (Insi[1-102]), a region important for binding to the telomerase RNA and

telomeric substrates, and the other a deletion in the C-terminus of the RT domain

(Dele12[1-40]) that generates a premature stop codon, encoding a protein lacking the C-

terminus. Structural analyses revealed that the terminal TEN domain has both DNA-

binding and nonspecific RNA-binding properties and the TRBD domain provides a

second RNA-interaction site. When it comes to RNA binding specifically, the TEN

domain contains RNA interacting domain 1 (RID1), a low-affinity binding site for the TR

template/pseudoknot domain (Lai, Mitchell et al. 2001). The TRBD contains RNA

interacting domain 2 (RID2), which is a high-affinity binding site for the TR CR4/5

domain. A large insertion in this region may interrupt its function in either DNA or RNA

binding. The deletion variant falls in a region which corresponds to the hTERT C-

Terminal Extension (CTE). The RT and CTE domains in hTERT have been shown to be

important for binding to telomeric DNA (Nakamura, Morin et al. 1997) but more

importantly for the reverse transcriptase catalytic activity. Deleting a sequence in such an

essential part of the mTERT may modify it slightly or it might render it completely

inactive.

Specifically, our objectives were to express the alternatively spliced mTERTs

with telomerase RNA using a transcription and translation rabbit reticulocyte lysate

(RRL) system (Fakhoury, Marie-Egyptienne et al. 2010)and to verify whether or not they

can reconstitute telomerase activity will be measured by a telomeric repeat amplification

protocol (TRAP). Reconstitution of telomerase activity requires efficient binding of

TERT to the telomerase RNA component and to its telomeric DNA substrate.

Determinants of telomerase activity have been mapped to both the N- and C-termini of

TERT. The RNA interaction domains of TERT are located in the N-terminus, as is the

domain which interacts with telomeric DNA substrates. Thus both alternatively spliced

mTERT variants were assessed for binding to the telomerase RNA and telomeric DNA

substrates using previously described RNA binding and DNA binding assays (Fakhoury,

Marie-Egyptienne et al. 2010).

The alternatively spliced mTERT mRNAs were also expressed in telomerase-

positive CB17 mouse cells (Marie-Egyptienne, Brault et al. 2008) in order to address the

potential negative-regulatory or dominant-negative function of these variants when

expressed in telomerase-positive cells such as CB17. Cell growth, telomerase activity and

telomere length (by telomere restriction fragment (TRF) length analysis and quantitative

fluorescent in-situ hybridization (qFISH)) were determined in these cells.

These variants were detected in various mouse cell lines and tissues. The insertion

variant has telomerase activity (albeit at lower levels the wildtype mTERT) while the

deletion variant cannot reconstitute activity in vitro and may be behaving in dominant

negative manner. The variants have decreased affinities for telomeric DNA and

telomerase RNA substrates. In telomerase-positive mouse fibroblasts, the C-terminus

deletion variant-expressing cells, at middle passage, grew more slowly than those

expressing wild-type mTERT without any significant effect on telomere length. Our

findings provide an initial identification and characterization mouse TERT alternative

splicing. A better understanding of both mouse and human TERT alternative splicing

may identify specific and effective therapeutic approaches to inhibit telomerase in tumor

cells.

2. Materials & Methods Outline

2.1 Cell Line Preparation for RNA Isolation and Cell Culture

Three mouse cell lines were analyzed (Figure 1B) for the detection of possible

alternatively-spliced variants of mouse telomerase reverse transcriptase (mTERT). These cell lines were CB17, a mouse fibroblast cell line, NIH3T3, a mouse embryonic fibroblast and FM3A, a mouse mammary carcinoma cell line. In culture, NIH 3T3 cell line (ATCC,

CRL-1658) were grown in DMEM (Invitrogen) supplemented with 10% FBS (Wisent,

Canada). FM3A cell line (gift from Carol Prives) were grown in RPMI (Invitrogen)

supplemented with 10% FBS (Wisent, Canada). CB17 cells were grown in Waymouth’s medium (Invitrogen) supplemented with 10% FBS (Wisent, Canada). Cells were collected from 10-cm culture dish at 85% confluency and RNA extraction was performed followed by reverse transcription and by a series of 2 sets of Polymerase Chain Reactions

(PCRs), termed Nested PCR (as discussed in the following sections).

CB17 cells were transfected with pcDNA3.1-hygromycin-mTERT, with pcDNA3.1-hygromycin-mTERT-insa1, with pcDNA3.1-hygromycin-mTERT-dele12, grown in Waymouth’s medium (Invitrogen). Alternatively-spliced variants were introduced into telomerase-positive mouse fibroblast cell line, CB17, through stable transfection. Two clones of each variant-expressing cells were selected from a pool of potential colonies with 800ug/ml Hygromycin B (Invitrogen) for 3-4 weeks and split in a

1:8 ratio at confluency. These were termed Insertion clones 2 and 8, Deletion clones 1 and 9 and Empty Vector clones 1 and 2. The expression of the transgenes were confirmed by RT-PCR (against epitope FLAG-tagged mTERT). The clones were then passaged for approximately 200 population doublings (PD). To ensure that transgene levels did not

fluctuate during culture, RT-PCR analysis (against epitope FLAG-tagged mTERT) was

performed at early, middle and late passages. RNA levels remained relatively unchanged

at all passages. Cells were collected and RNA, DNA and proteins were extracted (as discussed in the following sections) to monitor transgene expression, telomerase activity and telomere length.

2.2 Mouse tissue preparation for total RNA isolation

Seven different organs were surgically excised from C60 strain mice (gift from

Animal quarters, Lady Davis Institute of Medical Research): brain, heart, kidney, liver, lung, ovaries and spleen. 30 mg of fresh tissue was disrupted and homogenized using

RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instruction. Leftover

material was frozen and stored at -80°C.

2.3 RNA isolation, RT-PCR and Protein Extraction

Total RNA isolation was performed using TRIzol according to the manufacturer’s

instructions (Invitrogen) and RNeasy Mini Kit (QIAGEN). At times, when necessary,

total RNA was polyA-purified using Absolutely mRNA Purification Kit according to the

manufacturer’s insctructions (Stratagene). cDNA was then generated from RNA through

reverse transcription (RT). Briefly, 1 ug of RNA per 20 ul reaction was subjected to

DNAse I treatment (5X First Strand Buffer (Invitrogen), 2 unit DNAseI (Fermentas) and

12 unit RNAseOut Recombinant Ribonuclease Inhibitor (Invitrogen) and incubated at 37

°C for 30 min, followed by heat-inactivation of the DNAseI enzyme at 75 °C for 10 min.

Reverse transcription was then performed using 0.2 mmole DTT (Invitrogen), 100 ng

Oligo (dT) primer and 10 mmole dNTP mix and incubating the reaction at 42 °C for 2

min. 200 unit Superscript II Reverse Transcriptase (SS[II]RT) (Invitrogen) was then

added and the reaction was incubated at 42 °C for 1 hour, followed by heat-inactivation

of the SS[II]RT enzyme at 70 °C for 10 min. Finally, the reaction was subjected to 2.5

unit RNAse H (New England Biolabs) treatment at 37 °C for 20 min. Nested PCR

(Snounou, Viriyakosol et al. 1993) (Table 1, Table 2) was then performed in order to

detect alternatively-spliced variants in mouse cell lines CB17, FM3A and NIH3T3.

Several of the suspected products were gel extracted using QIAquick Gel Extraction Kit

according to the manufacturer’s instructions (Qiagen) and sent for sequencing. Materials sent were from each of the following sources: variant a from NIH 3T3 and FM3A cell lines, variant b from NIH 3T3, FM3A and CB17 cell lines, variant c from NIH 3T3 and

CB17 cell lines, variants d, e and f from NIH 3T3 cell line only and variant k from CB17

and FM3A cell lines. 5 of these variants were validated as mTERT alternatively-spliced isoforms and to confirm that these variants could be detected independently of full- length, wildtype mTERT, they were amplified using variant-specific primers (Table 5) under set PCR conditions (Table 3, Table 6) from the NIH 3T3 cell line. In general the following measures are taken while performing PCR: for detection purposes, conditions are optimized using Taq DNA Polymerase (Table 3); for amplification and cloning purposes, high-fidelity PfuTurbo DNA Polymerase (Agilent) is used (Table 4).

For protein extaction, lysates were collected by centrifuging trypsinized cells from one 10-cm culture dish at 85% confluency. Pellets were treated with 50 ul TRAP lysis buffer, enhanced with the following: 40 unit RNAseOut Recombinant Ribonuclease

Inhibitor, 0.35 ul 2-Mercaptoethanol (Sigma-Aldrich), 10 ul Igepal NP-40 (Bioshop) and

10 mmole phenylmethylsulfonyl fluoride (PMSF) (per 1 ml TRAP lysis buffer,

composition described in later section). Pellet was then incubated on ice for 15 min,

followed by centrifugation at 4 °C at 13 000 rpm for 15 min. The supernatant of the

mixture was then subjected to a standard Bradford assay for protein quantification. Until

ready to perform TRAP assays, lysates were stored at -80 °C.

Table 1: Primers used to detect presence of alternatively-spliced mouse TERT variants

Primer Name Sequence Product Length (base pairs)

(1) mTERT136-F 5'-ATCTACCGCACTTTGGTTGC-3' 3422

mTERTIns4-R3 5'-CTGAGGGCATCAGGAAAG-3'

(2) mTERT136-F 5'-ATCTACCGCACTTTGGTTGC-3' 1967

mTERT2092-R 5'-AGTACATCCTGGGTGTCTGGTC-3'

(3) mTERT806-F 5'-GCAAATCATGGGTGCCAAGTC-3 2251

mTERT3056-R 5'-CTAACACGC TGGTCAAAGGGA-3'

(4) Del-F2 5'-CTTATGGGGTCTTCTGTACTG-3' 1565

mTERTIns4-R3 5'-CTGAGGGCATCAGGAAAG-3'

a mTERT136-F 5'-ATCTACCGCACTTTGGTTGC-3' 354

mTERT500-R 5'-GGGCACCAGAAGATAAAGAG-3'

b mTERT806-F 5'-GCAAATCATGGGTGCCAAGTC-3 884

mTERT1689-R 5'-GAGCCTGTTCTTCTGGAATGTG-3'

c mTERT1599-F 5'-GTTCCTGTTCTGGCTGATG-3' 496

mTERT2092-R 5'-AGTACATCCTGGGTGTCTGGTC-3'

d Del-F1 5'-GCATTTCACCCAGCGTCTC-3' 628

Del-R2 5'-CTGCACCTCAGCAAACAG-3'

e mTERT2487-F 5'-CCTATCCACCCTGCTCTG-3' 570

mTERT3056-R 5'-CTAACACGCTGGTCAAAGGGA-3'

f mTERT2781-F 5'-GCTGGACACTCAGACTTTGG-3' 531

mTERTIns3-R2 5'-ATGGTCATTGTCGCCTCTG-3'

Table 1 (Continued): Primers used to detect presence of alternatively-spliced mouse TERT variants

Primer Name Sequence Product Length (base pairs)

k mTERT386F 5'-AGACCCTGCGTGTCAGTG-3' 684

mTERT1069R 5'-GCTGGAGGTTGCTGAGTAG-3'

g mTERT136-F 5'-ATCTACCGCACTTTGGTTGC-3' 1554

mTERT1689-R 5'-GAGCCTGTTCTTCTGGAATGTG-3'

h mTERT1599-F 5'-GTTCCTGTTCTGGCTGATG-3' 955

Del-R2 5'-CTGCACCTCAGCAAACAG-3'

i Del-F1 5'-GCATTTCACCCAGCGTCTC-3' 1131

mTERT3056-R 5'-CTAACACGCTGGTCAAAGGGA-3'

j mTERT2781-F 5'-GCTGGACACTCAGACTTTGG-3' 777

mTERTIns4-R3 5'-CTGAGGGCATCAGGAAAG-3'

Table 1: Primers used to detect presence of alternatively-spliced mouse TERT variants

Done through nested PCR, each primer pair in the first set (1-4) were used to amplify larger regions of mTERT in the first PCR. During the second PCR, the second set of primers (a-j) covered narrower regions of cDNA isolated from mouse cells. Each pair consists of a forward and a reverse primer. To the right of each pair is the number corresponding to the expected size of their product (see Figure 1A for schematic of primer design).

Table 2: PCR conditions used to detect alternatively-spliced mouse TERT variants

PCR Conditions Number of Annealing Amplification Cycles Temperature (°C) (First PCR)

(1) 60 30

(2) 62 32

(3) 61 32

(4) 57 30

a 1) 94°C for 5 min 54 35

b 2) x (specific to each reaction) 56 35 amplification cycles of c 54 35 94°C for 45 sec d y °C (specific to each 54 35 reaction) for 45 sec e 72°C for sec 55 35 3) 72°C for 5 min f 55 35

k 64 35

g 61 30

h 62 30

i 62 30

j 61 30

Table 2:Done through nested PCR, the first set of primers (1-4, Table 1) were used under

set PCR conditions (second column) first amplify larger regions of mTERT. During the

second PCR, the second set of primers (a – j, Table 1) allowed for amplification of

narrower regions of the mTERT gene to be amplified and for possible alternatively-

spliced products to be detected in CB17, FM3A and NIH3T3 cell lines.

Table 3: Amount of Materials Used During PCR Reactions with TAQ DNA Polymerase

Material Amount per PCR 25 µl reaction

cDNA/plasmid 1.0 µl / 5-25 ng

Forward Primer (Alpha DNA) 40 ng

Reverse Primer (Alpha DNA) 40 ng

1 0X Taq Reaction buffer (200 mM Tris 2.5 µl pH 8.4, 500 mM KCl) (Invitrogen)

50 mM MgCl2 (Invitrogen) 1.0 µl

10 mM dNTP mix (Invitrogen) 1.0 µl

Dimethyl Sulfoxide (DMSO) (Sigma- 1.0 µl Aldrich)

Taq DNA Polymerase, Recombinant (15 0.4 µl units/ µl, Invitrogen)

ddH2O Up to 25 µl

Table 3: In general, for the detection of a gene or for troubleshooting purposes,

conditions are optimized using Taq DNA Polymerase during PCR. Note that for

detections of genes in cDNA, 1 µl is used. Since cDNA cannot be quantified, this is

based on the assumption that 1 µg RNA was used as starting material during reverse

transcription.

Table 4: Amount of Materials Used During PCR Reactions with High-Fidelity PfuTurbo DNA Polymerase

Material Amount per PCR 50 µl reaction

cDNA / plasmid 2.0 µl / 10-50 ng

Forward Primer (Alpha DNA) 80 ng

Reverse Primer (Alpha DNA) 80 ng

10× cloned Pfu DNA polymerase reaction 5.0 µl buffer

10 mM dNTP mix 2.0 µl

DMSO ((Sigma-Aldrich) 2.0 µl

PfuTurbo DNA Polymerase (2.5 units/ µl) 1.0 µl

ddH2O Up to 50 µl

Table 4: The amplification of a gene, for example during cloning, PfuTurbo is the

polymerase of choice due to its high fidelity. As stated above, for amplification purposes of genes from cDNA, 2 µl is used, based on the assumption that 1 µg RNA was used as starting material during reverse transcription.

Table 5: Variant-Specific Primers for Isolation of mTERT Isoforms

Variant Primers (PCR 1) Sequence of Primer (PCR 1)

Ins-i1[1- InsaF 5'-CCGGGAGGACGTGGGATAG-3' 102] mTERT1069-R 5'-GCTGGAGGTTGCTGAGTAG-3'

Del-e2[289- DelkF 5'-GCCTACCAGGGGAGATGG-3' 797] mTERT2092-R 5'-AGTACATCCTGGGTGTCTGGTC-3'

Del- mTERT806-F 5'-GCAAATCATGGGTGCCAAGTC-3 e2[1132- 1324] Del-R2 5'-CTGCACCTCAGCAAACAG-3'

Del-e6[1- Del-dF 5'-CTTTGTTAAGGGTAAGCTGG-3' 36] mTERT-3056R 5'-CTAACACGCTGGTCAAAGGGA-3'

Del-e12[1- DeleF 5'-ACTACTCAGGAGTGTCTTCA-3' 40] mTERTIns4-R3 5'-CTGAGGGCATCAGGAAAG-3'

Variant Primers (PCR 2) Sequence of Primer (PCR 2) Product Length (base pairs)

Ins-i1[1- InsaF 5'-CCGGGAGGACGTGGGATAG-3' 291 102] mTERT500-R 5'-GGGCACCAGAAGATAAAGAG-3'

Del- DelkF 5'-GCCTACCAGGGGAGATGG-3' 682 e2[289- 797] mTERT1689-R 5'-GAGCCTGTTCTTCTGGAATGTG-3'

Del- DelbF 5'-AGTACATCCTGGGTGTCTGGTC-3' 558 e2[1132- 1324] 2092R 5'-AGTACATCCTGGGTGTCTGGTC-3'

Del-e6[1- DeldF 5'-CTTTGTTAAGGGTAAGCTGG-3' 427 36] Del-R2 5'-CTGCACCTCAGCAAACAG-3'

Del- DeleF 5'-ACTACTCAGGAGTGTCTTCA-3' 459 e12[1-40] mTERTIns3-R2 5'-ATGGTCATTGTCGCCTCTG-3'

Table 5: Variant-Specific Primers for Isolation of mTERT Isoforms

Done through nested PCR, each primer pair in the first table (top) was used to specifically amplify larger regions of mTERT in the first PCR. During the second PCR, the second set of primers (bottom) covered narrower regions of the alternatively-spliced variants isolated from mouse embryonic fibroblasts, NIH 3T3. Each pair consists of a forward and a reverse primer. At the bottom, to the right of each pair is the number corresponding to the expected size of the final product of the variant of interest.

Table 6: PCR conditions used to isolate alternatively-spliced mouse TERT variants

Variant of Interest PCR1 PCR2

1) 94°C 5 min 1) 94°C 5 min

2) 27 amplification cycles of 2) 30 amplification cycles of

94°C for 45 sec 94°C for 45 sec 60°C for 45 sec 58°C for 45 sec Ins-i1[1-102] 72°C for sec 72°C for sec 3) 72°C for 5 min 3) 72°C for 5 min

1) 94°C 5 min 1) 94°C 5 min

2) 24 amplification cycles of 2) 27 amplification cycles of

94°C for 45 sec 94°C for 45 sec 63°C for 45 sec 64.5°C for 45 sec Del-e2[289-797] 72°C for sec 72°C for sec 3) 72°C for 5 min 3) 72°C for 5 min

1) 94°C 5 min 1) 94°C 5 min

2) 24 amplification cycles of 2) 27 amplification cycles of

94°C for 45 sec 94°C for 45 sec 62°C for 45 sec 66.5°C for 45 sec Del-e2[1132-1324] 72°C for sec 72°C for sec 3) 72°C for 5 min 3) 72°C for 5 min

1) 94°C 5 min 1) 94°C 5 min

2) 27 amplification cycles of 2) 27 amplification cycles of

94°C for 45 sec 94°C for 45 sec 59°C for 45 sec 59°C for 45 sec Del-e6[1-36] 72°C for sec 72°C for sec 3) 72°C for 5 min 3) 72°C for 5 min

1) 94°C 5 min 1) 94°C 5 min

2) 27 amplification cycles of 2) 27 amplification cycles of

Del-e12[1-40] 94°C for 45 sec 94°C for 45 sec 59°C for 45 sec 60°C for 45 sec 72°C for sec 72°C for sec 3) 72°C for 5 min 3) 72°C for 5 min

Table 6: PCR conditions used to isolate alternatively-spliced mouse TERT variants

Done through nested PCR, the first PCR conditions (middle column) was performed to

specifically amplify larger regions of mTERT. During the second PCR, the second set of

conditions allowed for amplification of narrower regions of the alternatively-spliced

variants isolated from mouse embryonic fibroblast cell line, NIH 3T3.

2.4 Plasmid construction

Construction of pcDNA3.1-hygromycin-FLAG-hTERT and pcDNA3.1- hygromycin-FLAG-mTERT were previously described (Fakhoury, Marie-Egyptienne et al. 2010). Construction of pcDNA3.1-hygromycin-FLAG-mTERT-insi1 was performed using insertion-specific primers, InsaF and mTERT-1069R for the first PCR and InsaF and mTERT500-R for the second PCR (see Table 2 for primer sequences) to amplify mTERT containing the variants from NIH 3T3 cDNA via nested polymerase chain

reaction (PCR 1: denaturation at 95 °C for 5:00, followed by 35 amplification cycles of

95 °C for 45 sec, 62 °C for 45 sec and 72 °C for 2 min, followed by final extension at 72

°C for 10 min, PCR2: denaturation at 95 °C for 5 min, followed by 35 amplification

cycles of 95 °C for 45 sec, 56 °C for 45 sec and 72 °C for 2 min, followed by final

extension at 72 °C for 10 min). Construction of pcDNA3.1-hygromycin-FLAG-mTERT-

dele12 was done using deletion specific primers, DeleF and mTERTIns4-R3 for the first

PCR and DeleF and mTERTIns3-R2 for the second PCR (see Table 2 for primer sequences) to amplify mTERT containing the deletion variant from NIH 3T3 cDNA via

nested polymerase chain reaction (PCR 1: denaturation at 95 °C for 5 min, followed by

30 amplification cycles of 95 °C for 0:45, 56 °C for 45 sec and 72 °C for 3 min, followed

by final extension at 72 °C for 10 min, PCR 2: denaturation at 95 °C for 5 min, followed

by 40 amplification cycles of 95 °C for 45 sec, 56 °C for 45 sec and 72 °C for 2 min,

followed by final extension at 72 °C for 10 min). The PCR products were digested using

restriction enzymes NotI and NcoI (NEB) and ligated into the NotI/NcoI- digested pcDNA3.1-hygromycin-FLAG-mTERT plasmid and confirmed by sequencing.

2.5 In vitro transcription and translation, SDS-PAGE and TRAP Assay

In vitro transcription and translation were performed using the T7-coupled

transcription/translation RRL system (Promega) (Moriarty, Marie-Egyptienne et al.

2004). Full-length FLAG-hTERT, FLAG-mTERT, FLAG-mTERT-insi1 and truncated

FLAG-mTERT-dele12 were synthesized in RRL in the presence of 300 ng pcDNA3.1-

hygromycin-FLAG-hTERT, pcDNA3.1-hygromycin-FLAG-mTERT, pcDNA3.1-

hygromycin-FLAG-mTERT-insi1 or pcDNA3.1-hygromycin-FLAG-mTERT-dele12,

300 ng of purified hTR and 0.8 uCi L-[35S]methionine (1175 Ci/mmol, Perkin Elmer)).

hTR was synthesized from FspI-linearized phTR+1 (Autexier, Pruzan et al. 1996). The

reactions were incubated at 30 °C for 1 ½ hours. 1µl of the RRL reaction was separated

by SDS-PAGE. The remaining RRL was diluted in the PCR based telomerase assay,

telomeric repeat amplification protocol (TRAP) lysis buffer (10mM Tris-HCl pH 7.5,

1mM MgCl2, 1mM EGTA, 10% glycerol, 150mM NaCl) at 1/50. 1µl of this dilution was then used in a two-step TRAP assay. Reaction conditions for the first step were 20mM

Tris-HCl pH 8.3, 1.5mM MgCl2, 63mM KCl, 1mM EGTA, 0.005% Tween-20, 0.1 mg/ml BSA, 40 pmol TS primer (IDT), 20pmol NT primer (IDT), 1x10-13 M TSNT primer (IDT) (sequences of primers as described in (Kim and Wu 1997)) and dNTPs in

50µl reaction, incubated at 30 °C for 30 min. For the second step, 20 pmol of ACX

primer (IDT), 0.5µl of [α-32P]dGTP (3000 Ci/mmol, Perkin Elmer) and 2U Taq

(Invitrogen) were added to the tube and the reaction was amplified for 30 cycles at 94 °C

for 30 sec, 60 °C for 30 sec and 72°C for 30 sec. The products were separated on a 10%

acrylamide gel in 0.6X TBE, dried at 80 °C for 45 min and autoradiographed on a

phosphorimager cassette overnight and scanned the next morning using the Storm 840

(GE). Quantification was carried out as previously described (Moriarty, Dupuis et al.

2002).

To test for dominant-negative potentials of the alternatively-spliced variants,

mixed TRAP assays were performed. Briefly, in-vitro reconstituted wildtype mTERT and

variant enzymes synthesized separately in RRLwere used together as templates during

PCR elongation reaction at the 1/50 dilution. Increasing amounts of either the insertion or

the deletion variant proteins (reconstituted in vitro with in vitro transcribed telomerase

RNA) were added in the presence of a constant amount of wildtype mTERT protein

(reconstituted with in-vitro transcribed telomerase RNA) and their mixed telomerase

activity was assayed. TRAP assays were performed as previously stated.

The level of telomerase activity in cell extracts from CB17 cells expressing

pcDNA3.1-hygromycin-FLAG-mTERT, pcDNA3.1-hygromycin-FLAG-mTERT-insi1

or pcDNA3.1-hygromycin-FLAG-mTERT-dele12 and empty vector was evaluated using

the TRAP assay as described in (Hrdlickova, Nehyba et al. 2006) with a few exceptions.

Differing amounts of protein extracts in TRAP lysis buffer (100ng, 40ng, 20ng, 10ng,

4ng, 2ng of total protein) were first incubated with 0.5 μg of the TS primer and 2.5mM

each dNTP in 1× TRAP reaction buffer (see above) containing 0.8 mM spermidine and 5

mM β-mercaptoethanol in a total reaction volume of 50 μl for 30 min at 37oC. The

reactions were stopped by incubation at 94C for 2 min. Aliquots of synthesis (2.5 μl)

were then PCR amplified with 0.1 μg unlabeled TS primer, 20uM NT, 20uM ACX, 1x10-

13M TSNT, 0.5µl of [α-32P]dGTP (3000 Ci/mmol, Perkin Elmer) and 2U of Taq

(Invitrogen). PCR conditions were 94oC for 2 min followed by 36 cycles of 30 sec at

94oC, 30 sec at 50oC and 1 min at 72oC. The TRAP PCR products were then separated

on 7.5% polyacrylamide gels in 0.5X TBE.

2.6 In vitro DNA-binding assay and quantification

Primer-binding assays were performed as described (Wyatt, Lobb et al. 2007),

except streptavidin magnesphere paramagnetic particles were used for oligonucleotide

pull down (Promega, USA) and immobilization of biotinylated oligonucleotides. TERT

proteins were synthesized in RRL in the presence of 300 ng pcDNA3.1-hygromycin-

FLAG-mTERT, pcDNA3.1-hygromycin-FLAG-mTERT-insi1 or pcDNA3.1-

hygromycin-FLAG-mTERT-dele12 using L-[35S]methionine to visualize and quantify the

oligonucleotide-bound TERT. 5’-biotinylated oligonucleotides, purified through high-

performance liquid chromatography (HPLC) were prepared by Operon (USA). Often,

HPLC purification is recommended for oligonucleotides that require additional

purification, such as oligonucleotides bound to a biotin molecule, which are poorly

purified by standard purification methods. HPLC purification also helps to remove non-

specific oligonucleotides that aren’t tagged with biotin. Non-tagged oligonucleotides

were prepared by IDT (Canada) through standard salt-free (desalted) purification.

Quantification of primer-binding experiments was done as described (Wyatt, Lobb et al.

2007), except Imagequant (GE Healthcare) software was used for quantification.

2.7 In vitro RNA-binding assay and quantification

RNA-binding assays were performed as described (Moriarty et al., 2002), except

8.82 ug/ml of M2 anti-FLAG antibody (Sigma) and α-32UTP (1 mCi/ml) (Perkin-Elmer)

was used to immunoprecipitate FLAG-hTERT, FLAG-mTERT and FLAG-mTERT-insi1

and FLAG-mTERT-dele12. Quantification of FLAG-TERT-hTR interactions was

performed as described previously (Moriarty, Dupuis et al. 2002).

2.8 Telomere Restriction Fragment Assay

Telomere length was determined by terminal restriction fragment (TRF) analysis

(Cerone, Londono-Vallejo et al. 2001). Genomic DNA from CB17 cells at early, middle

and late passage was extracted as per Kimura, Stone et al. 2010, with the following

modifications: DNA lysis buffer (10mM TRIS pH 8.0, 0.1 M NaCl, 25 mM EDTA, 0.5%

SDS), resuspension in double-distilled water instead of suggested TE buffer, and

verifying DNA integrity on 1% agarose – TAE gel and digested with HinfI and RsaI.

Equal amounts of digested DNA were separated by pulse-field gel electrophoresis

(PFGE) on a 1% agarose gel in 1X TBE at 5V/cm for 12h at 14C. Gels were then

partially dried at room temperature for 30min, then at 50C for 1h, denatured for 30 min in

0.5M NaOH and 1.5M NaCl and neutralized for 30 min in 1M Tris-HCl pH 7.5 and 1.5M

NaCl. Hybridization was carried out according to (Counter, Avilion et al. 1992), except

that the three washes were performed in 2x SSC and the gel was autoradiographed on X-

ray film (Kodak) or a phosphorimager cassette for 72 h and scanned using the Storm 840

(GE).

3. Results

3.1 Identification of alternatively-spliced mTERT mRNA variants

In humans, a total of 13 alternative splicing sites have been identified within the TERT mRNA (Kilian, Bowtell et al. 1997). Various alternatively-spliced TERT mRNAs in other vertebrates and plants have also been identified, yet their role in the regulation of telomerase, telomere maintenance and cell survival is poorly characterized. In humans, two variants that are characterized more than the others, the alpha and the beta isoforms, correspond to an in-frame deletion in the RT motif, with the alpha variant appearing to be a dominant inhibitor of telomerase activity (Colgin, Wilkinson et al. 2000). In mus musculus, however, very little is known about the regulation of mTERT by alternative splicing.

We identified several novel mTERT variants using nested PCR (Zhu and Autexier, unpublished data, Figure 1A). Three mouse cell lines were analyzed (Figure 1B) to ensure that the identification of spliced variants was not cell line specific. These cell lines were CB17, a mouse fibroblast cell line, NIH3T3, a mouse embryonic fibroblast and FM3A, a mouse mammary carcinoma cell line. The templates for the first of two PCRs were cDNAs from the three cell lines synthesized using gene-specific primers and oligo(dT). Four pairs of primers spanning large regions of the mTERT gene were used for the first PCR and ten pairs of primers spanning shorter regions were used in total for the second PCR. The expected sizes of the PCR products derived from normally spliced mTERT mRNAs are listed in Table 1 for each primer pair. The presence of several products of different sizes with the same set of primers indicated that alternatively-spliced variants may exist in certain regions of mTERT (Zhu and Autexier, unpublished data) (Figure

1B). Twelve products derived from potential spliced variants from the three mouse cell lines were sequenced (see methods).

Five of the twelve suspected variants, Ins-i1[1-102] (found using primer pair a (table 1, figure 1A)), Del-e2[289-797] (found using primer pair k, (Table 1, Figure 1A)), Del-e2[1132-

1324] (found using primer pair b (Table 1, Figure 1A)), Del-e6[1-36] (found using primer pair d

(Table 1, Figure 1A)) and Del-e12[1-40] (found using primer pair e (Table 1, Figure 1A)) were validated by sequencing (Figure 2A). At this point, the mTERT alternatively-spliced variants were assigned descriptive names; for example, Ins-i1[1-102] would be an insertion of intronic sequence (in this case, intron 1 between nucleotides 1 and 40) and Del-e12[1-40], deletion of exonic sequence (in this case, exon 12 between nucleotides 1 and 40).

Ins-i1[1-102] is an in-frame insertion of intronic sequence in intron 1. The 102-nucleotide sequence falls in a region which corresponds to the N-terminal portion of the human TERT transcript, a region important for enzymatic activity as well as binding to both the DNA and RNA substrates. An insertion of this size would interrupt and change the amino acid sequence of the protein and may interfere with any of the above listed functions. In humans, ten insertion variants have been reported to date (Saeboe-Larssen, Fossberg et al. 2006), none of which correspond to the ins-i1[1-102]. In fact, this alternatively spliced variant is completely novel and has not been reported in any other species. Next, we detected two deletion variants in exon 2, Del-e2[289-797] and Del-e2[1132-1324]. Neither deletion maintains the original mTERT sequence in-frame; in fact, they both introduce premature stop codons that delete all of the RT and C-terminal regions, most likely leading to inactive proteins (further analysed in Discussion). Next, we detected an in- frame 36-nucleotide deletion at the beginning of exon 6, Del-e6[1-36]. Interestingly, this deletion corresponds to the alpha-deletion in the hTERT mRNA. This 36-base pair deletion falls within a region which corresponds to the human reverse transcriptase motif A (Kilian, Bowtell et al.

1997). While the alpha-deletion cannot reconstitute telomerase activity in human fibroblasts which do not express hTERT (Shay and Wright 2000), it’s been shown to inhibit telomerase activity in telomerase-positive cells (Colgin, Wilkinson et al. 2000; Yi, White et al. 2000). It’s

61 also been shown to cause telomere shortening and cell death in these cells, all in a dominant- negative manner. Since this variant was not novel, we opted not to characterize it further. Finally, the last alternatively-spliced variant confirmed by sequencing, del-e12[1-40], was a 40-nucleotide deletion detected at the beginning of exon 12 in a region which corresponds to the human C- terminal extension (CTE). Similar to the deletions in exon 2, this one is out-of-frame and causes a premature stop codon. However, this deletion variant, if translated, would lead to a longer protein than would Del-e2[289-797] and Del-e2[1132-1324]. We predict that this variant is inactive because it will yield a truncated protein lacking the C-terminus. Of great interest was whether this variant could act in a dominant-negative fashion and inhibit telomerase function similarly to the human alpha-deletion.

3.2 Mouse TERT alternatively-spliced variants exist in different mouse cells and tissues

To confirm that these variants could be detected independently of full-length, wildtype mTERT, nested PCR was once again performed, this time using variant-specific primers (Table

5) under specific conditions (Table 3, Table 6) (Zhu and Autexier, unpublished data, Figure 3).

To confirm that the variants are represented in cytoplasmic polyA+ preparations, we determined whether the alternatively-spliced products could be derived from purified polyA+ mRNA from the mouse NIH3T3 cell line. Products were indeed generated by RT-PCR from alternatively- spliced mTERT mRNAs. With this confirmed, we pursued further characterization of two variants: ins-i1[1-102], because it falls in a region important not only for enzymatic activity but also for binding to the telomerase RNA and telomeric DNA substrates in humans and del-e12[1-

40], because it is C-terminally located in the RT domain important for enzymatic activity and encodes a protein lacking the C-terminus. Structural analyses revealed that the terminal TEN domain has both DNA‐binding and nonspecific RNA‐binding properties and the TRBD domain provides a second RNA‐interaction site. When it comes to RNA binding specifically, the TEN domain contains RNA interacting domain 1 (RID1), a low‐affinity binding site for

62 the TR template/pseudoknot domain (Lai, Mitchell et al. 2001). The TRBD contains RNA interacting domain 2 (RID2) which is a high‐affinity binding site for the TR CR4/5 domain. A large insertion in this region may interrupt its function in either DNA or RNA binding. The deletion variant falls in a region which corresponds to the hTERT C‐Terminal Extension

(CTE). The RT and CTE domains in hTERT have been shown to be important for binding to telomeric DNA (Nakamura, Morin et al. 1997) but more importantly for the reverse transcriptase catalytic activity. Deleting a sequence in such an essential part of the mTERT may modify it slightly or it might render it completely inactive. First, we assessed if both variants could be detected in different mouse cell lines using variant specific primers. Using primers that were variant-specific (Table 5) under set conditions (Table 3, Table 6), the variants were detected in the mouse fibroblast cell line, CB17 and the mouse embryonic fibroblast cell line, NIH3T3, but absent in mouse mammary carcinoma cell line, FM3A (Figure 4A).

To validate that these spliced variants are not specific to cell lines, and might be physiologically relevant, we assessed if they were present in mouse tissues. Using the same variant-specific primers mentioned above, we detected ins-i1[1-102] and del-e12[1-40] in different mouse organs. Of the 7 organs surveyed, the insertion variant was detected in the brain and kidney while the deletion variant was detected only in the ovaries (Figure 4B). Here, the NIH

3T3 cell line used as a positive control. Collectively, the above data suggest a tissue-specific role for these isoforms.

3.3 The mTERT del-e12[1-40] variant behaves in a dominant-negative manner

Telomerase reverse transcriptase, TERT, is divided into four regions, the telomerase essential N-terminal (TEN) domain, the telomerase RNA binding domain (TRBD), the enzymatic

RT domain, and C-terminal extension (CTE) (Sykorova and Fajkus 2009). Alignment of the aforementioned motifs from diverse organisms indicates that these regions are well conserved

63 within the TERT family (Peng et al. 2001; Bryan et al. 2000; Gillis et al. 2008; Mitchell et al.

2010). We used this sequence conservation to our advantage in order to acquire a detailed understanding of the mouse telomerase reverse transcriptase (mTERT) alternative splicing and predict its functions in telomere length maintenance and cell survival.

We first sought to characterize the in vitro properties of the insertion and deletion variants. Having cloned the cDNA of each variant into plasmids (pcDNA3.1-FLAG-hygromicin) optimized for in vitro use, wildtype hTERT, wildtype mTERT, Ins-i1[1-102] and Del-e12[1-40] were expressed in vitro in rabbit reticulocyte lysate (RRL) in the presence of [35S] methionine.

Products of the expected molecular weights of approximately 130 kDa for wildtype hTERT and125 kDa for wildtype mTERT, corresponding to full-length proteins, were observed (Figure 5, bottom left panel). Products of the expected molecular weights were also recovered for the insertion and deletion variant, with the deletion variant, as expected, encoding a shorter protein lacking the C-terminus. The insertion and deletion variant proteins were expressed in the presence of in vitro transcribed telomerase RNA, and telomerase activity of a range of dilutions of the lysate (to determine the linear range) was assayed using the Telomeric Repeat Amplification

Protocol (TRAP) assay. The mTERT insertion variant can reconstitute telomerase activity while the mTERT deletion variant does not (Figure 5, left). Quantification of the data, performed for the

1/50 dilution, confirmed that the insertion variant reconstitutes 40% of the telomerase activity reconstituted by wildtype mTERT and that the deletion variant cannot reconstitute telomerase activity (n=3) (Figure 5, right). In humans, the alpha-deletion, when overexpressed, inhibits telomerase activity in telomerase-positive cells in a dominant-negative manner (Shay et al. 2000).

To unveil a similar mechanism in mouse, telomerase activity of mixed in vitro reconstituted wildtype mTERT and variant enzymes was measured using the TRAP assay at the 1/50 dilution.

Increasing amounts of either the insertion or the deletion variant proteins (reconstituted in vitro with in vitro transcribed telomerase RNA were mixed in the presence of a constant amount of

64 wildtype mTERT protein (reconstituted with in vitro transcribed telomerase RNA) and their mixed telomerase activity was assayed. While the insertion variant does not have a strong inhibitory effect on wildtype mTERT reconstituted enzyme (n=3) (Figure 6A), the deletion variant seems to be inhibiting the telomerase activity of wildtype telomerase and may have dominant negative effects on telomerase activity (n=3) (Figure 6B). Our results indicate that this is a dose-dependant effect. If this is the case, it could be used as a strategy to target distinct sites on the hTERT gene as a specific therapeutic approach to inhibit telomerase in tumour cells.

3.4 The variants have decreased binding affinities for DNA and RNA substrates

TERT has DNA- and RNA-binding domains localized in the TEN domain (Jacobs,

Podell et al. 2006) and the TRBD domain (Rouda and Skordalakes 2007). Structural analyses revealed that the terminal TEN domain has both DNA-binding and nonspecific RNA-binding properties and the TRBD domain provides a second RNA-interaction site. Previous studies have shown, through mutagenesis of the aforementioned domains, that hTERT mutants have reduced binding to their DNA substrates (Wyatt, Lobb et al. 2007). To determine whether a defect in primer binding could explain the reduced activity of the insertion and deletion variants, we used a primer-binding assay (Fakhoury, Marie-Egyptienne et al. 2010). We expressed L-[35S]- methionine-radiolabelled wildtype hTERT, wildtype mTERT, insertion and deletion variant proteins in vitro in RRL. We then incubated the reconstituted complexes with a biotinylated telomeric substrate, bio-(T2AG3)3 or a biotinylated non-telomeric substrate, bio-(A2TC3)3 (Figure

7A, C). To control for non-specific binding, in vitro reconstituted TERT proteins was confirmed to have a significantly higher affinity for the telomeric bio-(T2AG3)3 than for non-telomeric oligonucleotide bio-(A2TC3)3 (Figure 7B, D). As a last additional control, we used a non- biotinylated primer, (T2AG3)3, to ensure as much elimination of non-specific binding as possible,

In theory, these oligonucleotide sequences should not be able to bind the Streptavadin

Paramagnetic beads which are specific to biotin. However, as compared to wildtype mTERT, the

65 insertion variant had ~63% binding affinity for telomeric DNA and the deletion variant, ~74%

(n=2) (Figure 7B,D, experiment performed twice). Thus, the reduced activities of the insertion and deletion variants in vitro may be also partially due to decreased TERT binding to its DNA substrate.

To assess whether the decrease in activity of the proteins encoded by the alternatively- spliced variants was due to defective telomerase RNA-binding, we performed an in vitro RNA- binding assay. We expressed L-[35S]-methionine-radiolabelled wildtype hTERT, wildtype mTERT, insertion and deletion variant proteins in vitro in RRL in the presence of in vitro transcribed α-32UTP-labelled human telomerase RNA (hTR). To note, all assays requiring the presence of telomerase RNA (TR) (TRAP, RNA-Binding) have been performed using the human

TR because levels of in vitro reconstitution using mTERT and mTR are not optimal. FLAG- tagged wildtype hTERT, wildtype mTERT, insertion variant and deletion variant were immunoprecipitated (IP) using packed Protein G-Sepharose beads. Their interaction with the α-

32UTP-labelled hTR was confirmed by visualizing IP’d products on two pieces of autoradiographic film. Gel analysis of hTR coimmunoprecipitated with FLAG-tagged insertion and deletion variant proteins revealed significant defects in TERT-hTR interactions. Both variants have decreased binding affinities for RNA substrates, with the insertion variant having a more drastic effect with only 20.24% of the wildtype binding and the deletion variant, 45.76% (n=3)

(Figure 8). For the insertion variant, the decrease in binding may be due to the fact that the 102- nucleotide insert falls in a region which corresponds to the human N-terminus RNA Binding

Domain. This data confirmed that the reduced activity of in vitro reconstituted mTERT insertion and deletion variant proteins may also be due to their decreased binding to full-length hTR. Our data show that the reduced activity of the insertion and deletion variant enzymes can be attributed to reduced TERT-DNA or TERT-TR interactions.

3.5 Growth defects of mTERT deletion variant-expressing cells

Since mixed in vitro reconstituted TERT assays revealed that the deletion variant inhibited telomerase activity and might have dominant negative effects on telomerase activity, we sought to find a similar effect in vivo. First, we predicted that the mTERT deletion variant’s decrease in activity would confer a growth disadvantage in CB17 cells by decreasing their telomere lengths in a dominant negative manner. The alternatively-spliced variants were introduced into telomerase positive mouse fibroblasts, CB17, through stable transfection. Two clones each of the wildtype mTERT and the insertion and deletion variant-expressing cells were selected from a pool of potential colonies, WT mTERT clones 1 and 2 (data not shown), insertion clones 2 and 8 (Figure 9), Deletion clones 1 and 9 (Figure 9) and Empty Vector clones 1 and 2

(data not shown) and expression of the transgenes were confirmed by RT-PCR (against epitope

FLAG-tagged mTERT). The clones were then passaged for approximately 200 population doublings (PD) (Figure 10A). One of the del-e12[1-40] variant clones, clone 9, appeared to have a noticeable growth defect. To ensure that transgene levels did not fluctuate during culture, RT-

PCR analysis (against epitope FLAG-tagged mTERT) was performed at early, middle and late passages. RNA levels remained relatively unchanged at all passages (Figure 10B). Furthermore, there were no growth defects with Ins-i1[1-102] clones 2 and 8 nor with Del-e12[1-40] clone 1.

Finally, the disparity in growth between deletion variant clones 1 and 9 doesn’t appear to be as a result of less transgene expression (Figure 10B).

3.6 mTERT deletion variant-expressing cells, at middle and late passage, have reduced telomerase activity

TRAP assays were also performed to confirm that the deletion variant and wild type proteins were expressed and to determine their levels of activity. Since CB17 is a telomerase- positive line, it expresses both TERT as well as TR. Cell extracts were collected from transfected

67 clones at early, middle and late passages for both the wildtype mTERT as well as for the deletion variant clone 9. TRAPs were performed across a wide range of dilutions of wildtype (Figure 11A) and deletion (Figure 11B) protein extracts in TRAP lysis buffer (100ng, 40ng, 20ng, 10ng, 4ng,

2ng of total protein). At middle and late passage, mTERT deletion variant-expressing clones displayed lower levels of activity compared to wildtype mTERT-expressing cells (Figure 11B).

Interestingly, this is in agreement with our initial findings in which we showed the inhibitory properties of the deletion variant. In vitro, we showed that, in a dose-dependent manner, the deletion variant inhibits wildtype telomerase activity and may be doing so in a dominant-negative dominant negative fashion. In cells, this effect is seen with increasing population doublings.

Unlike in vitro, however, telomerase activity is not completely or nearly completely abolished, even at later passages. Finally, TRAP assays were performed for all clones initially selected (WT mTERT clones 1 and 2, insertion clones 2 and 8, Deletion clones 1 and 9 and Empty Vector clones 1 and 2) at early passage (Figure 12). No significant difference in telomerase activity was observed between the different clones at early passage.

3.7 The mTERT deletion variant expression shows no change in telomere length with increasing passage

The deletion variant, when transfected into telomerase-positive CB17 cells, confer a growth disadvantage by middle passage. To confirm that this slowed growth is due to decreased telomerase activity and perhaps shorter telomeres, we examined the effects of mTERT deletion variant expression on telomere length. Cells expressing the deletion variant clone 9 or wildtype mouse TERT were passaged for 200 passage doublings and genomic DNA was collected at early, middle and late passage. The collective telomere lengths of each clone were measured using

Telomere Restriction Fragment (TRF) Assay. Long telomeres, between 11-15 kb, were observed for deletion clone 9 at early, middle and late passages, similar to those of wildtype mTERT cells

(Figure 13). The initial observance of similar-sized telomeres indicates that the deletion variant’s

68 decrease in telomerase activity and cell growth defect is not caused by bulk telomere shortening.

It is possible that an increase in the number of short telomeres might trigger a growth defect

(Herrera, Samper et al. 1999). Thus telomere length distribution analysis by quantitative

Fluorescent in-situ Hybridization (qFISH) should be performed (see discussion).

A -

B -

Figure 1: Identification of alternatively-spliced mTERT mRNA variants

A: Schematic of experimental Design of Nested PCR for detection of alternatively-spliced mTERT variants in mouse cells. Templates for the first PCRs are cDNAs from the three cell lines synthesized using gene-specific primers or oligo(dT). Four pairs of primers were then used for the first PCR and 10 pairs in total for the second PCR covering shorter regions. B: Identification of alternatively spliced mTERT variants through nested PCR. Products were generated by RT-PCR from full length and alternatively spliced mTERT mRNAs. Three mouse cell lines were analyzed:

CB17, mouse fibroblast cell line, NIH3T3, Mouse embryonic fibroblast and FM3A, mouse mammary carcinoma. Orange arrowheads indicate the products of alternatively spliced mRNAs

(lanes 3, 5, 6, 8, 9,12,13, 16 and 22). Primer pairs g, h, i and j yielded only wildtype full length mTERT products (data not shown). Lanes 2 and 3 (with primer pairs b1 and b2) represent PCR products from different cDNA from the same cell line for that region of the mTERT gene. The same can be applied to lanes 9 and 10 as well as lanes 14 and 15. O'GeneRuler™ 1 kb DNA

Ladder used as DNA ladder marker (Zhu and Autexier, unpublished data)

Figure 2 - Alternatively-spliced variants of mouse TERT: Five of the suspected alternatively- spliced variants from Figure 1B were confirmed by sequencing. Exons are shown as white boxes with numbering inside. Two arrowheads above the full-length mTERT mRNA show the regions of each PCR product covered. Insertions are shown by black boxes, Deletions are marked by missing parts of exon sequence. The predicted ORF is indicated below each alternatively-spliced site (ASPS) by arrows. The mTERT ASPSs have been assigned descriptive names.

Figure 3: Specific PCR products of alternatively spliced mTERT variants

Following sequencing, nested PCR was performed using variant-specific primers (top). Products were generated by RT-PCR from alternatively-spliced mTERT mRNAs. Alternatively-spliced products were derived from purified polyA+ mRNA from the mouse NIH3T3 cell line (bottom).

O'GeneRuler™ 1 kb DNA Ladder used as DNA ladder marker (Zhu and Autexier, unpublished data).

Figure 4A: Presence of alternatively-spliced variants in mouse cell lines

Expression of two mTERT variants, Ins-i1[1-102] and Del -e12[1-40], were analysed in different mouse cell lines. Nested PCR was performed using variant-specific primers (Figure 2B) and cDNA prepared from different mouse cell lines. Here, cDNA (-) negative control was used in which reverse transcription of mTERT total RNA was performed without any RNA present prior the PCR step. As a second negative control, we used a plasmid containing wildtype mTERT plasmid, which should not contain any sequences amplifiable by the variant-specific primers

Finally, as positive controls, we used variant-specific plasmids containing either the insertion or the deletion variant sequences. O'GeneRuler™ 1 kb DNA Ladder used as DNA ladder marker.

Figure 4B: Presence of alternatively-spliced variants in adult mouse tissues

Expression of two mTERT variants identified in mouse cell lines, Ins-i1[1-102] and Del -e12[1-

40], were analysed in mouse tissues. Nested PCR was performed using variant-specific primers

(Figure 2B) in different mouse organs. Here, the NIH 3T3 cell line was used as a positive control.

O'GeneRuler™ 1 kb DNA Ladder used as DNA ladder marker.

Figure 5: Telomerase activity of WT TERT and alternatively-spliced variants reconstituted enzymes

The alternatively-spliced variants were assessed for activity by the Telomeric Repeat

Amplification Protocol (TRAP), an assay based on the ability of telomerase to add telomeric

TTAGGG repeats to a telomerase substrate (TS) primer. The TRAP semi-competitive internal control, TSNT is amplified by primer TS and its own unique reverse primer, NT, which is not a substrate for telomerase. Full-length FLAG-hTERT, FLAG-mTERT, FLAG-mTERT-insi1 and truncated FLAG-mTERT-dele12 were reconstituted in RRL in the presence of L-[35S]methionine with purified hTR was synthesized from FspI-linearized phTR+1 (Autexier et al., 1996). Our results indicate that the mTERT insertion variant can reconstitute telomerase activity while the mTERT deletion variant does not (left). The alternatively-spliced variants are all equally expressed as proteins (bottom left panel). Wildtype hTERT is ~127 kDa, wildtype mTERT is

~125 kDa, Ins-i1[1-102] is ~129 kDa and Del-e12[1-40] is ~125 kDa. Quantification of the data

(right) confirmed that the insertion variant reconstitutes about 40% of the telomerase activity reconstituted by wildtype mTERT and that the deletion variant cannot reconstitute telomerase activity.

Figure 6A: Telomerase Activity of mixed WT and Insertion Variant reconstituted telomerase enzymes

Telomerase activity of mixed in vitro reconstituted wildtype mTERT and the insertion variant was measured using the TRAP assay. Increasing amounts of insertion variant were added while keeping levels of wildtype mTERT constant. The numbers above each lane represent a ratio of

WT mTERT to insertion variant added, with a ratio of 1 representing 300 ng of DNA reconstitute in RRL. Increasing amounts of insertion variant appear to have no inhibitory effects on the constant levels wildtype mTERT activity. Black triangle (bottom, left) represent the increasing concentration of the insertion variant in the overall TRAP reaction. TRAP semi-competitive internal control, TSNT is amplified by primer TS and its own unique reverse primer, NT, which is not a substrate for telomerase.

Figure 6B: Telomerase Activity of mixed WT and Deletion Variant reconstituted telomerase enzymes

Telomerase activity of mixed in vitro reconstituted wildtype mTERT and the deletion variant was measured using the TRAP assay. Increasing amounts of deletion variant were added while keeping levels of wildtype mTERT constant. The numbers above each lane represent a ratio of

WT mTERT to insertion variant added, with a ratio of 1 representing 300 ng of DNA reconstitute in RRL. Increasing concentrations of deletion variant has an inhibitory effects on the constant levels wildtype mTERT activity. Black triangle (bottom, left) represent the increasing concentration of the insertion variant in the overall TRAP reaction. TRAP semi-competitive internal control, TSNT is amplified by primer TS and its own unique reverse primer, NT, which is not a substrate for telomerase.

A -

B -

Figure 7: In vitro DNA Binding Affinities of Alternatively-Spliced mTERT Variants (n=1)

A - L-[35S]methionine- mTERT (Wildtype and alternatively-spliced variants) proteins were synthesized in RRL and were then incubated with bio-(T2AG3)3 or bio-(A2TC3)3. Input protein represents the amount of protein before biotin binding and bound protein represents TERT bound to DNA primer (n=1). B - Quantification of wildtype mTERT, Ins-i1[1-102] and Del-e12[1-40] binding affinities when incubated with bio-(T2AG3)3 (lanes 1-3) or bio-(A2TC3)3 (lanes 4-6). Ins- i1[1-102] variant shows 61.67% of wildtype mTERT binding affinity (lane 2) and Del-e12[1-40] shows 71.3% wildtype mTERT binding affinity (lane 3).

C -

D -

Figure 7 (Continued): In vitro DNA Binding Affinities of Alternatively-Spliced mTERT Variants

(n=2)

C - L-[35S]methionine- mTERT (Wildtype and alternatively-spliced variants) proteins were synthesized in RRL and were then incubated with bio-(T2AG3)3 or bio-(A2TC3)3. Input protein represents the amount of protein before biotin binding and bound proteinrepresents TERT bound to DNA primer (n=2). D - Quantification of wildtype mTERT, Ins-i1[1-102] and Del-e12[1-40] binding affinities when incubated with bio-(T2AG3)3 (lanes 1-3) or bio-(A2TC3)3 (lanes 4-6). Ins- i1[1-102] variant shows 64.30% of wildtype mTERT binding affinity (lane 2) and Del-e12[1-40] shows 76.72% wildtype mTERT binding affinity (lane 3).

A-

B-

Figure 8: In vitro RNA Binding Affinities of Alternatively-Spliced mTERT Variants

A - In vitro-expressed alternatively spliced mTERT variants were assessed for binding to the telomerase RNA. This was done by assessing the co-immunoprecipitation of 32P-UTP labelled hTR with FLAG-tagged 35S-methionine labelled TERT. One of the IPs was performed without hTR to control for non-specific binding. B - Quantification shows that both the insertion and deletion variants of mTERT appear to be binding to the TR albeit with less affinity. The insertion and deletion variant reconstitute about 20.24% and 45.76% of the TR binding reconstituted by wildtype hTERT respectively (n=3).

Figure 9: Screening of clones following CB17 cell transfection

RT-PCR analysis (against epitope FLAG-tagged mTERT) was performed at early passage to confirm presence of exogenous Insi1[1-102] and Del-e12[1-40] plasmids. 2 clones were chosen for each variant: clones 2 and 8 for the insertion variant (lanes 2 and 4) and clones 1 and 9 for the deletion variant (lanes 10 and 11). As negative control, no template was added during the PCR reactions (lanes 1 and 9). we used variant-specific plasmids containing either the insertion or the deletion variant sequences. O'GeneRuler™ 1 kb DNA Ladder used as DNA ladder marker.

A-

B-

Figure 10: Growth Phenotype of CB17 cells transfected with TERT Variants

A - Mouse fibroblasts, CB17, were transfected with pcDNA3.1-hygromycin-mTERT, pcDNA3.1- hygromycin (Empty Vector), pcDNA3.1-hygromycin-mTERT-ins-i1[1-102], and pcDNA3.1- hygromycin-mTERT-del-e12[1-40] and their growth was recorded in culture. Two clones of each were selected with 800ug/ml hygromycin for 3-4 weeks (Empty vector 1, empty vector 2, WT mTERT 1, WT mTERT 2, Insertion 2, Insertion 8, Deletion 1, Deletion 9) and split in a 1:8 ratio at confluency for 200 population doublings. B - Presence of transgenes was confirmed via RT-

PCR analysis (against epitope FLAG-tagged mTERT), performed at early passage (lanes e,h,k and n). Later, RT-PCR was performed again to ensure that transgene levels did not fluctuate during culture at middle and late passages (lanes f,g,I,j,l,m,o and p). RNA levels remained relatively unchanged at all passages. 4 negative controls were used: a – cDNA (-): Reverse

Transcription of mTERT total RNA without any RNA present, b – SS(II)RT (-): Reverse transcription of of mTERT total RNA without any Superscript II enzyme present, c – PCR negative control: no cDNA template present during PCR, d – Non-transfected CB17 cDNA (with no transgene) used as template during PCR. . O'GeneRuler™ 1 kb DNA Ladder used as DNA ladder marker.

Figure 11A: Telomerase Activity of CB17 Cells Transfected with WT mTERT at Early, Middle and Late Passages

Protein lysates from CB17 cells which are stably expressing WT mTERT were assessed for activity at early, middle and late passages by the Telomeric Repeat Amplification Protocol

(TRAP). At all passages, telomerase activity of WT mTERT are indistinguishable from one another. At each passage, different starting amounts of protein, 100 ng, 40 ng, 20 ng, 10 ng, 4 ng and 2 ng, were used to assay for activity. Black triangles (top) represent this increase in dilution of each sample. The TRAP semi-competitive internal control, TSNT is amplified by primer TS and its own unique reverse primer, NT, which is not a substrate for telomerase.

Figure 11B: Telomerase Activity of CB17 Cells stably expressing Del-e12[1-40] Variant Clone 9 at Early, Middle and Late Passages

Protein lysates from CB17 cells which are stably expressing Del-e12[1-40] (clone 9) were assessed for activity at early, middle and late passages by the Telomeric Repeat Amplification

Protocol (TRAP). With increasing passage, there is a visible decrease in telomerase activity. At each passage, different starting amounts of protein, 100 ng, 40 ng, 20 ng, 10 ng, 4 ng and 2 ng, were used to assay for activity. Black triangles (top) represent this increase in dilution of each sample. The TRAP semi-competitive internal control, TSNT is amplified by primer TS and its own unique reverse primer, NT, which is not a substrate for telomerase.

Figure 11C: Quantification of Telomerase Activity of CB17 Cells stably expressing WT mTERT

(Clone 1) and Del-e12[1-40] Variant (Clone 9) at Early, Middle and Late Passages

Protein lysates from CB17 cells which are stably expressing WT mTERT or Del-e12[1-40] (clone

9) were assessed for activity at early, middle and late passages by the Telomeric Repeat

Amplification Protocol (TRAP). Quantification of the data confirmed that, as compared to WT mTERT (black bars), cells transfected with the Deletion variant clone 9 (white bars) show a decrease in telomerase activity at middle and late passages.

Figure 12: Telomerase Activity of early passage CB17 Cells stably expressing WT mTERT or

Alternatively-Spliced Variants

Protein lysates from cells stably expressing either WT mTERT, or alternatively-spliced variants, or transfected with empty vectors were assessed for activity by the Telomeric Repeat

Amplification Protocol (TRAP). At early passage, telomerase activity of insertion and deletion variants are indistinguishable from that of WT mTERT. At each passage, different starting amounts of protein, 100 ng, 40 ng, and 20 ng, were used to assay for activity. Grey triangles (top) represent this increase in dilution of each sample. The TRAP semi-competitive internal control,

TSNT is amplified by primer TS and its own unique reverse primer, NT, which is not a substrate for telomerase.

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Figure 13: Telomere length of CB17 Cells as measured by TRF protocol

Telomere length of CB17 cell transfected with either WT mTERT or Del-e12[1-40] as measured with a Terminal Restriction Fragment (TRF) southern blot. (C3TA2)3 oligonucleotide probe, radioactively end-labeled with γ-32P ATP was hybridized to HinfI/RsaI digested genomic DNA extracted from transfected CB17 cells after gel electrophoresis and transfer to a nylon membrane and visualized on an autoradiographic film.

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4. Discussion

Telomerase, the enzyme which maintains telomere length in 85% of cancer cells, is regulated mainly at the level of transcription, but also partly by alternative splicing of the TERT gene. Alternative splicing is one of the most powerful mechanisms used by the cell to increase protein diversity. In a very ergonomic fashion, alternative splicing works to generate a large number of protein isoforms from a very low number of DNA sequences (Stamm, Ben-Ari et al. 2005). As a result, their encoded proteins have an altogether different structure and this is often coupled to different functions. Alternative splicing of a gene may change various properties of a gene, such as those of interest for the purpose of our report: their binding properties, enzymatic activity and their downstream effects on cell survival. In this study, we sought to characterize alternatively- spliced variants of the mouse telomerase reverse transcriptase (mTERT) RNA and acquire a detailed understanding of their effect on telomere length and cell survival.

That the human telomerase reverse transcriptase (hTERT) gene could be regulated by alternative splicing was first discovered when multiple products were recovered by

RT-PCR using the same set of primers within the hTERT reverse transcriptase domain

(Kilian, Bowtell et al. 1997). Sequencing of these products explained that they came to be through alternative splicing. In a similar fashion, we have detected various isoforms of the mTERT gene, five of which were confirmed by sequencing (Figure 1A, Figure 2A).

Two of the variants, Ins-i1[1-102] and Del-e12[1-40], were more extensively characterized. In human, the analysis of different tissues, cell lines and various tumors showed substantial differences in the hTERT splicing patterns (Kilian, Bowtell et al.

1997). Strikingly, the alternatively-spliced mTERT insertion and deletion variants in our

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study were present in mouse fibroblasts CB17 and NIH 3T3 (embryonic fibroblasts), but absent in mouse mammary carcinoma cell line, FM3A (Figure 3A). As previously stated,

alternatively-spliced variants of the hTERT mRNA, mainly those containing deletions in

exon 7 and 8, were present in melanomas and absent in normal skin samples (Villa, Porta

et al. 2001). Furthermore, it has recently been shown that the β-deletion transcripts were

the most prevalent splicing variants in many lung carcinomas and showed a strong

correlation with telomerase activity in these tissues (Liu, Wu et al. 2012). These

contrasting results may be indicative of variant- or cell-specific functions. While the

findings in human melanoma samples could imply that those variants of hTERT are

involved in the advancement in the cancer, data from our study show that the mouse

TERT variants act to downregulate telomerase expression to potentially inhibit tumor

development.

Furthermore, in human, the splicing pattern of the hTERT mRNA seems to

change with different stages of development (Ulaner, Hu et al. 2001). In human

development, the specific expression of hTERT splice variants that are predicted to

encode catalytically-defective telomerases, correlates with telomere shortening and

suggests that these transcripts may have important physiological roles (Ulaner, Hu et al.

2001). Using variant-specific primers mentioned in the results, we screened seven

different adult tissues, brain, heart, kidney, liver, lungs, ovaries and spleen and found that

ins-i1[1-102] and del-e12[1-40] were not present in the same organs. Of the 7 organs

surveyed, the insertion variant was detected in the brain and kidney while the deletion

variant was detected only in the ovaries (Figure 3B). In human, the pattern of splicing was analyzed not only in adult tissue but also in developing and fetal organs.

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Interestingly, the expression of a 159-bp insertion variant was only found in fetal liver, a hematopoietic organ during fetal development (Nishimura 1983), indicating that alternate hTERT transcripts and telomerase activity could be expressed in blood cell precursors or by developing liver cells. In general, an increase in the presence of the alternatively- spliced variants was seen in fetal tissues rather than in adult ones. Thus, it would be interesting to do an expression profile of the mouse TERT insertion and deletion variants not only in adult organs but also fetal and developing ones. Finally, as was the case with the human study (Ulaner, Hu et al. 2001), it would be interesting to study the telomerase- related characteristics of the mouse tissue cells expressing the alternatively spliced variants (for example, by looking at their telomerase activity, telomere length, etc.) in order to determine whether their presence is of any physiological relevance. In many species, telomerase can add telomeric repeat sequences onto the 3' strand of linear chromosomes by reverse transcribing the RNA template region within its intrinsic telomerase RNA (TR). In doing so, telomerase is able to maintain telomere length in, amongst others, stem cells, germline cells and 85% of cancer cells. The telomerase RNA must recognize the telomeric DNA substrate and bind to it through complementary base pairing, following which the template region of TR is copied in a second step, elongation, resulting in addition of nucleotides onto the telomeric DNA. For the purpose of this study, this was of utmost importance as deviations in the function of the mTERT alternatively-spliced variants seemed to result from defects in the aforementioned steps.

With expression of the ins-i1[1-102] and del-e12[1-40] variants in both RRL as well as in

CB17 cells, the activity of mouse telomerase decreased (Figure 4).

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To begin with, for the insertion variant, the decrease in activity may be due to the amino acids required for binding to either the DNA substrates or the telomerase RNA, disrupted by the introduction of a 102-nucleotide sequence. When it comes to its 3- dimensional structure, interactions between TERT and the DNA substrate are largely facilitated via backbone interactions with the thumb loop and helix (Mitchell, Gillis et al.

2010), structures which fall in the N-terminus region. Previous work in Tribolium

Castaneum have demonstrated that the thumb helix lies in the minor groove of the RNA-

DNA duplex, allowing for contact with the phosphodiester backbone and the ribose groups of the RNA-DNA hybrid (Gillis et al. 2008). The above interactions have not been extensively studied in human or mouse TERT but one can make several postulations based on an analogous helix (helix H) in retroviral reverse transcriptases (Kohlstaedt,

Wang et al. 1992; Jacobo-Molina, Ding et al. 1993). Interactions between the DNA and the thumb loop, a conserved structure in the thumb domain, include the side chains of

Lysine-416 and Asparagine-423. Previously, these have been shown to be of importance for hydrogen bonding to the DNA backbone.

More recently, it has been shown that other regions are also essential for DNA binding. Together, these sites provide greater specificity and higher affinity for DNA binding than does the N-terminal portion alone (Bryan, Sperger et al. 1998). The 4

domains reported to be important for mediating this process are the TEN, the TRBD, RT

and C-terminal domains. In this study, the N-terminal TEN domain, which is the

conventional domain implicated in DNA binding, showed only low affinity binding.

However, there appears to be cross-talk between the TEN and the other domains, leading

to more efficient DNA binding with greater affinity. A few studies have merely suggested

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the idea, but no concrete links had been made. For example, deletion of the C-terminal

extension in yeast TERT led to a slight (~2-fold) decrease in primer-binding affinity

(Hossain et al. 2002). The novel finding in Tetrahymena that the C-terminal domain could be involved in telomeric substrate binding could account for the decrease in in vitro

DNA binding of the del-e12[1-40]. Deletion of an essential 233 amino acid sequence in the C-terminus which is unique to TERT increased TERT-DNA dissociation by about 4- fold (Bryan et al. 2008), indicating that the RT motifs in the C-terminus may play an important role in the primer binding. Furthermore, a TERT mutant which lacked almost its entire C-terminus was shown to be completely deficient in telomerase activity, with only a modest decrease in primer binding affinity. Our study has yielded similar results, with the deletion variant missing part of the C-terminal extension lacking telomerase activity in vitro, while still being able to bind telomeric DNA substrates with 65.2% of wildtype TERT binding (Figure 6). Thus the C-terminus of TERT may be important for interactions with the DNA substrate in order to fulfill its essential role in telomerase activity, and this is consistent across many different species. However, it is difficult to make exact conclusions on the link between the alternative splice sites of the insertion and deletion variants and their link to DNA binding because the domain in the mouse

TERT are yet to be characterized. Studies have been performed in order to understand

TERT protein function by creating chimeric mouse-human TERT proteins (Middleman,

Choi et al. 2006; Fakhoury, Marie-Egyptienne et al. 2010). Analysis of these chimeric

TERT proteins showed that sequences in the human CTE are crucial for telomere length

maintenance in human fibroblasts (Middleman, Choi et al. 2006). In fact, the replacement of human carboxy-terminal sequences in the place of the mouse one is enough to promote

107 immortalization and maintain telomere length. Furthermore, recent work shows that species-specific determinants of activity, processivity and telomere function map not only to the TR but also to the TERT component (Fakhoury, Marie-Egyptienne et al. 2010).

Further work needs to be done in order to clarify the different regions of the mouse TERT gene and to define the domains important in aspects such as DNA binding and enzymatic activity. Until then, our assumptions will be made based on the domains defined in other species.

Next, another determinant of telomerase activity, binding to the telomerase RNA

(TR), has also been mapped the N-terminus of TERT. In vitro, the insertion variant was able to bind human TR with merely 20.20% of wildtype TERT binding affinity (Figure

7). Previously, it has been found that, unlike the numerous DNA binding domains, high- affinity telomerase RNA binding requires only a small region in the N-terminus of TERT

(Lai, Mitchell et al. 2001). Furthermore, the small region necessary and for telomerase

RNA binding is self-sufficient and completely separable from the reverse transcriptase motifs. This work was first done in Tetrahymena and later confirmed in human. hTERT contains a similar region, similarly sized to that of Tetrahymena, within the TERT N- terminus which is essential for human telomerase RNA binding as well. This region falls in the N-terminus, preceding the reverse transcriptase active site. Interestingly, based on previous work, it can be predicted that the catalytically inactive hTERT isoform, the alpha deletion derived from mRNA alternative splicing, would still contain a completely functional RNA binding domain (Kilian, Bowtell et al. 1997; Ulaner, Hu et al. 1998). It is intriguing that a link has been proposed between the RNA binding domain and its involvement in the negative dominance of an alternatively-spliced TERT variant

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(suggested through the sequestration of the hTR from interaction will full-length hTERT)

(Lai, Mitchell et al. 2001). Though we have not tested for negative dominance with the insertion variant, a similar mechanism could explain the dominant negative effect of the deletion variant, Del-e12[1-40]. One limitation to this experiment as well as to our activity assessments was the use of human telomerase RNA instead of mouse telomerase

RNA. The above assays have been optimized using the human TR because levels of in vitro reconstitution using mTERT and mTR are not optimal. However, previous work in our laboratory has shown that mixed TERT-TR arrangements yield data which can be analyzed quantitatively and amongst these was the mTERT-hTR combination (Fakhoury,

Marie-Egyptienne et al. 2010).

A combination of the above RNA and DNA binding defects could account for the decreased telomerase activity observed with the N-terminal insertion variant. With the C- terminal deletion variant, however, a more plausible explanation is that this isoform yields a truncated protein missing essential RT residues. In vitro, while the mTERT insertion variant can reconstitute 40% of wildtype telomerase activity, the deletion variant is completely inactive. Consistently, when transfected into telomerase-positive mouse fibroblasts, by middle passage, the deletion variant appeared to have a noticeable growth defect with a decrease in telomerase activity (Figure 8A). To ensure that this deviation in growth was not due to fluctuating levels of transgene levels during culture,

RT-PCR analysis (against epitope FLAG-tagged mTERT) was performed at several passages. RNA levels remained relatively unchanged at all passages (Figure 8B). A limitation to consider here is that the presence of the exogenous TERT variants could only be confirmed at the level of RNA. In the future, it would be informative to also

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determine the levels of protein expression by Western analysis. Similar to many other

viral reverse transcriptases, the TERT RT domain contains seven conserved motifs

required for telomerase enzymatic activity in vitro (Counter, Hahn et al. 1998). The C- terminal extension of TERT is important for enzymatic activity, nucleotide repeat addition processivity and cellular immortalization. A portion of the C-terminus is also required for protein multimerization (Beattie, Zhou et al. 2001). Any of the above could justify the growth defect in activity of the truncated deletion variant protein. To begin with, by itself in RRL, Del-e12[1-40] yields a catalytically inactive protein unable to elongate telomeres (as shown by the in vitro TRAP assay, Figure 4). Furthermore, in vitro, in the presence of wildtype mTERT, the deletion variant inhibits telomerase activity while the insertion variant has little effect (Figure 5A,B). In cells, the deletion variant shows a slight inhibition of the telomerase activity (Figure 10). Since addition of

nucleotides to telomeric ends is determined by the activity of telomerase, telomere length

in the CB17 cells expressing Del-e12[1-40] clone 9 should be verified. Furthermore, it

has recently been shown that telomere length may be directly correlated

to hTERT expression and splice variant patterns (Wang, Meeker et al. 2011). We have

begun an investigation of telomere length measurement, first with a Telomere Restriction

Fragment (TRF) assay. Though there is no apparent difference in bulk telomere length

between WT and deletion-transfected CB17 cells (Figure 11), this needs to be confirmed

at the level of the single chromosome through quantitative Fluorescent in-situ

Hybridization (qFISH). With this, if we see a difference in length, namely a decrease in

the length of telomeres in the cells transfected with the deletion variant, we can confirm

that their growth impediment is due to a lack of telomerase activity.

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In these CB17 cells, only a partial inhibition of telomerase activity was observed

by the introduction of the deletion variant. Total telomerase activity was not abolished,

most likely due to the presence of endogenous mTERT in the telomerase-positive mouse

fibroblasts. It would thus be interesting to express the insertion and the deletion variants

in mTERT -/- cells and determine whether these variants can prevent the phenotypes of late passage mTERT-/- cells, including extremely short telomeres and chromosome fusions (Fakhoury, Marie-Egyptienne et al. 2010). In the telomerase-positive mouse

fibroblasts, TRAP assays were performed for insertion variant clones only at early

passage to confirm presence of the transgene. Telomerase activity was not verified at

later passages because insertion-expressing cells did not display a growth disadvantage.

These cells grew at a normal exponential rate for the entire 200 population doublings;

thus, since cell growth was intact, it was assumed that telomerase activity, as well, was

intact. However, it would be important to also verify if endogenous telomerase might

have been upregulated, as we have previously observed telomerase upregulation upon

expression of a dominant negative mTERT (Marie-Egyptienne, Brault et al. 2008). Such

an upregulation could have occurred if the expression of the insertion variant was

detrimental to the cell. Returning to the C-terminal deletion, it cannot be ignored that the

growth defect may also be due to a defect in dimerization or due to a decreased repeat

addition processivity (RAP). In humans, both dimerization and RAP have been mapped

to the RT domain in the TERT protein carboxy terminal (Beattie, Zhou et al.

2001; Moriarty, Marie-Egyptienne et al. 2004). Further work needs to be done in order to

uncover a complete mechanism.

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Functionally, the roles of alternatively-spliced TERT variants across vertebrate

species remain unknown. This is also the case with many of the variants discovered in

this study. Previously, there have been no reports of these spliced variants being translated, likely due to a lack of good antibodies or the low levels of these isoforms.

Because they exist as such low abundance, the transcripts of these variants had to be isolated via Nested PCR. Nested PCR is a modification of convention PCR intended to better amplify low-abundance transcripts while reducing the contamination in products due to the amplification of unexpected primer binding sites (Snounou, Viriyakosol et al.

1993). In two consecutive runs of PCR, nested PCR involves two sets of primers, the second set used to amplify a more specific target within the first PCR product. While this is indicative of their existence at the level of RNA, proof that the proteins are translated would be imperative to identifying functional roles for these isoforms. Two deletion variants, Del-e2[289-797] and Del-e2[1132-1324], introduce premature stop codons

(Figure 2A), resulting in what would be very short amino acid sequences. We predict that

the resulting proteins, if translated, are inactive because they lack the reverse

transcriptase domain. However, whether these proteins are too short to be of any

physiological importance or whether they fulfil some other function is yet to be

determined. The deletions in exon 2 are intriguing because similar deletions in the mouse

TERT have been reported previously (though this work is yet to be published). The

alternatively-spliced variants in accordance with our findings are a deletion

corresponding to Del-e2[289-797] and a combination of Del-e2[289-797] and Del- e2[1132-1324] (a double deletion in the same exon) (Strausberg et al. 2002, unpublished work).

112

In humans, the alpha-deletion, which is missing 36 nucleotides at the 5’ end of exon 6, inhibits telomerase activities in when overexpressed in telomerase-positive cells in a dominant-negative manner (Shay and Wright 2000). We have unveiled a similar mechanism both in vitro as well as in cells with a C-terminally located deletion variant,

Del-e12[1-40]. In vitro TRAP assays of mixed in vitro reconstituted wildtype mTERT and the deletion variants at the showed that the deletion variant seems to be inhibiting telomerase activity and may have dominant negative effects on telomerase activity

(Figure 5B), consistent with a phenotype also observed in CB17 cells (Figure 10B). Our in vitro results indicate that this is a dose-dependent effect and our work in cells show that this is a passage-dependent effect. If this is the case, it could be used as part of a strategy to target similar sites on the hTERT gene as a novel approach to inhibit telomerase in tumour cells.

The targeting of pre-mRNA splice sites reduce full length hTERT mRNA levels, increases alternatively-spliced hTERT transcript levels, resulting in telomerase inhibition and cell growth inhibition or apoptosis (Brambilla, Folini et al. 2004) suggesting a potential for modulating hTERT alternative splicing in anticancer therapy. It would be interesting, thus, to see the effects of targeting pre-mRNA mTERT splice sites using modified antisense oligonucleotides to reduce full-length mTERT mRNA levels and increase alternatively-spliced mTERT transcript levels in telomerase-positive CB17 cells.

By quantifying transcript levels by RT-qPCR, verifying telomerase inhibition with TRAP assays and assessing effects on cell growth and proliferation after antisense oligonucleotide treatment, we can gain insight on new ways to prevent telomerase- dependent tumor progression.

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Interestingly, we detected another deletion variant completely analogous to the

human alpha deletion variant, Del-e6[1-36] (Figure 2A). Often, sequence similarities

across species act as evidence for functional conservation and show an evolutionary relationship between the sequences. If this is the case, we can assume that the dominant negative function of the alpha variant must be an important for telomerase regulation.

Among the most highly conserved sequences in biological databases today are active binding sites of enzymes. In humans, not only has it been shown that the alpha-deletion can inhibit telomerase activity in human fibroblasts (Shay and Wright 2000), but it has been

extensively studied in other aspects and has since been very well-characterized. Since this variant was not a novel finding, no further characterization was done. It would be

interesting, however, to see the phenotypes of mouse telomerase-positive fibroblasts

should they be transfected with Del-e6[1-36]. Would it inhibit mouse TERT in a similar

dominant negative manner or is this a species-specific effect? Could these effects be seen

in in vitro competition assays? Also, since a link was recently made between the RNA

binding domain of hTERT and its involvement in the negative dominance of an alternatively-spliced TERT variant, it would be interesting to perform a binding assay with a protein containing this deletion and assay its affinity for telomerase RNA.

Much of the work presented above has never been studied before and are being considered for the first time. Our novel results show that alternatively-spliced variants do exist in the mTERT genome and that their presence is important for telomerase regulation. The sequence conservation of some of the mTERT variants detected is

indicative of their biological importance. Some were reported in other species (Del-e6[1-

36]), while others have been observed in mice but never published (Del-e2[289-797] and

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Del-e2[1132-1324]). By acquiring a detailed understanding of the mTERT alternative splicing, we have advanced the knowledge required in this field to improve clinical outcomes for the inhibition of telomerase in tumor cells.

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5. Conclusion

Telomerase is the enzyme responsible for maintaining telomeres and it does so by adding the hexameric TTAGGG sequences to the end of existing chromosomes. The core components of telomerase are a reverse transcriptase (RT) (hTERT in human) and a telomerase RNA component (TR) which serves as a template for the synthesis of telomeric sequences (tandem d(TTAGGG)n repeats in human and mouse). Telomerase is regulated mainly at the level of transcription but also, to a lesser extent, though alternative splicing of the TERT gene. We have identified various novel mTERT variants, five of which were confirmed by sequencing. Two of these variants, an insertion variant in the N-terminus Ins-i1[1-102] and a C-terminally located deletion Del-e12[1-

40], exist in different murine cells as well as in specific adult mouse tissues, an aspect which may be indicative of their physiological importance. Further characterization showed that, in vitro, while the insertion variant only has reduced telomerase activity, the

C-terminal mTERT deletion variant cannot reconstitute activity at all and appears to be behaving in a dominant-negative manner. Moreover, both the insertion and deletion variants have decreased binding affinities for both telomeric DNA and telomerase RNA substrates. In addition to having reduced activity, telomerase-positive mouse fibroblasts which contain the mTERT deletion variant-expressing, at middle passage, grow slower than those expressing wild-type mTERT.

In the future, it would be interesting to do an expression profile of the mouse

TERT insertion and deletion variants not only in adult organs but also fetal and developing ones. Also, as previously done by (Ulaner, Hu et al. 2001), we want to study the telomerase-related characteristics of the mouse tissue cells expressing the

116 alternatively spliced variants (for example, by looking at their telomerase activity, telomere length, etc.) in order to determine their physiological relevance. Next, because there is no apparent difference in bulk telomere length between wildtype and deletion- transfected CB17 cells (Figure 11), it would be interesting to study this at the level of the single chromosome through quantitative Fluorescent in-situ Hybridization (qFISH). With this, we can confirm that the growth defect of the deletion variant is due to a lack of telomerase activity. Along the same lines, expressing the insertion and the deletion variants in mTERT -/- cells would expose their ability (or inability) to prevent the phenotypes of late passage mTERT-/- cells, including extremely short telomeres and chromosome fusions (Fakhoury, Marie-Egyptienne et al. 2010). Finally, it would be interesting to uncover the effects of targeting pre-mRNA mTERT splice sites using modified antisense oligonucleotides to reduce full-length mTERT mRNA levels and increase alternatively-spliced mTERT transcript levels in telomerase-positive CB17 cells.

The above studies have helped get insight on the function of alternatively spliced mTERT mRNAs in mouse telomere function and cell survival as well as telomerase regulation as a whole.

117

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Table of Abbreviations

Abbreviation Term

ALT Alternative lengthening of telomeres ASPS alternatively‐spliced site ATM Ataxia telangiectasia mutated ATR Ataxia‐telangiectasia and Rad3‐related Bio- Biotinylated‐ C-DAT C‐Terminal domain cDNA Complementary DNA CTE C‐terminal extension D-loop Displacement loop FRAP Flourescence Recovery After Photobleaching HIV Human immunodeficiency virus HPLC High‐performance liquid chromatography hTERT human telomerase reverse transcription hTR Human Telomerase RNA IP Immunoprecipitation mRNA Messenger RNA mTERT Mouse telomerase reverse transcription mTR Mouse Telomerase RNA N-DAT N‐Terminal domain ORF Open reading frame p53 Protein 53 kilodaltons PCR Polymerase chain reaction PD Population doubling POT1 Protection of telomeres protein 1 Q-FISH Quantitative Fluorescent in‐situ Hybridization RAP1 Repressor activator protein 1 Rb Retinoblastoma rDNA Recombinant DNA RID1 RNA interacting domain 1 RID2 RNA interacting domain 2 RRL Rabbit reticulocyte lysate RT Reverse transcriptase RT-PCR Reverse‐transcription polymerase chain reaction SS(II)RT Superscript II Reverse Transcriptase T-loop Telomere loop T-motif Telomerase‐specific motif TEN (domain) Telomerase essential N‐terminal (domain)

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Abbreviation Term

TERC Telomerase RNA TERT human telomerase reverse transcription TIN2 TRF1‐interacting protein 2 TPP1 Tin1/PTOP/PIP1 TR Telomerase RNA TRAP Telomere Repeat Amplification Protocol TRBD Telomerase RNA binding domain TRF Telomere Restriction Fragment TRF1 Telomeric repeat‐binding factor 1 TRF2 Telomeric repeat‐binding factor 2 WT Wildtype

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