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A Three-State Model for the Regulation of Telomerase by TERRA and Hnrnpa1 Sophie Redon, Ivo Zemp and Joachim Lingner*

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A Three-State Model for the Regulation of Telomerase by TERRA and Hnrnpa1 Sophie Redon, Ivo Zemp and Joachim Lingner* Published online 8 August 2013 Nucleic Acids Research, 2013, Vol. 41, No. 19 9117–9128 doi:10.1093/nar/gkt695 A three-state model for the regulation of telomerase by TERRA and hnRNPA1 Sophie Redon, Ivo Zemp and Joachim Lingner* Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Frontiers in Genetics National Center of Competence in Research, Ecole Polytechnique Fe´ de´ rale de Lausanne (EPFL), 1015 Lausanne, Switzerland Received May 24, 2013; Revised July 15, 2013; Accepted July 16, 2013 Downloaded from ABSTRACT processing. Short telomeres induce cellular senescence. The telomerase enzyme can solve the end replication Telomeres, the physical ends of eukaryotic chromo- problem, re-extending telomere 30 ends by reverse somes, are transcribed into telomeric repeat- transcribing the template region of its tightly associated http://nar.oxfordjournals.org/ containing RNA (TERRA), a large non-coding RNA, RNA moiety into telomeric repeats (2–4). The regulation which forms an integral part of telomeric hetero- of telomerase at chromosome ends is not well understood chromatin. In vitro, naked TERRA molecules are ef- and is the subject of intensive investigations in several ficient inhibitors of human telomerase, base-pairing laboratories (5,6). via their 50-UUAGGG-30 repeats with the template Telomeres are transcribed in most or all eukaryotes into sequence of telomerase RNA, in addition to contact- the long non-coding RNA TERRA (7). TERRA transcrip- tion starts in the subtelomeric region and proceeds toward ing the telomerase reverse transcriptase protein 0 subunit. In vivo, however, TERRA-mediated inhib- chromosome ends. Thus, TERRA contains near its 3 end at Bibliotheque Commune De ChimieUNIL - EPFL on April 18, 2016 telomeric repeats in the form of RNA. The UUAGGG- ition of telomerase can be prevented by unknown repeat containing RNA molecules act as potent mixed- mechanisms. Also, heterogeneous nuclear ribo- type inhibitors of human telomerase in vitro (8,9). nucleoprotein A1 (hnRNPA1) has been implicated However, it is unclear under which circumstances in telomere length control. In vivo, TERRA is partially TERRA may regulate telomerase in vivo. Supporting a associated with hnRNPA1, and hnRNPA1 is also role of TERRA in telomerase control, it was found that detected at telomeres. We demonstrate that on in human cells TERRA is displaced or degraded at telo- binding of TERRA, hnRNPA1 can alleviate the meres by factors involved in nonsense mediated RNA TERRA-mediated inhibition of telomerase. decay (NMD). Although more abundant in the cytoplasm, However, when in excess over TERRA, hnRNPA1 NMD factors are detected at low levels in the nucleus, becomes itself an inhibitor of telomere extension, physically interacting with the telomeric chromatin (10) on binding of the telomeric DNA substrate. Yet, and also with telomerase (11–14). On the other hand, elevated TERRA transcription at human telomeres did hnRNPA1 has no notable direct effects on the tel- not prevent telomerase-mediated telomere extension (15). omerase catalysis. Our in vitro results suggest that In Saccharomyces cerevisiae, induction of TERRA leads to TERRA-mediated telomerase inhibition may be pre- telomere shortening. However, this involves activation of vented by hnRNPA1 in vivo. Telomere extension by the chromosome end trimming exonuclease 1 at chromo- telomerase may require balanced levels of TERRA some ends rather than inhibition of telomerase (16). and hnRNPA1 at telomeres. Thus, TERRA and HnRNPA1 is a multifunctional RNA-binding protein hnRNPA1 can function as a bimolecular regulator involved in the regulation of RNA biogenesis. Interes- to turn telomerase and the telomere on and off. tingly, hnRNPA1 is also involved in telomere mainten- ance (17). Mouse cells deficient for hnRNPA1 have short telomeres, and this phenotype was rescued upon ex- INTRODUCTION pression of hnRNPA1 from a cDNA. HnRNPA1 binds Telomeres protect chromosome ends from DNA repair single-stranded human d(TTAGGG)n telomeric DNA activities that reseal chromosome internal DNA breaks repeats in vitro (18,19) as well as r(UUAGGG)n telomeric that occur during DNA damage (1). Telomeric DNA RNA repeats that are contained within TERRA (8,19,20). shortens with every round of semiconservative DNA rep- HnRNPA1 (21,22) or fragments thereof (17) also associ- lication due to the end replication problem and nucleolytic ate with telomerase activity. Finally, it has been proposed *To whom correspondence should be addressed. Tel: +41 21 693 0721; Fax: +41 21 693 0720; Email: joachim.lingner@epfl.ch ß The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 9118 Nucleic Acids Research, 2013, Vol. 41, No. 19 that a switch at telomeres from replication protein A (RP- hnRNPA1 depletion A) for DNA replication in S phase to protection of The 293T cells were transfected for 48 h with siRNAs telomeres 1 (POT1)/TPP1 after S phase is triggered by against GFP or hnRNPA1 (sequences in Supplementary hnRNPA1 and TERRA (23). In vitro, hnRNPA1 can Table S1) using Interferin (Polyplus). compete with RP-A for the binding of the single- stranded G-strand (23), and an increase in TERRA abun- dance after S phase may sequester hnRNPA1 from Protein expression and purification telomeric DNA. Binding of hnRNPA1 to TERRA may Rosetta pLysS competent bacteria (Novagen) were trans- in turn free the telomeric 30 overhang for binding by formed with pGEX-hnRNPA1. For expression of recom- POT1-TPP1. Proteins with sequence homology to binant GST-hnRNPA1, 2 l of 2YT containing 34 mg/ml hnRNPA1 and roles at telomeres have also been chloramphenicol, 50 mg/ml ampicillin and 50 mg/ml car- reported in S. cerevisiae, but the involved mechanisms benicillin were inoculated with 50 ml of overnight culture Downloaded from are not known (24). and incubated at 37 C until the OD600 reached 0.8. Here, we show that TERRA and hnRNPA1 cooperate Protein expression was induced for 3 h at 37Cby in telomerase control in vitro. The telomerase-inhibitory adding 0.4 mM IPTG. After centrifugation, the bacteria effects of TERRA are alleviated on hnRNPA1 association were washed once with ice-cold 1x PBS. The pellet was with TERRA. On the other hand, we find that excess of resuspended in 60 ml lysis buffer (1x PBS, 1 mM DTT and hnRNPA1 also inhibits telomerase through binding and 1x protease inhibitor cocktail EDTA-free from Roche). http://nar.oxfordjournals.org/ sequestration of the DNA primer, although hnRNPA1 The lysate was sonicated five times for 10 s (Branson has no notable direct effects on the telomerase catalytic sonifier 250, setting 3, constant). Glycerol and Triton X- activity. Our data demonstrate that in vitro telomerase 100 were added to final concentrations of 10 (v/v) and 1% activity is maintained as long as the levels of TERRA (v/v), respectively. The extract was incubated on a rotating and hnRNPA1 are balanced. Thus, TERRA and wheel for 15 min at room temperature and then hnRNPA1 may provide a bimolecular switch to fine- centrifuged for 15 min at 12 000 g at 4C. The supernatant tune telomerase activity at chromosome ends. was incubated with 2 ml 50% slurry of GSH-coupled beads (GE Healthcare). After binding for 2 h at 4C, bead-bound proteins were washed once with 1x PBS, at Bibliotheque Commune De ChimieUNIL - EPFL on April 18, 2016 MATERIALS AND METHODS 10% (v/v) glycerol and three times with 1xPBS. GST- Plasmids and oligonucleotides hnRNPA1 was eluted for 30 min at 4 C with 2 ml 20 mM NaCl, 20 mM GSH and 200 mM Tris–HCl (pH 9.5). Plasmids for transient transfection of human telomerase Glycerol was added to a final concentration of 10% reverse transcriptase (hTERT) (pcDNA6-ZZ-3xFlag- (v/v). To remove the remaining beads, the eluate was hTERT) and hTR (pBS-U1-hTR) were described previ- passed through a 35 mM filter (Mobitec). The same ously (25). For bacterial expression of hnRNPA1, protocol was applied to express and purify GST alone pGEX-hnRNPA1 was generated by PCR amplification using pGEX-6P-1 vector with the only exception that of the hnRNPA1 open reading frame from pcDNA6- the elution steps were performed with 20 mM GSH and hnRNPA1 using BamH1-A1_F and A1-EcorRI_R 200 mM Tris–HCl (pH 7.5). The second purification step primers (Supplementary Table S1) and subcloned into was performed on a HiTrapQ HP 1 ml column using the the BamHI and EcoRI sites of pGEX-6P-1 vector (GE Akta purifier system and the Unicorn software (GE Healthcare). pcDNA6-hnRNPA1 was generated by PCR Healthcare). Before loading, each eluate from the first amplification of the hnRNPA1 open reading frame from purification step was diluted 10x in 20 mM Tris–HCl pCMV6-XL5-hnRNPA1 (Origene, clone NM_002136) (pH 8.0) and 25 mM NaCl. A 20 ml NaCl gradient was using HindIII-A1_F and EcoRI-A1_R primers. cDNAs applied from 25 mM to 1 M. 500 ml fractions were col- were subcloned into retroviral-based pCL vectors for ex- lected, glycerol was added to 10% (v/v) final concentra- pression of ZZ or hnRNPA1-ZZ proteins in HT1080 cells. tion and samples were quick-frozen. Oligonucleotides were purchased from Microsynth and are listed in Supplementary Table S1. Telomerase purification Antibodies For telomerase overexpression, Human Embyronic Kidney (HEK) 293E cells were grown in suspension Antibodies against Flag (F1804) and tubulin (T9026, for culture and transiently transfected with pcDNA6-ZZ- western blotting) were obtained from Sigma. The 9B11 3xFLAG-hTERT and pBS-U1-hTR at the Protein antibody against Myc was purchased from Cell Signaling.
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