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Single-Molecule Analysis of the Human Telomerase RNA Center Dot Dyskerin Interaction and the Effect of Dyskeratosis Congenita Mu

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Single-Molecule Analysis of the Human Telomerase RNA Center Dot Dyskerin Interaction and the Effect of Dyskeratosis Congenita Mu Single-Molecule Analysis of the Human Telomerase RNA center dot Dyskerin Interaction and the Effect of Dyskeratosis Congenita Mutations Ashbridge, B; Orte, A; Yeoman, JA; Kirwan, M; Vulliamy, T; Dokal, I; Klenerman, D; Balasubramanian, S © 2009 American Chemical Society http://pubs.acs.org/page/policy/authorchoice_termsofuse.html For additional information about this publication click this link. http://qmro.qmul.ac.uk/xmlui/handle/123456789/14851 Information about this research object was correct at the time of download; we occasionally make corrections to records, please therefore check the published record when citing. For more information contact [email protected] 10858 Biochemistry 2009, 48, 10858–10865 DOI: 10.1021/bi901373e Single-Molecule Analysis of the Human Telomerase RNA 3 Dyskerin Interaction and the Effect of Dyskeratosis Congenita Mutations† Beth Ashbridge,‡,^ Angel Orte,‡,^,@ Justin A. Yeoman,‡,^,# Michael Kirwan, ) Tom Vulliamy, ) Inderjeet Dokal, ) David Klenerman,*,‡,r and Shankar Balasubramanian*,‡,§,r ‡University Chemical Laboratories, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K., §School of Clinical Medicine, University of Cambridge, Cambridge CB2 0SP, U.K., and Centre) for Paediatrics, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, London E1 2AT, U.K. ^These authors contributed equally to this work. @Present address: Department of Physical Chemistry, Faculty of Pharmacy, Campus Cartuja, Granada 18071, Spain. #Present address: National Centre for Biological Sciences, Tata Institute of Fundamental Research, UAS GKVK, Bellary Road, Bangalore 560 065, India. rJoint senior authorship. Received August 7, 2009; Revised Manuscript Received October 1, 2009 ABSTRACT: It has been proposed that human telomerase RNA (hTR) interacts with dyskerin, prior to assembly of the telomerase holoenzyme. The direct interaction of dyskerin and hTR has not been demonstrated and is an experimentally challenging research problem because of difficulties in expressing and purifying dyskerin in quantities that are useful for biophysical analysis. By orthogonally labeling dyskerin and hTR, we have been able to employ single-molecule two-color coincidence detection (TCCD) to observe directly the formation of a dyskerin 3 hTR complex. By systematic deletion of hTR subdomains, we have gained insights into the RNA sites required for interaction with dyskerin. We then investigated mutated forms of hTR and dyskerin that are associated with dyskeratosis congenita (DC), on the basis of clinical genetics studies, for their effects on the dyskerin 3 hTR interaction. Dyskerin mutations associated with X-linked DC resulted in significant impairment of the dyskerin 3 hTR interaction, whereas mutations in hTR associated with autosomal dominant (AD) DC did not affect the interaction. We propose that disruption of the dyskerin 3 hTR interaction may contribute to X-linked DC. Dyskerin is a putative pseudouridine synthase that is expressed telomerase complex, but there has been no explicit physical constitutively and is required for correct modification of riboso- evidence of a direct interaction between hTR and dyskerin. mal and small nuclear RNA precursors (1). A complex capable of Dyskeratosis congenita (8, 9) is thought to be the first primary pseudouridylation is formed by dyskerin and three other pro- telomere maintenance disorder to be identified in humans. It is a teins, NOP10, NHP2, and GAR1, in association with a specific premature aging syndrome that can lead to a triad of muco- guide RNA containing the box H/ACA sequence motif (2, 3). cutaneous features, namely, abnormal skin pigmentation, nail The presence of this motif in the 30 half of the RNA component of dystrophy, and mucosal leukoplakia (10). DC adversely affects human telomerase (hTR)1 has been proposed to provide a highly proliferative tissues, with bone marrow failure being the discrete binding site for dyskerin within the telomerase com- major cause of death (11). Cells from patients with this disease plex (2, 4). hTR provides the template for telomere synthesis by generally display short telomeres, and the lack of efficient telomere human telomerase reverse transcriptase (hTERT) and acts as a maintenance has been attributed to reduced levels of active protein-binding scaffold for telomerase holoenzyme assembly (5). telomerase enzyme. DC is a genetically diverse condition and Recently, the protein dyskerin has been identified as a component arises from three possible inheritance patterns. The most common of active human telomerase purified from cells (6, 7)inaddition is the X-linked form, which is associated with mutations in the to hTERT and hTR. It has been hypothesized that dyskerin is DKC1 gene that encodes dyskerin (12, 13). Patients with linked in a spatiotemporal manner to the biogenesis of the pre- X-linked DC, mainly young males, go on to develop bone marrow telomerase RNP (7) and/or the stabilization of hTR within the failure before the age of 30 (14) and have an increased risk of cancer, which is thought to arise because shortened telomeres †This work was supported by the BBSRC, the Wellcome Trust (Grant promote genomic instability. More recently, an autosomal domi- 069399/Z/02), and the MRC (Grant G0400534). A.O. thanks the Marie nant form of the disease has been characterized, in which patients Curie IEF of the sixth EU Framework for financial support. *To whom correspondence should be addressed. (S. B.) Telephone: þ44 generally display milder symptoms that present later in life. (0) 1223 336347. Fax: þ44 (0) 1223 336913. E-mail: [email protected]. A major subset of cases, demonstrating a clinically heterogeneous (D.K.) Telephone: +44 (0) 1223 336481. E-mail: [email protected]. 1Abbreviations: DC, dyskeratosis congenita; AD DC, autosomal phenotype, are associated with mutations in hTR (15). dominant dyskeratosis congenita; hTR, human telomerase RNA; Physical analysis of the components of the telomerase complex hTERT, human telomerase reverse transcriptase; TCCD, two-color has been hampered by technical challenges because of low natural - coincidence detection; YFP, yellow fluorescent protein; 647N RNA, abundance, poor expression, and inefficient in vitro assembly RNA molecule conjugated to the maleimide derivative of ATTO-TEC 647N fluorescent dye; PUA, pseudouridine and archaeosine trans- of the recombinant enzyme. In our hands, human dyskerin glycosylase; PCR, polymerase chain reaction; RNP, ribonucleoprotein. has proven to be similarly difficult to express at high levels pubs.acs.org/Biochemistry Published on Web 10/17/2009 r 2009 American Chemical Society Article Biochemistry, Vol. 48, No. 46, 2009 10859 (greater than picomoles) in vitro, which precludes the use of most shRNA SHCLNG-NM_001363, TRCN0000039738 (Sigma-Al- classical biophysical methods that require at least 1000-fold greater drich, Gillingham, U.K.), using Lipofectamine LTX (Invitrogen) sample quantities for effective analysis. Our single-molecule according to the manufacturer’s protocol. A knockdown negative fluorescence approach two-color coincidence detection (TCCD) control consisted of transfection of 2 mg of pIRES2-EGFP alone. can overcome such limitations as it requires a minimal quantity Cells were cultured at 37 °C for 72 h before being harvested by (less than femtomoles) of labeled com- trypsinization. Successfully transfected, GFP-positive cells were ponents. Furthermore, it can be used to characterize a selected using a BD FACSAria cell sorter (BD Biosciences) and partially purified complex of orthogonally labeled species in the separated into two aliquots for further analysis. presence of a high background of labeled, unassociated Telomerase enzyme activity was measured using the telomer- molecules (16-18). The advantage of TCCD compared to other ase repeat amplification protocol (TRAP) assay (19) using the single-molecule techniques, such as FRET, is that the fluorophore TRAPeze RT Telomerase Detection Kit (Millipore, Watford, label can be placed at any convenient position on the molecule, U.K.) according to the manufacturer’s protocol. Briefly, lysates and that no prior knowledge of the complex structure is required. of 3,000 cells were incubated at 30 °C for 30 min in the presence of Furthermore, the cross-talk between channels is weak, making the a substrate oligonucleotide for the addition of telomeric repeats. method significantly more sensitive to low concentrations of The products of this incubation were amplified by real-time associated molecules. quantitative PCR, and the fluorescein-labeled products were Herein, we describe the use of single-molecule TCCD to detect detected on an ABI 7500 Real-Time PCR System (Applied and study the dyskerin 3 hTR complex. In particular, we evaluated Biosystems, Warrington, U.K.). the dependence of this protein 3 RNA interaction on subdo- To determine cellular hTR levels, RNA was prepared from mains of hTR and on mutations associated with the rare 50,000 cells using the RNeasy Mini Kit (Qiagen, Crawley, U.K.). human disease DC. Random hexamers (Qiagen) and M-MLV reverse transcriptase (Invitrogen) were used to synthesize first-strand cDNA according MATERIALS AND METHODS to the manufacturer’s protocol. Absolute RNA expression of the hTR gene and the ABL1 housekeeping gene was assessed by  Construction of the pET28a-3 FLAG-YFP-DKC1 quantitative real-time PCR on an ABI 7500 Real-Time PCR Plasmid. The DKC1 coding sequence had
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