Single-Molecule Analysis of the Human Telomerase RNA center dot Dyskerin Interaction and the Effect of Dyskeratosis Congenita Mutations

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 investigatedmutated 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 constitutively and is required for correct modification of ribosomal and small nuclear RNAprecursors (1). A complex capable of pseudouridylation is formed by dyskerin and three other proteins, NOP10, NHP2, and GAR1, in association with a specific guide RNA containing the box H/ACA sequence motif (2, 3). The presence of thismotif in the 30 half of theRNAcomponent of human telomerase (hTR) has been proposed to provide a discrete binding site for dyskerin within the telomerase complex (2, 4). hTR provides the template for telomere synthesis by human telomerase reverse transcriptase (hTERT) and acts as a protein-binding scaffold for telomerase holoenzyme assembly (5). Recently, the protein dyskerin has been identified as a component of active human telomerase purified from cells (6, 7) in addition to hTERT and hTR. It has been hypothesized that dyskerin is linked in a spatiotemporal manner to the biogenesis of the pretelomerase RNP (7) and/or the stabilization of hTR within the telomerase complex, but there has been no explicit physical evidence of a direct interaction between hTR and dyskerin. Dyskeratosis congenita (8, 9) is thought to be the first primary telomere maintenance disorder to be identified in humans. It is a premature aging syndrome that can lead to a triad of mucocutaneous features, namely, abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia (10). DC adversely affects highly proliferative tissues, with bone marrow failure being the major cause of death (11). Cells from patients with this disease generally display short telomeres, and the lack of efficient telomere maintenance has been attributed to reduced levels of active telomerase enzyme. DC is a genetically diverse condition and arises from three possible inheritance patterns. The most common is the X-linked form, which is associated with mutations in the DKC1 gene that encodes dyskerin (12, 13). Patients with X-linkedDC,mainly youngmales, go on to develop bonemarrow failure before the age of 30 (14) and have an increased risk of cancer, which is thought to arise because shortened telomeres promote genomic instability. More recently, an autosomal dominant form of the disease has been characterized, in which patients generally display milder symptoms that present later in life. A major subset of cases, demonstrating a clinically heterogeneous phenotype, are associated with mutations in hTR (15). Physical analysis of the components of the telomerase complex has been hampered by technical challenges because of lownatural abundance, poor expression, and inefficient in vitro assembly of the recombinant enzyme. In our hands, human dyskerin has proven to be similarly difficult to express at high levels This work was supported by the BBSRC, theWellcome Trust (Grant 069399/Z/02), and the MRC (Grant G0400534). A.O. thanks the Marie Curie IEF of the sixth EU Framework for financial support. *To whom correspondence should be addressed. (S. B.) Telephone:þ44 (0) 1223 336347. Fax: þ44 (0) 1223 336913. E-mail: sb10031@cam.ac. uk. (D.K.) Telephone: +44 (0) 1223 336481. E-mail: dk10012@cam.ac. uk. Abbreviations: DC, dyskeratosis congenita; AD DC, autosomal dominant dyskeratosis congenita; hTR, human telomerase RNA; hTERT, human telomerase reverse transcriptase; TCCD, two-color coincidence detection; YFP, yellow fluorescent protein; 647N-RNA, RNA molecule conjugated to the maleimide derivative of ATTO-TEC 647N fluorescent dye; PUA, pseudouridine and archaeosine transglycosylase; PCR, polymerase chain reaction; RNP, ribonucleoprotein. Article Biochemistry, Vol. 48, No. 46, 2009 10859 (greater than picomoles) in vitro, which precludes the use of most classical biophysicalmethods that require at least 1000-fold greater sample quantities for effective analysis. Our single-molecule fluorescence approach two-color coincidence detection (TCCD) can overcome such limitations as it requires a minimal quantity (less than femtomoles) of labeled components. Furthermore, it can be used to characterize a partially purified complex of orthogonally labeled species in the presence of a high background of labeled, unassociated molecules (16-18). The advantage of TCCD compared to other single-molecule techniques, such as FRET, is that the fluorophore label can be placed at any convenient position on the molecule, and that no prior knowledge of the complex structure is required. Furthermore, the cross-talk between channels is weak, making the method significantly more sensitive to low concentrations of associated molecules. Herein, we describe the use of single-molecule TCCD to detect and study the dyskerin 3 hTRcomplex. In particular, we evaluated the dependence of this protein 3RNA interaction on subdomains of hTR and on mutations associated with the rare human disease DC. MATERIALS AND METHODS Construction of the pET28a-3 FLAG-YFP-DKC1 Plasmid. The DKC1 coding sequence had previously been cloned from human cDNA and inserted into the pEF1 plasmid vector (Invitrogen, Paisley, U.K.). The sequence was subsequently amplified by PCR using the forward primer 50-CTT AGTGCTAGCATGGCGGATGCGGAAGTAATTATT TTG C-30, containing the NheI restriction site, and the reverse primer 50-AGA TCT GAA TTC CTA CTC AGA AAC CAA TTCTACC-30, containing the EcoRI restriction site. TheDKC1 PCR product was subjected to double-restriction endonuclease digestion with enzymes NheI and EcoRI and subcloned into a pET28a vector containing the 3 FLAG coding sequence to produce pET28a-3 FLAG-DKC1. The YFP coding sequence was amplified by PCR from plasmid pET28a-3 FLAG-YFPhTERTusing the forward primer 50-TATACAGCTAGCGTG AGC AAG GGC GAG GAG C-30 and the reverse primer 50-AGA TCT GCT AGC CTT GTA CAG CTC GTC CAT GCC-30, both containing theNheI restriction site. TheYFP PCR product was subjected to restriction endonuclease digestion with NheI and subcloned into the pET28a-3 FLAG-DKC1 plasmid to produce pET28a-3 FLAG-YFP-DKC1. The final construct was verified by DNA sequencing (Cogenics Lark,