X-linked Dyskeratosis Congenita Research Articles

Accelerated hematopoietic stem cell aging in a mouse model of dyskeratosis congenita responds to antioxidant treatment

Mutations in DKC1, encoding telomerase associated protein dyskerin, cause X-linked dyskeratosis congenita (DC), a bone marrow (BM) failure, and cancer susceptibility syndrome. Decreased accumulation of telomerase RNA resulting in excessive telomere shortening and premature cellular senescence is thought to be the primary cause of disease in X-linked DC. Affected tissues are those that require constant renewal by stem cell activity. We previously showed that in Dkc1(Δ15) mice, which contain a mutation that is a copy of a human mutation causing DC, mutant cells have a telomerase-dependent proliferative defect and increased accumulation of DNA damage in the first generation before the telomeres are short. We now demonstrate the presence of the growth defect in Dkc1(Δ15) mouse embryonic fibroblasts in vitro and show that accumulation of DNA damage and levels of reactive oxygen species increase with increasing population doublings. Treatment with the antioxidant, N-acetyl cysteine (NAC), partially rescued the growth disadvantage of mutant cells in vitro and in vivo. Competitive BM repopulation experiments showed that the Dkc1(Δ15) mutation is associated with a functional stem cell defect that becomes more severe with increasing age, consistent with accelerated senescence, a hallmark of DC hematopoiesis. This stem cell phenotype was partially corrected by NAC treatment. These results suggest that a pathogenic Dkc1 mutation accelerates stem cell aging, that increased oxidative stress might play a role in the pathogenesis of X-linked DC, and that some manifestations of DC may be prevented or delayed by antioxidant treatment.

Dermatological manifestations of inherited cancer syndromes in children

Various cutaneous signs presenting in childhood, for example café-au-lait macules, may have systemic cancer associations. Indeed, this may be the first manifestation of the underlying cancer predisposition. The syndromes covered in this review fall into four main categories: (i) DNA damage processing defects including Fanconi anaemia, ataxia telangiectasia, Bloom syndrome, Rothmund-Thomson syndrome, constitutional mismatch repair defects and xeroderma pigmentosum; (ii) signalling pathway defects, including naevoid basal cell carcinoma and Costello syndromes; (iii) primary immunodeficiency syndromes; and (iv) syndromes that do not fit this molecular classification, such as X-linked dyskeratosis congenita. This review focuses on the dermatological findings of these conditions. Some of these conditions exhibit a milder heterozygous phenotype and this should be elicited in the family history. Where the dermatological findings are subtle, a targeted family history can provide clues towards making a diagnosis. Nondermatological features of each condition are summarized too, together with molecular testing strategies, which will direct genetic counselling and screening. This review will enable the dermatologist and other clinicians in the early recognition and molecular confirmation of underlying cancer-predisposing syndromes. This allows the possibility of surveillance and prevention strategies to be initiated in a timely manner, in affected children and other at-risk family members.

Dyskerin and cancer: more than telomerase. The defect in mRNA translation helps in explaining how a proliferative defect leads to cancer.

Point mutations in the DKC1 gene that encodes dyskerin cause the rare inherited syndrome called X-linked dyskeratosis congenita, characterized by a failure of proliferating tissues and increased susceptibility to cancer. Dyskerin is a nucleolar protein with different functions, all fundamental to basic cellular events such as protein expression, growth, and proliferation. The two best-characterized dyskerin activities are the stabilization of the telomerase RNA component, allowing the proper function telomerase enzymatic complex, and the modification of specific uridine residues of ribosomal RNA by converting them to pseudouridine, thus allowing proper ribosome processing and function. In light of the recent findings, this review focuses on the molecular pathogenesis of dyskeratosis congenita, discussing how a defect in ribosomal function might impact on the translation of a subset of mRNAs encoding for tumour suppressors, thus providing an explanation for the apparent paradox of dyskeratosis congenita in which reduced cell proliferation is associated with cancer susceptibility. In addition, the current evidence pointing to a role played by dyskerin in tumours in the general population is also discussed.

Loss of Function of the Tumor Suppressor DKC1 Perturbs p27 Translation Control and Contributes to Pituitary Tumorigenesis

Mutations in DKC1, encoding for dyskerin, a pseudouridine synthase that modifies rRNA and regulates telomerase activity, are associated with ribosomal dysfunction and increased cancer susceptibility in the human syndrome, X-linked dyskeratosis congenita (X-DC). In a mouse model for X-DC, impairments in DKC1 function affected the translation of specific mRNAs harboring internal ribosomal entry site (IRES) elements, including the tumor suppressor, p27. However, how this translational deregulation contributes to tumor initiation and progression remains poorly understood. Here, we report that impairment in p27 IRES-mediated translation due to decreased levels of DKC1 activity markedly increases spontaneous pituitary tumorigenesis in p27 heterozygous mice. Using a new bioluminescent mouse model, we monitored p27 translation in vivo and show that p27 IRES-mediated translation is reduced in the pituitary of DKC1 hypomorphic mice (DKC1(m)). Furthermore, we show that DKC1 has a critical role in regulating the assembly of the 48S translational preinitiation complex mediated by the p27 IRES element. An analysis of human tumors identified a novel mutation of DKC1 (DKC1(S485G)) in a human pituitary adenoma. We show that this specific amino acid substitution significantly alters DKC1 stability/pseudouridylation activity, and this correlates with reductions in p27 protein levels. Furthermore, DKC1(S485G) mutation does not alter telomerase RNA levels. Altogether, these findings show that genetic alterations in DKC1 could contribute to tumorigenesis associated with somatic cancers and establish a critical role for DKC1 in tumor suppression, at least in part, through translational control of p27.

Novel dyskerin-mediated mechanism of p53 inactivation through defective mRNA translation.

In up to 60% of human cancers, p53 gene mutations are responsible for direct inactivation of the tumor suppressor function of p53. Alternative mechanisms of p53 inactivation described thus far mainly affect its posttranslational regulation. In X-linked dyskeratosis congenita, a multisystemic syndrome characterized by increased cancer susceptibility, mutations of the DKC1 gene encoding dyskerin cause a selective defect in the translation of a subgroup of internal ribosome entry site (IRES)-containing cellular mRNAs. In this study, we show that impairment of dyskerin function can cause p53 inactivation due to a defect in p53 mRNA translation. siRNA-mediated reduction of dyskerin levels caused a decrease of p53 mRNA translation, protein levels, and functional activity, both in human breast cancer cells and in primary mammary epithelial progenitor cells. These effects seemed to be independent of the known role of dyskerin in telomerase function, and they were associated with a specific impairment of translation initiation mediated by IRES elements present in p53 mRNA. In a series of human primary breast cancers retaining wild-type p53, we found that low levels of dyskerin expression were associated with reduced expression of p53-positive target genes. Our findings suggest that a dyskerin-mediated mechanism of p53 inactivation may occur in a subset of human tumors.

Deregulation of oncogene-induced senescence and p53 translational control in X-linked dyskeratosis congenita

Defects in ribosome biogenesis and function are present in a growing list of human syndromes associated with cancer susceptibility. One example is X-linked dyskeratosis congenita (X-DC) in which the DKC1 gene, encoding for an enzyme that modifies ribosomal RNA, is found to be mutated. How ribosome dysfunction leads to cancer remains poorly understood. A critical cellular response that counteracts cellular transformation is oncogene-induced senescence (OIS). Here, we show that during OIS, a switch between cap- and internal ribosome entry site (IRES)-dependent translation occurs. During this switch, an IRES element positioned in the 5'untranslated region of p53 is engaged and facilitates p53 translation. We further show that in DKC1(m) cells, p53 IRES-dependent translation is impaired during OIS ex vivo and on DNA damage in vivo. This defect in p53 translation perturbs the cellular response that counteracts oncogenic insult. We extend these findings to X-DC human patient cells in which similar impairments in p53 IRES-dependent translation are observed. Importantly, re-introduction of wild-type DKC1 restores p53 expression in these cells. These results provide insight into the basis for cancer susceptibility in human syndromes associated with ribosome dysfunction.

Dyskerin Ablation in Mouse Liver Inhibits rRNA Processing and Cell Division

Dyskerin is a component of small nucleolar ribonucleoprotein complexes and acts as a pseudouridine synthase to modify newly synthesized ribosomal, spliceosomal, and possibly other RNAs. It is encoded by the DKC1 gene, the gene mutated in X-linked dyskeratosis congenita, and is also part of the telomerase complex. The yeast ortholog, Cbf5, is an essential protein, but in mammals the effect of dyskerin ablation at the cellular level is not known. Here we show that mouse hepatocytes can survive after induction of a Dkc1 deletion. In the absence of dyskerin, rRNA processing is inhibited with the accumulation of large precursors, and fibrillarin does not accumulate in nucleoli. A low rate of apoptosis is induced in the hepatocytes, which show an induction of the p53-dependent cell cycle checkpoint pathway. Signs of liver damage including an increase in serum alanine aminotransferase activity and a disordered structure at the histological and macroscopic levels are observed. In response to carbon tetrachloride administration, when wild-type hepatocytes mount a rapid proliferative response, those without dyskerin do not divide. We conclude that hepatocytes can survive without dyskerin but that the role of dyskerin in RNA modification is essential for cellular proliferation.

Telomere Elongation in Dyskeratosis Congenita Induced Pluripotent Stem Cells.

Abstract 497Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer premature degeneration of multiple tissues. Bone marrow failure is the principal cause of mortality, and allogeneic stem cell transplantation is limited by increased treatment-related mortality. Somatic cells can be reprogrammed using defined genetic and chemical factors, yielding “induced pluripotent stem” (iPS) cell lines which have the capacity to differentiate into any tissue. Patient-specific iPS cells therefore hold promise as therapeutic agents and disease models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, and we have found that telomere length is increased in human iPS cells relative to the normal primary somatic cells from which they are derived. Here we investigated whether defects in telomerase function would limit derivation or self-renewal of iPS cells from patients with DC. We reprogrammed primary fibroblasts from patients with X-linked and autosomal dominant DC, caused by mutations in the genes encoding dyskerin and telomerase RNA component (TERC), respectively. We were able to establish multiple DC-specific iPS lines showing all hallmarks of pluripotency, including the formation of hematopoietic progenitors in vitro. Unexpectedly, DC-specific iPS cells were able to sustain continual proliferation in vitro, in contrast to the premature senescence displayed by the DC fibroblasts. Although early passage DC iPS cells had shorter telomeres than donor fibroblasts, we found that telomere length in DC iPS cells increased with continued passage in culture. To explain this finding, we discovered that steady state levels of TERC, which are critically limiting in several forms of DC, are upregulated in normal and DC iPS cells. We found that TERC upregulation is a feature of the pluripotent state, that the TERC locus is a target of pluripotency-associated transcription factors, and that transcriptional silencing accompanies a 3' deletion at the TERC locus in autosomal dominant DC. Our results demonstrate that reprogramming restores self-renewal capacity in DC cells despite genetic lesions affecting telomerase, and suggest that strategies to enhance endogenous TERC expression may be feasible and therapeutically beneficial in DC patients. The studies demonstrate the value of patient-specific iPS cells for basic and translational discovery, and further the rationale for autologous iPS based cellular therapy of genetic hematologic disorders. Disclosures:Daley:MPM Capital: Consultancy; Solasia: Consultancy; Epizyme: Consultancy; iPierian: Consultancy, Equity Ownership.

Single-molecule analysis of the human telomerase RNA.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·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·hTR interaction. Dyskerin mutations associated with X-linked DC resulted in significant impairment of the dyskerin·hTR interaction, whereas mutations in hTR associated with autosomal dominant (AD) DC did not affect the interaction. We propose that disruption of the dyskerin·hTR interaction may contribute to X-linked DC.

Pathogenic NAP57 mutations decrease ribonucleoprotein assembly in dyskeratosis congenita

X-linked dyskeratosis congenita (DC) is a rare bone marrow failure syndrome caused by mostly missense mutations in the pseudouridine synthase NAP57 (dyskerin/Cbf5). As part of H/ACA ribonucleoproteins (RNPs), NAP57 is important for the biogenesis of ribosomes, spliceosomal small nuclear RNPs, microRNAs and the telomerase RNP. DC mutations concentrate in the N- and C-termini of NAP57 but not in its central catalytic domain raising questions as to their impact. We demonstrate that the N- and C-termini together form the binding surface for the H/ACA RNP assembly factor SHQ1 and that DC mutations modulate the interaction between the two proteins. Pinpointing impaired interaction between NAP57 and SHQ1 as a potential molecular basis for X-linked DC has implications for therapeutic approaches, e.g. by targeting the NAP57-SHQ1 interface with small molecules.

Severe Variant of X‐linked Dyskeratosis Congenita (Hoyeraal‐Hreidarsson Syndrome) Causes Significant Enterocolitis in Early Infancy

Severe Variant of X‐linked Dyskeratosis Congenita (Hoyeraal‐Hreidarsson Syndrome) Causes Significant Enterocolitis in Early Infancy

Identification of a novel mutation in DKC1 in dyskeratosis congenita

Dyskeratosis congenita (DC) is a rare congenital syndrome characterized by the triad of reticular skin pigmentation, nail dystrophy and mucosal leukoplakia, and the predisposition to bone marrow failure and malignancy. DC is genetically heterogeneous and X-linked and autosomal forms of the disease exist. Here, we report the clinical description and mutation analysis of a Russian family with X-linked DC. A novel mutation in DKC1 raised de novo in the maternal grandmother's gamete was found; this mutation is a 2 bp inversion in exon 3: NM_001363:c.166_167invCT (Leu56Ser).

ATM in breast cancer susceptibility – results of a pooled analysis of case-control mutation screening data

Dyskeratosis congenita (DKC) is a rare and fatal congenital syndrome characterized by the triad of reticular skin pigmentation, nail dystrophy and mucosal leukoplakia, and the predisposition to bone marrow failure and malignancies. Mutations in DKC1 gene encoding dyskerin are responsible for the X-linked dyskeratosis congenita. Here we report mutation analysis of two Chinese pedigrees with dyskeratosis congenita. The 15 coding exons of DKC1 and their flanking regions were amplified from genomic DNA by PCR. DNA sequencing and restriction endonuclease digestion were used for mutation detection. Transition mutation of 1226C→T (P409L) found in the first pedigree is a novel mutation. In the second pedigree, the proband's mother phenotypically normal carried a de novo transition mutation of 1058C→T (A353 V) in one allele, and transmitted the mutant allele to her two sons who had typical manifestations of dyskeratosis congenita.

Telomerase reverse-transcriptase homozygous mutations in autosomal recessive dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome

Dyskeratosis congenita (DC) is a multisystem bone marrow failure syndrome characterized by a triad of mucocutaneous abnormalities and an increased predisposition to malignancy. X-linked DC is due to mutations in DKC1, while heterozygous mutations in TERC (telomerase RNA component) and TERT (telomerase reverse transcriptase) have been found in autosomal dominant DC. Many patients with DC remain uncharacterized, particularly families displaying autosomal recessive (AR) inheritance. We have now identified novel homozygous TERT mutations in 2 unrelated consanguineous families, where the index cases presented with classical DC or the more severe variant, Hoyeraal-Hreidarsson (HH) syndrome. These TERT mutations resulted in reduced telomerase activity and extremely short telomeres. As these mutations are homozygous, these patients are predicted to have significantly reduced telomerase activity in vivo. Interestingly, in contrast to patients with heterozygous TERT mutations or hemizygous DKC1 mutations, these 2 homozygous TERT patients were observed to have higher-than-expected TERC levels compared with controls. Collectively, the findings from this study demonstrate that homozygous TERT mutations, resulting in a pure but severe telomerase deficiency, produce a phenotype of classical AR-DC and its severe variant, the HH syndrome.

A dyskerin motif reactivates telomerase activity in X-linked dyskeratosis congenita and in telomerase-deficient human cells

Dyskerin gene is mutated in patients with X-linked dyskeratosis congenita (X-DC), which results in greatly reduced levels of telomerase activity. A genetic suppressor element (GSE) termed GSE24-2 has been isolated in a screening for cisplatin resistance. GSE24-2-expressing cells presented impaired telomerase inhibition following in vitro exposure to chemotherapies, such as cisplatin, or telomerase inhibitors. The promoter of the telomerase component hTERT was constitutively activated in GSE24-2 cells in a c-myc expression-dependent manner. Deletion analyses and mutagenesis of the human c-myc promoter demonstrated that the target sequence for activation was the nuclease hypersensitive element-III (NHEIII) site located upstream to the P1 region of the promoter. Further, expression of GSE24-2 in cell lines derived from patients with X-DC and in VA13 cells induced increased hTERT RNA and hTR levels and recovery of telomerase activity. Finally, expression of GSE24-2 was able to rescue X-DC fibroblasts from premature senescence. These data demonstrate that this domain of dyskerin plays an important role in telomerase maintenance following cell insults such as cisplatin treatment, and in telomerase-defective cells in patients with X-DC. The expression of this dyskerin fragment has a dominant function in X-DC cells and could provide the basis for a therapeutic approach to this disease.

Dyskeratosis congenita: a genetic disorder of many faces

Dyskeratosis congenita (DC) is an inherited syndrome exhibiting marked clinical and genetic heterogeneity. It is characterized by multiple features including mucocutaneous abnormalities, bone marrow failure and an increased predisposition to cancer. Three genetic subtypes are recognized: X-linked recessive DC bears mutations in DKC1, the gene encoding dyskerin, a component of H/ACA small nucleolar ribonucleoprotein particles; autosomal dominant (AD) DC has heterozygous mutations in either TERC or TERT, the RNA and enzymatic components of telomerase, respectively, and autosomal recessive DC in which the genes involved remain largely elusive. Disease pathology is believed to be a consequence of chromosome instability because of telomerase deficiency due to mutations in DKC1, TERC and TERT; in patients with DKC1 mutations, defects in ribosomal RNA modification, ribosome biogenesis, translation control or mRNA splicing may also contribute to disease pathogenesis. The involvement of telomerase complex components in X-linked and AD forms and the presence of short telomeres in DC patients suggest that DC is primarily a disease of defective telomere maintenance. Treatment is variable and complicated by the development of secondary cancers but, being a monogenic disorder, it could potentially be treated by gene therapy. DC overlaps both clinically and genetically with several other diseases including Hoyeraal-Hreidarsson syndrome, aplastic anaemia and myelodysplasia, among others and its underlying telomeric defect has implications for a broader range of biological processes including ageing and many forms of cancer.

Splenic peliosis and rupture in patients with dyskeratosis congenita on androgens and granulocyte colony‐stimulating factor

Splenic peliosis and rupture in patients with dyskeratosis congenita on androgens and granulocyte colony‐stimulating factor

Genetic heterogeneity in autosomal recessive dyskeratosis congenita with one subtype due to mutations in the telomerase-associated protein NOP10

Dyskeratosis congenita (DC) is characterized by multiple features including mucocutaneous abnormalities, bone marrow failure and an increased predisposition to cancer. It exhibits marked clinical and genetic heterogeneity. DKC1 encoding dyskerin, a component of H/ACA small nucleolar ribonucleoprotein (snoRNP) particles is mutated in X-linked recessive DC. Telomerase RNA component (TERC), the RNA component and TERT the enzymatic component of telomerase, are mutated in autosomal dominant DC, suggesting that DC is primarily a disease of defective telomere maintenance. The gene(s) involved in autosomal recessive DC remains elusive. This paper describes studies aimed at defining the genetic basis of AR-DC. Homozygosity mapping in 16 consanguineous families with 25 affected individuals demonstrates that there is no single genetic locus for AR-DC. However, we show that NOP10, a component of H/ACA snoRNP complexes including telomerase is mutated in a large consanguineous family with classical DC. Affected homozygous individuals have significant telomere shortening and reduced TERC levels. While a reduction of TERC levels is not a universal feature of DC, it can be brought about through a reduction of NOP10 transcripts, as demonstrated by siRNA interference studies. A similar reduction in TERC levels is also seen when the mutant NOP10 is expressed in HeLa cells. These findings identify the genetic basis of one subtype of AR-DC being due to the first documented mutations in NOP10. This further strengthens the model that defective telomere maintenance is the primary pathology in DC and substantiates the evidence in humans for the involvement of NOP10 in the telomerase complex and telomere maintenance.

Unbalanced X Chromosome Inactivation Independent of Telomere Shortening in Mice Heterozygous for a Mutant Dyskerin Allele.

X-linked Dyskeratosis Congenita (DC) is a rare recessive disorder caused by mutations in the DKC1 gene that encodes dyskerin. Dyskerin is part of ribonucleoprotein complexes that participate in two different pathways: ribosome biogenesis and telomere maintenance. It is the subject of intense debate whether disease manifestations in DC are due to dysfunctional telomere maintenances or are caused by a defect in ribosome biogenesis. Pathogenic mutations in dyskerin cause telomere shortening and patients with X-linked DC have critically short telomeres, However, whether there is an additional defect in ribosome biogenesis is difficult to investigate. To dissect the impact of a pathogenic dyskerin mutation on telomeres from the possible additional impact on ribosome biogenesis in an in vivo model, we generated mice expressing a mutant dyskerin protein. Because laboratory mice have very long telomeres a short telomere phenotype requires several generations of inbreeding, whereas a phenotype seen in the first generation is likely to be caused by the defect in ribosome biogenesis. To delete the last 21 amino acids of dyskerin (Del15) we used homologous recombination followed by conditional gene deletion in murine embryonic stem (ES) cells and in mice. Six independent ES cell clones with the deleted Dkc1 gene were obtained. In vitro analysis of the ES cells showed that the Del15 mutation led to dramatically decreased expression of a truncated dyskerin protein with decreased accumulation of the telomerase RNA. In addition, both reduction in telomerase activity and significant telomere shortening after 65 passages were observed. These findings indicate that the Del15 mutation impairs the telomerase maintenance pathway. After testing the accumulation of a series of mouse H/ACA snoRNA in Del15 ES cells, we found a decrease of the mU68 and mE1 snoRNAs suggesting the mutation may also confer effects which are outside the telomerase pathway. We therefore went on to produce a line of mice expressing the truncated Dkc1 protein and were able to obtain male mice hemizygous for the mutant Dkc1 gene as well as female heterozgotes. The male mice express the truncated dyskerin protein and show no gross abnormality up to 6 months of age. Interestingly, heterozygous female mice were healthy as well but the truncated dyskerin protein was dramatically decreased in expression compared to the wild type dyskerin in spleen, thymus, and bone marrow, but not in liver and brain. This result must derive from preferential proliferation of cells expressing wild type dyskerin after random X-inactivation in early embryogenesis. Our analysis indicates that the mutant dyskerin impairs the proliferation in hematopoietic tissues while it does not affect cells which are not rapidly proliferating such as those in liver and brain. Because of the early appearance of the skewed X-inactivation phenotype we conclude that skewing in these mice is caused by a telomere independent mechanism. Interestingly, the lack of overt DC-like abnormalities in the male hemizygous mice indicates that this proliferative disadvantage is insufficient to cause bone marrow failure but in combination with impaired telomere maintenance may accelerate the onset and severity of disease and thus explain the earlier and more severe manifestation in X-linked DC compared to autosomal dominant DC which only affects the telomerase pathway.

Dynamic association and localization of human H/ACA RNP proteins

Mammalian H/ACA RNPs are essential for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. To form mature RNA-protein complexes, one H/ACA RNA associates with four core proteins. In the cell, this process is assisted by at least one nuclear assembly factor, NAF1. Here we report several unanticipated dynamic aspects of H/ACA RNP proteins. First, when overexpressed, NAF1 delocalizes to the cytoplasm. However, its nucleocytoplasmic shuttling properties remain unaffected. These observations demonstrate a subtle equilibrium between NAF1 expression levels and the availability of NAF1 nuclear binding sites. Second, although NAF1 is excluded from mature RNPs in nucleoli and Cajal bodies, NAF1 associates with mature H/ACA RNA in cell lysates. This association occurs post-lysis because it is observed even when NAF1 and the H/ACA RNA are expressed in separate cells. This documents a protein-RNP association in cell lysates that is absent from intact cells. Third, in similar experiments, all H/ACA core proteins, except NAP57, exchange with their exogenous counterparts, portraying an unexpected dynamic picture of H/ACA RNPs. Finally, the irreversible association of only NAP57 with H/ACA RNA and the conundrum that only NAP57 is mutated in X-linked dyskeratosis congenita (even though most core proteins are required for maintaining H/ACA RNAs) may be more than a coincidence.