title: Dyskeratosis congenita mutations in the H/ACA domain of human telomerase RNA affect its assembly into a pre-RNP.
abstract: Dyskeratosis congenita (DC) is an inherited disorder that implicates defects in the biology of telomeres, which are maintained by telomerase, a ribonucleoprotein with reverse transcriptase activity. Like all H/ACA RNAs, the H/ACA domain of nascent human telomerase RNA (hTR) forms a pre-RNP with H/ACA proteins NAF1, dyskerin, NOP10, and NHP2 in vivo. To assess the pre-RNP assembly of hTR mutants that poorly accumulate in vivo, we developed an in vitro system that uses components of human origin. Pre-RNPs were reconstituted with synthetic (32)P-labeled RNAs and (35)S-labeled proteins produced in rabbit reticulocyte lysate, and immunoprecipitations were carried out to analyze RNP formation. We show that human NAF1 cannot bind directly to the H/ACA domain of hTR, and requires the core trimer dyskerin-NOP10-NHP2 to be efficiently incorporated into the pre-RNP. This order of assembly seems common to H/ACA RNAs since it was observed with snoRNA ACA36 and scaRNA U92, which are predicted to guide pseudouridylation of 18S rRNA and U2 snRNA, respectively. However, the processing H/ACA snoRNA U17 did not conform to this rule, as NAF1 alone was able to bind it. We also provide the first evidence that DC-related mutations of hTR C408G and Delta378-451 severely impair pre-RNP assembly. Integrity of boxes H and ACA of hTR are also crucial for pre-RNP assembly, while the CAB box is dispensable. Our results offer new insights into the defects caused by some mutations located in the H/ACA domain of hTR.
authors.affiliation: Département des Sciences Biologiques and Centre de Recherche BioMed, Université du Québec à Montréal, Canada.
authors.forename: Christian
authors.initials: C
authors.lastname: Trahan
authors.forename: François
authors.initials: F
authors.lastname: Dragon
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D020541:Coiled Bodies / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D019871:Dyskeratosis Congenita / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; C112148:DKC1 protein, human; D004268:DNA-Binding Proteins; C416929:NHP2 protein, human; C416930:NOP10 protein, human; D009687:Nuclear Proteins; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; C117046:TNIP1 protein, human; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase