title: Defects in mTR stability and telomerase activity produced by the Dkc1               A353V mutation in dyskeratosis congenita are rescued by a peptide from the dyskerin TruB domain.
abstract: BACKGROUND
  The predominant X-linked form of dyskeratosis congenita results from mutations in dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells.
  
  
  METHODS
  Here, we have generated F9 mouse cell lines expressing the most frequent mutation found in X-DC patients, A353V and study the effect of expressing the GSE24.2 cDNA or GSE24.2 peptide on telomerase activity by TRAP assay, and mTERT and mTR expression by Q-PCR. Point mutation in GSE24.2 residues were generated by site-directed mutagenesis.
  
  
  RESULTS
  Expression of GSE24.2 increases mTR and to a lesser extent mTERT RNA levels, and leads to recovery of telomerase activity. Point mutations in GSE24.2 residues known to be highly conserved and crucial for the pseudouridine-synthase activity of dyskerin abolished the effect of the peptide. Recovery of telomerase activity and increase in mTERT levels were found when the GSE24.2 peptide purified from bacteria was introduced into the cells. Moreover, mTR stability was also rescued by transfection of the peptide GSE24.2.
  
  
  CONCLUSIONS
  These data indicate that supplying GSE24.2, either from a cDNA vector, or as a peptide, can reduces the pathogenic effects of Dkc1 mutations and could form the basis of a novel therapeutic approach.
authors.affiliation: Instituto de Investigaciones Biomédicas CSIC/UAM, IDIPaz, Madrid, Spain. rosario_machado_pinilla@hotmail.com
authors.forename: Rosario
authors.initials: R
authors.lastname: Machado-Pinilla
authors.forename: Jaime
authors.initials: J
authors.lastname: Carrillo
authors.forename: Cristina
authors.initials: C
authors.lastname: Manguan-Garcia
authors.forename: Leandro
authors.initials: L
authors.lastname: Sastre
authors.forename: Alexander
authors.initials: A
authors.lastname: Mentzer
authors.forename: B-W
authors.initials: BW
authors.lastname: Gu
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Mason
authors.forename: Rosario
authors.initials: R
authors.lastname: Perona
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology / Q000627:therapeutic use; D002478:Cells, Cultured; D019871:Dyskeratosis Congenita / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D004789:Enzyme Activation / Q000235:genetics; D015316:Genetic Therapy; D006367:HeLa Cells; D006801:Humans; D019751:Intramolecular Transferases / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000502:physiology*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology / Q000627:therapeutic use; D010446:Peptide Fragments / Q000494:pharmacology / Q000627:therapeutic use; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D020871:RNA Stability / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D019098:Telomerase / Q000737:chemistry / Q000378:metabolism*; D014633:Valine / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; C510431:Dkc1 protein, mouse; D009687:Nuclear Proteins; D010446:Peptide Fragments; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase; C509187:Tert protein, mouse; D019751:Intramolecular Transferases; C096995:pseudouridine synthases; D014633:Valine; D000409:Alanine