title: Functional and cellular characterization of human Retinoic Acid Induced 1 (RAI1) mutations associated with Smith-Magenis Syndrome.
abstract: BACKGROUND
  Smith-Magenis Syndrome is a contiguous gene syndrome in which the dosage sensitive gene has been identified: the Retinoic Acid Induced 1 (RAI1). Little is known about the function of human RAI1.
  
  
  RESULTS
  We generated the full-length cDNA of the wild type protein and five mutated forms: RAI1-HA 2687delC, RAI1-HA 3103delC, RAI1 R960X, RAI1-HA Q1562R, and RAI1-HA S1808N. Four of them have been previously associated with SMS clinical phenotype. Molecular weight, subcellular localization and transcription factor activity of the wild type and mutant forms were studied by western blot, immunofluorescence and luciferase assays respectively. The wild type protein and the two missense mutations presented a higher molecular weight than expected, localized to the nucleus and activated transcription of a reporter gene. The frameshift mutations generated a truncated polypeptide with transcription factor activity but abnormal subcellular localization, and the same was true for the 1-960aa N-terminal half of RAI1. Two different C-terminal halves of the RAI1 protein (1038aa-end and 1229aa-end) were able to localize into the nucleus but had no transactivation activity.
  
  
  CONCLUSIONS
  Our results indicate that transcription factor activity and subcellular localization signals reside in two separate domains of the protein and both are essential for the correct functionality of RAI1. The pathogenic outcome of some of the mutated forms can be explained by the dissociation of these two domains.
authors.affiliation: John P Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, Florida, USA.
authors.forename: Paulina
authors.initials: P
authors.lastname: Carmona-Mora
authors.forename: Carolina A
authors.initials: CA
authors.lastname: Encina
authors.forename: Cesar P
authors.initials: CP
authors.lastname: Canales
authors.forename: Lei
authors.initials: L
authors.lastname: Cao
authors.forename: Jessica
authors.initials: J
authors.lastname: Molina
authors.forename: Pamela
authors.initials: P
authors.lastname: Kairath
authors.forename: Juan I
authors.initials: JI
authors.lastname: Young
authors.forename: Katherina
authors.initials: K
authors.lastname: Walz
mesh_terms: D000818:Animals; D002460:Cell Line; D002874:Chromosome Mapping; D004252:DNA Mutational Analysis; D018628:Gene Dosage; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D058496:Smith-Magenis Syndrome / Q000235:genetics* / Q000503:physiopathology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: C431552:RAI1 protein, human; C502844:Rai1 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors