title: Intron-derived aberrant splicing of A20 transcript in rheumatoid arthritis.
abstract: OBJECTIVE
  Aberrant splicing is one of the most significant components generating functional diversity in many pathological conditions. The objective of this study was to analyse the mutations or aberrant splicing of A20 transcript, the region encompassing the ovarian tumour (OTU) domain [which is functionally important as an inhibitor of nuclear factor (NF)-κB activation] in fibroblast-like synoviocytes (FLSs) from RA patients.
  
  
  METHODS
  Alterations in A20 transcripts were determined through sequence analysis of 10 clones of A20 cDNA in FLSs from each of the five RA patients. The levels of aberrant A20 transcript were measured by quantitative real-time RT-PCR with primers to specifically recognize the inserted introns. The functional role of A20 and its aberrant variants were examined by analysing NF-κB luciferase reporter activity and NF-κB-dependent target gene expression.
  
  
  RESULTS
  In RA FLSs, we discovered four novel aberrant A20 transcripts, most of which resulted from insertion of partial intron 2, intron 4 and/or deletion of exon 4. In each of these FLSs, sequence analysis revealed that these aberrant insertional sequences were flanked by consensus splice donor and acceptor sequences without nucleotide substitution, suggesting alternative splicing as the likely mutational mechanism. These variants elicited a codon frame shift by creating a premature translational stop codon, and eventually, disruption of the OTU domain (which is functionally important as an inhibitor of NF-κB activation) of A20. The expression level of aberrant A20 transcript was correlated well with persisitently enhanced status of NF-κB signalling, as evident by the phosphorylation of inhibitor of NF-κB (IκB)-α and transcription of NF-κB target genes.
  
  
  CONCLUSIONS
  The results suggest that A20 inactivation by the novel aberrant splicing may contribute to RA progression by inducing persistent NF-κB activation.
authors.affiliation: Department of Pharmacology, College of Medicine, Chungnam National University, 6 Munhwa-dong, Jung-gu, Daejeon, Korea.
authors.forename: Hyun Kyung
authors.initials: HK
authors.lastname: Yoon
authors.forename: Hee Sun
authors.initials: HS
authors.lastname: Byun
authors.forename: Hyunji
authors.initials: H
authors.lastname: Lee
authors.forename: Juhee
authors.initials: J
authors.lastname: Jeon
authors.forename: Yoonjung
authors.initials: Y
authors.lastname: Lee
authors.forename: Yuwen
authors.initials: Y
authors.lastname: Li
authors.forename: Eun-Heui
authors.initials: EH
authors.lastname: Jin
authors.forename: JaeWoo
authors.initials: J
authors.lastname: Kim
authors.forename: Jang Hee
authors.initials: JH
authors.lastname: Hong
authors.forename: Jin Hyun
authors.initials: JH
authors.lastname: Kim
authors.forename: Jeong Ho
authors.initials: JH
authors.lastname: Seok
authors.forename: Seong Wook
authors.initials: SW
authors.lastname: Kang
authors.forename: Won Hyung
authors.initials: WH
authors.lastname: Lee
authors.forename: Gang Min
authors.initials: GM
authors.lastname: Hur
mesh_terms: D017398:Alternative Splicing; D001172:Arthritis, Rheumatoid / Q000235:genetics*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000502:physiology; D016328:NF-kappa B / Q000502:physiology; D009687:Nuclear Proteins / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D015398:Signal Transduction / Q000235:genetics; D013583:Synovial Membrane / Q000166:cytology; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3
chemical_list: D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D009687:Nuclear Proteins; D012333:RNA, Messenger; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3