title: Correct mRNA processing at a mutant TT splice donor in FANCC ameliorates the clinical phenotype in patients and is enhanced by delivery of suppressor U1 snRNAs.
abstract: The U1 small nuclear RNA (U1 snRNA) as a component of the major U2-dependent spliceosome recognizes 5' splice sites (5'ss) containing GT as the canonical dinucleotide in the intronic positions +1 and +2. The c.165+1G>T germline mutation in the 5'ss of exon 2 of the Fanconi anemia C (FANCC) gene commonly predicted to prevent correct splicing was identified in nine FA patients from three pedigrees. RT-PCR analysis of the endogenous FANCC mRNA splicing pattern of patient-derived fibroblasts revealed aberrant mRNA processing, but surprisingly also correct splicing at the TT dinucleotide, albeit with lower efficiency. This consequently resulted in low levels of correctly spliced transcript and minute levels of normal posttranslationally processed FANCD2 protein, indicating that this naturally occurring TT splicing might contribute to the milder clinical manifestations of the disease in these patients. Functional analysis of this FANCC 5'ss within splicing reporters revealed that both the noncanonical TT dinucleotide and the genomic context of FANCC were required for the residual correct splicing at this mutant 5'ss. Finally, use of lentiviral vectors as a delivery system to introduce expression cassettes for TT-adapted U1 snRNAs into primary FANCC patient fibroblasts allowed the correction of the DNA-damage-induced G2 cell-cycle arrest in these cells, thus representing an alternative transcript-targeting approach for genetic therapy of inherited splice-site mutations.
authors.affiliation: Institute of Virology, Heinrich-Heine-University, D-40225 Düsseldorf, Germany.
authors.forename: Linda
authors.initials: L
authors.lastname: Hartmann
authors.forename: Kornelia
authors.initials: K
authors.lastname: Neveling
authors.forename: Stephanie
authors.initials: S
authors.lastname: Borkens
authors.forename: Hildegard
authors.initials: H
authors.lastname: Schneider
authors.forename: Marcel
authors.initials: M
authors.lastname: Freund
authors.forename: Elke
authors.initials: E
authors.lastname: Grassman
authors.forename: Stephan
authors.initials: S
authors.lastname: Theiss
authors.forename: Angela
authors.initials: A
authors.lastname: Wawer
authors.forename: Stefan
authors.initials: S
authors.lastname: Burdach
authors.forename: Arleen D
authors.initials: AD
authors.lastname: Auerbach
authors.forename: Detlev
authors.initials: D
authors.lastname: Schindler
authors.forename: Helmut
authors.initials: H
authors.lastname: Hanenberg
authors.forename: Heiner
authors.initials: H
authors.lastname: Schaal
mesh_terms: D005199:Fanconi Anemia / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D052218:Fanconi Anemia Complementation Group C Protein / Q000235:genetics*; D016195:G2 Phase / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus; D009154:Mutation / Q000235:genetics; D010375:Pedigree; D010641:Phenotype; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics / Q000502:physiology*; D022821:RNA Splice Sites / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000502:physiology*; D012342:RNA, Small Nuclear / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C497651:FANCC protein, human; D052218:Fanconi Anemia Complementation Group C Protein; D022821:RNA Splice Sites; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA