Analysis of the LAMB3 gene in a junctional epidermolysis bullosa patient reveals exonic splicing and allele-specific nonsense-mediated mRNA decay.
How splicing, the process of intron removal in pre-messenger RNA (mRNA), is carried out with such fidelity in human cells is still not understood, although some general rules are being proposed mainly by in vitro experiments. These rules are currently being redefined by analysis of splicing mechanisms in patients presenting splicing defects. We analysed material of a patient suffering from junctional epidermolysis bullosa, a heritable blistering skin disease. Absence of laminin-5 protein together with hypoplastic hemidesmosomes at the dermo-epidermal junction in the patient's skin was shown by immunohistochemical analysis and immunoelectron microscopy. Subsequent DNA analysis revealed heterozygosity for the mutations R635X and 3009C-->T in the LAMB3 gene. The latter did not alter codon translation, but introduced an exonic splice site in exon 20. Interestingly, this exonic splice site, which presented a splice score of only 68.6, was preferentially used by the spliceosome over the wild-type splice site at the exon 20-intron 20 border, which showed a splice score of 92.2. LAMB3 mRNA was still detectable in RT-PCR analysis although the aberrantly spliced mRNA leads to a stop codon in exon 21, 5' of the commonly assumed 3' border for nonsense-mediated mRNA decay. These results describe an exception to the proposed rules of pre-mRNA splicing and RNA degradation.
Buchroithner
Birgit
B
Department of Dermatology, Paracelsus Private Medical University of Salzburg, Salzburg, Austria.
Klausegger
Alfred
A
Ebschner
Ulrike
U
Anton-Lamprecht
Ingrun
I
Pohla-Gubo
Gabriele
G
Lanschuetzer
Christoph M
CM
Laimer
Martin
M
Hintner
Helmut
H
Bauer
Johann W
JW
D000328:Adult; D017398:Alternative Splicing; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000235:genetics*; D018389:Codon, Nonsense; D004252:DNA Mutational Analysis; D016109:Epidermolysis Bullosa, Junctional / Q000235:genetics* / Q000473:pathology; D005091:Exons / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009679:Nuclear Family; D010375:Pedigree; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000097794:Kalinin
D015815:Cell Adhesion Molecules; D018389:Codon, Nonsense; D012333:RNA, Messenger; D000097794:Kalinin