TY - JOUR
T1 - A frameshifting mutation in CHRNE unmasks skipping of the preceding exon
AU - Ohno, Kinji
AU - Milone, Margherita
AU - Shen, Xin Ming
AU - Engel, Andrew G.
N1 - Funding Information:
This work was supported by the National Institutes of Health grant NS6277 and by a Muscular Dystrophy Association research grant to A.G.E. We are grateful to Dr Susan T. Iannaccone for referral of Pts 1 and 7, Dr John N. Whittaker for Pt 4, Dr Shin J. Oh for Pts 5 and 9, and Dr John Chapin for Pt 6.
PY - 2003/12/1
Y1 - 2003/12/1
N2 - A frameshifting 7 bp deletion (E553del7) in exon 7 of CHRNE encoding the acetylcholine receptor E subunit, observed in seven congenital myasthenic syndrome patients, enhances expression of an aberrantly spliced transcript that skips the preceding 101 bp exon 6. To recapitulate the aberrant splicing, we cloned the entire CHRNE spanning 12 exons and 11 introns and expressed it in COS cells. Scanning mutagenesis revealed that E553del7 does not disrupt an exonic splicing enhancer. Inhibition of protein synthesis and of nonsense-mediated mRNA decay (NMD) by anisomycin shows that even wild-type CHRNE produces an exon 6-skipped transcript, and that even E553del7-CHRNE yields a normally spliced transcript. Both transcripts, however, are degraded by NMD due to a premature stop codon. In contrast, the normally spliced transcript from wild-type CHRNE and the exon 6-skipped transcript from E553del7-CHRNE carry no premature stop codon and hence are immune to NMD. Optimization of splicing signals for exon 6 prevents it being skipped even in the presence of anisomycin and/or E553del7, indicating that inherently weak splicing signals for exon 6 account for its skipping. We suggest that a similar mechanism probably operates in other genes in skipping of remote exons. The presence of weak splicing signals for exon 6 also prompted us to search for mutations in exon 6 that disrupt an exonic splicing enhancer. Indeed, we found that EEF157V and EE154X in exon 6, observed in two other patients, caused aberrant splicing of exon 6.
AB - A frameshifting 7 bp deletion (E553del7) in exon 7 of CHRNE encoding the acetylcholine receptor E subunit, observed in seven congenital myasthenic syndrome patients, enhances expression of an aberrantly spliced transcript that skips the preceding 101 bp exon 6. To recapitulate the aberrant splicing, we cloned the entire CHRNE spanning 12 exons and 11 introns and expressed it in COS cells. Scanning mutagenesis revealed that E553del7 does not disrupt an exonic splicing enhancer. Inhibition of protein synthesis and of nonsense-mediated mRNA decay (NMD) by anisomycin shows that even wild-type CHRNE produces an exon 6-skipped transcript, and that even E553del7-CHRNE yields a normally spliced transcript. Both transcripts, however, are degraded by NMD due to a premature stop codon. In contrast, the normally spliced transcript from wild-type CHRNE and the exon 6-skipped transcript from E553del7-CHRNE carry no premature stop codon and hence are immune to NMD. Optimization of splicing signals for exon 6 prevents it being skipped even in the presence of anisomycin and/or E553del7, indicating that inherently weak splicing signals for exon 6 account for its skipping. We suggest that a similar mechanism probably operates in other genes in skipping of remote exons. The presence of weak splicing signals for exon 6 also prompted us to search for mutations in exon 6 that disrupt an exonic splicing enhancer. Indeed, we found that EEF157V and EE154X in exon 6, observed in two other patients, caused aberrant splicing of exon 6.
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U2 - 10.1093/hmg/ddg334
DO - 10.1093/hmg/ddg334
M3 - Article
C2 - 14532324
AN - SCOPUS:0345530998
SN - 0964-6906
VL - 12
SP - 3055
EP - 3066
JO - Human molecular genetics
JF - Human molecular genetics
IS - 23
ER -