Journal of Biochemistry Advance Access originally published online on November 26, 2007
Journal of Biochemistry 2008 143(3):303-310; doi:10.1093/jb/mvm227
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© 2007 The Japanese Biochemical Society.
A Strong Exonic Splicing Enhancer in Dystrophin Exon 19 Achieve Proper Splicing Without an Upstream Polypyrimidine Tract
1Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe 650-0017; and 2Kobe Pharmaceutical University, Kobe 658-8558, Japan
*To whom correspondence should be addressed. Tel: +81-78-382-6090, Fax: +81-78-382-6099, E-mail: myagi{at}med.kobe-u.ac.jp
Received October 17, 2007; Accepted November 3, 2007
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Abstract |
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Proper splicing is known to proceed under the control of conserved cis-elements located at exon–intron boundaries. Recently, it was shown that additional elements, such as exonic splicing enhancers (ESEs), are essential for the proper splicing of certain exons, in addition to the splice donor and acceptor site sequences; however, the relationship between these cis-elements is still unclear. In this report, we utilize dystrophin exon 19 to analyse the relationship between the ESE and its upstream acceptor site sequences. Dystrophin exon 19, which maintains adequate splicing donor and acceptor consensus sequences, encodes exonic splicing enhancer (dys-ESE19) sequences. Splice pattern analysis, using a minigene reporter expressed in HeLa cells, showed that either a strong polypyrimidine tract (PPT) or a fully active dys-ESE19 is sufficient for proper splicing. Each of these two cis-elements has enough activity for proper exon 19 splicing suggesting that the PPT, which is believed to be an essential cis-element for splicing, is dispensable when the downstream exon contains a strong ESE. This compensation was only seen in living cells but not in ‘in vitro splicing’. This suggests the possibility that the previous splicing experiments using an in vitro splicing system could underestimate the activity of ESEs.
Key Words: acceptor site sequences, dystrophin, exonic splicing enhancer, polypyrimidine tract, splicing
Abbreviations: AO, antisense oligonucleotide; AR, ampicillin resistant gene; BMD, Becker muscular dystrophy; BPS, branch point sequence; CV, Shapiro's splice site consensus value; DK, dystrophin Kobe; DMD, Duchenne muscular dystrophy; dys-ESE19, dystrophin exonic splicing enhancer 19; ESE, exonic splicing enhancer; PPT, polypyrimidine tract; RT–PCR, reverse transcription–PCR
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