Specific short oligonucleotide sequences that enhance pre-mRNA splicing when present in exons, termed exonic splicing enhancers (ESEs), play important roles in constitutive and alternative splicing (ESE References). A hybrid computational/experimental method, RESCUE-ESE, was recently developed for identifying sequences with ESE activity. In this approach, specific hexanucleotide sequences are identified as candidate ESEs on the basis that they have both significantly higher frequency of occurrence in exons than in introns and also significantly higher frequency in exons with weak (non-consensus) splice sites than in exons with strong (consensus) splice sites. Representative hexamers from ten different classes of candidate ESEs, together with 6 or 7 bases of flanking sequence context on each side, were introduced into a weak (poorly spliced) exon in a splicing reporter construct. These reporter minigenes were then transfected into cultured cells, where they are transcribed and spliced, and the relative level of inclusion of the test exon was assayed by quantitative (radio-labeled) RT-PCR. Point mutants of these sequences were also analyzed to confirm the precise motifs responsible for ESE activity.
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Specific short oligonucleotide seq …
nce of these candidate ESE hexamers.
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|Is part of||Massachusetts Institute of Technology; Massachusetts; USA +|
|ModifiedDate||21 June 2013 +|
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