Role and Mechanism of Action of Exonic Splicing Regulatory Sequences

Goina, Elisa (2008). Role and Mechanism of Action of Exonic Splicing Regulatory Sequences. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f279

Abstract

Exonic mutations can result in altered protein function by affecting exon recognition during splicing. To understand this mechanism I have extensively evaluated the exonic regulatory elements affected by the disease G to T mutation at position +6 of BRCA1 exon 18. This substitution induces the exclusion of the exon and it has been suggested that it disrupts an ASF/SF2-dependent enhancer. Using a pulldown assay with an internal standard, I show that WT and T6 sequences bind ASF/SF2 with similar efficiency, which is significantly lower compared to the binding to a typical enhancer derived from the fibronectin EDA exon. Consistent with the absence of an ASF/SF2 enhancing effect, siRNA depletion of ASF/SF2 did not induce exon WT exclusion indicating that ASF/SF2 is not essential for BRCA1 exon 18 splicing. However, depletion or overexpression of ASF/SF2 resulted in a decrease or increase of T6 exon 18 inclusion, respectively. By a series of exonic deletions in the WT and T6 BRCA1 minigenes, an enhancer sequence was identified in position 23-32 which mediates the observed T6-specific, ASF/SF2-dependent splicing activation. Furthermore, extensive mutations analysis indicated that the T6 mutant creates a sequence with a predominantly inhibitory function. Double site-directed mutations showed that the point mutations affecting the “TAG” sequence, between +6 and +8 positions, completely restore normal splicing. Indeed, RNA protein interaction and siRNA experiments showed that the skipping of T6 BRCA1 exon 18 is due to the creation of a silencer element. This sequence specifically binds to the proteins hnRNP A1/A2 and to DAZAP1. By siRNA experiments, DAZAP1 and hnRNP A1/A2 are shown to be involved redundantly in the regulation of the defective BRCA1 exon 18. The present results support a gain-of-function model for the BRCA1 T6 exon 18 and indicate that the binding of the hnRNP A1/A2 and DAZAP1 is the primary determinant of exon skipping.

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