Characterisation of a Composite Exonic Regulatory Element of Splicing (CERES) in CFTR Exon 12: Functional Properties and Evolutionary Constraints

Haque, Ariful (2010). Characterisation of a Composite Exonic Regulatory Element of Splicing (CERES) in CFTR Exon 12: Functional Properties and Evolutionary Constraints. PhD thesis The Open University.



Genotype screening in human disease frequently results in the identification of exon/intron sequence variations whose direct connection with occurrence of disease is often unclear, especially if they occur within exons but do not alter the amino acid coding sequence. However, it is now clear that many of these seemingly harmless changes very often can exert flaws in the splicing process by affecting splicing regulatory elements (SREs). Usually, SREs are classified based on their mode of action with regards to exon inclusion, either positive (enhancer) or negative (silencer). In addition to these clear cut definitions, using systematic site directed mutagenesis experiments in previous analyses from our lab we have identified a novel type of splicing controlling element that we called CERES (for Composite Exonic Regulatory Element of Splicing). The distinguishing feature of CERES elements resides in the fact that they represent an extreme physical overlap of enhancer and silencer sequence. As a result, the functional effect at the level of exon inclusion/skipping of a single nucleotide change in a CERES element is hard to predict.

In this study I have addressed both issues in the context of the functional CERES2 element in CFTR exon 12. The result show that CERES2 can bind to a number of SR (SF2/ASF and SRp55) and hnRNP (A1, A2, C2, U, DAZAP1) factors in a small stretch of RNA in close proximity to each other. In particular, one of the disease causing mutations, G48C and a synonymous substitution next to it (A49G) showed reduced binding with SF2/ASF, whereas another natural mutation, A51T showed that the SF2/ASF interaction was increased compared to the wild type exon 12 sequences. Functional assays confirmed the potential regulatory role of the SF2/ASF and hnRNP Al interactions.

Two synonymous mouse substitutions (T40C and C52T) near the CERES2 region were observed to cause skipping in human exon 12 but had no effect if the exon was truncated in a reduced context. Restoration of the truncated sequences restored skipping of the exon. However, if these flanking sequences were replaced with mouse sequences then no skipping occurred. This observation suggested that the human exon 12 sequences have ESS regions in both flanks of the exon whereas in the mouse sequence the flanking exon sequences contain ESE elements. Affinity purification of these flanking sequence showed that both of the mouse flanking sequences bind to SR proteins (SF/ASF, SRp 75, SRp 55 and SRp 40) but not in human. The consequences of this situation were then checked at the evolutionary level by comparing the distribution of SREs in different species. Altogether, our results suggest that in several species other than human the entire sequence of CFTR exon 12 is involved in its definition.

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