Compensatory Relationship Between Exonic Splicing Enhancer, Splice Site and Protein Function

Falanga, Alessia (2012). Compensatory Relationship Between Exonic Splicing Enhancer, Splice Site and Protein Function. PhD thesis The Open University.



The process of pre-mRNA splicing involves the removal of intronic sequences from the pre-mRNA and it is directed by intronic cis acting elements know as the 5’ and 3’ splice sites that mark the boundaries of the exons. Over the two decades, however, it has become clear that exons encode for auxiliary splicing signals that either enhance or perturb their inclusion in the final mRNA product. It is possible that the evolution of mRNA sequences could be conditioned by the presence of these exonic cis-acting splicing regulatory elements and not mainly by the selection of optimal protein function.

To explore this hypothesis, I have investigated how the need for ESE influences the gene evolution of a paralogous gene family, specifically the human Alkaline Phosphatases (ALPs). In this work, I have identified in correspondence to a weak 3’splice site, two ESE sequences in the placental ALP exon 4, and demonstrate that the ESE are necessary for the exon inclusion in the mRNA due to the weak 3’splice sites. Furthermore, I show that they are absent in the corresponding exon of the non-tissue specific ALP transcript, specifically exon 5 that carries a strong 3’ splice site. Most importantly, the localization of the ESEs correspond to an area that in the paralogous non-tissue specific ALP gene differs in amino acid composition with respect, not only to the placental ALP where I mapped the ESEs but also to the other members of the family, where this area is well conserved. These amino acid changes may represent a possible evolutionary constraint on enzymatic activity, in keeping with this hypothesis, substituting the amino acids in the region of the ESE for those of the paralogous non-tissue specific ALP gene increases the enzymatic activity. Thus splicing-related constraints challenge the primacy of biochemical function in rates of protein evolution.

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