Frequent Gain and Loss of Intronic Splicing Regulatory Elements during the Evolution of Vertebrates

Splicing regulatory elements (SREs) are sequences bound by proteins that influence splicing of nearby splice sites. Constitutively spliced introns have evolved to utilize many different splicing factors. The evolutionary processes that influenced which splicing factors are used for splicing of individual introns are generally unclear. We demonstrate that in the lineage that gave rise to mammals, many introns lost U-rich sequences and gained G-rich sequences, both of which resemble known SREs. The apparent conversion of U-rich to G-rich SREs suggests that the associated splicing factors are functionally equivalent. In support of this we demonstrated that U-rich and G-rich SREs are both capable of promoting splicing of an SRE-dependent splicing reporter. Furthermore, we demonstrate, using the heterologous MS2 tethering system (bacterial MS2 coat fusion-protein and its RNA stem-loop binding site), that both the U-rich SRE-binding protein (TIA1) and the G-rich SRE-binding protein (HNRNPF) can promote splicing of the same intron. We also observed that gain of G-rich SREs is significantly associated with G/C-rich genomic isochores, suggesting that gain or loss of SREs was driven by the same processes that ultimately resulted in the formation of mammalian genomic isochores. We propose the following model for the gain and loss of mammalian SREs. Ancestral U-rich SREs located in genomic regions that were experiencing high rates of A/T to G/C conversion would have suffered frequent deleterious mutations. However, this same process resulted in increased formation of functionally equivalent G-rich SREs, and acquisition of new G-rich SREs decreased purifying selection on the U-rich SREs, which were then free to decay.