Testing the "proto-splice sites" model of intron origin: evidence from analysis of intron phase correlations.

A few nucleotide sites of nuclear exons that flank introns are often conserved. A hypothesis has suggested that these sites, called "proto-splice sites," are remnants of recognition signals for the insertion of introns in the early evolution of eukaryotic genes. This notion of proto-splice sites has been an important basis for the insertional theory of introns. This hypothesis predicts that the distribution of proto-splice sites would determine the distribution of intron phases, because the positions of introns are just a subset of the proto-splice sites. We previously tested this prediction by examining the proportions of the phases of proto-splice sites, revealing nothing in these proportion distributions similar to observed proportions of intron phases. Here, we provide a second independent test of the proto-splice site hypothesis, with regard to its prediction that the proto-splice sites would mimic intron phase correlations, using a CDS database we created from GenBank. We tested four hypothetical proto-splice sites G / G, AG / G, AG / GT, and C/AAG / R. Interestingly, while G / G and AG / GT site phase distributions are not consistent with actual introns, we observed that AG / G and C/AAG / R sites have a symmetric phase excess. However, the patterns of the excess are quite different from the actual intron phase distribution. In addition, particular amino acid repeats in proteins were found to partially contribute to the excess of symmetry at these two types of sites. The phase associations of all four sites are significantly different from those of intron phases. Furthermore, a general model of intron insertion into proto-splice sites was simulated by Monte Carlo simulation to investigate the probability that the random insertion of introns into AG / G and C/AAG / R sites could generate the observed intron phase distribution. The simulation showed that (1) no observed correlation of intron phases was statistically consistent with the phase distribution of proto-splice sites in the simulated virtual genes; (2) most conservatively, no simulation in 10,000 Monte Carlo experiments gave a pattern with an excess of symmetric (1, 1) exons larger than those of (0, 0) and (2, 2), a major statistical feature of intron phase distribution that is consistent with the directly observed cases of exon shuffling. Thus, these results reject the null hypothesis that introns are randomly inserted into preexisting proto-splice sites, as suggested by the insertional theory of introns.