Figure 7. The ROSG model. According to the ROSG model, mRNA splicing evolved to overcome the problem of the frequent occurrence of stop codons in primordial random DNA that severely restricted ORF lengths. (A) Stop codons occurred too frequently to allow functional proteins to be encoded in random DNA. Long contiguous coding sequences were made by the splicing together of short coding-pieces occurring within short ORFs (which became exons) and the elimination of the intervening random sequences (which became introns). (B) Consistent with this model, stop codons are present at exon borders at uniquely high frequencies. The majority of the codons that border the 39 end of exons are stop codons and all three stop-codons occur at this position (Table S1). Meanwhile one stop-codon (TAG) is predominant at the 59 end. doi:10.1371/journal.pone.0003456.g007 sequence, a long ORF could only be derived by the splicing together of these short ORFs containing the short exons. If a large genome with intron-rich genes began to lose introns, there would be a drastic reduction in the size of the genome—as approximately 95% of a gene constitutes introns in the large intron-rich genomes such as the human, the content of introns being 20–25 fold higher than that of exons. Simultaneously, such intron loss may also lead to a significant increase in the average length of the exons in the genome. The results here reflect this theme by the reduction of the genome size from the large (human) to the small (Plasmodium) genome, as their average exon length increased, and as the fraction of the non-conforming ORFs increased. Even though the variations of the fraction of non-conforming ORFs among different genomes (human to E. coli) occurred within a tiny range of less than 1%, the increase in the average length of ORFs in the different genomes correlated clearly with the corresponding increase in the average length of exons, which in turn correlated with the reduction in genome size. This result also indicates that a majority of the non-conforming ORFs in each of the genomes was accounted for by the non-conforming exons in the genome. The present results suggest that the preexistence of long non- random coding sequences in random DNA is exceedingly improbable. Thus, under the ROSG model, prokaryotic genes may not have been possible to occur in a random sequence. The possible conclusion is that introns were present in the most primitive genes, the very first genes were intron-dense, and that the genes of the smaller intron-poor (eukaryotic parasites and yeast) and intron- Origin of Split Gene Structure less (bacteria) genomes were derived from the intron-rich original genes by loss of introns. Our results are supported by the recent findings that introns may have been numerous before the divergence of eukaryotes and prokaryotes [2,3,5]. The current results suggest that more introns may have been lost over the course of evolution in the genomes which are presently smaller in size, and which have more non-conforming ORFs and exons (Table 1). Interestingly, among the genomes examined here, the human seems to be the closest to random (retaining most of the original introns) and the E. coli seems to be the farthest from random. It is remarkable that all the characteristics of random DNA are still essentially present in the split genes of present day intron-dense large genomes such as those in the human. The Arabidopsis (120 M bases) and Caenorhabditis (100 M bases) genomes are exceptional in that they are relatively small but have high intron densities, owing to their distinctively short introns. It is possible that their introns were originally long, and that they were perhaps shortened during evolution by yet unknown biological processes. This may perhaps mean that the prokaryotic genes may have lost all their introns. It may also mean that the introns present in microbes such as the archeabacteria are the residual remnants of the leftover introns. The recent discovery of the possible extensive transcription of non-coding RNAs (ncRNAs) from the genome [34] supports the ROSG model. These ncRNAs occur at very high probability in random DNA (data not shown), as most of these RNAs such as the miRNAs and siRNAs are very short. Sequences such as the exon- splice enhancers (that help the removal of introns) within introns are also highly probable in random DNA. Several functional sequences could have fortuitously occurred in these originally non- functional sequences that were useful in the evolution of the genome and the organism. Consistent with the ROSG model, the present study may show that the original reason for the gene to be split was the stop-codon interference problem in a random sequence, and that functional sequences may have fortuitously occurred with high probability within these long non-coding introns (and intergenic sequences) subsequent to their origin in random DNA. Consistent multiple evidence for the ROSG model The current study brings out three major lines of corroborating evidence for the random-sequence origin of protein coding split- genes. First, the very high congruence of the negative exponential distribution of ORF lengths between the intron-rich genomes (e.g., human) and the random DNA sequence (.99.97%, R = 0.9986). Second, the upper limit for the length of exons in intron-rich genomes (that codes for ,200 amino acid long protein) was found to be the same as the longest coding sequence in random sequence. Third, the distribution of actual exon lengths in intron- rich genomes is congruent with and falls below the distribution of the lengths of random ORFs. The average exon length from the intron-rich genomes is about 170 bases whereas that expected from random ORF lengths is 60 bases. This may indicate that there has been a selection for longer exons within the allowed maximum ORF length of 600 bases for optimizing the frequency of suitable exon lengths. Fourth, the very small fraction of the non- conforming exons (,2%) in intron-rich genomes (e.g., human) are contained within the extremely small fraction of the non- conforming ORFs (0.03%), and a majority of the non-conforming ORFs are filled with non-conforming exons. This phenomenon is observed in every genome examined, even where the fraction of the non-conforming exons increases. Thus the current study may provide mathematical evidence that the long non-conforming ORFs and exons in smaller genomes were derived by splicing PLoS ONE | www.plosone.org 9 October 2008 | Volume 3 | Issue 10 | e3456