short exons from the pre-existing intron-rich genes of larger genomes. The fifth evidence is that the stop-codons that ought to mark out introns do in fact mark them out. The long-observed presence of GURA and YAG at the intron ends has been taken for granted that they ought to be the integral parts of splice junctions, without asking the fundamental question as to why the stop codons that comprise them got to be there in the first place. The current work provides a reasonable mechanism for their origin from the random sequences. Origin of the spliceosomal machinery for splicing short ORFs The ROSG model proposes that protein-coding sequences simply occurred intrinsically in random DNA sequences in a split form that led to the evolution of split-genes for particular proteins. Our other recent research has shown that all of the authentic structures of complex split genes occur intrinsically within random genetic sequences such that an abundant number of split genes were found in a small amount of random DNA (A. Bhasi, B. Balan, B. Kumar and P. Senapathy, Unpublished). Furthermore, numerous split coding genes for complex protein sequences were found to exist intrinsically in random genetic sequences because of the high variability of the amino acid sequences in proteins and the high degeneracy of the codons in coding sequences (P. Senapathy, Unpublished) [36]. If this is the case, a vast number of split genes, each coding for a unique complex protein, could occur indigenously in random genetic sequences. This may enable the trial of different permutations and combinations of genes for various biological structures and functions, and thus pave the way for the evolution of biological systems and possibly complete genomes [36]. Essentially, the ROSG model suggests that split-genes for complex proteins were available in a relatively small amount of random genetic sequences, and these genes could assemble in various combinations to evolve genomes [36]. For example, many complex proteins must have been required for the cellular processes, such as the ribosome or the spliceosome, to have come into existence. These proteins must have been present all at once, or there would have been no meaningful structure of the ribosome or the spliceosome. Recent research shows that almost all of the 110 proteins that constitute the spliceosome must have been present in the eukaryotic ancestor [35]. There has been no organism including the microbes that has any simpler version of the spliceosome. In essence, when the spliceosome came into existence in the first living cell, it must have been as complex as that is found in modern living cells. The ROSG model suggests that this was possible because complete split-genes coding for numerous complex proteins may have simultaneously occurred indigenously in random genetic sequences, from which the correct combination of genes for a particular structure such as the spliceosome was highly probable to be assembled and evolved. The present findings may suggest a plausible basis for the origin of spliceosomal introns. They indicate that introns may have been the random sequences that needed to be eliminated in order for the coding sequence pieces available within unconnected short ORFs in random DNA to be joined. The spliceosomal machinery may have ultimately evolved to accomplish the ejection of the unneeded introns. The facts that introns rarely exhibit biological function and that the spliceosome functions only to eliminate introns support this interpretation. The results may thus corroborate the ROSG model that the occurrence of the essentially non-coding and useless intron sequences within genes was necessitated due to their origin from random genetic sequences, and that a machinery was required for removing the genetic waste. The results may also suggest that the spliceosomal Origin of Split Gene Structure machinery may have possibly originated within the earliest living cells, which required long functional proteins to be produced from the original genome that contained intron rich split-genes. The recent findings that almost all of the proteins that constitute the modern spliceosome may have been present in the eukaryotic ancestor [35] corroborate the ROSG model’s suggestion that the spliceosome necessarily originated within the first eukaryotic cells. The results here may thus explain why introns are essentially non- functional, why they are ubiquitous in eukaryotic genes, why they should have co-originated with the earliest nascent eukaryotic genes, and why they are dispensed off at the expense of the large and complex spliceosomal machinery. These interesting possibilities are supported by the recent studies of phylogenetic intron distributions, showing that the large intron-dense genomes may have been structurally closer to that of the eukaryotic ancestor [1–3]. More recently, several entirely different approaches, based on the comparative genomics of gene content, protein content, protein domain content and the organization of protein domains, have been undertaken for determining the root of the evolutionary tree. These studies have pointed to the intriguing possibility that the genome of the last universal common ancestor (LUCA) of life may have had a complex, eukaryotic-like, gene-rich, sophisticated, and relatively modern organization [37–40]. However, these studies have not addressed the fundamental questions: How, when and wherefrom did the genes of the earliest eukaryotic ancestor originate? What was the source and mechanism of the origin of the complex structures of split-genes in the eukaryotic ancestor? The present results based on the ROSG concept may provide credible answers to these questions. In conclusion, the results show that the spliceosomal genes may have originated indigenously in primordial random genetic sequences, which may explain the origin of both the intervening sequences and the split structure of the original protein-coding genes. This study may provide a reasonable explanation for why the introns are non-coding and non-functional, and why the original genes may have been rich in introns. It also suggests that the large, intron-rich genes containing short exons were perhaps closer to the original genes present in the eukaryotic ancestor, and that the shorter, intron-sparse genes containing relatively longer exons may have been derived by successive loss of introns. This study thus shows the possibility that the genome of the very first living cell may have prebiotically originated from complex split- genes occurring in primordial random genetic sequences— explaining why the LUCA must have been a eukaryote with a complex genome. Methods Datasets Reference sequence assemblies of different organisms were downloaded (Jan 2006) from NCBI (ftp://ftp.ncbi.nih.gov/ genomes/) in FASTA and GBK formats. All gaps within genome sequences were removed as a preliminary step. Generation of ORF and ARF frequencies from the genome and random sequences Only continuous sequences were extracted from genomes. For each reading frame, the lengths of successive ORFs were computed by counting the number of bases between successive stop codons (TAG, TGA, TAA). This was done in all the 6 frames (3 in each strand). The frequencies of these lengths were plotted as a scatter graph and compared with those from a random sequence. The random sequences were generated by a random base PLoS ONE | www.plosone.org 10 October 2008 | Volume 3 | Issue 10 | e3456