Origination of the Split Structure of Spliceosomal Genes from Random Genetic Sequences Rahul Regulapati1., Ashwini Bhasi2., Chandan Kumar Singh3, Periannan Senapathy2,3* 1 Department of Biotechnology, Indian Institute of Technology Madras, Chennai, India, 2 Department of Human Genetics, Genome International Corporation, Madison, Wisconsin, United States of America, 3 Department of Bioinformatics, International Center for Advanced Genomics and Proteomics, Nehru Nagar, Chennai, India Abstract The mechanism by which protein-coding portions of eukaryotic genes came to be separated by long non-coding stretches of DNA, and the purpose for this perplexing arrangement, have remained unresolved fundamental biological problems for three decades. We report here a plausible solution to this problem based on analysis of open reading frame (ORF) length constraints in the genomes of nine diverse species. If primordial nucleic acid sequences were random in sequence, functional proteins that are innately long would not be encoded due to the frequent occurrence of stop codons. The best possible way that a long protein-coding sequence could have been derived was by evolving a split-structure from the random DNA (or RNA) sequence. Results of the systematic analyses of nine complete genome sequences presented here suggests that perhaps the major underlying structural features of split-genes have evolved due to the indigenous occurrence of split protein-coding genes in primordial random nucleotide sequence. The results also suggest that intron- rich genes containing short exons may have been the original form of genes intrinsically occurring in random DNA, and that intron-poor genes containing long exons were perhaps derived from the original intron-rich genes. Citation: Regulapati R, Bhasi A, Singh CK, Senapathy P (2008) Origination of the Split Structure of Spliceosomal Genes from Random Genetic Sequences. PLoS ONE 3(10): e3456. doi:10.1371/journal.pone.0003456 Editor: Dawn Field, NERC Centre for Ecology and Hydrology, United Kingdom Received February 21, 2008; Accepted August 1, 2008; Published October 20, 2008 Copyright: 2008 Senapathy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project has been internally funded by Genome International Corporation. This project is purely an academic project, fulfilling the academic interest of the corresponding author, who owns the company. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ps@genome.com . These authors contributed equally to this work. Introduction Despite intense investigation since the discovery of introns and the split structure of spliceosomal genes nearly 30 years ago [1– 11], the mechanism of their origin has remained a mystery. The complex spliceosomal machinery is geared towards the linking together of exons and the elimination of introns, which are rarely functional [12,13]. The rare functional role of introns is also geared mainly towards splicing, during which such functional introns are themselves eliminated [14,15]. Thus the evolution of introns together with the highly complex spliceosomal machinery to concomitantly eliminate them is perplexing, and the primary molecular mechanisms underlying the origin of introns and the split-gene structure remain unresolved [1,2,11]. Evolutionary studies of introns have primarily been based on the Introns-Early (IE) or the Introns-Late (IL) views. The IE view assumes that introns were present in primitive cells, but does not provide a mechanism for their origination [6,16–18]. The IL view assumes that introns were inserted into preexisting contiguously coding genes, without addressing how the contiguous genes first originated [19,20]. Proponents of both views have focused their work on the phylogenetic distribution of spliceosomal introns, but the results have varied disparately depending upon the particular assumptions and methodologies adopted [1–11,21–25]. Moreover, while these studies explore the history of introns across organisms by comparing the ages of different introns, they have not resolved basic questions about their origin. The ‘‘exon theory of genes’’ states that the introns in early genes were sequence spacers that facilitated efficient recombination between short coding regions (exons) that represented protein domains [6,16,26], and that this led to the evolution of new genes capable of coding for complex multi-domain proteins. This theory assigns a logical role for introns in abetting evolution subsequent to their origin, but does not offer a mechanism of their origin. In recent years, both IE and IL proponents have suggested that introns present in primitive eukaryotes may have arisen by a process distinct from that underlying the evolution of more recently acquired introns [2,3,8,11,25]. It is increasingly realized that a fundamental solution capable of explaining the nascent origin of introns in original split-genes may be possible [1,2,9]. Here we have applied an innovative bioinformatics approach to investigate the basic mechanism by which the split-structure of spliceosomal genes originated. We present a comprehensive comparative analysis of open reading frame (ORF) length constraints in the genomes of nine species, including a mammal, two invertebrate animals, a plant, a protist, fungi and bacteria. We tested whether the genome sequence and ORF properties of these diverse species corroborate or disprove the random-sequence origin of the split-gene (ROSG) model. The ROSG model posits that the indigenous occurrence of protein-coding genes in primordial random DNA sequences may underlie the origin of the intron and the split structure of original genes [27]. The occurrence of stop codons in random DNA would have been too frequent to allow functional proteins to be encoded. A logical PLoS ONE | www.plosone.org 1 October 2008 | Volume 3 | Issue 10 | e3456