The split gene theory offers an explanation for the origin of eukaryotic introns. It suggests that random primordial DNA sequences would only permit short (< 600bp) open reading frames (ORFs) due to frequent stop codons. The short ORFs could have contained the short protein-coding exons observed in eukaryotic genes, whereas the intervening sequences with numerous stop codons could have formed long non-coding introns. In this introns-first framework, the spliceosomal machinery evolved due to the necessity to join exons into longer protein-coding sequences, and intron-less bacterial genes were derived from split eukaryotic genes through the loss of introns. The theory was introduced by Periannan Senapathy.
The theory provides solutions for the origin of split gene architecture, including exons, introns, splice junctions, and branch points from random genetic sequences. It also provides possible solutions for the origin of the spliceosomal machinery, the nuclear boundary, and the eukaryotic cell from prebiotic chemistry.
This theory led to the Shapiro–Senapathy algorithm, which provides a methodology for detecting splice sites in eukaryotic DNA, and has been used to find splice site mutations that cause hundreds of diseases.
The split gene theory contradicts the scientific consensus about the formation of eukaryotic cells by endosymbiosis of bacteria. In 1994, Senapathy wrote a book about this aspect of his theory - The Independent Birth of Organisms. It proposed that multiple eukaryotic genomes originated independently from a primordial pool of split genes. Dutch biologist Gert Korthoff criticized the theory by posing various problems that cannot be explained by a theory of independent origins. He pointed out that various eukaryotes need nurturing and called this the 'boot problem', in that even the initial eukaryote needed parental care. Korthoff notes that a large fraction of eukaryotes are parasites. Senapathy's theory would require a coincidence to explain their existence. Senapathy's theory cannot explain the strong evidence for common descent (homology, universal genetic code, embryology, fossil record.)
Background
Genes of all organisms, except bacteria, consist of short protein-coding regions (exons) interrupted by long sequences (introns). When a gene is expressed, its DNA sequence is copied into a "primary RNA" sequence by the enzyme RNA polymerase. Then the "spliceosome" machinery physically removes the introns from the RNA copy of the gene by the process of splicing, leaving only a contiguously connected series of exons, which becomes messenger RNA (mRNA). This mRNA is now read by the ribosome, which produces the encoded protein. Thus, although introns are not physically removed from a gene, a gene's sequence is read as if introns were not present.
Exons are usually short, with an average length of about 120 bases (e.g. in human genes). Intron lengths vary widely from 10 to 500,000, but exon lengths have an upper bound of about 600 bases in most eukaryotes. Because exons code for protein sequences, they are important for the cell, yet constitute only ~2% of the sequences. Introns, in contrast, constitute 98% of the sequences but seem to have few crucial functions, except for enhancer sequences and developmental regulators in rare instances.
Until Philip Sharp and Richard Roberts discovered introns within eukaryotic genes in 1977, it was believed that the coding sequence of all genes was always in one single stretch, bounded by a single long ORF. The discovery of introns was a profound surprise, which instantly brought up the questions of how, why and when the introns came into the eukaryotic genes.
It soon became apparent that a typical eukaryotic gene was interrupted at many locations by introns, dividing the coding sequence into many short exons. Also surprising was that the introns were long, as long as hundreds of thousands of bases. These findings prompted the questions of why many introns occur within a gene (for example, ~312 introns occur in the human gene TTN), why they are long, and why exons are short.
+The longest introns in the human genomeGene symbolGene length(bases)Longest Intron length(bases)Number of introns in the geneROBO2 1,743,2691,160,411104KCNIP41,220,1831,097,90376ASIC21,161,8771,043,91118NRG11,128,573956,398177DPP101,403,453866,399142 It was also discovered that the spliceosome machinery was large and complex with ~300 proteins and several SnRNA molecules. The questions extended to the origin of the spliceosome. Soon after the discovery of introns, it became apparent that the junctions between exons and introns on either side exhibited specific sequences that directed the spliceosome machinery to the exact base position for splicing. How and why these splice junction signals came into being was another important question.
History The discovery of introns and the split gene architecture of the eukaryotic genes started a new era of eukaryotic biology. The question of why eukaryotic genes had fragmented genes prompted speculation and discussion almost immediately.
Ford Doolittle published a paper in 1978 in which he stated that most molecular biologists assumed that the eukaryotic genome arose from a 'simpler' and more 'primitive' prokaryotic genome rather like that of Escherichia coli. However, this type of evolution would require that introns be introduced into the coding sequences of bacterial genes. Regarding this requirement, Doolittle said, "It is extraordinarily difficult to imagine how informationally irrelevant sequences could be introduced into pre-existing structural genes without deleterious effects." He stated "I would like to argue that the eukaryotic genome, at least in that aspect of its structure manifested as 'genes in pieces' is in fact the primitive original form."
James Darnell expressed similar views in 1978. He stated, "The differences in the biochemistry of messenger RNA formation in eukaryotes compared to prokaryotes are so profound as to suggest that sequential prokaryotic to eukaryotic cell evolution seems unlikely. The recently discovered non-contiguous sequences in eukaryotic DNA that encode messenger RNA may reflect an ancient, rather than a new, distribution of information in DNA and that eukaryotes evolved independently of prokaryotes."
However, in an apparent attempt to reconcile with the idea that RNA preceded DNA in evolution, and with the concept of the three evolutionary lineages of archea, bacteria and eukarya, both Doolittle and Darnell deviated from their original speculation in a joint paper in 1985. They suggested that the ancestor of all three groups of organisms, the 'progenote,' had a genes-in-pieces structure, from which all three lineages evolved. They speculated that the precellular stage had primitive RNA genes which had introns, which were reverse transcribed into DNA and formed the progenote. Bacteria and archea evolved from the progenote by losing introns, and 'urkaryote' evolved from it by retaining introns. Later, the eukaryote evolved from the urkaryote by evolving a nucleus and absorbing mitochondria from bacteria. Multicellular organisms then evolved from the eukaryote.
These authors predicted that the distinctions between the prokaryote and the eukaryote were so profound that the prokaryote to eukaryote evolution was not tenable, and had different origins. However, other than the speculations that the precellular RNA genes must have had introns, they did not address the key questions of intron origin. No explanations described why exons were short and introns were long, how the splice junctions originated, what the structure and sequence of the splice junctions meant, and why eukaryote genomes were large.
Around the same time that Doolittle and Darnell suggested that introns in eukaryotic genes could be ancient, Colin Blake and Walter Gilbert published their views on intron origins independently. In their view, introns originated as spacer sequences that enabled convenient recombination and shuffling of exons that encoded distinct functional domains in order to evolve new genes. Thus, new genes were assembled from exon modules that coded for functional domains, folding regions, or structural elements from preexisting genes in the genome of an ancestral organism, thereby evolving genes with new functions. They did not specify how exons or introns originated. In addition, even after many years, extensive analysis of thousands of proteins and genes showed that only extremely rarely do genes exhibit the supposed exon shuffling phenomenon. Furthermore, molecular biologists questioned the exon shuffling proposal, from a purely evolutionary view for both methodological and conceptual reasons, and, in the long run, this theory did not survive.
Hypothesis Around the time introns were discovered, Senapathy was asking how genes themselves could have originated. He surmised that for any gene to come into being, genetic sequences (RNA or DNA) must have been present in the prebiotic environment. A basic question he asked was how protein-coding sequences could have originated from primordial DNA sequences at the origin of the first cells.
To answer this, he made two basic assumptions:
before a self-replicating cell could come into existence, DNA molecules were synthesized in the primordial soup by random addition of the 4 nucleotides without the help of templates and the nucleotide sequences that code for proteins were selected from these preexisting random DNA sequences in the primordial soup, and not by construction from shorter coding sequences.
He also surmised that codons must have been established prior to the origin of the first genes. If primordial DNA did contain random nucleotide sequences, he asked: Was there an upper limit in coding-sequence lengths, and, if so, did this limit play a crucial role in the formation of the structural features of genes at the origin of genes?
His logic was the following. The average length of proteins in living organisms, including the eukaryotic and bacterial organisms, was ~400 amino acids. However, much longer proteins existed, even longer than 10,000-30,000 amino acids in both eukaryotes and bacteria. Thus, the coding sequence of thousands of bases existed in a single stretch in bacterial genes. In contrast, the coding sequence of eukaryotes existed only in short segments of exons of ~120 bases regardless of the length of the protein. If the coding sequence ORF lengths in random DNA sequences were as long as those in bacterial organisms, then long, contiguous coding genes were possible in random DNA. This was not known, as the distribution of ORF lengths in a random DNA sequence had never been studied.
As random DNA sequences could be generated in the computer, Senapathy thought that he could ask these questions and conduct his experiments in silico. Furthermore, when he began studying this question, sufficient DNA and protein sequence information existed in the National Biomedical Research Foundation (NBRF) database in the early 1980s.
Testing the hypothesis
Origin of introns/split genes