Split gene theory

Transcription, splicing and translation of a eukaryotic gene. A eukaryotic gene consists of a promoter, exons, introns and a poly-A addition site. It is transcribed into a primary RNA transcript (or pre-mRNA) by the RNA polymerase enzyme. This RNA undergoes the process of editing by the spliceosome for the precise removal of the introns and joining of the exons, which produces the mRNA molecule. This mRNA contains the complete coding sequence without any interrupting stop codons that is translated by the ribosome into the protein encoded by the gene. In the figure, the lengths of introns are short, but in reality, they are extremely long, on average 20 times longer than the exons, and often even much longer up to around 500,000 bases. The exons are usually short with an average of ~120 bases, and with an upper maximum of ~600 bases.[1] Also shown is an example protein structure (PDB ID: 2VUX) for the Human ribonucleotide reductase, subunit M2 B.

Genes of all organisms, except bacteria, consist of short protein-coding regions (exons) interrupted by long sequences (introns).[1][2] 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.[7][8]

Until Philip Sharp[9][10] and Richard Roberts[11] discovered introns[12] 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.

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.

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.[13] 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.”[14]

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.[15] 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[16] and Walter Gilbert[17][18] 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.[19][20] 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.

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.[21] 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.

Research based on the split gene theory sheds light on other basic questions of exons and introns. The exons of eukaryotes are generally short (human exons average ~120 bases, and can be as short as 10 bases) and introns are usually long (average of ~3,000 bases, and can be several hundred thousands bases long), for example genes RBFOX1, CNTNAP2, PTPRD and DLG2. Senapathy provided a plausible answer to these questions, the only explanation to date. If eukaryotic genes originated from random DNA sequences, they have to match the lengths of ORFs from random sequences, and possibly should be around 100 bases (close to the median length of ORFs in random sequence). The genome sequences of living organisms exhibit exactly the same average lengths of 120 bases for exons, and the longest exons of 600 bases (with few exceptions), which is the same length as that of the longest random ORFs.[1][2][3][22]

If split genes originated in random DNA sequences, then introns would be long for several reasons. The stop codons occur in clusters leading to numerous consecutive short ORFs: longer ORFs that could be defined as exons would be rarer. Furthermore, the best of the coding sequence parameters for functional proteins would be chosen from the long ORFs in random sequence, which may occur rarely. In addition, the combination of donor and acceptor splice junction sequences within short lengths of coding sequence segments that would define exon boundaries would occur rarely in a random sequence. These combined reasons would make introns long compared to exons.

This work also explains why genomes such as the human genome have billions of bases, and why only a small fraction (~2%) codes for proteins and other regulatory elements.[29][30] If split genes originated from random primordial DNA sequences, they would contain a significant amount of DNA that represented by introns. Furthermore, a genome assembled from random DNA containing split genes would also include intergenic random DNA. Thus, genomes that originated from random DNA sequences had to be large, regardless of the complexity of the organism.

The observation that several organisms such as the onion (~16 billion bases[31]) and salamander (~32 billion bases[32]) have much larger genomes than humans (~3 billion bases[33][34]) while the organisms are no more complex than humans comports with the theory. Furthermore, the fact that several organisms with smaller genomes have a similar number of genes as human, such as C. elegans (genome size ~100 million bases, ~19,000 genes)[35] and Arabidopsis thaliana (genome size ~125 million bases, ~25,000 genes),[36] supports the theory. The theory predicts that the introns in the split genes in these genomes could be the “reduced” (or deleted) form compared to larger genes with long introns, thus leading to reduced genomes.[1][22] In fact, researchers have recently proposed that these smaller genomes are actually reduced genomes.[37]

Senapathy addressed the origin of the spliceosomal machinery that edits out the introns from RNA transcripts. If the split genes had originated from random DNA, then the introns would have become an unnecessary but integral part of eukaryotic genes along with the splice junctions. The spliceosomal machinery would be required to remove them and to enable the short exons to be linearly spliced together as a contiguously coding mRNA that can be translated into a complete protein. Thus, the split gene theory argues that spliceosomal machinery exists to remove the unnecessary introns.[1][2]

Blake states, “Work by Senapathy, when applied to RNA, comprehensively explains the origin of the segregated form of RNA into coding and noncoding regions. It also suggests why a splicing mechanism was developed at the start of primordial evolution.”[16]

Senapathy proposed a plausible mechanistic and functional rationale why the eukaryotic nucleus originated, a major question in biology.[1][2] If the transcripts of the split genes and the spliced mRNAs were present in a cell without a nucleus, the ribosomes would try to bind to both the un-spliced primary RNA transcript and the spliced mRNA, which would result in chaos. A boundary that separates the RNA splicing process from the mRNA translation avoids this problem. The nuclear boundary provides a clear separation of the primary RNA splicing and the mRNA translation.

These investigations thus led to the possibility that primordial DNA with essentially random sequence gave rise to the complex structure of the split genes with exons, introns and splice junctions. Cells that harbored split genes had to be complex with a nuclear cytoplasmic boundary, and must have a spliceosomal machinery. Thus, it was possible that the earliest cell was complex and eukaryotic.[1][2][3][22] Surprisingly, findings from extensive comparative genomics research from several organisms since 2007 overwhelmingly show that the earliest organisms could have been highly complex and eukaryotic, and could have contained complex proteins,[38][39][40][41][42][43][44] as predicted by Senapathy's theory.

The spliceosome is a highly complex mechanism, containing ~200 proteins and several SnRNPs. Collins and Penny stated, “We begin with the hypothesis that ... the spliceosome has increased in complexity throughout eukaryotic evolution. However, examination of the distribution of spliceosomal components indicates that not only was a spliceosome present in the eukaryotic ancestor but it also contained most of the key components found in today's eukaryotes. ... the last common ancestor of extant eukaryotes appears to show much of the molecular complexity seen today.” This suggests that the earliest eukaryotic organisms were complex and contained sophisticated genes and proteins.[45]

Genes with uninterrupted coding sequences that are thousands of bases long - up to 90,000 bases - that occur in many bacterial organisms[21] were practically impossible to have occurred. However, the bacterial genes could have originated from split genes by losing introns, the only proposed way to arrive at long coding sequences. It is also a better way than by increasing the lengths of ORFs from short random ORFs to long ORFs by specifically removing the stop codons by mutation.[1][2][3]

Origin of bacterial genes from split genes. The split genes of modern eukaryotes with short exons (average length of 120 bases, and maximum of ~600 bases) interrupted by long introns are extremely probable in random DNA sequences due to the reasons described in the section Origin of introns and the split gene structure, above. In contrast, the long contiguously coding bacterial genes (that can be as long and 10,000 bases, and longer up to 90,000 bases) without introns are practically impossible to occur in random sequences. Thus, the only way that bacterial genes could originate was to delete the introns from the split genes that occurred in random DNA sequences and produce contiguously coding genes. The example protein shown with its 3D structure is from PDB database (ID:1UNF).

According to the split gene theory, this process of intron loss could have happened from prebiotic random DNA. These contiguously coding genes could be tightly organized in the bacterial genomes without any introns and be more streamlined. According to Senapathy, the nuclear boundary that was required for a cell containing split genes would not be required for a cell containing only uninterrupted genes. Thus, the bacterial cells did not develop a nucleus. Based on split gene theory, the eukaryotic genomes and bacterial genomes could have independently originated from the split genes in primordial random DNA sequences.

Senapathy developed algorithms to detect donor and acceptor splice sites, exons and a complete split gene in a genomic sequence. He developed the position weight matrix (PWM) method based on the frequency of the four bases at the consensus sequences of the donor and acceptor in different organisms to identify the splice sites in a given sequence. Furthermore, he formulated the first algorithm to find the exons based on the requirement of exons to contain a donor sequence (at the 5’ end) and an acceptor sequence (at the 3’ end), and an ORF in which the exon should occur, and another algorithm to find a complete split gene. These algorithms are collectively known as the Shapiro-Senapathy algorithm (S&S).[46][47]

This algorithm aids in the identification of splicing mutations that cause disease and adverse drug reactions.[46][47] Scientists used the algorithm to identify mutations and genes that cause cancers, inherited disorders, immune deficiency diseases and neurological disorders. It is increasingly used in clinical practice and research to find mutations in known disease-causing genes in patients and to discover novel genes that are causal of different diseases. Furthermore, it is used in defining the cryptic splice sites and deducing the mechanisms by which mutations can affect normal splicing and lead to different diseases. It is also employed in basic research.

Findings based on S&S have impacted major questions in eukaryotic biology and in human medicine.[48]

The split gene theory implies that structural features of split genes predicted from computer-simulated random sequences occur in eukaryotic split genes. This is borne out in most known split genes. The sequences exhibit a nearly perfect negative exponential distribution of ORF lengths.[1][2][22][3] With rare exceptions, eukaryotic gene exons fall within the predicted 600 base maximum.

The theory correctly predicts that exons are delimited by stop codons, especially at the 3’ ends of exons. Actually they are precisely delimited more strongly at the 3’ ends of exons and less strongly at the 5’ ends in most known genes, as predicted.[1][2][22][3] These stop codons are the most important functional parts of both splice junctions. The theory thus provides an explanation for the “conserved” splice junctions at the ends of exons and for the loss of these stop codons along with introns when they are spliced out. The theory correctly predicts that splice junctions are randomly distributed in eukaryotic DNA sequences.[3][25][46][47] The theory correctly predicts that splice junctions present in transfer RNA genes and ribosomal RNA genes, do not contain stop codons. The lariat signal, another sequence involved in the splicing process, also contains stop codons.[1][2][3][22][25][46][47]

The theory correctly predicts that introns are non-coding and that they are mostly non-functional. Except for some intron sequences including the donor and acceptor splice signal sequences and branch point sequences, and possibly the intron splice enhancers that occur at the ends of introns, which aid in the removal of introns, the vast majority of introns are devoid of any functions. The theory does not exclude rare sequences within introns that could be used by the genome and the cell, especially because introns are so long.

Thus, the theory's predictions are precisely corroborated by the major elements in modern eukaryotic genomes.

Comparative analysis of the modern genome data from several living organisms found that the characteristics of split genes trace back to the earliest organisms. These organisms could have contained the split genes and complex proteins that occur in today's living organisms.[49][50][51][52][53][54][55][56][57]

Studies employing maximum likelihood analysis found that the earliest eukaryotic organisms contained the same genes as modern organisms with yet a higher intron density.[58] Comparative genomics of many organisms including basal eukaryotes[59] (considered to be primitive eukaryotic organisms such as Amoeboflagellata, Diplomonadida, and Parabasalia) showed that intron-rich split genes accompanied and spliceosome from modern organisms were present in their earliest forebears, and that the earliest organisms came with all the eukaryotic cellular components.[60][49][61][62][63][58]

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• Regulapati, R.; Bhasi, A.; Singh, C.K.; Senapathy, P. (2008). "Origination of the Split Structure of Spliceosomal Genes from Random Genetic Sequences". PLOS ONE. 3 (10): 10. Bibcode:2008PLoSO...3.3456R. doi:10.1371/journal.pone.0003456. PMC 2565106. PMID 18941625.
• Senapathy, P. (1995). "Introns and the origin of protein-coding genes". Science. 268 (5215): 1366–7. Bibcode:1995Sci...268.1366S. doi:10.1126/science.7761858. PMID 7761858.