Exon shuffling

Exon shuffling follows certain splice frame rules. Introns can interrupt the reading frame of a gene by inserting a sequence between two consecutive codons (phase 0 introns), between the first and second nucleotide of a codon (phase 1 introns), or between the second and third nucleotide of a codon (phase 2 introns). Additionally exons can be classified into nine different groups based on the phase of the flanking introns (symmetrical: 0-0, 1-1, 2-2 and asymmetrical: 0-1, 0-2, 1-0, 1-2, etc.) Symmetric exons are the only ones that can be inserted into introns, undergo duplication, or be deleted without changing the reading frame. Exon shuffling was first introduced in 1978 when Walter Gilbert discovered that the existence of introns could play a major role in the evolution of proteins. It was noted that recombination within introns could help assort exons independently and that repetitive segments in the middle of introns could create hotspots for recombination to shuffle the exonic sequences. However, the presence of these introns in eukaryotes and absence in prokaryotes created a debate about the time in which these introns appeared. Two theories arose: the 'introns early' theory and the 'introns late' theory. Supporters of the 'introns early theory' believed that introns and RNA splicing were the relics of the RNA world and therefore both prokaryotes and eukaryotes had introns in the beginning. However, prokaryotes eliminated their introns in order to obtain a higher efficiency, while eukaryotes retained the introns and the genetic plasticity of the ancestors. On the other hand, supporters of the 'introns late' theory believe that prokaryotic genes resemble the ancestral genes and introns were inserted later in the genes of eukaryotes. What is clear now is that the eukaryotic exon-intron structure is not static, introns are continually inserted and removed from genes and the evolution of introns evolves parallel to exon shuffling. In order for exon shuffling to start to play a major role in protein evolution the appearance of spliceosomal introns had to take place. This was due to the fact that the self-splicing introns of the RNA world were unsuitable for exon-shuffling by intronic recombination. These introns had an essential function and therefore could not be recombined. Additionally there is strong evidence that spliceosomal introns evolved fairly recently and are restricted in their evolutionary distribution. Therefore, exon shuffling became a major role in the construction of younger proteins. Moreover, to define more precisely the time when exon shuffling became significant in eukaryotes, the evolutionary distribution of modular proteins that evolved through this mechanism were examined in different organisms (i.e., Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, etc.) These studies suggested that there was an inverse relationship between the genome compactness and the proportion of intronic and repetitive sequences. As well as the fact that exon shuffling became significant after metazoan radiation. Evolution of eukaryotes is mediated by sexual recombination of parental genomes and since introns are longer than exons most of the crossovers occur in noncoding regions. In these introns there are large numbers of transposable elements and repeated sequences which promote recombination of nonhomologous genes. In addition it has also been shown that mosaic proteins are composed of mobile domains which have spread to different genes during evolution and which are capable of folding themselves. There is a mechanism for the formation and shuffling of said domains, this is the modularization hypothesis. This mechanism is divided into three stages. The first stage is the insertion of introns at positions that correspond to the boundaries of a protein domain. The second stage is when the 'protomodule' undergoes tandem duplications by recombination within the inserted introns. The third stage is when one or more protomodules are transferred to a different nonhomologous gene by intronic recombination. All states of modularization have been observed in different domains such as those of hemostatic proteins. A potential mechanism for exon shuffling is the long interspersed element (LINE) -1 mediated 3' transduction. However it is important first to understand what LINEs are. LINEs are a group of genetic elements that are found in abundant quantities in eukaryotic genomes. LINE-1 is the most common LINE found in humans. It is transcribed by RNA polymerase II to give an mRNA that codes for two proteins: ORF1 and ORF2, which are necessary for transposition. Upon transposition, L1 associates with 3' flanking DNA and carries the non-L1 sequence to a new genomic location. This new location does not have to be in a homologous sequence or in close proximity to the donor DNA sequence. The donor DNA sequence remains unchanged throughout this process because it functions in a copy-paste manner via RNA intermediates; however, only those regions located in the 3' region of the L1 have been proven to be targeted for duplication.