Introns may be lost or gained over evolutionary time, as shown by many comparative studies of orthologous genes. Subsequent analyses have identified thousands of examples of intron loss and gain events, and it has been proposed that the emergence of eukaryotes, or the initial stages of eukaryotic evolution, involved an intron invasion. Two definitive mechanisms of intron loss, reverse transcriptase-mediated intron loss (RTMIL) and genomic deletions, have been identified, and are known to occur. The definitive mechanisms of intron gain, however, remain elusive and controversial. At least seven mechanisms of intron gain have been reported thus far: intron transposition, transposon insertion, tandem genomic duplication, intron transfer, intron gain during double-strand break repair (DSBR), insertion of a group II intron, and intronization. In theory it should be easiest to deduce the origin of recently gained introns due to the lack of host-induced mutations, yet even introns gained recently did not arise from any of the aforementioned mechanisms. These findings thus raise the question of whether or not the proposed mechanisms of intron gain fail to describe the mechanistic origin of many novel introns because they are not accurate mechanisms of intron gain, or if there are other, yet to be discovered, processes generating novel introns.
In intron transposition, the most commonly purported intron gain mechanism, a spliced intron is thought to reverse splice into either its own mRNA or another mRNA at a previously intron-less position. This intron-containing mRNA is then reverse transcribed and the resulting intron-containing cDNA may then cause intron gain via complete or partial recombination with its original genomic locus. Transposon insertions can also result in intron creation. Such an insertion could intronize the transposon without disrupting the coding sequence when a transposon inserts into the sequence AGGT, resulting in the duplication of this sequence on each side of the transposon. It is not yet understood why these elements are spliced, whether by chance, or by some preferential action by the transposon. In tandem genomic duplication, due to the similarity between consensus donor and acceptor splice sites, which both closely resemble AGGT, the tandem genomic duplication of an exonic segment harboring an AGGT sequence generates two potential splice sites. When recognized by the spliceosome, the sequence between the original and duplicated AGGT will be spliced, resulting in the creation of an intron without alteration of the coding sequence of the gene. Double-stranded break repair via non-homologous end joining was recently identified as a source of intron gain when researchers identified short direct repeats flanking 43% of gained introns in Daphnia. These numbers must be compared to the number of conserved introns flanked by repeats in other organisms, though, for statistical relevance. For group II intron insertion, the retrohoming of a group II intron into a nuclear gene was proposed to cause recent spliceosomal intron gain.
Intron transfer has been hypothesized to result in intron gain when a paralog or pseudogene gains an intron and then transfers this intron via recombination to an intron-absent location in its sister paralog. Intronization is the process by which mutations create novel introns from formerly exonic sequence. Thus, unlike other proposed mechanisms of intron gain, this mechanism does not require the insertion or generation of DNA to create a novel intron.
The only hypothesized mechanism of recent intron gain lacking any direct evidence is that of group II intron insertion, which when demonstrated in vivo, abolishes gene expression. Group II introns are therefore likely the presumed ancestors of spliceosomal introns, acting as site-specific retroelements, and are no longer responsible for intron gain. Tandem genomic duplication is the only proposed mechanism with supporting in vivo experimental evidence: a short intragenic tandem duplication can insert a novel intron into a protein-coding gene, leaving the corresponding peptide sequence unchanged. This mechanism also has extensive indirect evidence lending support to the idea that tandem genomic duplication is a prevalent mechanism for intron gain. The testing of other proposed mechanisms in vivo, particularly intron gain during DSBR, intron transfer, and intronization, is possible, although these mechanisms must be demonstrated in vivo to solidify them as actual mechanisms of intron gain. Further genomic analyses, especially when executed at the population level, may then quantify the relative contribution of each mechanism, possibly identifying species-specific biases that may shed light on varied rates of intron gain amongst different species.