Massive intron gain in the most intron-rich eukaryotes is driven by introner-like transposable elements of unprecedented diversity and flexibility

SummarySpliceosomal introns, which interrupt nuclear genes and are removed from RNA transcripts by machinery termed spliceosomes, are ubiquitous features of eukaryotic nuclear genes [1]. Patterns of spliceosomal intron evolution are complex, with some lineages exhibiting virtually no intron creation while others experience thousands of intron gains [2–5]. One possibility is that this punctate phylogenetic distribution is explained by intron creation by Introner-Like Elements (ILEs), transposable elements capable of creating introns, with only those lineages harboring ILEs undergoing massive intron gain [6–10]. However, ILEs have been reported in only four lineages. Here we study intron evolution in dinoflagellates. The remarkable fragmentation of nuclear genes by spliceosomal introns reaches its apex in dinoflagellates, which have some twenty introns per gene [11,12]. Despite this, almost nothing is known about the molecular and evolutionary mechanisms governing dinoflagellate intron evolution. We reconstructed intron evolution in five dinoflagellate genomes, revealing a dynamic history of intron loss and gain. ILEs are found in 4/5 studied species. In one species, Polarella glacialis, we find an unprecedented diversity of ILEs, with ILE insertion leading to creation of some 12,253 introns, and with 15 separate families of ILEs accounting for at least 100 introns each. These ILE families range in mobilization mechanism, mechanism of intron creation, and flexibility of mechanism of intron creation. Comparison within and between ILE families provides evidence that biases in so-called intron phase, the distribution of introns relative to codon periodicity, are driven by ILE insertion site requirements [9,13,14]. Finally, we find evidence for multiple additional transformations of the spliceosomal system in dinoflagellates, including widespread loss of ancestral introns, and alterations in required, tolerated and favored splice motifs. These results reveal unappreciated intron creating elements diversity and spliceosomal evolutionary capacity, and suggest complex evolutionary dependencies shaping genome structures.

Intron gain by tandem genomic duplication: a novel case in a potato gene encoding RNA-dependent RNA polymerase

The origin and subsequent accumulation of spliceosomal introns are prominent events in the evolution of eukaryotic gene structure. However, the mechanisms underlying intron gain remain unclear because there are few proven cases of recently gained introns. In anRNA-dependent RNA polymerase(RdRp) gene, we found that a tandem duplication occurred after the divergence of potato and its wild relatives among otherSolanumplants. The duplicated sequence crosses the intron-exon boundary of the first intron and the second exon. A new intron was detected at this duplicated region, and it includes a small previously exonic segment of the upstream copy of the duplicated sequence and the intronic segment of the downstream copy of the duplicated sequence. The donor site of this new intron was directly obtained from the small previously exonic segment. Most of the splicing signals were inherited directly from the parental intron/exon structure, including a putative branch site, the polypyrimidine tract, the 3′ splicing site, two putative exonic splicing enhancers, and the GC contents differed between the intron and exon. In the widely cited model of intron gain by tandem genomic duplication, the duplication of an AGGT-containing exonic segment provides the GT and AG splicing sites for the new intron. Our results illustrate that the tandem duplication model of intron gain should be diverse in terms of obtaining the proper splicing signals.

Spliceosomal intronogenesis

The presence of intervening sequences, termed introns, is a defining characteristic of eukaryotic nuclear genomes. Once transcribed into pre-mRNA, these introns must be removed within the spliceosome before export of the processed mRNA to the cytoplasm, where it is translated into protein. Although intron loss has been demonstrated experimentally, several mysteries remain regarding the origin and propagation of introns. Indeed, documented evidence of gain of an intron has only been suggested by phylogenetic analyses. We report the use of a strategy that detects selected intron gain and loss events. We have experimentally verified, to our knowledge, the first demonstrations of intron transposition in any organism. From our screen, we detected two separate intron gain events characterized by the perfect transposition of a reporter intron into the yeast genes RPL8B and ADH2, respectively. We show that the newly acquired introns are able to be removed from their respective pre-mRNAs by the spliceosome. Additionally, the novel allele, RPL8Bint, is functional when overexpressed within the genome in a strain lacking the Rpl8 paralogue RPL8A, demonstrating that the gene targeted for intronogenesis is functional.