Evolutionary Convergence on Highly-Conserved 39 Intron Structures in Intron-Poor Eukaryotes and Insights into the Ancestral Eukaryotic Genome Manuel Irimia1.*, Scott William Roy2.* 1 Departament de Genetica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain, 2 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, United States of America Abstract The presence of spliceosomal introns in eukaryotes raises a range of questions about genomic evolution. Along with the fundamental mysteries of introns’ initial proliferation and persistence, the evolutionary forces acting on intron sequences remain largely mysterious. Intron number varies across species from a few introns per genome to several introns per gene, and the elements of intron sequences directly implicated in splicing vary from degenerate to strict consensus motifs. We report a 50-species comparative genomic study of intron sequences across most eukaryotic groups. We find two broad and striking patterns. First, we find that some highly intron-poor lineages have undergone evolutionary convergence to strong 39 consensus intron structures. This finding holds for both branch point sequence and distance between the branch point and the 39 splice site. Interestingly, this difference appears to exist within the genomes of green alga of the genus Ostreococcus, which exhibit highly constrained intron sequences through most of the intron-poor genome, but not in one much more intron-dense genomic region. Second, we find evidence that ancestral genomes contained highly variable branch point sequences, similar to more complex modern intron-rich eukaryotic lineages. In addition, ancestral structures are likely to have included polyT tails similar to those in metazoans and plants, which we found in a variety of protist lineages. Intriguingly, intron structure evolution appears to be quite different across lineages experiencing different types of genome reduction: whereas lineages with very few introns tend towards highly regular intronic sequences, lineages with very short introns tend towards highly degenerate sequences. Together, these results attest to the complex nature of ancestral eukaryotic splicing, the qualitatively different evolutionary forces acting on intron structures across modern lineages, and the impressive evolutionary malleability of eukaryotic gene structures. Citation: Irimia M, Roy SW (2008) Evolutionary Convergence on Highly-Conserved 39 Intron Structures in Intron-Poor Eukaryotes and Insights into the Ancestral Eukaryotic Genome. PLoS Genet 4(8): e1000148. doi:10.1371/journal.pgen.1000148 Editor: Barbara J. Trask, Fred Hutchinson Cancer Research Center, United States of America Received January 31, 2008; Accepted July 1, 2008; Published August 8, 2008 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: Manuel Irimia was funded by the Spanish Ministerio of Educaci o´n y Ciencia, through the FPI grant (BFU2005-00252), and Scott William Roy by the Intramural Research Program of the National Library of Medicine at National Institutes of Health/DHHS. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mirimia@gmail.com (MI); scottwroy@gmail.com (SWR) . These authors contributed equally to this work. Introduction Spliceosomal introns are genomically-encoded sequences that are removed from RNA transcripts by the spliceosome, a massive RNA-protein complex. The spliceosome and spliceosomal introns are common and ancestral to eukaryotes [1–4], however spliceosomal organization shows striking divergence across species. Intron number per genome differs by orders of magnitude, from fewer than ten known introns in the genomes of some protist species [4,5] to nearly ten per gene in some metazoans [6,7]. Introns also vary dramatically in length, from the ‘bonzaied’ 19 nt introns of the Bigelowiella natans nucleomorph [8] to the giant kilobases-long introns of humans and other mammals. Intron sequence elements, which are important for intron recognition by the RNA and protein components of the spliceosome, also vary significantly across species. In some species, the consensus of an intron sequence is largely restricted to an initial 59 GT dinucleotide (the ‘‘donor’’ site), a terminal 39 AG (the ‘‘acceptor’’), and a degenerate few nt ‘‘branch point’’ site, lying somewhere within the intron. In other species, sequences are more conserved. For instance, in the apicomplexan parasite Cryptospo- ridium, 84% of introns begin with the most common sixmer GTAAGT, enabling close complementary base pairing with the U1 RNA of the spliceosome (compared with only 14% for humans; see examples in Figure 1). We previously studied the phylogenetic pattern of conservation of 59 intronic sequences and found a strong correspondence between species with very few introns and those with such strong 59 consensus sequences [9]. Clear differences are also observed in other structures. For instance, branch points vary across species from a highly conserved seven-mer (TACTAAC) in hemiascomycetous yeasts (e.g., Saccharomyces cerevisiae) to several lineages in which previous studies have failed to find branch points [10,11]. Here we report studies of the evolution of 39 intron structures including the branch point, poly-pyrimidine tract and 39 splice site across 50 species spanning all major eukaryotic kingdoms (opisthokonts, amoebozo- ans, red and green plants, chromalveolates, excavates and rhizarians, Figure 2). PLoS Genetics | www.plosgenetics.org 1 August 2008 | Volume 4 | Issue 8 | e1000148