PT - JOURNAL ARTICLE AU - Graham E. Larue AU - Scott W. Roy TI - Where the Minor Things Are: A Pan-Eukaryotic Survey Suggests Neutral Processes May Dominate Minor Spliceosomal Intron Evolution AID - 10.1101/2022.09.24.509304 DP - 2022 Jan 01 TA - bioRxiv PG - 2022.09.24.509304 4099 - http://biorxiv.org/content/early/2022/12/04/2022.09.24.509304.short 4100 - http://biorxiv.org/content/early/2022/12/04/2022.09.24.509304.full AB - Spliceosomal introns are segments of eukaryotic pre-mRNA that are removed (“spliced”) during creation of mature mRNA, and are one of the defining and domain-specific features of gene structure in eukaryotes. Introns are spliced by large, multi-subunit ribonucleoprotein machineries called the spliceosomes. Some eukaryotic genomes contain two distinct sets of this machinery—the major (or U2-type) spliceosome is responsible for removal of the vast majority (usually > 99%) of introns in a given genome, with the minor (or U12-type) spliceosome processing the remaining minuscule fraction. Despite in some cases only being responsible for the removal of single-digit numbers of introns, the minor splicing system has been maintained in many species over the roughly 1.5 billion years of eukaryotic evolution since the last eukaryotic common ancestor, and a number of recent studies have suggested that minor introns may be involved in certain aspects of cell cycle regulation and cancer development. It is in this context that we present a broad bioinformatic survey of minor introns across more than 3000 eukaryotic genomes, providing a dramatic increase in information about their distribution in extant species. In contrast to the general association of minor introns with plants and animals, we find surprisingly high numbers of minor introns in certain fungi and green algae, and find notable cases of loss in mites, Dipterans and crustaceans, underscoring the dynamic and diverse histories of minor introns in different lineage. In addition, these data allow us to answer many long-standing questions about these mysterious genomic elements. We find several previously overlooked biases in minor introns’ positional and genic distribution. In addition, we use provide the first estimates of ancestral intron densities. Estimated densities are comparable to those in most minor intron-rich modern species (excluding the single case of recent massive gain), suggesting these species largely retain ancestral minor intron complements. Finally, we find several findings that suggest that much of minor intron evolution is explained by neutral processes. First, contrary to previous reports, we find no evidence for preferential conservation of minor introns; however, we also find no evidence for preferential loss of minor introns from the genome, even in those lineages undergoing the most dramatic reduction in minor intron numbers. Second, we find that observed functional biases in minor introncontaining genes are largely explain these genes’ general greater age, in turn reflecting minor introns’ creation early in evolution. Third, in contrast to reports in animal and possibly plants, we find that patterns of gene expression in an intron-rich fungus do not support an association of minor intron splicing with cell pro-liferation. This finding suggests that these regulatory roles may be more recently evolved, in which case these functional roles could not explain minor introns’ persistence in diverse ancient and recent lineages. These data constitute the most comprehensive views to date of modern minor introns, their evolutionary history, and the forces shaping minor splicing, and provide a foundation for future studies of these remarkable genomic elements.Competing Interest StatementThe authors have declared no competing interest.