Introns are noncoding DNA sequences that interrupt genes in higher organisms, and are removed prior to the manufacture of a protein. Some species have just a couple of introns in their entire genome, and others have many introns inserted into almost every gene. The causes of these differences are unknown despite much prior work on the subject, and solving the problem remains a major challenge for evolutionary genetics. Currently, we know very little about how new introns arise; this is because in nearly all species that have been studied, all the introns are of ancient origin. Active loss and gain of introns is known in only one species, the waterflea Daphnia pulex. The PIs will conduct genomic analyses of this species and a related one in which introns are stable. They will use extensive surveys of populations at the genomic level to learn where the new introns come from, the mechanisms of insertion, whether newly inserted introns persist, and rates at which new introns arise and are lost. This project has a high likelihood of substantially improving our understanding of the origin, mechanisms, and fates of new introns in Daphnia - results that will be applicable to higher organisms in general.
The PIs will mentor undergraduates through independent supervised projects using molecular bench work and bioinformatic analyses, as well as giving the students opportunities to develop communication skills. This research will also provide opportunities for international collaboration.
This dissertation award enabled Wenli Li to pursue the first study ever to observe the origin of introns in action. Introns, sequences inserted into protein-coding genes that must be spliced out from messenger RNAs, remain one of the greatest mysteries in genome evolution, primarily because they appear to be nonadaptive, and yet are extremely abundant in most genomes (>100,000 in the human genome, for example). Essentially nothing is known about the mechanisms by which they arise. The astonishing harvest of intron gains documented in Li et al. (2009) strongly suggested that further work in the microcrustacean Daphnia pulex would provide many more cases of intron gain/loss, allowing us to test a number of hypotheses. First, if exogenous filler DNA following double-strand break (DSB) repair is responsible for intron gains, as we originally postulated, we should find sequences (such as direct repeats) consistent with this model. Second, if NUMTs (mitochondrial genome fragments transferred to the nuclear genome) are a common filler DNA in DSB NHEJ (nonhomologous end-joining), we expected to find numerous neo-introns with homology to Daphnia mitochondrial DNA. Third, if intron colonization is opposed by natural selection, we expected to see an enrichment of newly inserted introns in populations with small effective population sizes, as well as within obligately asexual clones, because in both cases random genetic drift has an elevated role in shaping genome architecture relative to purifying selection. Finally, if recently inserted introns are transient and deleterious, the frequency distribution of intron-containing alleles should be more skewed towards lower frequencies than in the situation for neutral SNPs. Performing comparative studies on several fully sequenced genomes, we were able to address all of these questions, and quite a few more. We identified nearly 150 newly arisen introns in the species (so young that they have not yet spread through the species), most of which bear a very strong signal of having arisen by insertions of exogenous DNA into double-strand breaks, in other words as apparent accidents. This is an astonishing finding, as no study had previously identified the emergence of a new intron. Remarkably, nearly a third of these cases appear to represent parallel (independent) gains at exactly the same sites in different sublineages of the study species. This suggests the presence of fragile genomic sites, with perhaps fortuitous surrounding signatures enabling the cell to splice out inserts. In the vast majority of cases, we are unable to identify the source of exogenous DNA, although a few cases appear to be examples of insertion of DNA from microbial sources (horizontal transfer). In a very small minority of cases, the novel intron-bearing gene has been able to rapidly rise to a high frequency, suggesting the possibility of a benefical effect. In summary, this project has utilized results from a new model system in evolutionary genomics, Daphnia pulex, to reveal numerous insights into the cellular and population-genetic mechanisms that promote vs. inhibit the proliferation of spliceosomal introns, one of the major components of eukaryotic genomes. Reference: LI, W., A. TUCKER, W. SUNG, W. THOMAS and M. LYNCH, 2009 Extensive, recent intron gains in Daphnia populations. Science 326: 1260-1262.
