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Dynamic genome architecture in ciliates: an analysis of the evolution of internal eliminated sequences (IESs) in the Paramecium aurelia species-complex

Final Report Summary - DNA SPLICING (Dynamic genome architecture in ciliates: an analysis of the evolution of internal eliminated sequences (IESs) in the Paramecium aurelia species-complex)

A key issue in evolutionary biology is to understand the dynamic processes that shape genome content and architecture. By combining the remarkable biological features and molecular tools of the ciliate Paramecium with its peculiar genomic architecture, the proposed research work aimed at gaining insights into this issue.

Eukaryotic genomes are dynamic units that often undergo developmentally regulated genome rearrangements (DRGRs), the most famous case being the modification of vertebrates' immunoglobulin genes during lymphocyte development, a phenomenon termed somatic or V(D)J recombination. An intriguing example of DRGR is found in ciliate protozoa. In these organisms, extraordinary levels of DNA splicing lead to the massive elimination of 'internal eliminated sequences' (IESs) following sexual reproduction. Such processing is possible because of the binucleate nature of ciliate cells-the diploid, germline nucleus contains the entire genome and is transcriptionally silent; but after meiosis and syngamy, the germline DNA regenerates the new somatic nucleus, from which all gene expression occurs, while the old, maternal somatic nucleus degrades. IES elimination is one of the major events leading to the development of the new somatic DNA.

While a study of the evolution of DNA splicing and possible function of the IESs is currently lacking, a recent study found that IES excision is not an entirely accurate process (Duret et al., Genome Research 2008). The imperfection of nature reveals evolution (Gould, Discover 2 1981), and the discovery of an imperfect process of DNA splicing hints at a hypothetical but verifiable scenario in which the imperfection of the DNA splicing process has evolutionary implications. More specifically, the proposed research addressed the following question: Can IESs become integral and permanent parts of the somatic DNA as a result of DNA splicing inaccuracies? As IESs reside both within and outside genes in the germline DNA, imperfect-splicing events that lead to the permanent incorporation of IESs into the somatic DNA could alter primary gene structures or regulatory domains. When non-lethal and heritable, the alteration of the somatic DNA could lead to the emergence of functional genetic novelties and to key biological events (e.g. genetic isolation, if differential IES retention occurs within or nearby loci that are involved in mating). Once established within a cell, the newly generated IES-allele would have a non-zero probability of spreading through a population or a species.

By using a comparative genomic approach, the proposed study aimed to provide insights into
1) the mechanisms of origin, the evolution, and the loss of IESs from protein-coding genes and intergenic regions across Paramecium species, and
2) the potential functions of IESs, such as the capacity to modulate gene expression. More broadly, the aim of the proposed project was to elucidate the mutational and selective processes that shape genomes and provide hints about how effectively non-coding DNA can contribute to the emergence of organismal complexity.

The work involved the study of three Paramecium species (P. biaurelia, P. tetraurelia and P. sexaurelia). The unpublished genome sequences of P. biaurelia and P. sexaurelia were generously provided by Prof. Michael Lynch (Dept. of Biology, Indiana University, Bloomington, USA). The design of a bioinformatics pipeline and a set of comparative genomic analyses led to detection of greater than 5,000 putative IESs across the genome of the three species. Several physical and selective characteristics of these IESs as well as the conservation of the IES excision profile across species were examined.

Two major results were obtained.

(1) The IES excision profile partially differs, as hypothesized, between the three closely related Paramecium species that were used in the investigation, i.e. IESs that are excised from the somatic DNA of one species are retained in the somatic DNA of another Paramecium species.
(2) IESs may be either retained into the somatic DNA (within and nearby genes) or emerge from the somatic DNA.

While IES emergence is presumably a result of mutations triggering excision of somatic DNA regions, IES retentions are most often independent of the individual genotype and presumably arise as result of maternal effects.

The current sets of results are consistent with the idea that non-coding DNA plays an important role in evolution; that is by altering coding or regulatory sequences, non-coding DNA may contribute to the emergence of organismal complexity. Moreover, coding and non-coding DNA sequences do not appear to be entirely separate compartments, as traditionally thought, but to interconvert dynamically. These initial findings promise to have a significant impact on the understanding of the mechanisms of genome evolution in Paramecium. Functional analyses and additional studies will be required to determine the extent to which retained or emerged IESs alter protein sequences or gene regulatory domains. Also, additional studies will be needed to determine if other eukaryotic species, such as humans for example, exhibit similar patterns. That inter-conversion between coding and non-coding DNA is a common aspect of eukaryotic genomes has been previously hypothesized (Catania and Lynch, PloS Biology, 2008).

There is an apparent resemblance between the processes of DNA splicing in ciliates and the initial events of somatic recombination in vertebrates. Despite a net difference in the scale of the two events-DNA splicing involves the whole germline nuclear DNA in ciliates, whereas somatic recombination is limited to immunoglobulin and T-cell receptor genes in vertebrates-the two processes exhibit a number of intriguing similarities. Both processes, for example, contribute to the formation of functional somatic genes and both are developmentally and epigenetically regulated (DNA splicing takes place during the process of germline/soma differentiation and is directed by non-coding RNAs; somatic recombination occurs during the differentiation and maturation of B and T lymphocytes and is governed by multiple epigenetic activation and silencing mechanisms). Additionally, both in ciliates and vertebrates, DNA excision events are triggered by DNA double-strand breaks and involve intervening sequences that are flanked by conserved motifs. The presence of flanking motifs helps guide the excision machinery, which in either case is a domesticated transposase: PiggyMac in Paramecium and Rag1/Rag2 in vertebrates.

While it is still unclear how far the inventory of similarities between DNA splicing in ciliates and somatic recombination in vertebrates can be extended, it is possible that novel findings concerning either process could contribute to advances in the understanding of the other. Under this hypothesis, findings associated with this work may provide hints about the mechanism and evolution of somatic recombination and serve as working hypotheses for immunologists.