Collaborative Research: RNA-guided DNA recombination through assembly graphs

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Genome rearrangements occur on an evolutionary scale, as well as on a developmental scale, in a wide range of eukaryotes. Even in cancer cells, dramatic DNA rearrangements are frequently observed in somatic cell lineages. This project uses certain species of ciliates as a model system to study gene rearrangements. These organisms undergo massive recombination during differentiation of an archival germline micronucleus into a somatic macronucleus capable of gene expression. The objective of this project is to increase the understanding of this process of assembly, starting from the basic principles of molecular DNA - RNA - enzyme interactions, through general knowledge of the possible pathways and intermediate steps that lead to the final product. Through mutual reinforcement, the project interlaces biological experiments with mathematical findings. The theoretical model utilizes spatial graphs with 4-valent vertices as a physical representation of the DNA at the time of recombination, and smoothing of the vertices models the actual recombination. Specific aims include: (a) examine the interactions of the template RNA and the involvement of possible cutting and splicing enzymes at the moment of recombination, (b) identify steps in the process by pinpointing putative intermediate molecules during unscrambling a variety of genes, and theoretically analyze sets of pointers leading to these intermediates (c) characterize theoretically and confirm experimentally all possible gene unscrambling pathways for a subset of genes, and (d) develop mathematical techniques based on methods from knot theory and formal languages to study the theoretical model.Our understanding of genome-wide DNA rearrangements is still very limited. These processes appear on an evolutionary scale, where they can lead to species-specific differences and even the creation of species boundaries. On a developmental scale, genome rearrangements appear in a wide range of differentiating eukaryotic cells. The findings of this project will offer insight into the process of gene descrambling in ciliates and may also extend more generally to other model systems. This project seeks to learn from both theory and experiments how a mechanism for RNA-templated DNA recombination sculpts the assembly of genetic information in ciliates. This mechanism of template-mediated DNA rearrangement can be visualized as a scaffold for controlling and programming certain types of genetic information within a cell. Such control of information processing could facilitate biomolecular computing in vivo, as well as methods for epigenetic control of development.