Towards Genome Engineering: Principles of precision in RNA-binding and cDNA-synthesis for a site-specifically targeted non-LTR retroelement

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ABSTRACT Transposable elements are selfish genes widespread in all domains of life that are capable of moving to new sites in the genome. Non-long terminal repeat (non-LTR) retroelements, the ancestral and most diverse eukaryotic retrotransposons and the most prevalent and active transposable elements in the human genome, are implicated in a number of host cell processes including development, gene expression, cancer progression and genome evolution. Non-LTR retroelements integrate through a process called target-primed reverse transcription (TPRT) in which the retroelement protein reverse transcribes the retroelement RNA directly into the genome. This mechanism bypasses the creation of a double-stranded DNA break and avoids the need for homologous recombination, providing incentive to thoroughly understand protein and RNA features that facilitate this process for future gene therapy endeavors. The site-specific R2 non-LTR retroelement has been the primary model for studying TPRT. The evolutionary persistence of R2 mobility, despite changes in protein and template RNA sequence, provides an opportunity to elucidate fundamental requirements for, as well as phylogenetic diversification of, the TPRT mechanism. This work proposes a combination of biochemical, molecular and cellular approaches to elucidate the principles of the affinity between the R2 retroelement protein and RNA, to decipher the principles of the very high specificity of their interaction and to achieve complete TPRT-mediated transgene insertion. This project will build upon my foundation in RNA biology and advance my research career as I receive rigorous training under Dr. Kathleen Collins, who has expertise in RNA-protein interaction, ribonucleoprotein complex formation, and reverse transcriptases. This work will contribute to a greater understanding of how evolution has honed and diversified the pairwise specificity of a retroelement protein and RNA in addition to furthering the eventual goal of exploiting non-LTR retrotransposons for transgene introduction.