Retrotransposons are ubiquitous genomic parasites found in nearly all eukaryotes and constitute a major component of these genomes. Replication of retrotransposons results in chromosomal aberrations including insertions, deletions, and double stranded breaks. Upon integration into the genome, retrotransposons can affect cellular gene expression and can act as a source of new genes. Non-long terminal repeat retrotransposons (NLRs) in particular have played a central role in this process. The integration machinery of these elements is responsible for most of the reverse transcribed material in animal genomes, including short interspersed nucleotide elements (SINEs), pseudogenes, and retrogenes. For example, over 34% of the human genome is the direct result of NLR activity. In addition, NLR elements' or their progenitors' played a role in the origin of retroviruses. A detailed knowledge of the replication mechanism of NLRs is essential to understanding how retrotransposons interact with the genome. Certain aspects of element integration, especially target primed reverse transcription (a process whereby an RNA transcript is copied into a DNA strand at the site of insertion), are reasonably well understood; however, how the integration process is completed (e.g., how double stranded DNA is made from the products of target primed reverse transcription), and how NLRs can change their integration site specificity is a mystery. A mechanistic understanding of full integration has eluded study until now because of the complexity of the reaction and the lack of the right tools to address the question. In vivo systems using mutant NLR elements to the search for structure function relationships are often hard to interpret when the mutant fails to integrate. In vitro models have been missing a key RNA component that is required for complete integration to occur. This research will explore the second half of the integration reaction by restoring the missing RNA component - enabling full integration events to occur in vitro as well as in vivo. How site specific NLRs interact with DNA and change site specificity over time will also be addressed with the hopes of developing NLRs into a site specific gene targeting vector. Both aims will provide information on not only the integration mechanism of the elements used in this study but also on the integration of all NLRs. The core mechanism of integration is believed to be conserved for this class of retroelements and shares mechanistic similarities to (and evolutionary connections with) telomere elongation, group II homing introns, and retroviruses.
Broader Impacts. The biochemical study of (NLRs) will be of interest to people in transposon biology, genome biology, evolutionary biology, and to those in the retroviral and biomedical fields. The University of Texas Arlington has high numbers of underrepresented groups in its graduate and undergraduate programs. This diversity is reflected in the makeup of the investigator's lab; the majority of the graduate and undergraduate researchers in the investigator's lab are from underrepresented groups. This research will be disseminated through peer reviewed journals and scientific meetings. The investigator is a guest lecturer in continuing education classes for local K-12 teachers where he talks about transposons, gene formation, and genomics; topics central to this research. These topics are also covered in the undergraduate and graduate courses the investigator teaches using research from the investigators lab.
