PROJECT SUMMARY/ABSTRACT Non-long terminal repeat (non-LTR) retroelements and spliceosomal introns comprise ~70% of the human genome. Both of these genetic elements are thought to have evolved from a group II intron ancestor. Group II introns are catalytic RNAs that are able to engage in both retrotransposition and pre-mRNA splicing. Group II introns engage in retrotransposition and function as retroelements using a copy-and-paste mechanism that allows insertion into new locations in DNA genomes using a reverse transcriptase (RT) and an intron RNA template. Despite the prevalence of retroelements in eukaryotic genomes, relatively little is known about the precise molecular mechanism of retrotransposition. Group II introns are also ancestral to the splicesome, which is responsible for catalyzing pre-mRNA splicing in eukaryotes. This evolutionary linkage is supported by the fact that the active site of the group II intron is conserved with that of the spliceosome. Group II introns can catalyze self-splicing reactions that results in the excision of intron lariat and ligation of the adjacent exons. There are still many unanswered questions regarding the precise mechanism of pre-mRNA splicing and the function of highly conserved nucleotides within the active site. Group II introns consist of two major components that form a ribonucleoprotein (RNP) complex: 1) a self-splicing catalytic RNA and 2) a multi- functional maturase protein that has RT activity. We have isolated a thermostable group II intron RNP that exhibits high levels of retrotransposition and splicing activity. Our group II intron complex is very amenable to high-resolution structure determination and in vitro biochemical studies. To gain mechanistic insight into both retrotransposition and pre-mRNA splicing, we aim to use single-particle cryo-EM, x-ray crystallography, genetics and single-molecule approaches to characterize and capture the different stages of catalysis in this group II intron system. The knowledge gained from these studies will provide direct insight into the mechanisms of both retroelements and the splicing machinery found in higher eukaryotes. This work also lays the foundation for the future biochemical and structural investigation of mammalian retroelements. In summary, the goals of this proposal will further knowledge of the structure and function of genetic elements that comprise a majority of the human genome.