Characterization of LINE-1 reverse transcriptase activity

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Project Summary The objective of this research proposal is to characterize the enzymatic activities of the reverse transcriptase (RT) of the human long interspersed element 1 (LINE-1, L1 RT). LINE-1 sequences constitute 17% of the human genome, and L1 RT activity is responsible for approximately 40% of our genome because it has also led to the proliferation of short interspersed elements (e.g., Alu), and other retroelements. L1 RT activity is also implicated as a driving force behind a variety of human diseases such as macular degeneration, Aicardi- Goutières syndrome, and systemic lupus erythematosus. LINE-1 expression and retrotransposition are commonplace in numerous cancers, including 90-100% of breast, colon, and esophageal cancers, making understanding the basic biochemical activity of L1 RT relevant to public health research. Reverse transcription is key to the ability of LINE-1 to self-propagate in our genome, through a process known as target-primed reverse transcription (TPRT). L1 RT is encoded by the second open reading frame (ORF2) of L1, residing in the protein known as ORF2p. In this proposal, I show that I have isolated the ORF2p RT domain, and that this has RT activity in vitro. I intend to use this protein domain, as well as full-length ORF2p, and L1 RNPs to fully characterize the biochemical and enzymatic properties of L1 RT. In my first Specific Aim, I will characterize its processivity, which is an indication of whether the RT stops and pauses, or whether it keeps going until it reaches the end of a template. I will also measure the fidelity of L1 RT, which defines how faithfully it copies DNA from an RNA template. Finally, I present preliminary sequencing data showing that L1 RT has the unexpected ability to begin processing an RNA template without a primer and propose rigorous experiments to study this feature. In my second Specific Aim, I focus on what happens when L1 RT reaches the end of its template, which may be crucial to understanding how cells repair intermediates of transposition. I present preliminary data showing that L1 RT adds extra nucleotides to the end of its cDNA and propose studies to fully characterize this activity. Additionally, I will study the ability of L1 RT to switch to a different template at the end of one template and whether LINE-1 RT can use modified RNA as a template, such as the pseudouridine found in mRNA vaccines or the naturally occurring N6-methyladenosine modification. This work will advance our understanding of how LINE-1 operates inside cells and how LINE-1 completes its life cycle. The proposed research will have implications for cancer biology and innate immune signaling where L1 RT is increasingly recognized as a DNA damaging agent and trigger for pattern recognition receptors, respectively. Finally, this research will give insight into the evolution of LINE-1-like retroelements that are found across all kingdoms of life.