Mechanisms and dynamics of cis preference in LINE-1 replication

About This Grant

Project Summary Mobile genetic elements use strategies that closely resemble those of viruses to replicate, spread, and persist. The long interspersed element-1 (LINE-1, L1) retrotransposon is the only autonomously active retrotransposon in humans and accounts for nearly one-fifth of the genome. L1 replicates through an RNA intermediate and encodes two proteins—ORF1p and ORF2p—that assemble into a ribonucleoprotein (RNP) complex to reverse transcribe the encoding RNA into DNA and insert a new L1 copy into the genome. Despite existing among hundreds of thousands of defective relatives and competing with parasitic elements such as Alu, a small number of full-length L1s continue to replicate. This success relies on cis preference, in which L1 proteins preferentially act on the RNA that encodes them. Similarly, cis preference is observed to varying degrees in viruses. Hepatitis B virus polymerase exhibits a translation-coupled cis preference when interacting with its pregenomic RNA via the epsilon stem-loop, a crucial step in viral genome packaging and reverse transcription. RNA viruses, such as alphaviruses and flaviviruses, exhibit a related bias, with evidence of localized, co-translational coupling between viral proteins and replication templates. The central hypothesis of this proposal is that L1 cis preference is enforced co-translationally, through ribosome-linked mechanisms and/or co-assembly of RNP complexes, analogous to strategies employed by viruses. To test this, we have developed a novel RNA launch system that initiates L1 replication directly from synthetic RNA, with distinct advantages over classical DNA-based retrotransposition assays. Using this platform, we have established a quantitative barcoded assay for L1 cis preference. We will extend the system to study Alu retrotransposition and employ a retron-based surrogate system to probe the generalizability of our findings. Aim 1 will quantify L1 cis preference and determine how Alu subverts it, addressing the dynamics of competition between autonomous and parasitic elements. Aim 2 will dissect the molecular basis of ORF1p’s cis preference using in vitro translation, ribosome profiling, and deep mutational scanning. Aim 3 will define the molecular basis of ORF2p’s cis preference and test whether cis preference can be transferred to a heterologous reverse transcriptase using a bacterial retron system. The impact of this project is to resolve a long-standing mystery in retroelement biology: how cis preference is achieved. By combining innovative RNA-based tools with high-throughput discovery methods, this work will reveal general principles of viral and retroelement replication, illuminate strategies by which selfish genetic elements maintain a fitness advantage in competitive environments, and provide new entry points for biotechnology and therapeutic development.