Revealing and Live Imaging the LINE-1-RNA Intractome

Brown University, Providence RI

Investigators

Abstract

The LINE-1 retrotransposon (L 1} is a virus-liKe genomic parasite that is conserved throughout euKaryotes and has written at least a third of the human genome. L 1 is thus in a ~2-billion-year arms race with our genome, wherein the transposon attempts to mobilize through a ·copy and paste" mechanism, and the host defends through multiple redundant processes. L 1 is a streamlined machine, encoding only two proteins in a bicistronic arrangement from a single Pol 11 transcribed RNA: the highly expressed homotrimeric ORF 1 p RNA chaperone and the poorly expressed ORF2p multifunctional enzyme, which contains both endonuclease and reverse transcriptase (RT) activities. Through insertional mutagenesis, one in every ~80 babies has a new genomic L 1 copy not in either parent, a rare cause of sporadic genetic disease. L 1 activities including innate immune agonism and altering cell signaling liKely contribute broadly to common disease pathology, but the extent of this is unclear. Both L 1 proteins are required for L 1's mutagenic lifecycle, and to enable specific mobilization of the L 1 RNA. both L 1 proteins exhibit striKing cis preference, binding preferentially to the very RNA that encoded them. Dozens of host factors have evolved to repress this genotoxic process. and as a streamlined parasite, L 1 coopts many host proteins including PCNA. RNase H2, topoisomerase 1, and poly(A) binding proteins to mobilize. The emerging paradigm is that there are both common and celltype- specific defenses against L 1, however our knowledge of these remains limited. Building on my lab's recent advances in understanding L 1 structure and biochemical mechanisms and grounded in our early discoveries of Key L 1 interactors. this COBRE proposal see Ks to understand how L 1 co-opts host machinery, how the cell attempts to defend, and now L 1 evades cellular defenses. Specifically, we will develop the first high fidelity imaging systems to visualize L 1 proteins and RNA in both live and fixed cells, allowing us to study these interactions and L1 nuclear entry, which has been elusive. We will also study the L 1 RNP-RNA interactome in colorectal cancer and model cells using a suite of developed complementary tools. which appear to be a uniquely privileged environment for L 1 mobilization. Together, our studies will illuminate fundamental basic mechanisms that shape our genomes and enable translational medicine that benefits human health.