COMPREHENSIVE SCANNING MUTAGENESIS OF A HUMAN RETROTRANSPOSON IDENTIFIES MOTIFS ESSENTIAL FOR FUNCTION
Embargo until
2020-05-01
Date
2018-12-10
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Johns Hopkins University
Abstract
Long Interspersed Nuclear Element-1 (LINE-1, L1) is the only autonomous active transposable element in the human genome. In general, we strive for molecular level understanding of how the L1- encoded proteins ORF1p and ORF2p facilitate retrotransposition as they are essential for enabling this element to jump from one locus to another via a “copy and paste” mechanism. In this work, we aimed to develop a variety of tools to probe specific intermolecular interactions that form with RNA, proteins and target DNA throughout the L1 lifecycle. ORF1p is an RNA-binding protein and ORF2p has both endonuclease and reverse transcriptase activities. These proteins bind the L1 RNA to form L1 ribonucleoprotein complexes (RNPs). As a streamlined parasite, the L1 retrotransposon requires a variety of host factors to complete a successful lifecycle and the host has likely mainly evolved to limit the mutagenic potential of novel L1 insertions. First, we study L1 RNP formation in vivo and, second, we study the L1 encoded proteins’ sensitivity to mutation. As a follow-up to studying the composition of RNPs in tissue culture, we established customized tools for isolating active L1 RNP complexes from live mammalian tissues. This necessitated the establishment of novel transgenic mouse lines highly expressing tagged mouse L1 proteins, as well as the production of a high-quality antibody against mouse ORF1p. Prior studies used human L1 in mouse, and thus these studies represent a truly homologous system. We also successfully conducted a mutagenic scan of human L1 by constructing a library consisting of 538 consecutive trialanine substitutions, scanning along ORF1p and ORF2p. We describe the construction of the library, its initial characterization, and its use as a resource for future studies. For each variant, we measured retrotransposition efficiency. We also measured both total ORF1p and RNA produced by each variant. We also started to develop an RNA sequencing-based method to quantify how well each ORF1 variant protein was able to bind its own L1 RNA for proper RNP formation. Retrotransposition was extremely sensitive to mutations in ORF1p and ORF2p. The library provides comprehensive information on which regions are most critical to retrotransposition and which are dispensable.
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Keywords
LINE-1, retrotransposon