R2 3’RNA determinants for function of the Bombyx mori retroelement protein

Bombyx mori R2 (R2Bm) is the model for a non-LTR retrotransposon gene insertion reaction called target-primed reverse transcription or TPRT. R2Bm is amenable to manipulation in vitro and has been shown to perform highly-specific contacts with target DNA and RNA template. Revealing the RNA features for the first portion to be copied (3’UTR) and that activates TPRT, can help us elucidate species or R2 clade-specific determinants for RNP assembly. Here, we test the hypothesis that defined R2Bm RNA structural feature(s) mediate specificity of RNP formation by generating mutant 3’UTR RNAs, then performing retroelement protein binding, template binding competition, and TPRT assays in vitro. Ultimately, in addition to expanding our understanding of eukaryotic retroelement evolution, generating functional and structurally stable RNA handles improves TPRT manipulation in vivo for the purpose of transgene delivery with therapeutic purposes.