Structural RNA components supervise the sequential DNA cleavage in R2 retrotransposon

Summary Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determined the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we revealed two essential DNA regions, Drr and Dcr, required for R2 recognition and cleavage. The association of 3’ regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the polyA tail. Removing 3’ regulatory RNA by reverse transcription allows the association of 5’ regulatory RNA and initiates the second-strand cleavage. Our work explained the DNA recognition and supervised sequential retrotransposition mechanisms by R2 machinery, providing novel insights into the retrotransposon and application reprogramming.