Assessing Retrotransposition of Keno, a Site-specific Transposable Element in Zebrafish
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Abstract
Transposable elements (TEs) constitute a significant portion of eukaryotic genomes, yet their roles in genome function, disease and evolution remains understudied. Keno is a site-specific non-LTR retrotransposon that inserts into a conserved region of the U2 small nuclear RNA (U2 snRNA), which is an essential component of the spliceosomal machinery. Intrigued by these properties, we set out to investigate whether Keno is transpositionally active and what cellular properties affect its transposition in zebrafish. By observing GFP expression, marking insertion of a copy from injected RNA in zebrafish embryos, I reveal no detectable activity of the cloned Keno element. By whole plasmid sequencing of the cloned Keno element, I discovered that it was significantly (10.8%) diverged from the consensus sequence. I attempted to isolate Keno copies that were potentially more likely to be active by cloning from zebrafish cDNA separately, but this method was unsuccessful in isolating more recent Keno copies. We also assayed transposition with Keno ORF1 and ORF2 RNAs introduced with the reporter RNA, but observed no transposition. To assess if the synthesized Keno ORF2 RNA was being translated into protein during development, we monitored translation through a split luciferase tag (HiBiT epitope tag). I observed low levels of luminescence, suggesting low levels of translation of the injected Keno ORF2 HiBiT RNA. Taken together, these results suggest that the copies of Keno we isolated thus far are not capable of transposition. We discuss future directions including the possibility that other Keno copies not yet isolated are transpositionally active.