Native R2 Rz sequences confer different efficiencies of RNA template function. (A) Depiction of R2 retrotransposon insertion mechanism. (1) R2-encoded protein nicks first-strand DNA at insertion site. (2) R2-encoded protein generates a cDNA copy of its bound transcript by TPRT. (3) Second-strand nicking and second-strand synthesis complete R2 insertion. (B) HDV Rz secondary structure showing helices (gray, denoted P) and single-stranded joining regions (black, denoted J). C labels the position of the essential catalytic base. (C) Predicted cleavage positions (dashed vertical lines) and rRNA lengths of R2 HDV-like ribozymes aligned to the rDNA region upstream of the R2 insertion site denoted as number 0. (D) Plasmid construct used as a template for generation of in vitro transcription (IVT) RNAs for Rz cleavage and transgene insertion assays. (E) Analysis of native R2 Rz self-cleavage from a PP7 hairpin (hp) leader during a 3 h IVT reaction. Reaction products were separated by 6% Urea-PAGE and stained with SYBR Gold. Here and subsequently, uncleaved and cleaved products are indicated by red and white circles, respectively, and the fraction cleaved (fc) is quantified below each lane. (F) Depiction of transgene insertion assay. ZoAl R2p mRNA and green fluorescent protein (GFP) RNA template are codelivered to hTERT RPE-1 cells by lipofection. (G) Representative flow cytometry plots show the percentage of cells expressing GFP 1-day post-transfection of both ZoAl R2p RNA and DroSi Rz RNA template (top), RNA template only (middle), or ZoAl R2p RNA only (bottom). Forward scatter (FSC-A) on the y-axis is a proxy for cell size. (H) Quantification of percentage of cells expressing GFP (left y-axis, green bars) and median GFP intensity (right y-axis, black dots) as assessed by flow cytometry 1-day post-transfection of ZoAl R2p mRNA and the indicated 5′ module RNA templates. GFP% was quantified by subtracting template-only GFP% from the corresponding 2-RNA delivery GFP%. Values indicate mean ± SD, n = 3. (I) PCR amplification of 3′ (top) and 5′ (bottom) rDNA-transgene junctions from gDNA in a representative replicate of the experiment in H. 5′ junctions were amplified with the same rDNA-specific forward primer and different, 5′ module-specific reverse primers. Products were separated by 2% agarose gel. Here and subsequently, expected 5′ junction product bands from annealing of transgene cDNA with upstream rDNA are indicated by green circles. (J) Depiction of 5′ junction formation mechanisms. (Top) cDNA anneals to upstream rDNA. (Bottom) cDNA is end-joined to rDNA.
