Ta.it.I1 in vitro retromobility. (A) Cartoon representation of the fluorescently dilabeled DNA substrate engineered for fluorescent in vitro retromobility assays. (B) Schematic diagram of reverse splicing and cDNA synthesis. Each expected intermediate and product are numbered in the order they appear in the reaction. The dashed line box insert shows a close-up view of the short intervening sequence containing a polydT stretch engineered to allow limited cDNA synthesis with dATP addition. (C) WT and YAAA mutant Ta.it.I1 RNP retromobility into DNA substrate; assay was performed with or without addition of dATP/dNTPs. Pseudocoloring of product bands on the gel correspond to which fluorophore was detected: Cy5 = red, FAM = green, both = yellow. (D) RNase H digestion of mobility products confirms the presence of extensive cDNA synthesis. (E) Time-course of WT and π mutant Ta.it.I1 RNP retromobility into DNA substrate.
