Analysis of the SRE-dependent repressor complex assembled on chimeric RNAs. (A) Rapid sedimentation of repressed RNAs. Radiolabeled chimeric RNAs were incubated for 30 min in Drosophila embryo extract under preincubation conditions. Radiolabeled regulatory RNAs not fused to the nLuc reporter (top left) were used as positive controls. At the end of the incubation, 200 µL aliquots were analyzed by sucrose gradient centrifugation as previously described (Götze et al. 2017). Radioactivity in each fraction was measured by scintillation counting. Formation of the repressor complex resulted in accelerated sedimentation, as reported before (Götze et al. 2017). (B) Partial RNase resistance of repressed RNAs. Regulatory RNA by itself as well as flipped and forward chimeric constructs, all in the SRE+ and SRE− versions, were incubated in Drosophila embryo extract under preincubation conditions, then a time-course of RNase digestion was performed as described in Materials and Methods. Time points were taken at 5 min intervals. A radiolabeled synthetic RNA oligonucleotide was added before RNA purification and served as a recovery control (bottom part of the gel). White and black arrowheads indicate the nLuc and the regulatory RNA, respectively. Stabilization factors were calculated by comparison of the SRE+ RNA and SRE− RNA half-lives and are listed at the bottom.
