Extended Data Fig. 8: Stabilization of dMCP by RNA containing reduced-affinity MS2 loops.
(a) Generalized ribonucleotide sequences for high affinity C-variant and reduced affinity U-variant MS2 loops. ‘S’ corresponds to G or C, ‘H’ corresponds to A,U, or C, and ‘N’ corresponds to any nucleotide. (b) Representative flow cytometry data of the dMCP stabilization response to C-variant MS2 RNA (BFP-24xMS2(C)-pA, red), U-variant MS2 RNA (BFP-24xMS2(U)-pA, blue), or control BFP RNA (BFP-pA, gray). mScarlet-dMCP intensity is plotted over BFP intensity. (c) Median mNG-normalized mScarlet-dMCP intensities for the BFP positive population of cells in the experiment described in (e). Quantification performed by flow cytometry in HEK293FT cells co-transfected to express BFP mRNA containing C-variant MS2 stem loops (red circles), U-variant MS2 stem loops (blue circles) or no MS2 stem loops (gray circles) in combination with the dMCP reporter construct. Each point represents the median mNG-normalized mScarlet expression for independent transfections. Bars and error bars represent the mean and S.D of three independent transfections (n = 3). (d) Widefield fluorescence microscopy images of live U2OS cells transfected to express BFP-24xMS2(C)-pA or BFP-24xMS2(U)-pA with 4xmNG-dMCP. Images are single frames extracted from live recordings that were obtained using the same microscope settings.
