Supplementary Figure 9: The MBSV6-MCP system reporters the expression of ASH1 and DOA1 mRNAs tagged with 24xMBSV6.
From: An improved MS2 system for accurate reporting of the mRNA life cycle
(a) Quantification of single ASH1 mRNAs of cell shown in Figure 6b during the cell cycle (black dots connected by green line). Black curve indicates cytoplasmic ASH1 mRNA profiles fitted to a single exponential decay model.t1/2 = 5.6 min.
(b) Top, schematic representation of ASH1 locus with 24xMBSV6 followed by the kanamycin coding sequence inserted in the 3’UTR. As RNAPII transcribes, the MBS are bound by MCP, marking nascent mRNAs at the transcription site. Bottom, representative images of Supplementary Video 5. Simultaneous two-color imaging of cells co-expressing ASH1 24xMBSV6-KAN-MCP (gray) and NLS-2xmCherry (red). Time indicates the minutes after imaging starts. Images were acquired every 2 minutes.
(c) Schematic representation of DOA1 locus tagged with 24xMBSV6 inserted in the 3’UTR. The dotted lines designate the position of the probes that recognize the CDS (green) or the MBS sequence (red).
(d) Two color smFISH for DOA1 mRNA tagged with 24xMBSV6 in cells expressing MCP or a control plasmid. DIC/MERGE shows the overlap of the DAPI (blue), smFISH for the DOA1 CDS (green) and the MBS (red) with the DIC image. Yellow lines define the shape of a single cell. For each cell is indicated the stage of the cell cycle. Scale bar = 5 μm.
(e) Quantification of smFISH represented in Figure Supplementary 9d with CDS probes (green plots) or MBS probes (red plots) reported as frequency distribution of mature DOA1 mRNAs per cell. Mean and s.d. of two independent cell cultures, n≍500 cells per experiment, distribution of the mRNAs was generated using the same binning.
