Figure 1.

An Assay for Real-Time Visualization of NMD of Single mRNA Molecules

(A) Schematic of NMD single-molecule imaging assay before (top) or after (bottom) NMD induction. Green and red spots (insets) show nascent proteins and reporter mRNA, respectively, as observed through the microscope.

(B) Schematic of the NMD reporter constructs. e, exon; in, intron. PTC160 and PTC1 indicate PTC at amino acids 160 and 1, respectively.

(C–J) U2OS cells expressing scFv-sfGFP and either PCP-mCherry-CAAX (C–G, I, and J) or PCP-HaloTag (H) were transfected with indicated reporter constructs (C–J) and siRNAs (G and J) and were analyzed by time-lapse microscopy.

(C) Representative images of mRNA molecules of indicated reporters are shown. Scale bar, 1 μm. Time is shown in min:s.

(D–J) The time from first detection of translation until separation of red and green foci (i.e., mRNA cleavage) (D–H) or the time from mRNA cleavage until disappearance of the 3′ cleavage fragment (red spot) (I and J) was quantified.

Solid lines and corresponding shaded regions in (D)–(J) represent mean ± SEM. Dotted lines in (H) indicate that the data are replotted from an earlier figure panel for comparison. Number of measurements for each experiment are listed in Table S1. See also Figures S1 and S2 and Videos S1, S2, S3, and S4.