Figure 3.

NMD Does Not Occur Preferentially on CBC-Bound mRNAs

(A–G) U2OS cells expressing scFv-sfGFP, PCP-mCherry-CAAX, and the translation reporter shown in (A) were transfected with ha4E-BP1 or mock transfected and analyzed by time-lapse microscopy.

(A) (top) Schematic of standardized translation reporter. Representative images of a single mRNA molecule of either mock (upper image panel) or ha4E-BP1 (lower image panel) transfected cells are shown. Scale bar, 1 μm. Time is shown in min:s.

(B–E) GFP fluorescence intensity over time of a representative mRNA (B, D, and E) or of the average of all mRNAs (C). Blue lines (D and E) indicate the best fit from simulations. Blue triangles indicate translation initiation events.

(F) Quantification of the mean translation initiation rate determined by the fitting approach illustrated in (D) and (E).

(G) Quantification of the number of ribosomes that initiated in the first burst of translation. Data were fit with a single exponential decay distribution (blue line).

(H) The calculated fraction of mRNAs that is targeted for NMD while CBC is bound to the mRNA cap (mean ± SEM).

Dotted lines in (D) and (E) indicate that the data are replotted from an earlier figure panel for comparison. Solid lines and corresponding shaded regions in (C), (F), and (G) represent mean ± SEM. Number of measurements for each experiment are listed in Table S1. See also Figure S4.