Figure 6. Dynein 1 heavy chain mRNA accumulates in blobs that are translation sites. (A) Localization of endogenous DYNC1H1 mRNAs in HeLa cells. Image displays the smFISH signal of HeLa cells labeled with probes recognizing DYNC1H1 mRNAs. Blue arrow: single RNA molecules; red arrow: RNA blob. The green dashed outline represents the position of the nucleus. Bar, 6 µm. (B) Localization of BAC-tagged DYNC1H1 mRNAs in HeLa cells. Legend as in A, except that the probes labeled the GFP sequences. Left panel: smFISH images; right panel: GFP signal. Bar, 6 µm.(C) Localization of endogenous DYNC1H1 mRNAs in NIH3T3 cells. Legend as in A. Bar, 10 µm. (D) Quantification of the number of blobs per cell (left graph), the number of DYNC1H1 mRNAs per blob (middle graph), and the fraction of DYNC1H1 mRNAs present in blobs (right graph). 3T3: NIH3T3 mouse cells (n = 60 cells); BAC: HeLa cells expressing the DYNC1H1 BAC (n = 220 cells); HeLa: HeLa cells expressing the SunTagx32-DYNC1H1 allele (n = 385 cells). The error bars represent the SD of the mean between experimental replicates (n = 3). (E) Colocalization among the bright protein foci labeled by the scFv-sfGFP, the endogenous DYNCH1 mRNAs, and the tagged DYNC1H1 mRNAs. Panels represent microscopy images of cells stably expressing the scFv-sfGFP and containing the SunTagx32-DYNC1H1 allele. Left panel: signal of the scFv; middle left panel: signal from smFISH probes recognizing the DYNC1H1 sequences; middle right panel: signal from smFISH probes recognizing the SunTag-Puro sequences; right panel: color overlay of the scFv signal (green), the endogenous DYNC1H1 mRNAs (red), and the tagged DYNC1H1 mRNA (green). (inset) A 2.5 × 2.5-µm zoom of the boxed area. Bar, 10 µm. (F) Percentage of translated SunTagx32-DYNC1H1 mRNAs, for mRNAs localizing in blobs or being single mRNAs (n = 260 cells). The error bars represent the SD of the mean between experimental replicates (n = 3). (G) Effect of inhibition of translation initiation on the accumulation of DYNC1H1 in blobs. (left) Number of DYNC1H1 polysome per cell, in cells untreated or incubated with Torin1. (right) Fraction of DYNCH1 mRNA in blobs, in cells untreated or incubated with Torin1. The error bars represent the SD of the mean between experimental replicates (n = 4).
Figure 6.

Dynein 1 heavy chain mRNA accumulates in blobs that are translation sites. (A) Localization of endogenous DYNC1H1 mRNAs in HeLa cells. Image displays the smFISH signal of HeLa cells labeled with probes recognizing DYNC1H1 mRNAs. Blue arrow: single RNA molecules; red arrow: RNA blob. The green dashed outline represents the position of the nucleus. Bar, 6 µm. (B) Localization of BAC-tagged DYNC1H1 mRNAs in HeLa cells. Legend as in A, except that the probes labeled the GFP sequences. Left panel: smFISH images; right panel: GFP signal. Bar, 6 µm.(C) Localization of endogenous DYNC1H1 mRNAs in NIH3T3 cells. Legend as in A. Bar, 10 µm. (D) Quantification of the number of blobs per cell (left graph), the number of DYNC1H1 mRNAs per blob (middle graph), and the fraction of DYNC1H1 mRNAs present in blobs (right graph). 3T3: NIH3T3 mouse cells (n = 60 cells); BAC: HeLa cells expressing the DYNC1H1 BAC (n = 220 cells); HeLa: HeLa cells expressing the SunTagx32-DYNC1H1 allele (n = 385 cells). The error bars represent the SD of the mean between experimental replicates (n = 3). (E) Colocalization among the bright protein foci labeled by the scFv-sfGFP, the endogenous DYNCH1 mRNAs, and the tagged DYNC1H1 mRNAs. Panels represent microscopy images of cells stably expressing the scFv-sfGFP and containing the SunTagx32-DYNC1H1 allele. Left panel: signal of the scFv; middle left panel: signal from smFISH probes recognizing the DYNC1H1 sequences; middle right panel: signal from smFISH probes recognizing the SunTag-Puro sequences; right panel: color overlay of the scFv signal (green), the endogenous DYNC1H1 mRNAs (red), and the tagged DYNC1H1 mRNA (green). (inset) A 2.5 × 2.5-µm zoom of the boxed area. Bar, 10 µm. (F) Percentage of translated SunTagx32-DYNC1H1 mRNAs, for mRNAs localizing in blobs or being single mRNAs (n = 260 cells). The error bars represent the SD of the mean between experimental replicates (n = 3). (G) Effect of inhibition of translation initiation on the accumulation of DYNC1H1 in blobs. (left) Number of DYNC1H1 polysome per cell, in cells untreated or incubated with Torin1. (right) Fraction of DYNCH1 mRNA in blobs, in cells untreated or incubated with Torin1. The error bars represent the SD of the mean between experimental replicates (n = 4).

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