Fig. 3: Luciferase RNA is modestly recruited to stress granules when tethered with G3BP1, YTHDF1, or YTHDF2.
From: Limited effects of m6A modification on mRNA partitioning into stress granules

A Cartoon schematic of tethering YTHDF proteins to luciferase mRNA using the BoxB-lambdaN (λN) approach. B Number of 0 or 25 BoxB luciferase mRNA in U-2 OS cells when expressing λN, G3BP1-GFP-λN, YTHDF1-GFP-λN, and YTHDF2-GFP-λN transgene. U-2 OS cells were stressed for 1 h with arsenite. Each dot represents a cell. N = 45, 29, 25, and 13 for λN, G3BP1-GFP-λN, YTHDF1-GFP-λN, and YTHDF2-GFP-λN with 0 BoxB luciferase mRNA. N = 21, 33, 37, and 25 for 15 for λN, G3BP1-GFP-λN, YTHDF1-GFP-λN, and YTHDF2-GFP-λN with 25 BoxB luciferase mRNA. The box plots are defined by minima, 25% percentile, median, 75% percentile, and maximum. For λN, the number of 0 boxB RNAs/cell are 10, 18, 27, 41.5, and 87 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For G3BP1-GFP-λN, the number of 0 boxB RNAs/cell are 16, 26, 38, 48, and 74 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For YTHDF1-GFP-λN, the number of 0 boxB RNAs/cell are 16, 24, 38, 72.5, and 109 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For YTHDF2-GFP-λN, the number of 0 boxB RNAs/cell are 13, 20, 29, 40, and 81 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For λN, the number of 25 boxB RNAs/cell are 131, 174.5, 214, 313.5, and 466 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For G3BP1-GFP-λN, the number of 25 boxB RNAs/cell are 96, 204.5, 255, 356.5, and 574 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For YTHDF1-GFP-λN, the number of 25 boxB RNAs/cell are 39, 119.5, 175, 207, and 360 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For YTHDF2-GFP-λN, the number of 25 boxB RNAs/cell are 56, 104, 126, 145.5, and 228 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. C Representative images of arsenite stressed-U-2 OS cells stably expressing 0 or 25 BoxB luciferase mRNA and transiently expressing GFP-λN, G3BP1-GFP-λN, YTHDF1-GFP-λN, and YTHDF2-GFP-λN. Single-molecule FISH probes are illustrated in white, endogenous G3BP antibody staining in green, while tethered proteins are in red. The nuclei are stained with DAPI (blue). The scale bar is 3 µm. D Box plot of the fraction of 0 and 25 BoxB luciferase RNA molecules in stress granules in wild-type U-2 OS cells. Each dot represents an experimental data point (or cell). N = 45, 29, 25, and 13 for λN, G3BP1-GFP-λN, YTHDF1-GFP-λN, and YTHDF2-GFP-λN with 0 BoxB luciferase mRNA. N = 21, 33, 37, and 25 for λN, G3BP1-GFP-λN, YTHDF1-GFP-λN, and YTHDF2-GFP-λN with 25 BoxB luciferase mRNA. The box plots are defined by minima, 25% percentile, median, 75% percentile, and maximum. For λN, the fraction of 0 boxB RNAs in stress granules/cell are 0, 0.068, 0.087, 0.117, and 0.286 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For G3BP1-GFP-λN, the fraction of 0 boxB RNAs in stress granules/cell are 0, 0.082, 0.105, 0.158, and 0.257 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For YTHDF1-GFP-λN, the fraction of 0 boxB RNAs in stress granules/cell are 0, 0.095, 0.128, 0.178, and 0.280 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For YTHDF2-GFP-λN, the fraction of 0 boxB RNAs in stress granules/cell are 0, 0.079, 0.130, 0.160, and 0.194 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For λN, the fraction of 25 boxB RNAs in stress granules/cell are 0.028, 0.077, 0.108, 0.144, and 0.171 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For G3BP1-GFP-λN, the fraction of 25 boxB RNAs in stress granules/cell are 0.080, 0.114, 0.160, 0.216, and 0.302 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For YTHDF1-GFP-λN, the fraction of 25 boxB RNAs in stress granules/cell are 0.015, 0.112, 0.203, 0.298, and 0.519 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. For YTHDF2-GFP-λN, the fraction of 25 boxB RNAs in stress granules/cell are 0.024, 0.134, 0.167, 0.209, and 0.415 for minima, 25% percentile, median, 75% percentile, and maximum, respectively. ns denotes not significant (P > 0.05) (unpaired two-tailed student t-test). Source data are provided for this Figure (see data availability).