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. Author manuscript; available in PMC: 2024 Jun 3.
Published in final edited form as: Mol Cell. 2023 Dec 21;83(24):4509–4523.e11. doi: 10.1016/j.molcel.2023.11.025

Figure 4. Experimental validation of regulators of subcytoplasmic mRNA transcript distribution.

Figure 4.

(A) TG+ mRNAs are shown and are color-coded based on their change in compartment localization. No change (N = 508).

(B) Length distribution of mRNAs from (A). Mann-Whitney test p values as in Figure 2A.

(C) As in (B) but for protein size distribution. Boxplots depict median, 25th and 75th percentiles (box), and 5% and 95% confidence intervals (error bars).

(D) As in (B) but for CDS exon length distribution.

(E) As in Figure 3E but for mRNA features of TG+ mRNAs that change their localization upon TIS11B KO.

(F) Schematic of mRNA reporter for validation of a 3′ UTR-bound RBP on mRNA localization. The GFP-THAP1 reporter mRNA contains MS2 hairpins as 3′ UTR, which bind to cotransfected MS2 coat protein (mCherry-tagged MCP). TIAL1-MCP fusion tethers TIAL1 to the reporter 3′ UTR. mC, mCherry.

(G) Confocal live cell imaging of HeLa cells expressing the indicated constructs. Scale bars, 5 μm.

(H) RNA-FISH (teal) of the GFP reporter mRNA from (F) in HeLa cells. GFP-SEC61B visualizes the rough ER (magenta). Representative images are shown. Scale bars, 5 μm.

(I) Pearson’s correlation coefficients of fluorescence intensities at arrows in (H).

(J) Quantification of (H) and (I). MCP (n = 26 cells), MCP-TIAL1 (n = 21). Horizontal line: median, error bars: 25th, 75th percentiles. Mann-Whitney test, ****p < 0.0001.