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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 5. 3′ UTR-bound TIAL1 cooperates with the rough ER membrane environment to increase protein expression.

Figure 5.

(A) Protein abundance of mRNAs stratified by RBP binding. No RBP (N = 126), bound by TIS11B (N = 267), bound by TIA1/L1 (N = 232). Mann-Whitney test p values as in Figure 2A. Boxplots depict median, 25th and 75th percentiles (box), and 5% and 95% confidence intervals (error bars).

(B) GFP protein expression in HeLa cells using the GFP-THAP1 reporter mRNA with and without TIAL1 tethering. Representative histograms are shown. Dotted lines: GFP-negative cells.

(C) Quantification of (B) as mean ± SD of five independent experiments. t test for independent samples, ****p = 0.0003.

(D) Quantification of mRNA level from (B) as mean ± SD of three independent experiments. t test for independent samples.

(E) Schematic of GFP-THAP1 mRNA reporter to investigate the influence of subcellular mRNA localization on protein expression. MCP-SEC61B fusion localizes the reporter mRNA (as in Figure 4F) to the rough ER membrane, MCP localizes it to the cytosol.

(F) Confocal live cell imaging of HeLa cells. Scale bars, 5 μm.

(G) As in (B), but reporter mRNA was used with and without SEC61B tethering.

(H) Quantification of (G) as mean ± SD of four independent experiments. t test for independent samples, **p = 0.0026.

(I) Quantification of mRNA level from (G) as mean ± SD of three independent experiments. t test for independent samples, NS, not significant.

(J) As in Figure 4F. Addition of prenylation signal (CAAX) localizes the TIAL1-bound reporter mRNA to the plasma membrane. In the absence of CAAX, the TIAL1-bound reporter mRNA localizes to the rough ER.

(K) Confocal live cell imaging of HeLa cells. Scale bars, 5 μm.

(L) As in (B) but the reporter mRNA was tethered with the indicated constructs.

(M) Quantification of (L) as mean ± SD of four independent experiments. t test for independent samples, ****p < 0.0006, **p = 0.002.

(N) Endogenous mRNAs bound by TIA1/L1 encode higher expressed proteins than mRNAs not bound by any RBP. The largest TIA1/L1-associated increase was observed for ER+ mRNAs. Mann-Whitney test p values as in Figure 2A.