Fig 2: Experimental validation of regulators of subcytoplasmic mRNA transcript distribution(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.

Fig 3: Localization of cytosolic mRNAs to the rough ER membrane increases their protein expression(A) Schematic of a GFP-THAP1 reporter mRNA bound by TIS11B to investigate localization-dependent GFP protein expression. MCP-TIS11B fusion localizes the mRNA reporter to the cytosol. TIS11B-MCP-SEC61B fusion localizes the mRNA reporter to the rough ER membrane.(B) Confocal live cell imaging of HeLa cells expressing constructs from (A). Scale bars, 5 μm.(C) As in Figure 5B.(D) Quantification of (C) as mean ± SD of four independent experiments. t test for independent samples, ****p < 0.0001, **p = 0.003.(E) Quantification of mRNA level in the experiment from (C). Shown is the mean ± SD of three independent experiments. t test for independent samples, *p = 0.037; NS, not significant.

Fig 4: 3′ UTR-bound TIAL1 cooperates with the rough ER membrane environment to increase protein expression(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.

Fig 5: Characteristics of compartment-enriched mRNAs(A) Steady-state mRNA abundance levels obtained from whole-cell lysates. TG+, N = 1,246; ER+, N = 919, CY+, N = 1,481; unbiased, N = 3,369. Mann-Whitney test: *0.05 > p > 10−9; **10−10 >p> 10−20; ***10−21 >p> 10−80; ****10−81 > p > 0. Exact p values are listed in Table S3. Boxplots depict median, 25th and 75th percentiles (box), and 5% and 95% confidence intervals (error bars).(B) As in (A), but steady-state protein levels obtained from whole-cell lysates are shown. TG+, N = 469; ER+, N = 638; CY+, N = 833; unbiased, N = 2,001.(C) As in (B), but Pro-seq levels are shown, which indicate transcription rates. TG+, N = 1,222; ER+, N = 896; CY+, N = 1,425; unbiased, N = 3,268.(D) As in (C), but estimated mRNA half-lives are shown.(E) As in (A), but protein size distributions are shown. AA, amino acid.(F) As in (A), but mRNA length distributions are shown.(G) As in (A), but 3′ UTR length distributions are shown.(H) As in (A), but average CDS exon length distributions are shown.(I) ZFP36L1 (TIS11B) mRNA model. Tall boxes: CDS exons, narrow boxes: 5′ and 3′ UTRs.(J) Gene ontology analysis for TG+ mRNAs. Top six functional gene classes uniquely enriched in TG+ mRNAs and Benjamini Hochberg-adjusted p values are shown.(K) As in (J), but for ER+ mRNAs.(L) As in (J), but for CY+ mRNAs.