Fig 2: Loss of 4EBP1/2 is sufficient to alleviate 5′TOP translational repression during Torin1 treatment.(A) Quantification of translation site intensities of reporter mRNAs in 4EBP1/2 KD cells ± Torin1 (250 nM, 1 hour). SunTag intensites are plotted for all mRNAs (colored circles) overlaid with the mean ± SD (≥1344 mRNAs per condition, n = 5). (B) Fraction of mRNAs undergoing translation quantified per cell for reporter mRNAs in 4EBP1/2 KD cells ± Torin1 (250 nM, 1 hour). Values are plotted for each cell (colored circles) overlaid with the mean ± SD (≥204 cells per condition, n = 5). (C) Quantification of translation site intensities of reporter mRNAs in 4EBP1/2 KD_LARP1/1B KO cells ±1 hour Torin1 (250 nM). SunTag intensities are plotted for all mRNAs overlaid with the mean ± SD (≥783 mRNAs per condition, n = 3). (D) Fraction of mRNAs undergoing translation quantified per cell of reporter mRNAs in 4EBP1/2 KD_LARP1/1B KO cells ± Torin1 (250 nM, 1 hour). Values are plotted for each cell (colored circles) overlaid with the mean ± SD (≥97 cells per condition, n = 3). For statistics, unpaired t tests were performed, with statistical significance claimed when P < 0.05. (E) Time course of fraction of translating mRNAs per cell for canonical and 5′TOP reporters in 4EBP1/2 KD cells with WT LARP1 or with LARP1/1B KO. Cells were treated with 0-, 30-, 60-, 120-, and 240-min Torin1. Values are plotted as the mean ± SEM (≥97 cells per condition, n = 3 to 5).

Fig 3: Loss of LARP1 results in selective destabilization of 5′TOP mRNAs.(A) Differential gene expression analysis of LARP1 KO cells compared to WT cell lines. Significantly down- and up-regulated mRNAs are highlighted (−log10 P value ≥5 and absolute log2 FC ≥ 0.5) indicating all classical 5′TOP mRNAs (blue circles) as well as all significant non-5′TOP mRNAs (orange circles). For WT, data are the average of n = 2 biological replicates (canonical and 5′TOP reporter cell lines, averaged). For LARP1 KO, data are the average of n = 8 biological replicates (four KO clones of each reporter cell line). (B) Differential gene expression analysis of 4EBP1/2 KD reporter cell lines compared to WT reporter cell lines. Significantly down- and up-regulated mRNAs are highlighted (orange circles, −log10 P value ≥5 and absolute log2 FC ≥ 0.5). 5′TOP mRNAs (blue) are unaffected by 4EBP1/2 KD. Data shown for n = 2 biological replicates (canonical and 5′TOP reporter cell lines, averaged). (C) FC of 5′TOP mRNAs shown in (A) and (B). Mean FC (log2) in LARP1 KO cells = −0.68, in 4EBP1/2 KD cells = −0.06. (D) Correlation plot of mRNA half-lives in LARP1 KO compared to WT reporter cells. Experimental setup of global mRNA stability analysis using s4U labeling (SLAM-seq) shown on top, with 24 hours s4U labeling, washout, and time point collection. RNAs with significantly decreased stability (log2 FC ≤ −1) are highlighted for 5′TOP mRNAs (blue circles) and non-5′TOP mRNAs (orange circles), as well as nonsignificantly down-regulated 5′TOP mRNAs (magenta circles). (E) List of all RNAs highlighted in (D), ranked by log2 FC for 5′TOP mRNAs and non-5′TOP mRNAs separately.

Fig 4: Loss of LARP1 partially alleviates 5′TOP translational repression during Torin1 treatment.(A) Quantification of translation site intensities in LARP1 KO cells ± Torin1 (250 nM, 1 hour). SunTag intensities are plotted for all mRNAs overlaid with the mean ± SD (≥652 mRNAs per condition, n = 3). (B) Fraction of mRNAs undergoing translation quantified per cell in LARP1 KO cells ± Torin1 (250 nM, 1 hour). Values are plotted for each cell (colored circles) overlaid with the mean ± SD (≥91 cells per condition, n = 3). (C) Quantification of translation site intensities in LARP1/1B KO cells ± Torin1 (250 nM, 1 hour). SunTag intensities are plotted for all mRNAs (colored circles) overlaid with the mean ± SD (≥218 mRNAs per condition, n = 3). (D) Fraction of mRNAs undergoing translation quantified per cell in LARP1/1B KO cells ± Torin1 (250 nM, 1 hour). Values are plotted for each cell (colored circles) overlaid with the mean ± SD (≥30 cells per condition, n = 3). For statistics, unpaired t tests were performed, with statistical significance claimed when P < 0.05 (*P < 0.05, **P < 0.01, ****P < 0.0001). (E) Time course of fraction of translating mRNAs per cell for canonical and 5′TOP reporter cell lines in the presence (WT) or absence of LARP1/1B (KO). Cells were treated with 0-, 30-, 60-, 120-, and 240-min Torin1. Values are plotted as the mean ± SEM (≥30 cells per condition, n = 3).

Fig 5: 5’ TOP motifs target transcripts for LARP1-dependent lysosomal degradation.a, Schematic of the GFP reporter. Variants differ only in their first seven nucleotides. b, Effect of 5’ TOP motif on lysosomal targeting of the GFP reporter. RNASET2, PLD3, RNASE4 tKO HEK293T cells stably expressing TMEM192-3x-HA grown in serum-free media were transfected with plasmids encoding the indicated GFP reporter transcripts. Four days after transfection, equal cell numbers were subjected to the LysoIP protocol, and whole-cell and lysosomal RNA were analyzed by quantitative PCR. The amount of GFP mRNA was normalized to that of the 18S rRNA in both whole-cell and lysosomal fractions. 3 independent transfections were done for each reporter. Significance was evaluated by one-way ANOVA (ns, P value >.05; *, P value <.05; **, P value <.01). c, Effect of LARP1 KO on whole-cell 5’ TOP stability. The number above each violin plot indicates how many unique RNAs were considered in that plot. d, Effect of LARP1 KO on lysosomal enrichment for different mRNA types. Protein localization data is from the Human Protein Atlas39, mitochondrial proteins were defined by Mitocarta340, and half-life data are from Agarwal and Kelly41. The number above each violin plot indicates how many unique mRNAs were considered in that plot. e, Effect of LARP1 KO on lysosomal enrichment for different RNA types. The number above each violin plot indicates how many unique RNAs were considered in that plot. f, Effect of LARP1 KO on lysosomal enrichment as a function of TOPscore45. The number above each violin plot indicates how many unique RNAs were considered in that plot.