Translation regulation of specific mRNAs by RPS26 C-terminal RNA-binding tail integrates energy metabolism and AMPK-mTOR signaling

Abstract

Increasing evidence suggests that ribosome composition and modifications contribute to translation control. Whether direct mRNA binding by ribosomal proteins regulates the translation of specific mRNA and contributes to ribosome specialization has been poorly investigated. Here, we used CRISPR-Cas9 to mutate the RPS26 C-terminus (RPS26dC) predicted to bind AUG upstream nucleotides at the exit channel. RPS26 binding to positions -10 to -16 of short 5' untranslated region (5'UTR) mRNAs exerts positive and negative effects on translation directed by Kozak and Translation Initiator of Short 5'UTR (TISU), respectively. Consistent with that, shortening the 5'UTR from 16 to 10 nt diminished Kozak and enhanced TISU-driven translation. As TISU is resistant and Kozak is sensitive to energy stress, we examined stress responses and found that the RPS26dC mutation confers resistance to glucose starvation and mTOR inhibition. Furthermore, the basal mTOR activity is reduced while AMP-activated protein kinase is activated in RPS26dC cells, mirroring energy-deprived wild-type (WT) cells. Likewise, the translatome of RPS26dC cells is correlated to glucose-starved WT cells. Our findings uncover the central roles of RPS26 C-terminal RNA binding in energy metabolism, in the translation of mRNAs bearing specific features and in the translation tolerance of TISU genes to energy stress.

Figure 1.

Generation and characterization of RPS26 C-terminal deletion mutant clones. (A) Human RPS26 [Protein Data Bank (PDB) ID: 4V6X] was structurally aligned to RPS26 from yeast 48S PIC (PDB ID: 3J81) using PyMol (shown in cartoon format). Human RPS26 is colored in turquoise, yeast RPS26 in green, mRNA backbone in orange and side chains are colored in green with blue and red. The human RPS26 C-terminus is shown in pink, R82 from yeast in red and K82 from human in purple. mRNA positions −10, −6, −5 and −4 are highlighted in black, dark gray, gray and light gray, respectively. (B) Western blot analysis of WT and RPS26dC cells using anti-RPS26 and anti-GAPDH antibodies. The graph shows the RPS26 protein levels normalized to GAPDH (N = 3). (C) WT (red) and RPS26dC (blue) cells were seeded and cell proliferation was measured using a luminescent cell viability assay at the indicated time points. Luminescence values were normalized to the values of day 1 (N ≥ 4). (D) Polysome profiling of WT and RPS26dC cells. Cell lysates of WT (red) or RPS26dC (blue) were subjected to sucrose gradient sedimentation followed by a fraction collection to obtain polysome profiles. Representative profiles are shown. The ratios of polysome to monosome (P/M) and 40S to 60S of three independent repeats are indicated. The asterisk denotes a statistically significant difference (P < 0.05). The free, 40S, 60S and 80S fractions were subjected to western blot analysis using anti-RPS26 (E), anti-RPS3 (F) and anti-RPL17 (G). Percentages of association were quantified (N = 3). Representative immunoblots are shown. Asterisks denote statistically significant differences (*P < 0.05, ***P < 0.005).

Figure 2.

RPS26 C-terminal deletion effect on start codon fidelity of different 5′UTR length and AUG contexts. (A) A schematic representation of the GFP reporter gene with an AUG in either a sub-optimal Kozak (GGAAUGU) or a TISU context (CAAGAUGGCGGC), preceded by a long or short 5′UTR and downstream in-frame AUG. US and DS (in red) denote the upstream and downstream translation initiation sites, respectively. Arrow indicates the position of the TSS. WT or RPS26dC cells were co-transfected with a GFP plasmid bearing an AUG in a strong (B) or TISU context (C) preceded by a short 5′UTR or a long 5′UTR and a strong context (D) together with a firefly luciferase as a normalizing control. Cells were harvested 24 h after transfection and normalized amounts were loaded on SDS–PAGE followed by western blot analysis using an anti-GFP antibody. US and DS denote upstream and downstream initiation sites, respectively. Representative immunoblots are shown. Quantified results are shown in the graphs in which the relative intensity of the upstream translation site is presented by orange bars and the downstream translation site by yellow bars. The overall translation of 16 or 94 nt 5′UTR in the WT cells was set to 1. Gray and black asterisks on the graphs denote a statistically significant difference in overall translation or US/DS ratio of translation directed by strong context with short 5′UTR, respectively (N ≥ 3). (E) A schematic representation of the reporter gene with a 5′UTR of 16 nt of original Renilla luciferase or mutated 5′UTR. (F) A total of five different 5′UTR reporters were transcribed and capped in vitro and the resultant mRNAs were then transfected into WT and RPS26dC cells where they underwent in vivo translation. Cells were harvested 24 h after transfection and luciferase levels were measured. Luciferase levels of the original 5′UTR in WT and RPS26dC were set to 1. Red and blue asterisks denote a statistically significant difference between original and mutated 5′UTR luciferase levels in WT and RPS26dC, respectively. Black asterisks denote a statistically significant difference in luciferase levels between WT and RPS26dC cells (N = 5). Asterisks denote statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001).

Figure 3.

The effects of stresses on RPS26dC viability and association with the ribosome. (A–C) Cell viability assay after different treatments of WT, RPS26dC and RPS26dC2 cells for 24 h. Cells were treated with 0.9 g/l glucose or without glucose (A), with 25 or 75 nM Torin-1 (B), or with 500 or 1000 nM thapsigargin (C). Luminescence values were normalized to the values of control conditions (N ≥ 3). (D) Polysome profiling of WT cells and RPS26dC cells with and without glucose. Cell lysates of WT and RPS26dC cells with and without glucose for 4 h were subjected to sucrose gradient sedimentation followed by fraction collection to obtain polysome profiles. Representative profiles are shown (WT: red and light gray, respectively; RPS26dC: blue and gray, respectively). (E) The free, 40S, 60S, 80S and two ribosomes fractions of the glucose-starved WT and RPS26dC cells were subjected to western blot analysis using anti-RPS26. Representative immunoblots are shown. Asterisks denote statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001).

Figure 4.

AMPK-dependent inhibition of mTOR in RPS26 C-terminus deletion cells. (A, B) WT and RPS26dC cells were treated with 0.9 g/l or without glucose. Cells were harvested after 4 h and subjected to western blot analysis using anti-4EBP and anti-phosphorylated 4EBP (p-4EBP) (A) or anti-AMPK and anti-phosphorylated AMPK (p-AMPK) (B) and anti-tubulin antibodies (N = 3). (C) Cells were treated with or without glucose in the presence or absence of 20 µM compound C. Cells were harvested after 3 h and subjected to western blot analysis using anti-4EBP, anti-p-4EBP and anti-tubulin antibodies. Representative immunoblots are shown. Quantified results are shown in the graphs (N = 3). (D) ATP/ADP ratio per cell of WT (red) and RPS26dC (blue) cells (N = 5). The graphs represent the mean ± SE from three to five independent experiments. Asterisks denote statistically significant differences (*P < 0.05, ****P < 0.001).

Figure 5.

Global translation analysis of RPS26dC cells revealed energy stress signature. (A) Regression analysis between log₂(RPS26dC TE/WT TE) and log₂(WT GS TE/WT TE). R = 0.435, P< 2.2 × 10⁻¹⁶. Boxplots presenting the TE distributions of TISU (B), OXPHOS pathway (C), glycolysis pathway (D), fatty acid oxidation pathway (E) and TOP-containing RP genes (F) in all samples. Asterisks denote statistically significant differences (*P < 0.05, ***P < 0.005, ****P < 0.001).