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mTORC1-Rps15 Axis Contributes to the Mechanisms Underlying Global Translation Reduction During Senescence of Mouse Embryonic Fibroblasts

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mTORC1-Rps15 Axis Contributes to the Mechanisms Underlying Global Translation Reduction During Senescence of Mouse Embryonic Fibroblasts

Su Wu et al. Front Cell Dev Biol.

Abstract

The reduction of protein translation is a common feature in senescent cells and aging organisms, yet the underlying mechanisms are not fully understood. Here we show that both global mRNA translation and mammalian/mechanistic target of rapamycin complex 1 (mTORC1) kinase activity are declined in a senescent model of mouse embryonic fibroblasts (MEFs). Furthermore, RNA-seq analyses from polysomal versus total mRNA fractions identify TOP-like mRNA of Rps15 whose translation is regulated by mTORC1 during MEF senescence. Overexpression of Rps15 delays MEF senescence, possibly through regulating ribosome maturation. Together, these findings indicate that the activation of mTORC1-Rps15 axis ameliorate senescence by regulating ribosome biogenesis, which may provide further insights into aging research.

Keywords: MEF; Rps15; cell senescence; mRNA translation; mTORC1.

Figures

FIGURE 1
FIGURE 1
Protein synthesis is globally reduced in senescent MEFs. (A) Growth curve of MEFs from passage 0 to passage 5. PRE and SEN refer to presenescent and senescent, separately. (B) Relative quantification of p16INK4a, p21, and p53 mRNAs in young and senescent MEFs. β-actin was used as internal control (Mean ± SEM, n = 3, ∗∗∗p < 0.001, p < 0.05). (C,D) Representative western blot and quantification of phosphorylated protein and total protein levels of p38 in cell extracts from passage 1 and passage 5 MEFs. Total p38 protein was used as internal loading control (Mean ± SEM, n = 3, p < 0.05). (E,F) SA-β-gal staining and SA-β-gal positive rate of MEFs in passage 1 and passage 5 (Mean ± SEM, n = 3, ∗∗∗p < 0.001). (G) Puromycin incorporation assay of presenescent and senescent MEFs. SYPRO Ruby staining was used to visualize total proteins (left) and immunoactivity of puromycin indicates nascent peptide synthesis rate (right). β-actin was used as internal loading control. (H) Polysomal profiles of young and senescent MEFs with continuous sucrose gradient of 10–50% were fractioned and measured with absorbance of light at 260 nm. Peaks belonged to small subunit of 40S, large subunit of 60S, intact ribosome of 80S and polysomes were labeled.
FIGURE 2
FIGURE 2
Polysomal RNA-seq reveals that the ribosome biogenesis is deficient in senescent cells. (A) RNA-seq samples are collected using total mRNA extract and polysomal mRNA fraction in young and senescent MEFs. (B) Gene numbers of transcriptional and translational changes were calculated from comparing total and polysomal RNA-seq data. The threshold of fold-changes was set to 1.5 for the trend of up-regulation and 0.667 for the trend of down-regulation. (C) Volcano plot shows significantly regulated genes on translational level. The threshold of significance was set to FPKM SEN/PRE >1.5, FDR q < 0.05 for up-regulation and FPKM SEN/PRE <0.67, FDR q < 0.05 for down-regulation. (D) Biological process analysis of genes which were only down-regulated in translational level shown in panel (C). (E) Translationally changed genes of ribosomal protein calculated from polysomal RNA-seq data. All 46 large subunit ribosomal proteins were illustrated in the upper part, while 30 small subunit ribosomal protein were shown in the lower part.
FIGURE 3
FIGURE 3
mTORC1 regulates Rps15 mRNA translation during senescence. (A,B) Representative western blot and quantification of phosphorylated protein and total protein levels of S6K, S6, 4E-BP1 in cell extracts from young and senescent MEFs. Relative total proteins were used as internal loading control (Mean ± SEM, n = 3, ∗∗p < 0.01, p < 0.05). (C,D) Relative quantification of Rps15 mRNA using polysomal mRNAs (C) and total mRNAs (D) extracted from young and senescent MEFs. β-actin was used as internal control (Mean ± SEM, n = 3, p < 0.05). (E,F) Relative quantification of β-actin (E) and Rps15 (F) mRNA using polysomal mRNAs extracted from young and senescent MEFs with adding spike RNA of mScarlet after polysome profiling. Spike RNA of mScarlet mRNA was used as internal control (Mean ± SEM, n = 3, ∗∗p < 0.01). (G) Relative quantification of Rps15 mRNA-spike ratio to β-actin-spike RNA ratio from the results of panels (A,B) (Mean ± SEM, n = 3, p < 0.05). (H) Transcription start site (TSS) annotations for TOP-like mRNA Rps15. Arrow head indicates TSS. Bold letters refer to TOP-like structure. Rps15 5’UTR sequence was from Ribosomal Protein Gene Database (RPG). (I,J) Relative quantification of Rps15 mRNA using total mRNAs (I) and polysomal mRNAs (J) extracted from DMSO or rapamycin treated young MEFs (250 nM, 2 h). β-actin was used as internal control (Mean ± SEM, n = 3, p < 0.05).
FIGURE 4
FIGURE 4
Rps15 is important for ribosome biogenesis and overexpression of Rps15 ameliorates senescent phenotypes in MEFs. (A) Relative quantification of Rps15 mRNA after knockdown Rps15 in MEFs. β-actin was used as internal control (Mean ± SEM, n = 3, ∗∗p < 0.01). (B) Polysomal profiles of shRps15 MEFs and negative control with continuous sucrose gradient of 10–50% were fractioned and measured with absorbance of light at 260 nm. Peaks belonged to large subunit of 60S, intact ribosome of 80S and polysomes were labeled. (C) Representative western blot analysis of untransfected control MEFs (CT) and overexpression of GFP, Rps15, Rps14, and Rps24 in extracts from respective stable-expression MEFs, recognized by anti-HA (mouse). β-actin was used as internal loading control. (D) Growth curves of Rps15, Rps24, Rps14, GFP stable transgenic MEFs and untransfected control MEF (CT) starting from passage 1 (Mean ± SEM, n = 3, p < 0.05, ∗∗p < 0.01). (E) SA-β-gal positive rate of senescent Rps15, Rps24, Rps14, GFP stable-expression MEFs and untransfected control MEFs (CT) (Mean ± SEM, n = 3, ∗∗∗p < 0.001). (F) Relative quantification of p16INK4a mRNAs in senescent Rps15, Rps24, Rps14, GFP stable-expression MEFs and untransfected control MEFs (CT). β-actin was used as internal control (Mean ± SEM, n = 3). (G) A schematic model of mTORC1-Rps15 axis contributes in senescence-associated translation reduction.

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