RNA methyltransferase NSUN2 promotes stress-induced HUVEC senescence

Abstract

The tRNA methyltransferase NSUN2 delays replicative senescence by regulating the translation of CDK1 and CDKN1B mRNAs. However, whether NSUN2 influences premature cellular senescence remains untested. Here we show that NSUN2 methylates SHC mRNA in vitro and in cells, thereby enhancing the translation of the three SHC proteins, p66SHC, p52SHC, and p46SHC. Our results further show that the elevation of SHC expression by NSUN2-mediated mRNA methylation increased the levels of ROS, activated p38MAPK, thereby accelerating oxidative stress- and high-glucose-induced senescence of human vascular endothelial cells (HUVEC). Our findings highlight the critical impact of NSUN2-mediated mRNA methylation in promoting premature senescence.

Keywords: Gerotarget; HUVEC; NSUN2; SHC mRNA methylation; premature senescence; translational regulation.

Figure 1. NSUN2 regulates SHC expression

A. HeLa cells were transfected with a vector expressing NSUN2 or with a siRNA targeting NSUN2. Forty eight hours later, cell lysates were prepared and subjected to Western blot analysis to assess the levels of proteins NSUN2, p66SHC, p52SHC, p46SHC, TP53, p16, cyclin A, cyclin B1, and GAPDH. Data are representative from three independent experiments. B. HUVECs were transfected with a siRNA targeting NSUN2. Forty eight h after transfection, Western blot analysis was performed to assess the protein levels of NSUN2, p66SHC, p52SHC, p46SHC, TP53, p16, cyclin A, cyclin B1, and GAPDH. Data are representative from three independent experiments. C.,D. RNA samples described in panels A (C) and B (D) were subjected to RT-qPCR analysis to assess the levels of SHC mRNA. Data represent mean ± SD from 3 independent experiments. E. The cellular ROS level in cells described in Figure 1B were determined. Data represent mean ± SD from 3 independent experiments; significance was analyzed by using Students' t test (**, p < 0.01).

Figure 2. NSUN2 methylates SHC mRNA in vitro

A. Schematic representation depicting the p66SHC mRNA fragments used for in vitro methylation assays. B. Incorporation of ³H-labeled SAM into p66SHC 5′UTR, CR, and 3′UTR fragments (left) as well as 5′UTR, 5′UTR1, 5′UTR2, CR1, CR2, CR3, CR4, CR5, 3′UTR1, 3′UTR2, 3′UTR3, 3′UTR4, and 3′UTR5 fragments (right). The incorporation of ³H-labeled SAM into p66SHC cDNA (DNA) and p16-CR (coding region) served as negative controls. The incorporation of ³H-labeled SAM into bacteria tRNA served as a positive control. C. p66SHC 5′UTR, CR3, 3′UTR1, 3′UTR2, and 3′UTR3 fragments were in vitro methylated by non-isotopic SAM (Met) or kept untreated (Unmet), whereupon these fragments were subjected to HPLC-MS analysis to determine the formation of m5C. Data present the peak value of m5C.

Figure 3. NSUN2 methylates SHC mRNA in cells

A. In vitro methylated 5′UTR1, CR3, 3′UTR1, 3′UTR2, and 3′UTR5 fragments were subjected to bisulfate RNA sequencing analysis to identify the methylation sites, as described in the Materials and Methods section. The percentage of methylation of each identified site (more than 20%) was indicated. B. Incorporation of ³H-labeled SAM into the 5′UTR, CR, and 3′UTR and their variants with methylated sites (5′UTRm, CRm, and 3′UTRm). P16 3′UTR and CR fragments were served as a negative and a positive control, respectively. C. Left, RNA isolated from HUVECs was subjected to IP using anti-m5C or IgG antibodies. The presence of SHC mRNA in the IP materials was analyzed using RT-qPCR. Right, RNA isolated from cells described in Fig. 1B was subjected to IP assays by using anti-m5C antibody, the presence p66SHC mRNA in the IP materials was analyzed by RT-qPCR Data represent the means ± SD from 3 independent experiments; significance was analyzed by Student's t test (**, p < 0.01).

Figure 4. Methylation of SHC mRNA by NSUN2 enhances SHC translation

A. Schematic representation depicting the pGL3-derived reporter vectors used for reporter gene assays. B. HeLa cells were transfected with each of the reporter vectors described in Figure 4A together with a pRL-CMV control reporter. Twenty-four hours later, cells were further transfected with with a siRNA targeting NSun2 and cultured for an additional 48 h. Firefly luciferase activity against Renilla luciferase activity was analyzed. Data represent the means ± SD from 3 independent experiments; significance was analyzed by Student's t test (**, p < 0.01). C. In vitro methylated (Met) or unmethylated (Unmet) luc-5′UTR, luc-5′UTRm, luc-CR, luc-CRm, luc-3′UTR, and luc-3′UTRm reporter transcripts were used for in vitro translation assays. Firefly luciferase activity was measured to reflect the translation efficiency. Data represent the means ± SD from 3 independent experiments; significance was analyzed by Student's t test (**, p < 0.01). D. Cells described in Figure 1B were used for isolating the polysomal fractions. RNA prepared from the fractions was subjected to RT-qPCR analysis to assess the presence of SHC mRNA and β-Actin (ACTB) mRNA in the polysomal fraction. Data represent the means ± SD from 3 independent experiments; significance was analyzed by Student's t test (**, p < 0.01).

Figure 5. The NSUN2-SHC regulatory axis impacts on oxidative stress-induced cellular senescence

A. HUVECs were transfected with an NSUN2 siRNA or a control siRNA. Twenty-four h later, cells were exposed to H₂O₂ (30 μM) and cultured for an additional 48 h. Cell lysates were prepared and subjected to Western blot analysis to assess the levels of p66SHC, p52SHC, p46SHC, p38, p-p38, p16, TP53 and GAPDH. B. Cells described in Figure 5A were subjected to FACS analysis. Data are representative from 3 independent experiments. C. Cellular ROS levels in cells described in Figure 5A were analyzed. Data shown are the mean ± SD from 3 independent experiments and statistical significance was analyzed by Student's t test (**, p < 0.01). D. Cells described in Fig. 5A were subjected to SA-β-gal analysis. Data represent the mean ± SD from 3 independent experiments and statistical significance was analyzed by Student's t test (**, p < 0.01).

Figure 6. The NSUN2-mediated regulation of SHC impacts on high glucose-induced cellular senescence

A. HUVECs were transfected with an NSUN2 siRNA or a control siRNA. Twenty-four h later, cells were exposed to glucose (33 mM) and cultured for an additional 48 h. Cell lysates were prepared and subjected to Western blot analysis to assess the protein levels of p66SHC, p52SHC, p46SHC, p38, p-p38, p16, TP53 and GAPDH. B. Cells described in Figure 6A were subjected to FACS analysis. Data are representative from 3 independent experiments. C. Cellular ROS levels in cells described in Figure 6A were analyzed. Data shown are the mean ± SD from 3 independent experiments and statistical significance was analyzed by Student's t test (**, p < 0.01). D. Cells described in Figure 6A were subjected to SA-β-gal analysis. Data represent the mean ± SD from 3 independent experiments and statistical significance was analyzed by Student's t test (**, p < 0.01).