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7SL RNA represses p53 translation by competing with HuR.

Abdelmohsen K, Panda AC, Kang MJ, Guo R, Kim J, Grammatikakis I, Yoon JH, Dudekula DB, Noh JH, Yang X, Martindale JL, Gorospe M - Nucleic Acids Res. (2014)

Bottom Line: The interaction of 7SL with TP53 mRNA reduced p53 translation, as determined by analyzing p53 expression levels, nascent p53 translation and TP53 mRNA association with polysomes.We propose that the competition between 7SL and HuR for binding to TP53 3'UTR contributes to determining the magnitude of p53 translation, in turn affecting p53 levels and the growth-suppressive function of p53.Our findings suggest that targeting 7SL may be effective in the treatment of cancers with reduced p53 levels.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA abdelmohsenk@mail.nih.gov.

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Opposite regulation of p53 translation by 7SL and HuR. (A) HeLa cells were transfected with the indicated siRNAs; 48 h later, the levels of p53, HuR and loading control β-actin were assessed by western blot analysis and p53 signals were quantified by densitometry and plotted. (B) Top, luciferase reporter constructs pmirGLO-p53(3′UTR), bearing partial sequence of TP53 3′UTR (nucleotides 1421–2629) that includes 7SL and HuR binding sites, and pmirGLO-p53(3′UTRΔ) that lacks the 7SL interaction site (nucleotides 2161–2300). Thirty-six hours after transfection of the indicated siRNAs, luciferase plasmids were transfected for 6 h and followed by luciferase assay 12 h thereafter. (C) RIP analysis of HuR interaction with the FL mRNAs expressed from the reporters in panel (B); data represent enrichment levels relative to FL mRNA abundance in IgG IP, using GAPDH mRNA for normalization. (D) De novo translation of p53 as well as housekeeping control protein β-Tubulin (Tub), as assessed by 35S-p53 IP and 35S-Tub IP 48 h after transfection of the indicated siRNAs (details in Materials and Methods section); ‘Fold’ quantified 35S-p53 signals, relative to signals in the Ctrl siRNA group. (E) and (F) Cells were transfected as described in (A) followed by fractionation through sucrose gradients. Global RNA profile for these transfection conditions are shown (E). The relative distribution of TP53 mRNA (and housekeeping GAPDH mRNA) was studied by RT-qPCR analysis of RNA in each of 10 gradient fractions (F). Data in (A)–(C) represent the means and S.D. from three independent experiments; data in (D)–(F) are representative of three independent experiments; P values are shown.
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Figure 5: Opposite regulation of p53 translation by 7SL and HuR. (A) HeLa cells were transfected with the indicated siRNAs; 48 h later, the levels of p53, HuR and loading control β-actin were assessed by western blot analysis and p53 signals were quantified by densitometry and plotted. (B) Top, luciferase reporter constructs pmirGLO-p53(3′UTR), bearing partial sequence of TP53 3′UTR (nucleotides 1421–2629) that includes 7SL and HuR binding sites, and pmirGLO-p53(3′UTRΔ) that lacks the 7SL interaction site (nucleotides 2161–2300). Thirty-six hours after transfection of the indicated siRNAs, luciferase plasmids were transfected for 6 h and followed by luciferase assay 12 h thereafter. (C) RIP analysis of HuR interaction with the FL mRNAs expressed from the reporters in panel (B); data represent enrichment levels relative to FL mRNA abundance in IgG IP, using GAPDH mRNA for normalization. (D) De novo translation of p53 as well as housekeeping control protein β-Tubulin (Tub), as assessed by 35S-p53 IP and 35S-Tub IP 48 h after transfection of the indicated siRNAs (details in Materials and Methods section); ‘Fold’ quantified 35S-p53 signals, relative to signals in the Ctrl siRNA group. (E) and (F) Cells were transfected as described in (A) followed by fractionation through sucrose gradients. Global RNA profile for these transfection conditions are shown (E). The relative distribution of TP53 mRNA (and housekeeping GAPDH mRNA) was studied by RT-qPCR analysis of RNA in each of 10 gradient fractions (F). Data in (A)–(C) represent the means and S.D. from three independent experiments; data in (D)–(F) are representative of three independent experiments; P values are shown.

Mentions: Consistent with earlier evidence (23), HuR promoted p53 expression (Figure 5A), but the enhancement of p53 levels after silencing 7SL was inhibited when HuR was silenced (Figure 5A). These data suggested that HuR and 7SL competed for binding to TP53 mRNA and that HuR was required for enhancing p53 expression in 7SL-silenced cells. Since modulating 7SL or HuR did not alter TP53 mRNA levels [Figure 2D; (23)], we hypothesized that 7SL RNA and HuR may regulate p53 mRNA translation by binding to the TP53 3′UTR. To test this hypothesis, we cloned the TP53 3′UTR [nucleotides 1421–2629 (23)] into a dual luciferase construct [pmirGlo-p53(3′UTR)] and prepared a TP53 3′UTR reporter plasmid lacking the 7SL interaction region (nucleotides 2161–2300) [pmirGlo-p53(3′UTRΔ). In cells transfected with pmirGlo-p53(3′UTR), luciferase activity [FL/RL] increased in 7SL-silenced cells and decreased in HuR-silenced cells (Figure 5B). Importantly, the induction in luciferase activity by 7SL silencing was attenuated in cells with both 7SL and HuR silenced and in cells expressing pmirGlo-p53(3′UTRΔ). Moreover, HuR binding to TP53 3′UTR, as determined by ribonucleoprotein immunoprecipitation (RIP) analysis of HuR binding to FL RNA expressed from the reporter plasmids in Figure 5B, revealed that removing the 7SL site increased the interaction of HuR with the TP53 3′UTR. Since the precise TP53 3′UTR nucleotides within the HuR PAR-CLIP sites with which HuR interacts are not known, we were unable to test the impact of introducing point mutations upon 7SL binding to TP53 3′UTR. Collectively, these results indicate that 7SL repressed reporter activity by interacting with the TP53 3′UTR region complementary to 7SL and that derepression by silencing 7SL required the presence of HuR.


7SL RNA represses p53 translation by competing with HuR.

Abdelmohsen K, Panda AC, Kang MJ, Guo R, Kim J, Grammatikakis I, Yoon JH, Dudekula DB, Noh JH, Yang X, Martindale JL, Gorospe M - Nucleic Acids Res. (2014)

Opposite regulation of p53 translation by 7SL and HuR. (A) HeLa cells were transfected with the indicated siRNAs; 48 h later, the levels of p53, HuR and loading control β-actin were assessed by western blot analysis and p53 signals were quantified by densitometry and plotted. (B) Top, luciferase reporter constructs pmirGLO-p53(3′UTR), bearing partial sequence of TP53 3′UTR (nucleotides 1421–2629) that includes 7SL and HuR binding sites, and pmirGLO-p53(3′UTRΔ) that lacks the 7SL interaction site (nucleotides 2161–2300). Thirty-six hours after transfection of the indicated siRNAs, luciferase plasmids were transfected for 6 h and followed by luciferase assay 12 h thereafter. (C) RIP analysis of HuR interaction with the FL mRNAs expressed from the reporters in panel (B); data represent enrichment levels relative to FL mRNA abundance in IgG IP, using GAPDH mRNA for normalization. (D) De novo translation of p53 as well as housekeeping control protein β-Tubulin (Tub), as assessed by 35S-p53 IP and 35S-Tub IP 48 h after transfection of the indicated siRNAs (details in Materials and Methods section); ‘Fold’ quantified 35S-p53 signals, relative to signals in the Ctrl siRNA group. (E) and (F) Cells were transfected as described in (A) followed by fractionation through sucrose gradients. Global RNA profile for these transfection conditions are shown (E). The relative distribution of TP53 mRNA (and housekeeping GAPDH mRNA) was studied by RT-qPCR analysis of RNA in each of 10 gradient fractions (F). Data in (A)–(C) represent the means and S.D. from three independent experiments; data in (D)–(F) are representative of three independent experiments; P values are shown.
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Figure 5: Opposite regulation of p53 translation by 7SL and HuR. (A) HeLa cells were transfected with the indicated siRNAs; 48 h later, the levels of p53, HuR and loading control β-actin were assessed by western blot analysis and p53 signals were quantified by densitometry and plotted. (B) Top, luciferase reporter constructs pmirGLO-p53(3′UTR), bearing partial sequence of TP53 3′UTR (nucleotides 1421–2629) that includes 7SL and HuR binding sites, and pmirGLO-p53(3′UTRΔ) that lacks the 7SL interaction site (nucleotides 2161–2300). Thirty-six hours after transfection of the indicated siRNAs, luciferase plasmids were transfected for 6 h and followed by luciferase assay 12 h thereafter. (C) RIP analysis of HuR interaction with the FL mRNAs expressed from the reporters in panel (B); data represent enrichment levels relative to FL mRNA abundance in IgG IP, using GAPDH mRNA for normalization. (D) De novo translation of p53 as well as housekeeping control protein β-Tubulin (Tub), as assessed by 35S-p53 IP and 35S-Tub IP 48 h after transfection of the indicated siRNAs (details in Materials and Methods section); ‘Fold’ quantified 35S-p53 signals, relative to signals in the Ctrl siRNA group. (E) and (F) Cells were transfected as described in (A) followed by fractionation through sucrose gradients. Global RNA profile for these transfection conditions are shown (E). The relative distribution of TP53 mRNA (and housekeeping GAPDH mRNA) was studied by RT-qPCR analysis of RNA in each of 10 gradient fractions (F). Data in (A)–(C) represent the means and S.D. from three independent experiments; data in (D)–(F) are representative of three independent experiments; P values are shown.
Mentions: Consistent with earlier evidence (23), HuR promoted p53 expression (Figure 5A), but the enhancement of p53 levels after silencing 7SL was inhibited when HuR was silenced (Figure 5A). These data suggested that HuR and 7SL competed for binding to TP53 mRNA and that HuR was required for enhancing p53 expression in 7SL-silenced cells. Since modulating 7SL or HuR did not alter TP53 mRNA levels [Figure 2D; (23)], we hypothesized that 7SL RNA and HuR may regulate p53 mRNA translation by binding to the TP53 3′UTR. To test this hypothesis, we cloned the TP53 3′UTR [nucleotides 1421–2629 (23)] into a dual luciferase construct [pmirGlo-p53(3′UTR)] and prepared a TP53 3′UTR reporter plasmid lacking the 7SL interaction region (nucleotides 2161–2300) [pmirGlo-p53(3′UTRΔ). In cells transfected with pmirGlo-p53(3′UTR), luciferase activity [FL/RL] increased in 7SL-silenced cells and decreased in HuR-silenced cells (Figure 5B). Importantly, the induction in luciferase activity by 7SL silencing was attenuated in cells with both 7SL and HuR silenced and in cells expressing pmirGlo-p53(3′UTRΔ). Moreover, HuR binding to TP53 3′UTR, as determined by ribonucleoprotein immunoprecipitation (RIP) analysis of HuR binding to FL RNA expressed from the reporter plasmids in Figure 5B, revealed that removing the 7SL site increased the interaction of HuR with the TP53 3′UTR. Since the precise TP53 3′UTR nucleotides within the HuR PAR-CLIP sites with which HuR interacts are not known, we were unable to test the impact of introducing point mutations upon 7SL binding to TP53 3′UTR. Collectively, these results indicate that 7SL repressed reporter activity by interacting with the TP53 3′UTR region complementary to 7SL and that derepression by silencing 7SL required the presence of HuR.

Bottom Line: The interaction of 7SL with TP53 mRNA reduced p53 translation, as determined by analyzing p53 expression levels, nascent p53 translation and TP53 mRNA association with polysomes.We propose that the competition between 7SL and HuR for binding to TP53 3'UTR contributes to determining the magnitude of p53 translation, in turn affecting p53 levels and the growth-suppressive function of p53.Our findings suggest that targeting 7SL may be effective in the treatment of cancers with reduced p53 levels.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA abdelmohsenk@mail.nih.gov.

Show MeSH
Related in: MedlinePlus