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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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Competitive regulation of p53 expression by 7SL and HuR. (A) Schematic of TP53 mRNA depicting potential binding sites for 7SL (green) and HuR (yellow). The thick grey lines and numbers above and below TP53 3′UTR depict regions of complementarity with 7SL. (B) HeLa cells were transfected with 7SL siRNA; 48 h later, HuR association with the indicated mRNAs was quantified by RIP analysis. Data were normalized to the levels of GAPDH mRNA in each IP sample and represented as the enrichment of each mRNA relative to the levels in IgG IP. (C) Top, partial in vitro TP53 3′UTR biotinylated transcripts bearing the sites of interaction with HuR and 7SL [biot-TP53(3′)] or bearing only the sites of interaction with HuR [biot-TP53(3′Δ)]. Bottom, biotinylated RNAs were incubated with non-biotinylated 7SL and the levels of 7SL in the pulldown were measured by RT-qPCR analysis. (D) GST or GST-HuR were incubated in vitro with the biotinylated RNAs shown; following pulldown, western blot analysis was carried out to detect HuR levels. (E) GST-HuR was incubated in vitro with the biotinylated and non-biotinylated RNAs shown and its presence in the pulldown material was assessed by western blot analysis. Data in (B) are the means + S.D. from three independent experiments; data in (C) and (E) are average of two repeats showing similar results; data in (D) are representative of three repeats.
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Figure 4: Competitive regulation of p53 expression by 7SL and HuR. (A) Schematic of TP53 mRNA depicting potential binding sites for 7SL (green) and HuR (yellow). The thick grey lines and numbers above and below TP53 3′UTR depict regions of complementarity with 7SL. (B) HeLa cells were transfected with 7SL siRNA; 48 h later, HuR association with the indicated mRNAs was quantified by RIP analysis. Data were normalized to the levels of GAPDH mRNA in each IP sample and represented as the enrichment of each mRNA relative to the levels in IgG IP. (C) Top, partial in vitro TP53 3′UTR biotinylated transcripts bearing the sites of interaction with HuR and 7SL [biot-TP53(3′)] or bearing only the sites of interaction with HuR [biot-TP53(3′Δ)]. Bottom, biotinylated RNAs were incubated with non-biotinylated 7SL and the levels of 7SL in the pulldown were measured by RT-qPCR analysis. (D) GST or GST-HuR were incubated in vitro with the biotinylated RNAs shown; following pulldown, western blot analysis was carried out to detect HuR levels. (E) GST-HuR was incubated in vitro with the biotinylated and non-biotinylated RNAs shown and its presence in the pulldown material was assessed by western blot analysis. Data in (B) are the means + S.D. from three independent experiments; data in (C) and (E) are average of two repeats showing similar results; data in (D) are representative of three repeats.

Mentions: The RBP HuR binds the 3′UTR of TP53 mRNA and promotes its translation following irradiation with ultraviolet light (23). Recent HuR PAR-CLIP data revealed that the sites where HuR binds within TP53 3′UTR (11) were adjacent to the site of interaction with 7SL (Figure 4A; yellow, HuR interaction sites; green, putative 7SL sites). Since HuR enhances p53 abundance, 7SL lowers it, and both HuR and 7SL bind TP53 3′UTR, we hypothesized that HuR and 7SL specifically compete for binding to TP53 mRNA. To test this possibility, we first silenced 7SL and studied if this intervention affected HuR binding to target mRNAs (15,18,23). As shown, silencing 7SL selectively enhanced HuR binding to TP53 mRNA, but not to VHL, SIRT1 or NCL mRNAs (Figure 4B).


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)

Competitive regulation of p53 expression by 7SL and HuR. (A) Schematic of TP53 mRNA depicting potential binding sites for 7SL (green) and HuR (yellow). The thick grey lines and numbers above and below TP53 3′UTR depict regions of complementarity with 7SL. (B) HeLa cells were transfected with 7SL siRNA; 48 h later, HuR association with the indicated mRNAs was quantified by RIP analysis. Data were normalized to the levels of GAPDH mRNA in each IP sample and represented as the enrichment of each mRNA relative to the levels in IgG IP. (C) Top, partial in vitro TP53 3′UTR biotinylated transcripts bearing the sites of interaction with HuR and 7SL [biot-TP53(3′)] or bearing only the sites of interaction with HuR [biot-TP53(3′Δ)]. Bottom, biotinylated RNAs were incubated with non-biotinylated 7SL and the levels of 7SL in the pulldown were measured by RT-qPCR analysis. (D) GST or GST-HuR were incubated in vitro with the biotinylated RNAs shown; following pulldown, western blot analysis was carried out to detect HuR levels. (E) GST-HuR was incubated in vitro with the biotinylated and non-biotinylated RNAs shown and its presence in the pulldown material was assessed by western blot analysis. Data in (B) are the means + S.D. from three independent experiments; data in (C) and (E) are average of two repeats showing similar results; data in (D) are representative of three repeats.
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Figure 4: Competitive regulation of p53 expression by 7SL and HuR. (A) Schematic of TP53 mRNA depicting potential binding sites for 7SL (green) and HuR (yellow). The thick grey lines and numbers above and below TP53 3′UTR depict regions of complementarity with 7SL. (B) HeLa cells were transfected with 7SL siRNA; 48 h later, HuR association with the indicated mRNAs was quantified by RIP analysis. Data were normalized to the levels of GAPDH mRNA in each IP sample and represented as the enrichment of each mRNA relative to the levels in IgG IP. (C) Top, partial in vitro TP53 3′UTR biotinylated transcripts bearing the sites of interaction with HuR and 7SL [biot-TP53(3′)] or bearing only the sites of interaction with HuR [biot-TP53(3′Δ)]. Bottom, biotinylated RNAs were incubated with non-biotinylated 7SL and the levels of 7SL in the pulldown were measured by RT-qPCR analysis. (D) GST or GST-HuR were incubated in vitro with the biotinylated RNAs shown; following pulldown, western blot analysis was carried out to detect HuR levels. (E) GST-HuR was incubated in vitro with the biotinylated and non-biotinylated RNAs shown and its presence in the pulldown material was assessed by western blot analysis. Data in (B) are the means + S.D. from three independent experiments; data in (C) and (E) are average of two repeats showing similar results; data in (D) are representative of three repeats.
Mentions: The RBP HuR binds the 3′UTR of TP53 mRNA and promotes its translation following irradiation with ultraviolet light (23). Recent HuR PAR-CLIP data revealed that the sites where HuR binds within TP53 3′UTR (11) were adjacent to the site of interaction with 7SL (Figure 4A; yellow, HuR interaction sites; green, putative 7SL sites). Since HuR enhances p53 abundance, 7SL lowers it, and both HuR and 7SL bind TP53 3′UTR, we hypothesized that HuR and 7SL specifically compete for binding to TP53 mRNA. To test this possibility, we first silenced 7SL and studied if this intervention affected HuR binding to target mRNAs (15,18,23). As shown, silencing 7SL selectively enhanced HuR binding to TP53 mRNA, but not to VHL, SIRT1 or NCL mRNAs (Figure 4B).

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