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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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7SL binds TP53 mRNA and lowers p53 abundance. (A) Sequence alignment of 7SL and TP53 mRNA using BLAST shows potential sense–antisense interactions. (B) TP53 mRNA pulldown using the biotin-ASOs shown (see the Materials and Methods section) was followed by RT-qPCR analysis to quantify TP53 mRNA and 7SL levels; GAPDH mRNA was used for normalization. (C) HeLa cells were transfected with the indicated siRNAs; 48 h later, the levels of p53, p21 and loading control β-actin were assessed by western blot analysis. (D) The levels of TP53 and CDKN1A mRNA (encoding p21) and normalization control 18S rRNA were quantified by RT-qPCR. (E) Forty-eight hours after transfecting plasmids pcDNA3 or pcDNA3-7SL, total cellular 7SL levels were measured by RT-qPCR analysis (left) and the levels of p53 and loading control HSP90 by western blot analysis (right). Data in (B) and (E) are the means + S.D. from three independent experiments. In (C) and (E), the means ± standard deviation of p53 signals are indicated and the p values are shown.
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Figure 2: 7SL binds TP53 mRNA and lowers p53 abundance. (A) Sequence alignment of 7SL and TP53 mRNA using BLAST shows potential sense–antisense interactions. (B) TP53 mRNA pulldown using the biotin-ASOs shown (see the Materials and Methods section) was followed by RT-qPCR analysis to quantify TP53 mRNA and 7SL levels; GAPDH mRNA was used for normalization. (C) HeLa cells were transfected with the indicated siRNAs; 48 h later, the levels of p53, p21 and loading control β-actin were assessed by western blot analysis. (D) The levels of TP53 and CDKN1A mRNA (encoding p21) and normalization control 18S rRNA were quantified by RT-qPCR. (E) Forty-eight hours after transfecting plasmids pcDNA3 or pcDNA3-7SL, total cellular 7SL levels were measured by RT-qPCR analysis (left) and the levels of p53 and loading control HSP90 by western blot analysis (right). Data in (B) and (E) are the means + S.D. from three independent experiments. In (C) and (E), the means ± standard deviation of p53 signals are indicated and the p values are shown.

Mentions: Long ncRNAs can enhance or suppress gene expression by interacting directly with mRNAs (2). A BLAST survey to identify possible interactions between 7SL and regulators of cell growth revealed that 7SL was predicted to form extensive regions of sense–antisense interaction with the 3′UTR of the TP53 mRNA (nucleotides 2209–2367; Figure 2A, Supplementary Figure S2); sequence identities were 85–93%. Other putative interaction partners of 7SL are listed (Supplementary Table S1). To verify if endogenous 7SL and TP53 mRNAs formed hybrid RNA interactions, we designed biotinylated AS oligomers complementary to TP53 mRNA (TP53-biot-ASO), to a negative control (the lncRNA MIAT, MIAT-biot-ASO), and to mRNAs encoding insulin (INS-biot-ASO) and Sirtuin-1 (SIRT1-biot-ASO). Incubation of lysates prepared from HeLa cells with biot-ASOs revealed that 7SL was enriched in TP53-biot-ASO pulldown samples, but not in the other biot-ASO pulldowns, indicating that 7SL selectively associated with TP53 mRNA (Figure 2B). The enrichment of GAPDH mRNA in the pulldown samples was measured to assess background RNA binding to biot-ASOs.


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)

7SL binds TP53 mRNA and lowers p53 abundance. (A) Sequence alignment of 7SL and TP53 mRNA using BLAST shows potential sense–antisense interactions. (B) TP53 mRNA pulldown using the biotin-ASOs shown (see the Materials and Methods section) was followed by RT-qPCR analysis to quantify TP53 mRNA and 7SL levels; GAPDH mRNA was used for normalization. (C) HeLa cells were transfected with the indicated siRNAs; 48 h later, the levels of p53, p21 and loading control β-actin were assessed by western blot analysis. (D) The levels of TP53 and CDKN1A mRNA (encoding p21) and normalization control 18S rRNA were quantified by RT-qPCR. (E) Forty-eight hours after transfecting plasmids pcDNA3 or pcDNA3-7SL, total cellular 7SL levels were measured by RT-qPCR analysis (left) and the levels of p53 and loading control HSP90 by western blot analysis (right). Data in (B) and (E) are the means + S.D. from three independent experiments. In (C) and (E), the means ± standard deviation of p53 signals are indicated and the p values are shown.
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Figure 2: 7SL binds TP53 mRNA and lowers p53 abundance. (A) Sequence alignment of 7SL and TP53 mRNA using BLAST shows potential sense–antisense interactions. (B) TP53 mRNA pulldown using the biotin-ASOs shown (see the Materials and Methods section) was followed by RT-qPCR analysis to quantify TP53 mRNA and 7SL levels; GAPDH mRNA was used for normalization. (C) HeLa cells were transfected with the indicated siRNAs; 48 h later, the levels of p53, p21 and loading control β-actin were assessed by western blot analysis. (D) The levels of TP53 and CDKN1A mRNA (encoding p21) and normalization control 18S rRNA were quantified by RT-qPCR. (E) Forty-eight hours after transfecting plasmids pcDNA3 or pcDNA3-7SL, total cellular 7SL levels were measured by RT-qPCR analysis (left) and the levels of p53 and loading control HSP90 by western blot analysis (right). Data in (B) and (E) are the means + S.D. from three independent experiments. In (C) and (E), the means ± standard deviation of p53 signals are indicated and the p values are shown.
Mentions: Long ncRNAs can enhance or suppress gene expression by interacting directly with mRNAs (2). A BLAST survey to identify possible interactions between 7SL and regulators of cell growth revealed that 7SL was predicted to form extensive regions of sense–antisense interaction with the 3′UTR of the TP53 mRNA (nucleotides 2209–2367; Figure 2A, Supplementary Figure S2); sequence identities were 85–93%. Other putative interaction partners of 7SL are listed (Supplementary Table S1). To verify if endogenous 7SL and TP53 mRNAs formed hybrid RNA interactions, we designed biotinylated AS oligomers complementary to TP53 mRNA (TP53-biot-ASO), to a negative control (the lncRNA MIAT, MIAT-biot-ASO), and to mRNAs encoding insulin (INS-biot-ASO) and Sirtuin-1 (SIRT1-biot-ASO). Incubation of lysates prepared from HeLa cells with biot-ASOs revealed that 7SL was enriched in TP53-biot-ASO pulldown samples, but not in the other biot-ASO pulldowns, indicating that 7SL selectively associated with TP53 mRNA (Figure 2B). The enrichment of GAPDH mRNA in the pulldown samples was measured to assess background RNA binding to biot-ASOs.

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