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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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(A) HeLa cells were transfected with siRNAs directed to SRP proteins and 48 h later the levels of the mRNAs encoding each SRP protein were measured by RT-qPCR (left) and the levels of protein were assessed by western blot analysis (right; the SRP19 antibody was not adequate for this analysis, not shown) and loading was monitored by detecting the housekeeping control protein β-actin. (B) In cells processed as in (A), the levels of TP53 mRNA were measured by RT-qPCR (left) and the levels of p53 protein (as well as loading control β-actin) were assessed by western blot analysis (right). This group included analysis of cells in which SRP68, SRP54 and SRP19 were silenced simultaneously (‘ALL SRP siRNAs’). (C) In cells processed as in (A), the levels of 7SL were measured by RT-qPCR analysis. (D) Forty-eight hours after transfection as in (A), cells were counted using a hemocytometer. In (A)–(D), data are shown as the means + S.D. from three independent experiments. (E)–(G) From each of 12 fractions from glycerol gradients shown in the global RNA profile (10-40% glycerol) (E), RNA was isolated and used for RT-qPCR analysis of 7SL levels (F), and protein was precipitated and used for western blot analysis of SRP54 and SRP68. Data are representative of three independent experiments.
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Figure 3: (A) HeLa cells were transfected with siRNAs directed to SRP proteins and 48 h later the levels of the mRNAs encoding each SRP protein were measured by RT-qPCR (left) and the levels of protein were assessed by western blot analysis (right; the SRP19 antibody was not adequate for this analysis, not shown) and loading was monitored by detecting the housekeeping control protein β-actin. (B) In cells processed as in (A), the levels of TP53 mRNA were measured by RT-qPCR (left) and the levels of p53 protein (as well as loading control β-actin) were assessed by western blot analysis (right). This group included analysis of cells in which SRP68, SRP54 and SRP19 were silenced simultaneously (‘ALL SRP siRNAs’). (C) In cells processed as in (A), the levels of 7SL were measured by RT-qPCR analysis. (D) Forty-eight hours after transfection as in (A), cells were counted using a hemocytometer. In (A)–(D), data are shown as the means + S.D. from three independent experiments. (E)–(G) From each of 12 fractions from glycerol gradients shown in the global RNA profile (10-40% glycerol) (E), RNA was isolated and used for RT-qPCR analysis of 7SL levels (F), and protein was precipitated and used for western blot analysis of SRP54 and SRP68. Data are representative of three independent experiments.

Mentions: Since 7SL is the RNA component of the signal recognition particle (SRP), we studied if the reduction in p53 protein levels was affected by other SRP components. Silencing protein components of the SRP (SRP19, SRP54 or SRP68; Figure 3A) lowered p53 abundance (Figure 3B) but did not affect the levels of TP53 mRNA (Figure 3B) or 7SL (Figure 3C). In addition, silencing SRP proteins did not significantly affect cell viability by 48 h after silencing SRP proteins (Figure 3D). These findings suggest that silencing SRP proteins does not recapitulate the increase in p53 elicited by silencing 7SL in HeLa cells. To test this possibility further, glycerol gradients were studied in order to study if 7SL is present in fractions devoid of SRP proteins. As shown in Figure 3E–G, SRP54 and SRP68 overlapped substantially with 7SL (particularly in fractions 5 through 9 of the gradient), but a sizeable amount of 7SL was found outside of these fractions. In fact, >30% of 7SL was detected in fractions that did not have SRP54 or SRP68. These results support the notion that a subset of 7SL exists outside of the SRPs and further indicate that the influence of 7SL on p53 expression is likely independent of its function as part of the SRP.


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)

(A) HeLa cells were transfected with siRNAs directed to SRP proteins and 48 h later the levels of the mRNAs encoding each SRP protein were measured by RT-qPCR (left) and the levels of protein were assessed by western blot analysis (right; the SRP19 antibody was not adequate for this analysis, not shown) and loading was monitored by detecting the housekeeping control protein β-actin. (B) In cells processed as in (A), the levels of TP53 mRNA were measured by RT-qPCR (left) and the levels of p53 protein (as well as loading control β-actin) were assessed by western blot analysis (right). This group included analysis of cells in which SRP68, SRP54 and SRP19 were silenced simultaneously (‘ALL SRP siRNAs’). (C) In cells processed as in (A), the levels of 7SL were measured by RT-qPCR analysis. (D) Forty-eight hours after transfection as in (A), cells were counted using a hemocytometer. In (A)–(D), data are shown as the means + S.D. from three independent experiments. (E)–(G) From each of 12 fractions from glycerol gradients shown in the global RNA profile (10-40% glycerol) (E), RNA was isolated and used for RT-qPCR analysis of 7SL levels (F), and protein was precipitated and used for western blot analysis of SRP54 and SRP68. Data are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 3: (A) HeLa cells were transfected with siRNAs directed to SRP proteins and 48 h later the levels of the mRNAs encoding each SRP protein were measured by RT-qPCR (left) and the levels of protein were assessed by western blot analysis (right; the SRP19 antibody was not adequate for this analysis, not shown) and loading was monitored by detecting the housekeeping control protein β-actin. (B) In cells processed as in (A), the levels of TP53 mRNA were measured by RT-qPCR (left) and the levels of p53 protein (as well as loading control β-actin) were assessed by western blot analysis (right). This group included analysis of cells in which SRP68, SRP54 and SRP19 were silenced simultaneously (‘ALL SRP siRNAs’). (C) In cells processed as in (A), the levels of 7SL were measured by RT-qPCR analysis. (D) Forty-eight hours after transfection as in (A), cells were counted using a hemocytometer. In (A)–(D), data are shown as the means + S.D. from three independent experiments. (E)–(G) From each of 12 fractions from glycerol gradients shown in the global RNA profile (10-40% glycerol) (E), RNA was isolated and used for RT-qPCR analysis of 7SL levels (F), and protein was precipitated and used for western blot analysis of SRP54 and SRP68. Data are representative of three independent experiments.
Mentions: Since 7SL is the RNA component of the signal recognition particle (SRP), we studied if the reduction in p53 protein levels was affected by other SRP components. Silencing protein components of the SRP (SRP19, SRP54 or SRP68; Figure 3A) lowered p53 abundance (Figure 3B) but did not affect the levels of TP53 mRNA (Figure 3B) or 7SL (Figure 3C). In addition, silencing SRP proteins did not significantly affect cell viability by 48 h after silencing SRP proteins (Figure 3D). These findings suggest that silencing SRP proteins does not recapitulate the increase in p53 elicited by silencing 7SL in HeLa cells. To test this possibility further, glycerol gradients were studied in order to study if 7SL is present in fractions devoid of SRP proteins. As shown in Figure 3E–G, SRP54 and SRP68 overlapped substantially with 7SL (particularly in fractions 5 through 9 of the gradient), but a sizeable amount of 7SL was found outside of these fractions. In fact, >30% of 7SL was detected in fractions that did not have SRP54 or SRP68. These results support the notion that a subset of 7SL exists outside of the SRPs and further indicate that the influence of 7SL on p53 expression is likely independent of its function as part of the SRP.

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