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p16(INK4a) translation suppressed by miR-24.

Lal A, Kim HH, Abdelmohsen K, Kuwano Y, Pullmann R, Srikantan S, Subrahmanyam R, Martindale JL, Yang X, Ahmed F, Navarro F, Dykxhoorn D, Lieberman J, Gorospe M - PLoS ONE (2008)

Bottom Line: Increased p16 expression with replicative senescence was associated with decreased levels of miR-24, a microRNA that was predicted to associate with the p16 mRNA coding and 3'-untranslated regions.Ectopic miR-24 overexpression reduced p16 protein but not p16 mRNA levels.Together, our results suggest that miR-24 represses the initiation and elongation phases of p16 translation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Biology, National Institute on Aging-IRP, National Institutes of Health, Baltimore, Maryland, United States of America. alal@cbrinstitute.org

ABSTRACT

Background: Expression of the tumor suppressor p16(INK4a) increases during aging and replicative senescence.

Methodology/principal findings: Here, we report that the microRNA miR-24 suppresses p16 expression in human diploid fibroblasts and cervical carcinoma cells. Increased p16 expression with replicative senescence was associated with decreased levels of miR-24, a microRNA that was predicted to associate with the p16 mRNA coding and 3'-untranslated regions. Ectopic miR-24 overexpression reduced p16 protein but not p16 mRNA levels. Conversely, introduction of antisense (AS)-miR-24 blocked miR-24 expression and markedly enhanced p16 protein levels, p16 translation, and the production of EGFP-p16 reporter bearing the miR-24 target recognition sites.

Conclusions/significance: Together, our results suggest that miR-24 represses the initiation and elongation phases of p16 translation.

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Microarray analysis of miRNAs showing reduced expression with senescence.(A) Three independent preparations of total RNA from early-passage (Young, pdl 25) and from late-passage (Senescent, pdl 54) WI-38 cells were subjected to microarray analysis (Exiqon, Materials and Methods). Thirty-one miRNAs showed reduced levels in the senescent cultures (by two-fold in at least two samples), 9 miRNAs showed increased levels (Supplemental Fig. S2). (B) The differential expression of ten downregulated miRNAs was validated using RT-qPCR analysis; the relative levels of each miRNA in Y and S WI-38 cells are shown (means+SEM from three independent experiments).
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pone-0001864-g002: Microarray analysis of miRNAs showing reduced expression with senescence.(A) Three independent preparations of total RNA from early-passage (Young, pdl 25) and from late-passage (Senescent, pdl 54) WI-38 cells were subjected to microarray analysis (Exiqon, Materials and Methods). Thirty-one miRNAs showed reduced levels in the senescent cultures (by two-fold in at least two samples), 9 miRNAs showed increased levels (Supplemental Fig. S2). (B) The differential expression of ten downregulated miRNAs was validated using RT-qPCR analysis; the relative levels of each miRNA in Y and S WI-38 cells are shown (means+SEM from three independent experiments).

Mentions: To test these possibilities, we examined whether RBPs implicated in translational control (like TIAR, TIA-1 or elav/Hu RBPs) bound the p16 mRNA, but no such pdl-dependent RNP interactions were observed (not shown). Therefore, we hypothesized that the translation of p16 mRNA might be influenced by its association with microRNAs. RNA was prepared from Y and S and used for miRNA microarray analysis (Exiqon, Materials and Methods); the complete array report is available from the authors. As shown (Fig. 2A), thirty-one miRNAs were found to be lower in S populations. Importantly, all of the miRNAs for which we were able to amplify PCR products (ten in total) were found to be downregulated in S cells, supporting the accuracy of the microarray analysis (Fig. 2B). The microRNA miR-24 was predicted to bind to the p16 mRNA both in the coding region (CR) and the 3′-untranslated region (UTR) (Fig. 3A), based on analysis using the Miranda and RNA22 programs ([28] Supplemental Fig. S1). Miranda also predicted miR-337 to associate with p16 mRNA, but miR-337 levels were not found to be altered during senescence (Fig. 2A). An additional nine miRNAs that were more highly expressed in S cells (Supplemental Fig. S2) will be investigated separately.


p16(INK4a) translation suppressed by miR-24.

Lal A, Kim HH, Abdelmohsen K, Kuwano Y, Pullmann R, Srikantan S, Subrahmanyam R, Martindale JL, Yang X, Ahmed F, Navarro F, Dykxhoorn D, Lieberman J, Gorospe M - PLoS ONE (2008)

Microarray analysis of miRNAs showing reduced expression with senescence.(A) Three independent preparations of total RNA from early-passage (Young, pdl 25) and from late-passage (Senescent, pdl 54) WI-38 cells were subjected to microarray analysis (Exiqon, Materials and Methods). Thirty-one miRNAs showed reduced levels in the senescent cultures (by two-fold in at least two samples), 9 miRNAs showed increased levels (Supplemental Fig. S2). (B) The differential expression of ten downregulated miRNAs was validated using RT-qPCR analysis; the relative levels of each miRNA in Y and S WI-38 cells are shown (means+SEM from three independent experiments).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2274865&req=5

pone-0001864-g002: Microarray analysis of miRNAs showing reduced expression with senescence.(A) Three independent preparations of total RNA from early-passage (Young, pdl 25) and from late-passage (Senescent, pdl 54) WI-38 cells were subjected to microarray analysis (Exiqon, Materials and Methods). Thirty-one miRNAs showed reduced levels in the senescent cultures (by two-fold in at least two samples), 9 miRNAs showed increased levels (Supplemental Fig. S2). (B) The differential expression of ten downregulated miRNAs was validated using RT-qPCR analysis; the relative levels of each miRNA in Y and S WI-38 cells are shown (means+SEM from three independent experiments).
Mentions: To test these possibilities, we examined whether RBPs implicated in translational control (like TIAR, TIA-1 or elav/Hu RBPs) bound the p16 mRNA, but no such pdl-dependent RNP interactions were observed (not shown). Therefore, we hypothesized that the translation of p16 mRNA might be influenced by its association with microRNAs. RNA was prepared from Y and S and used for miRNA microarray analysis (Exiqon, Materials and Methods); the complete array report is available from the authors. As shown (Fig. 2A), thirty-one miRNAs were found to be lower in S populations. Importantly, all of the miRNAs for which we were able to amplify PCR products (ten in total) were found to be downregulated in S cells, supporting the accuracy of the microarray analysis (Fig. 2B). The microRNA miR-24 was predicted to bind to the p16 mRNA both in the coding region (CR) and the 3′-untranslated region (UTR) (Fig. 3A), based on analysis using the Miranda and RNA22 programs ([28] Supplemental Fig. S1). Miranda also predicted miR-337 to associate with p16 mRNA, but miR-337 levels were not found to be altered during senescence (Fig. 2A). An additional nine miRNAs that were more highly expressed in S cells (Supplemental Fig. S2) will be investigated separately.

Bottom Line: Increased p16 expression with replicative senescence was associated with decreased levels of miR-24, a microRNA that was predicted to associate with the p16 mRNA coding and 3'-untranslated regions.Ectopic miR-24 overexpression reduced p16 protein but not p16 mRNA levels.Together, our results suggest that miR-24 represses the initiation and elongation phases of p16 translation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Biology, National Institute on Aging-IRP, National Institutes of Health, Baltimore, Maryland, United States of America. alal@cbrinstitute.org

ABSTRACT

Background: Expression of the tumor suppressor p16(INK4a) increases during aging and replicative senescence.

Methodology/principal findings: Here, we report that the microRNA miR-24 suppresses p16 expression in human diploid fibroblasts and cervical carcinoma cells. Increased p16 expression with replicative senescence was associated with decreased levels of miR-24, a microRNA that was predicted to associate with the p16 mRNA coding and 3'-untranslated regions. Ectopic miR-24 overexpression reduced p16 protein but not p16 mRNA levels. Conversely, introduction of antisense (AS)-miR-24 blocked miR-24 expression and markedly enhanced p16 protein levels, p16 translation, and the production of EGFP-p16 reporter bearing the miR-24 target recognition sites.

Conclusions/significance: Together, our results suggest that miR-24 represses the initiation and elongation phases of p16 translation.

Show MeSH
Related in: MedlinePlus