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MicroRNA-130a associates with ribosomal protein L11 to suppress c-Myc expression in response to UV irradiation.

Li Y, Challagundla KB, Sun XX, Zhang Q, Dai MS - Oncotarget (2015)

Bottom Line: Yet, mechanism underlying UV-induced c-Myc reduction is not completely understood.Overexpression of miR-130a promotes the Ago2 binding to c-myc mRNA, significantly reduces the levels of both c-Myc protein and mRNA and inhibits cell proliferation.Inhibiting miR-130a significantly suppresses UV-mediated c-Myc reduction.

View Article: PubMed Central - PubMed

Affiliation: Departments of Molecular & Medical Genetics, School of Medicine and the OHSU Knight Cancer Institute, Oregon Health & Science University, Portland, OR 97239, USA.

ABSTRACT
The oncoprotein c-Myc is essential for cell growth and proliferation while its deregulated overexpression is associated with most human cancers. Thus tightly regulated levels and activity of c-Myc are critical for maintaining normal cell homeostasis. c-Myc is down-regulated in response to several types of stress, including UV-induced DNA damage. Yet, mechanism underlying UV-induced c-Myc reduction is not completely understood. Here we report that L11 promotes miR-130a targeting of c-myc mRNA to repress c-Myc expression in response to UV irradiation. miR-130a targets the 3'-untranslated region (UTR) of c-myc mRNA. Overexpression of miR-130a promotes the Ago2 binding to c-myc mRNA, significantly reduces the levels of both c-Myc protein and mRNA and inhibits cell proliferation. UV treatment markedly promotes the binding of L11 to miR-130a, c-myc mRNA as well as Ago2 in cells. Inhibiting miR-130a significantly suppresses UV-mediated c-Myc reduction. We further show that L11 is relocalized from the nucleolus to the cytoplasm where it associates with c-myc mRNA upon UV treatment. Together, these results reveal a novel mechanism underlying c-Myc down-regulation in response to UV-mediated DNA damage, wherein L11 promotes miR-130a-loaded miRISC to target c-myc mRNA.

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miR-130a targets c-myc mRNA through its 3′-UTR(A) Overexpression of miR-130a reduces the activity of luciferase reporter with c-myc 3′-UTR. 293 cells transfected with control pGL3 or pGL3-myc-3′UTR in the presence of β-gal plasmid together with control or miR-130a mimic as indicated were assayed for the relative luciferase activity normalized to β-gal expression. *p < 0.01, compared with cells transfected with pGL3-myc-3′UTR and control miRNA mimic. (B) Three putative miR-130a binding sites (BS-1, BS-2 and BS-3) in the c-myc 3′-UTR predicted by RNA22 program. (C) Schematic diagram of the pGL3-myc-3′UTR vectors. The first nucleotide after stop codon is indicated as “1”. (D) miR-130a regulates c-Myc via BS-1. 293 cells transfected with control or miR-130a mimic together with the indicated pGL3 or pGL3-myc-3′UTR vectors were assayed for the relative luciferase activity normalized to β-gal expression. *p < 0.01, compared with cells transfected with control miRNA mimic and corresponding luciferase reporters. (E–F) Ago2 associates with miR-130a at c-myc mRNA. U2OS cells transfected with control or miR-130a mimic were subjected to RNA-IP using control IgG or anti-Ago2 antibody, followed by RT-qPCR detection of c-myc and GAPDH mRNA (E) as well as U6 and miR-130a (F).
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Figure 3: miR-130a targets c-myc mRNA through its 3′-UTR(A) Overexpression of miR-130a reduces the activity of luciferase reporter with c-myc 3′-UTR. 293 cells transfected with control pGL3 or pGL3-myc-3′UTR in the presence of β-gal plasmid together with control or miR-130a mimic as indicated were assayed for the relative luciferase activity normalized to β-gal expression. *p < 0.01, compared with cells transfected with pGL3-myc-3′UTR and control miRNA mimic. (B) Three putative miR-130a binding sites (BS-1, BS-2 and BS-3) in the c-myc 3′-UTR predicted by RNA22 program. (C) Schematic diagram of the pGL3-myc-3′UTR vectors. The first nucleotide after stop codon is indicated as “1”. (D) miR-130a regulates c-Myc via BS-1. 293 cells transfected with control or miR-130a mimic together with the indicated pGL3 or pGL3-myc-3′UTR vectors were assayed for the relative luciferase activity normalized to β-gal expression. *p < 0.01, compared with cells transfected with control miRNA mimic and corresponding luciferase reporters. (E–F) Ago2 associates with miR-130a at c-myc mRNA. U2OS cells transfected with control or miR-130a mimic were subjected to RNA-IP using control IgG or anti-Ago2 antibody, followed by RT-qPCR detection of c-myc and GAPDH mRNA (E) as well as U6 and miR-130a (F).

Mentions: We next asked whether miR-130a directly targets c-myc mRNA at its 3′-UTR. 293 cells were co-transfected with control or miR-130a mimic together with control pGL3-promoter vector or pGL3-myc 3′UTR, which contains a full-length c-myc 3′-UTR at the 3′ end of luciferase mRNA, followed by measuring relative luciferase activity. As shown in Fig. 3A, overexpression of miR-130a significantly reduced the luciferase activity in cells transfected with pGL3-myc-3′UTR, but not the control pGL3 vector, suggesting that miR-130a targets c-myc mRNA through its 3′-UTR. We then searched for the potential miR-130a binding sites at the c-myc 3′-UTR. Although no conserved seed sequence for miR-130a binding was noted, analysis using RNA22 program as described [23], which allows seed mismatches [49], identifies three putative non-canonical “seedless” miR-130a binding sites (BS-1, BS-2, and BS-3) in the 5′ of the c-myc 3′-UTR with the miRNA:mRNA free folding energy cutoff –20 Kcal/mol (Fig. 3B). Therefore, we tested whether miR-130a targets c-myc mRNA at these sites using luciferase reporters containing different fragments of c-myc 3′-UTR (Fig. 3C). As shown in Fig. 3D, overexpression of miR-130a significantly reduced the luciferase activity in cells expressing pGL3-myc-3′UTR-FL or pGL3-myc-3′UTR-F1 plasmid containing the three putative miR-130a binding sites, whereas it did not significantly affect such activity in cells transfected with other pGL3 reporter containing c-myc 3′-UTR fragments lacking these sites (F2, F3 or F4) (Fig. 3D). Further, deletion of the first putative binding sites (BS-1, nt 21–42) (pGL3-myc-3′UTRΔBS1) with folding energy below the stringent cutoff (–25 Kcal/mol) [49] completely abolished the inhibition of luciferase activity upon miR-130a overexpression (Fig. 3D). Together, these results suggest that miR-130a targets c-myc mRNA through binding to the BS-1 site. To further confirm the miR-130a targeting of c-myc mRNA, we performed miR-130a transfection followed by RNA-IP using anti-Ago2 antibodies. As shown in Figs. 3E and 3F, both miR-130a and c-myc mRNA, but not U6 or GAPDH mRNA, were significantly enriched in the anti-Ago2, but not the control IgG, immunoprecipitates in cells transfected with miR-130a mimic. Thus, miR-130a directly targets c-myc mRNA at its 3′-UTR.


MicroRNA-130a associates with ribosomal protein L11 to suppress c-Myc expression in response to UV irradiation.

Li Y, Challagundla KB, Sun XX, Zhang Q, Dai MS - Oncotarget (2015)

miR-130a targets c-myc mRNA through its 3′-UTR(A) Overexpression of miR-130a reduces the activity of luciferase reporter with c-myc 3′-UTR. 293 cells transfected with control pGL3 or pGL3-myc-3′UTR in the presence of β-gal plasmid together with control or miR-130a mimic as indicated were assayed for the relative luciferase activity normalized to β-gal expression. *p < 0.01, compared with cells transfected with pGL3-myc-3′UTR and control miRNA mimic. (B) Three putative miR-130a binding sites (BS-1, BS-2 and BS-3) in the c-myc 3′-UTR predicted by RNA22 program. (C) Schematic diagram of the pGL3-myc-3′UTR vectors. The first nucleotide after stop codon is indicated as “1”. (D) miR-130a regulates c-Myc via BS-1. 293 cells transfected with control or miR-130a mimic together with the indicated pGL3 or pGL3-myc-3′UTR vectors were assayed for the relative luciferase activity normalized to β-gal expression. *p < 0.01, compared with cells transfected with control miRNA mimic and corresponding luciferase reporters. (E–F) Ago2 associates with miR-130a at c-myc mRNA. U2OS cells transfected with control or miR-130a mimic were subjected to RNA-IP using control IgG or anti-Ago2 antibody, followed by RT-qPCR detection of c-myc and GAPDH mRNA (E) as well as U6 and miR-130a (F).
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Figure 3: miR-130a targets c-myc mRNA through its 3′-UTR(A) Overexpression of miR-130a reduces the activity of luciferase reporter with c-myc 3′-UTR. 293 cells transfected with control pGL3 or pGL3-myc-3′UTR in the presence of β-gal plasmid together with control or miR-130a mimic as indicated were assayed for the relative luciferase activity normalized to β-gal expression. *p < 0.01, compared with cells transfected with pGL3-myc-3′UTR and control miRNA mimic. (B) Three putative miR-130a binding sites (BS-1, BS-2 and BS-3) in the c-myc 3′-UTR predicted by RNA22 program. (C) Schematic diagram of the pGL3-myc-3′UTR vectors. The first nucleotide after stop codon is indicated as “1”. (D) miR-130a regulates c-Myc via BS-1. 293 cells transfected with control or miR-130a mimic together with the indicated pGL3 or pGL3-myc-3′UTR vectors were assayed for the relative luciferase activity normalized to β-gal expression. *p < 0.01, compared with cells transfected with control miRNA mimic and corresponding luciferase reporters. (E–F) Ago2 associates with miR-130a at c-myc mRNA. U2OS cells transfected with control or miR-130a mimic were subjected to RNA-IP using control IgG or anti-Ago2 antibody, followed by RT-qPCR detection of c-myc and GAPDH mRNA (E) as well as U6 and miR-130a (F).
Mentions: We next asked whether miR-130a directly targets c-myc mRNA at its 3′-UTR. 293 cells were co-transfected with control or miR-130a mimic together with control pGL3-promoter vector or pGL3-myc 3′UTR, which contains a full-length c-myc 3′-UTR at the 3′ end of luciferase mRNA, followed by measuring relative luciferase activity. As shown in Fig. 3A, overexpression of miR-130a significantly reduced the luciferase activity in cells transfected with pGL3-myc-3′UTR, but not the control pGL3 vector, suggesting that miR-130a targets c-myc mRNA through its 3′-UTR. We then searched for the potential miR-130a binding sites at the c-myc 3′-UTR. Although no conserved seed sequence for miR-130a binding was noted, analysis using RNA22 program as described [23], which allows seed mismatches [49], identifies three putative non-canonical “seedless” miR-130a binding sites (BS-1, BS-2, and BS-3) in the 5′ of the c-myc 3′-UTR with the miRNA:mRNA free folding energy cutoff –20 Kcal/mol (Fig. 3B). Therefore, we tested whether miR-130a targets c-myc mRNA at these sites using luciferase reporters containing different fragments of c-myc 3′-UTR (Fig. 3C). As shown in Fig. 3D, overexpression of miR-130a significantly reduced the luciferase activity in cells expressing pGL3-myc-3′UTR-FL or pGL3-myc-3′UTR-F1 plasmid containing the three putative miR-130a binding sites, whereas it did not significantly affect such activity in cells transfected with other pGL3 reporter containing c-myc 3′-UTR fragments lacking these sites (F2, F3 or F4) (Fig. 3D). Further, deletion of the first putative binding sites (BS-1, nt 21–42) (pGL3-myc-3′UTRΔBS1) with folding energy below the stringent cutoff (–25 Kcal/mol) [49] completely abolished the inhibition of luciferase activity upon miR-130a overexpression (Fig. 3D). Together, these results suggest that miR-130a targets c-myc mRNA through binding to the BS-1 site. To further confirm the miR-130a targeting of c-myc mRNA, we performed miR-130a transfection followed by RNA-IP using anti-Ago2 antibodies. As shown in Figs. 3E and 3F, both miR-130a and c-myc mRNA, but not U6 or GAPDH mRNA, were significantly enriched in the anti-Ago2, but not the control IgG, immunoprecipitates in cells transfected with miR-130a mimic. Thus, miR-130a directly targets c-myc mRNA at its 3′-UTR.

Bottom Line: Yet, mechanism underlying UV-induced c-Myc reduction is not completely understood.Overexpression of miR-130a promotes the Ago2 binding to c-myc mRNA, significantly reduces the levels of both c-Myc protein and mRNA and inhibits cell proliferation.Inhibiting miR-130a significantly suppresses UV-mediated c-Myc reduction.

View Article: PubMed Central - PubMed

Affiliation: Departments of Molecular & Medical Genetics, School of Medicine and the OHSU Knight Cancer Institute, Oregon Health & Science University, Portland, OR 97239, USA.

ABSTRACT
The oncoprotein c-Myc is essential for cell growth and proliferation while its deregulated overexpression is associated with most human cancers. Thus tightly regulated levels and activity of c-Myc are critical for maintaining normal cell homeostasis. c-Myc is down-regulated in response to several types of stress, including UV-induced DNA damage. Yet, mechanism underlying UV-induced c-Myc reduction is not completely understood. Here we report that L11 promotes miR-130a targeting of c-myc mRNA to repress c-Myc expression in response to UV irradiation. miR-130a targets the 3'-untranslated region (UTR) of c-myc mRNA. Overexpression of miR-130a promotes the Ago2 binding to c-myc mRNA, significantly reduces the levels of both c-Myc protein and mRNA and inhibits cell proliferation. UV treatment markedly promotes the binding of L11 to miR-130a, c-myc mRNA as well as Ago2 in cells. Inhibiting miR-130a significantly suppresses UV-mediated c-Myc reduction. We further show that L11 is relocalized from the nucleolus to the cytoplasm where it associates with c-myc mRNA upon UV treatment. Together, these results reveal a novel mechanism underlying c-Myc down-regulation in response to UV-mediated DNA damage, wherein L11 promotes miR-130a-loaded miRISC to target c-myc mRNA.

Show MeSH
Related in: MedlinePlus