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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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Related in: MedlinePlus

L11 recruits miR-130a-loaded miRISC to c-myc mRNA in response to UV irradiation(A) UV treatment increases the L11 binding to c-myc mRNA. U2OS cells treated without or with UV were subjected to RNA-IP using control IgG or anti-L11 antibodies, followed by RT-qPCR assays. (B–C) L11 inhibition of c-Myc in response to UV requires its binding to the c-myc 3′-UTR. 293 cells transfected with pGL3, pGL3-myc 3′-UTR-FL, or pGL3-myc 3′-UTR-F1 were treated with or without UV. The cells were then assayed for the relative luciferase activity normalized to β-gal expression (B) and subjected to RNA-IP using anti-L11 antibodies, followed by RT-qPCR detection of the luciferase mRNA. (D) UV treatment increases Ago2 binding to c-myc mRNA. U2OS cells treated with or without UV were subjected to RNA-IP using control IgG or anti-Ago2 antibodies, followed by RT-qPCR detection of c-myc mRNA (D). (E–F) UV treatment increases the binding of L11 and Ago2 to miR-130a, and, to a less extent, to miR-24. U2OS cells treated with or without UV were subjected to RNA-IP using control IgG or anti-Ago2 (E) or anti-L11 (F) antibodies, followed by RT-qPCR detection of miR-130a, miR-24 and the control U6 RNA. (G–H) UV treatment increases the L11 binding to Ago2, but not eIF4G. U2OS cells treated with or without UV were subjected to co-IP with anti-Ago2 (G) and anti-eIF4G (H) antibodies followed by IB. (I–J) Inhibiting miR-130a abolishes c-Myc reduction by UV treatment. U2OS cells transfected with control or miR-130a inhibitor were treated with or without UV. The cells were assayed for the expression of c-myc mRNA by RT-qPCR (I) and c-Myc protein by IB (J). *p < 0.05, compared the ratio of lane 4 to lane 3 with the ratio of lane 2 to lane 1. In all above assays, cells were treated with 40 J/m2 UV and harvested at 6 hours post-treatment.
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Figure 6: L11 recruits miR-130a-loaded miRISC to c-myc mRNA in response to UV irradiation(A) UV treatment increases the L11 binding to c-myc mRNA. U2OS cells treated without or with UV were subjected to RNA-IP using control IgG or anti-L11 antibodies, followed by RT-qPCR assays. (B–C) L11 inhibition of c-Myc in response to UV requires its binding to the c-myc 3′-UTR. 293 cells transfected with pGL3, pGL3-myc 3′-UTR-FL, or pGL3-myc 3′-UTR-F1 were treated with or without UV. The cells were then assayed for the relative luciferase activity normalized to β-gal expression (B) and subjected to RNA-IP using anti-L11 antibodies, followed by RT-qPCR detection of the luciferase mRNA. (D) UV treatment increases Ago2 binding to c-myc mRNA. U2OS cells treated with or without UV were subjected to RNA-IP using control IgG or anti-Ago2 antibodies, followed by RT-qPCR detection of c-myc mRNA (D). (E–F) UV treatment increases the binding of L11 and Ago2 to miR-130a, and, to a less extent, to miR-24. U2OS cells treated with or without UV were subjected to RNA-IP using control IgG or anti-Ago2 (E) or anti-L11 (F) antibodies, followed by RT-qPCR detection of miR-130a, miR-24 and the control U6 RNA. (G–H) UV treatment increases the L11 binding to Ago2, but not eIF4G. U2OS cells treated with or without UV were subjected to co-IP with anti-Ago2 (G) and anti-eIF4G (H) antibodies followed by IB. (I–J) Inhibiting miR-130a abolishes c-Myc reduction by UV treatment. U2OS cells transfected with control or miR-130a inhibitor were treated with or without UV. The cells were assayed for the expression of c-myc mRNA by RT-qPCR (I) and c-Myc protein by IB (J). *p < 0.05, compared the ratio of lane 4 to lane 3 with the ratio of lane 2 to lane 1. In all above assays, cells were treated with 40 J/m2 UV and harvested at 6 hours post-treatment.

Mentions: We then examined whether L11 promotes the recruitment of the miR-130a-loaded miRISC to c-myc mRNA in response to UV treatment. First, U2OS cells treated with or without UV were subjected to RNA-IP using control IgG or anti-L11 antibodies, followed by RT-qPCR assays. As shown in Fig. 6A, L11 binding to c-myc mRNA was drastically increased in cells treated with UV compared to the control cells, confirming that UV treatment promotes the L11 binding to c-myc mRNA. Our previous study showed that L11 binds to the 3′-end (nt 361 to 470) of the c-myc 3′-UTR [22]. To verify that UV treatment promotes L11 binding to the c-myc 3′-UTR, we transfected 293 cells with pGL3, pGL3-myc-3′UTR-FL, or pGL3-myc-3′UTR-F1 plasmid as diagramed in Fig. 3C (the F1 fragment contains the miR-130a binding site, but lacks the L11-binding site), followed by UV treatment. As shown in Fig. 6B, UV treatment significantly reduced the luciferase activity in cells expressing pGL3-myc-3′UTR-FL, but not the control pGL3 or pGL3-myc-3′UTR-F1 plasmid lacking the L11 binding site. Consistently, UV treatment significantly increased the binding of L11 to the luciferase mRNA in cells transfected with pGL3-myc 3′-UTR, but not the control pGL3 or pGL3-myc-3′UTR-F1 (Fig. 6C). These data reveal that UV treatment increases the L11 binding to the c-myc 3′-UTR and inhibits c-Myc expression.


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)

L11 recruits miR-130a-loaded miRISC to c-myc mRNA in response to UV irradiation(A) UV treatment increases the L11 binding to c-myc mRNA. U2OS cells treated without or with UV were subjected to RNA-IP using control IgG or anti-L11 antibodies, followed by RT-qPCR assays. (B–C) L11 inhibition of c-Myc in response to UV requires its binding to the c-myc 3′-UTR. 293 cells transfected with pGL3, pGL3-myc 3′-UTR-FL, or pGL3-myc 3′-UTR-F1 were treated with or without UV. The cells were then assayed for the relative luciferase activity normalized to β-gal expression (B) and subjected to RNA-IP using anti-L11 antibodies, followed by RT-qPCR detection of the luciferase mRNA. (D) UV treatment increases Ago2 binding to c-myc mRNA. U2OS cells treated with or without UV were subjected to RNA-IP using control IgG or anti-Ago2 antibodies, followed by RT-qPCR detection of c-myc mRNA (D). (E–F) UV treatment increases the binding of L11 and Ago2 to miR-130a, and, to a less extent, to miR-24. U2OS cells treated with or without UV were subjected to RNA-IP using control IgG or anti-Ago2 (E) or anti-L11 (F) antibodies, followed by RT-qPCR detection of miR-130a, miR-24 and the control U6 RNA. (G–H) UV treatment increases the L11 binding to Ago2, but not eIF4G. U2OS cells treated with or without UV were subjected to co-IP with anti-Ago2 (G) and anti-eIF4G (H) antibodies followed by IB. (I–J) Inhibiting miR-130a abolishes c-Myc reduction by UV treatment. U2OS cells transfected with control or miR-130a inhibitor were treated with or without UV. The cells were assayed for the expression of c-myc mRNA by RT-qPCR (I) and c-Myc protein by IB (J). *p < 0.05, compared the ratio of lane 4 to lane 3 with the ratio of lane 2 to lane 1. In all above assays, cells were treated with 40 J/m2 UV and harvested at 6 hours post-treatment.
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Figure 6: L11 recruits miR-130a-loaded miRISC to c-myc mRNA in response to UV irradiation(A) UV treatment increases the L11 binding to c-myc mRNA. U2OS cells treated without or with UV were subjected to RNA-IP using control IgG or anti-L11 antibodies, followed by RT-qPCR assays. (B–C) L11 inhibition of c-Myc in response to UV requires its binding to the c-myc 3′-UTR. 293 cells transfected with pGL3, pGL3-myc 3′-UTR-FL, or pGL3-myc 3′-UTR-F1 were treated with or without UV. The cells were then assayed for the relative luciferase activity normalized to β-gal expression (B) and subjected to RNA-IP using anti-L11 antibodies, followed by RT-qPCR detection of the luciferase mRNA. (D) UV treatment increases Ago2 binding to c-myc mRNA. U2OS cells treated with or without UV were subjected to RNA-IP using control IgG or anti-Ago2 antibodies, followed by RT-qPCR detection of c-myc mRNA (D). (E–F) UV treatment increases the binding of L11 and Ago2 to miR-130a, and, to a less extent, to miR-24. U2OS cells treated with or without UV were subjected to RNA-IP using control IgG or anti-Ago2 (E) or anti-L11 (F) antibodies, followed by RT-qPCR detection of miR-130a, miR-24 and the control U6 RNA. (G–H) UV treatment increases the L11 binding to Ago2, but not eIF4G. U2OS cells treated with or without UV were subjected to co-IP with anti-Ago2 (G) and anti-eIF4G (H) antibodies followed by IB. (I–J) Inhibiting miR-130a abolishes c-Myc reduction by UV treatment. U2OS cells transfected with control or miR-130a inhibitor were treated with or without UV. The cells were assayed for the expression of c-myc mRNA by RT-qPCR (I) and c-Myc protein by IB (J). *p < 0.05, compared the ratio of lane 4 to lane 3 with the ratio of lane 2 to lane 1. In all above assays, cells were treated with 40 J/m2 UV and harvested at 6 hours post-treatment.
Mentions: We then examined whether L11 promotes the recruitment of the miR-130a-loaded miRISC to c-myc mRNA in response to UV treatment. First, U2OS cells treated with or without UV were subjected to RNA-IP using control IgG or anti-L11 antibodies, followed by RT-qPCR assays. As shown in Fig. 6A, L11 binding to c-myc mRNA was drastically increased in cells treated with UV compared to the control cells, confirming that UV treatment promotes the L11 binding to c-myc mRNA. Our previous study showed that L11 binds to the 3′-end (nt 361 to 470) of the c-myc 3′-UTR [22]. To verify that UV treatment promotes L11 binding to the c-myc 3′-UTR, we transfected 293 cells with pGL3, pGL3-myc-3′UTR-FL, or pGL3-myc-3′UTR-F1 plasmid as diagramed in Fig. 3C (the F1 fragment contains the miR-130a binding site, but lacks the L11-binding site), followed by UV treatment. As shown in Fig. 6B, UV treatment significantly reduced the luciferase activity in cells expressing pGL3-myc-3′UTR-FL, but not the control pGL3 or pGL3-myc-3′UTR-F1 plasmid lacking the L11 binding site. Consistently, UV treatment significantly increased the binding of L11 to the luciferase mRNA in cells transfected with pGL3-myc 3′-UTR, but not the control pGL3 or pGL3-myc-3′UTR-F1 (Fig. 6C). These data reveal that UV treatment increases the L11 binding to the c-myc 3′-UTR and inhibits c-Myc expression.

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