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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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L11 associates with miR-130a(A) Identification of miR-130a as a L11-associated miRNA. 293 cells transfected with control or Flag-L11 were subjected to RNA-IP using anti-Flag antibody followed by detection of indicated miRNAs using RT-qPCR. The expression of Flag-L11 is shown in the right panel. (B–C) L11 associates with miR-130a in cells. Lysates from 293 (B) and U2OS (C) cells transfected with Flag-L11 were immunoprecipitated with control mouse IgG or anti-Flag antibody, followed by RT-qPCR detection of miR-130a.
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Figure 1: L11 associates with miR-130a(A) Identification of miR-130a as a L11-associated miRNA. 293 cells transfected with control or Flag-L11 were subjected to RNA-IP using anti-Flag antibody followed by detection of indicated miRNAs using RT-qPCR. The expression of Flag-L11 is shown in the right panel. (B–C) L11 associates with miR-130a in cells. Lysates from 293 (B) and U2OS (C) cells transfected with Flag-L11 were immunoprecipitated with control mouse IgG or anti-Flag antibody, followed by RT-qPCR detection of miR-130a.

Mentions: We have previously shown that L11 associates with miR-24, but not other Myc-targeting miRNAs including let-7b and miR-34c, to repress c-Myc expression in response to ribosomal stress [22]. To further elucidate the role of L11 in the regulation of c-Myc, we sought to examine whether it could associate with other miRNAs that negatively regulate cell growth and proliferation. We performed RNA-IP assays with anti-Flag antibody using lysates from 293 cells transfected with control or Flag-L11 plasmid. RNAs extracted from the immunoprecipitates were assayed by RT-qPCR for a panel of miRNAs with potential tumor suppressor function, including miR-15a, miR-16, miR-130a, miR-107, miR-200b, and several let-7 family members including let-7a, let-7c, let-7f and miR-98 [35, 43–48]. As shown in Fig. 1A, L11 bound strongly to miR-130a and to a less extent to miR-16, but not other tested miRNAs. To verify this L11-miR-130a association, we performed similar RNA-IP experiments in cells transfected with Flag-L11 using IgG control. Indeed, miR-130a was specifically immunoprecipitated by anti-Flag antibody, but not control IgG, in both 293 (Fig. 1B) and U2OS (Fig. 1C) cells, suggesting that L11 associates with miR-130a in cells.


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 associates with miR-130a(A) Identification of miR-130a as a L11-associated miRNA. 293 cells transfected with control or Flag-L11 were subjected to RNA-IP using anti-Flag antibody followed by detection of indicated miRNAs using RT-qPCR. The expression of Flag-L11 is shown in the right panel. (B–C) L11 associates with miR-130a in cells. Lysates from 293 (B) and U2OS (C) cells transfected with Flag-L11 were immunoprecipitated with control mouse IgG or anti-Flag antibody, followed by RT-qPCR detection of miR-130a.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4359220&req=5

Figure 1: L11 associates with miR-130a(A) Identification of miR-130a as a L11-associated miRNA. 293 cells transfected with control or Flag-L11 were subjected to RNA-IP using anti-Flag antibody followed by detection of indicated miRNAs using RT-qPCR. The expression of Flag-L11 is shown in the right panel. (B–C) L11 associates with miR-130a in cells. Lysates from 293 (B) and U2OS (C) cells transfected with Flag-L11 were immunoprecipitated with control mouse IgG or anti-Flag antibody, followed by RT-qPCR detection of miR-130a.
Mentions: We have previously shown that L11 associates with miR-24, but not other Myc-targeting miRNAs including let-7b and miR-34c, to repress c-Myc expression in response to ribosomal stress [22]. To further elucidate the role of L11 in the regulation of c-Myc, we sought to examine whether it could associate with other miRNAs that negatively regulate cell growth and proliferation. We performed RNA-IP assays with anti-Flag antibody using lysates from 293 cells transfected with control or Flag-L11 plasmid. RNAs extracted from the immunoprecipitates were assayed by RT-qPCR for a panel of miRNAs with potential tumor suppressor function, including miR-15a, miR-16, miR-130a, miR-107, miR-200b, and several let-7 family members including let-7a, let-7c, let-7f and miR-98 [35, 43–48]. As shown in Fig. 1A, L11 bound strongly to miR-130a and to a less extent to miR-16, but not other tested miRNAs. To verify this L11-miR-130a association, we performed similar RNA-IP experiments in cells transfected with Flag-L11 using IgG control. Indeed, miR-130a was specifically immunoprecipitated by anti-Flag antibody, but not control IgG, in both 293 (Fig. 1B) and U2OS (Fig. 1C) cells, suggesting that L11 associates with miR-130a in cells.

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