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Transformer 2? and miR-204 regulate apoptosis through competitive binding to 3' UTR of BCL2 mRNA.

ABSTRACT: RNA-binding proteins and microRNAs are potent post-transcriptional regulators of gene expression. Human transformer 2? (Tra2?) is a serine/arginine-rich-like protein splicing factor and is now implicated to have wide-ranging roles in gene expression as an RNA-binding protein. RNA immunoprecipitation (RIP) with an anti-Tra2? antibody and microarray analysis identified a subset of Tra2?-associated mRNAs in HCT116 human colon cancer cells, many of which encoded cell death-related proteins including Bcl-2 (B-cell CLL/lymphoma 2). Tra2? knockdown in HCT116 cells decreased Bcl-2 expression and induced apoptosis. Tra2? knockdown accelerated the decay of BCL2? mRNA that encodes Bcl-2 and full-length 3' UTR, while it did not affect the stability of BCL2? mRNA having a short, alternatively spliced 3' UTR different from BCL2? 3' UTR. RIP assays with anti-Tra2? and anti-Argonaute 2 antibodies, respectively, showed that Tra2? bound to BCL2? 3' UTR, and that Tra2? knockdown facilitated association of miR-204 with BCL2? 3' UTR. The consensus sequence (GAA) for Tra2?-binding lies within the miR-204-binding site of BCL2 3' UTR. Mutation of the consensus sequence canceled the binding of Tra2? to BCL2 3' UTR without disrupting miR-204-binding to BCL2 3' UTR. Transfection of an anti-miR-204 or introduction of three-point mutations into the miR-204-binding site increased BCL2 mRNA and Bcl-2 protein levels. Inversely, transfection of precursor miR-204 reduced their levels. Experiments with Tra2?-silenced or overexpressed cells revealed that Tra2? antagonized the effects of miR-204 and upregulated Bcl-2 expression. Furthermore, TRA2? mRNA expression was significantly upregulated in 22 colon cancer tissues compared with paired normal tissues and positively correlated with BCL2 mRNA expression. Tra2? knockdown in human lung adenocarcinoma cells (A549) increased their sensitivity to anticancer drugs. Taken together, our findings suggest that Tra2? regulates apoptosis by modulating Bcl-2 expression through its competition with miR-204. This novel function may have a crucial role in tumor growth.

SUBMITTER: Kuwano Y

PROVIDER: S-EPMC4392078 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Transformer 2β and miR-204 regulate apoptosis through competitive binding to 3' UTR of BCL2 mRNA.

Kuwano Y Y Nishida K K Kajita K K Satake Y Y Akaike Y Y Fujita K K Kano S S Masuda K K Rokutan K K

Cell death and differentiation 20141024 5

RNA-binding proteins and microRNAs are potent post-transcriptional regulators of gene expression. Human transformer 2β (Tra2β) is a serine/arginine-rich-like protein splicing factor and is now implicated to have wide-ranging roles in gene expression as an RNA-binding protein. RNA immunoprecipitation (RIP) with an anti-Tra2β antibody and microarray analysis identified a subset of Tra2β-associated mRNAs in HCT116 human colon cancer cells, many of which encoded cell death-related proteins including ...[more]

PMID: 25342468

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Project description:Aims/hypothesisThe role of beta cell microRNA (miR)-21 in the pathophysiology of type 1 diabetes has been controversial. Here, we sought to define the context of beta cell miR-21 upregulation in type 1 diabetes and the phenotype of beta cell miR-21 overexpression through target identification.MethodsIslets were isolated from NOD mice and mice treated with multiple low doses of streptozotocin, as a mouse model of diabetes. INS-1 832/13 beta cells and human islets were treated with IL-1β, IFN-γ and TNF-α to mimic the milieu of early type 1 diabetes. Cells and islets were transfected with miR-21 mimics or inhibitors. Luciferase assays and polyribosomal profiling (PRP) were performed to define miR-21-target interactions.ResultsBeta cell miR-21 was increased in in vivo models of type 1 diabetes and cytokine-treated cells/islets. miR-21 overexpression decreased cell count and viability, and increased cleaved caspase 3 levels, suggesting increased cell death. In silico prediction tools identified the antiapoptotic mRNA BCL2 as a conserved miR-21 target. Consistent with this, miR-21 overexpression decreased BCL2 transcript and B cell lymphoma 2 (BCL2) protein production, while miR-21 inhibition increased BCL2 protein levels and reduced cleaved caspase 3 levels after cytokine treatment. miR-21-mediated cell death was abrogated in 828/33 cells, which constitutively overexpress Bcl2. Luciferase assays suggested a direct interaction between miR-21 and the BCL2 3' untranslated region. With miR-21 overexpression, PRP revealed a shift of the Bcl2 message towards monosome-associated fractions, indicating inhibition of Bcl2 translation. Finally, overexpression in dispersed human islets confirmed a reduction in BCL2 transcripts and increased cleaved caspase 3 production.Conclusions/interpretationIn contrast to the pro-survival role reported in other systems, our results demonstrate that miR-21 increases beta cell death via BCL2 transcript degradation and inhibition of BCL2 translation.

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Transformer 2? and miR-204 regulate apoptosis through competitive binding to 3' UTR of BCL2 mRNA.

Publications

Transformer 2β and miR-204 regulate apoptosis through competitive binding to 3' UTR of BCL2 mRNA.

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