RESUMO
Ku heterodimer is a DNA binding protein with a prominent role in DNA repair. Here, we investigate whether and how Ku impacts the DNA damage response by acting as a post-transcriptional regulator of gene expression. We show that Ku represses p53 protein synthesis and p53-mediated apoptosis by binding to a bulged stem-loop structure within the p53 5' UTR However, Ku-mediated translational repression of the p53 mRNA is relieved after genotoxic stress. The underlying mechanism involves Ku acetylation which disrupts Ku-p53 mRNA interactions. These results suggest that Ku-mediated repression of p53 mRNA translation constitutes a novel mechanism linking DNA repair and mRNA translation.
Assuntos
Dano ao DNA/fisiologia , Reparo do DNA , Autoantígeno Ku/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/genética , Regiões 5' não Traduzidas , Acetilação , Apoptose , Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Autoantígeno Ku/genética , Ligação Proteica , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The activation of translation contributes to malignant transformation and is an emerging target for cancer therapies. RNA G-quadruplex structures are general inhibitors of cap-dependent mRNA translation and were recently shown to be targeted for oncoprotein translational activation. In contrast however, the G-quadruplex within the 5'UTR of the human vascular endothelial growth factor A (VEGF) has been shown to be essential for IRES-mediated translation. Since VEGF has a pivotal role in tumor angiogenesis and is a major target of anti-tumoral therapies, we investigated the structure/function relationship of the VEGF G-quadruplex and defined whether it could have a therapeutic potential. We found that the G-quadruplex within the VEGF IRES is dispensable for cap-independent function and activation in stress conditions. However, stabilization of the VEGF G-quadruplex by increasing the G-stretches length or by replacing it with the one of NRAS results in strong inhibition of IRES-mediated translation of VEGF. We also demonstrate that G-quadruplex ligands stabilize the VEGF G-quadruplex and inhibit cap-independent translation in vitro. Importantly, the amount of human VEGF mRNA associated with polysomes decreases in the presence of a highly selective stabilizing G-quadruplex ligand, resulting in reduced VEGF protein expression. Together, our results uncover the existence of functionally silent G-quadruplex structures that are susceptible to conversion into efficient repressors of cap-independent mRNA translation. These findings have implications for the in vivo applications of G-quadruplex-targeting compounds and for anti-angiogenic therapies.
Assuntos
Regiões 5' não Traduzidas , Regulação da Expressão Gênica , Sítios Internos de Entrada Ribossomal , Biossíntese de Proteínas , Fator A de Crescimento do Endotélio Vascular/genética , Sequência de Bases , Quadruplex G , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Genes Reporter , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Polirribossomos/genética , Polirribossomos/metabolismo , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The H2A.Z histone variant plays major roles in the control of gene expression. In human, H2A.Z is encoded by two genes expressing two isoforms, H2A.Z.1 and H2A.Z.2 differing by three amino acids. Here, we undertook an integrated analysis of their functions in gene expression using endogenously-tagged proteins. RNA-Seq analysis in untransformed cells showed that they can regulate both distinct and overlapping sets of genes positively or negatively in a context-dependent manner. Furthermore, they have similar or antagonistic function depending on genes. H2A.Z.1 and H2A.Z.2 can replace each other at Transcription Start Sites, providing a molecular explanation for this interplay. Mass spectrometry analysis showed that H2A.Z.1 and H2A.Z.2 have specific interactors, which can mediate their functional antagonism. Our data indicate that the balance between H2A.Z.1 and H2A.Z.2 at promoters is critically important to regulate specific gene expression, providing an additional layer of complexity to the control of gene expression by histone variants.