RESUMO
UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 5' untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos da radiação , Transporte Proteico/efeitos da radiação , RNA Mensageiro/metabolismo , Raios Ultravioleta , Linhagem Celular Tumoral , Dano ao DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Regulação da Expressão Gênica/efeitos da radiação , Células HeLa , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta , Biossíntese de Proteínas/genéticaRESUMO
The DNA damage response activates several pathways that stall the cell cycle and allow DNA repair. These consist of the well-characterized ATR (Ataxia telangiectasia and Rad-3 related)/CHK1 and ATM (Ataxia telangiectasia mutated)/CHK2 pathways in addition to a newly identified ATM/ATR/p38MAPK/MK2 checkpoint. Crucial to maintaining the integrity of the genome is the S-phase checkpoint that functions to prevent DNA replication until damaged DNA is repaired. Inappropriate expression of the proto-oncogene c-Myc is known to cause DNA damage. One mechanism by which c-Myc induces DNA damage is through binding directly to components of the prereplicative complex thereby promoting DNA synthesis, resulting in replication-associated DNA damage and checkpoint activation due to inappropriate origin firing. Here we show that following etoposide-induced DNA damage translation of c-Myc is repressed by miR-34c via a highly conserved target-site within the 3(') UTR. While miR-34c is induced by p53 following DNA damage, we show that in cells lacking p53 this is achieved by an alternative pathway which involves p38 MAPK signalling to MK2. The data presented here suggest that a major physiological target of miR-34c is c-Myc. Inhibition of miR-34c activity prevents S-phase arrest in response to DNA damage leading to increased DNA synthesis, DNA damage, and checkpoint activation in addition to that induced by etoposide alone, which are all reversed by subsequent c-Myc depletion. These data demonstrate that miR-34c is a critical regulator of the c-Myc expression following DNA damage acting downstream of p38 MAPK/MK2 and suggest that miR-34c serves to remove c-Myc to prevent inappropriate replication which may otherwise lead to genomic instability.
Assuntos
Dano ao DNA , Replicação do DNA/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MicroRNAs/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Replicação do DNA/genética , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , MicroRNAs/genética , Proto-Oncogene Mas , Fase S/genética , Fase S/fisiologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
MicroRNAs (miRNAs) are noncoding RNAs that base pair imperfectly to homologous regions in target mRNAs and negatively influence the synthesis of the corresponding proteins. Repression is mediated by a number of mechanisms, one of which is the direct inhibition of protein synthesis. Surprisingly, previous studies have suggested that two mutually exclusive mechanisms exist, one acting at the initiation phase of protein synthesis and the other at a postinitiation event. Here, we resolve this apparent dichotomy by demonstrating that the promoter used to transcribe the mRNA influences the type of miRNA-mediated translational repression. Transcripts derived from the SV40 promoter that contain let-7 target sites in their 3' UTRs are repressed at the initiation stage of translation, whereas essentially identical mRNAs derived from the TK promoter are repressed at a postinitiation step. We also show that there is a miR-34 target site within the 3' UTR of c-myc mRNA and that promoter dependency is also true for this endogenous 3' UTR. Overall, these data establish a link between the nuclear history of an mRNA and the mechanism of miRNA-mediated translational regulation in the cytoplasm.
Assuntos
MicroRNAs/genética , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas , Regiões 3' não Traduzidas/genética , Sequência de Bases , Cicloeximida/farmacologia , Células HeLa , Humanos , Dados de Sequência Molecular , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The proto-oncogenes c-, L-, and N-myc can all be translated by the alternative method of internal ribosome entry whereby the ribosome is recruited to a complex structural element (an internal ribosome entry segment [IRES]). Ribosome recruitment is dependent upon the presence of IRES-trans-acting factors (ITAFs) that act as RNA chaperones and allow the mRNA to attain the correct conformation for the interaction of the 40S subunit. One of the major challenges for researchers in this area is to determine whether there are groups of ITAFs that regulate the IRES-mediated translation of subsets of mRNAs. We have identified four proteins, termed GRSF-1 (G-rich RNA sequence binding factor 1), YB-1 (Y-box binding protein 1), PSF (polypyrimidine tract binding protein-associated splicing factor), and its binding partner, p54nrb, that bind to the myc family of IRESs. We show that these proteins positively regulate the translation of the Myc family of oncoproteins (c-, L-, and N-Myc) in vivo and in vitro. Interestingly, synthesis from the unrelated IRESs, BAG-1 and Apaf-1, was not affected by YB-1, GRSF-1, or PSF levels in vivo, suggesting that these three ITAFs are specific to the myc IRESs. Myc proteins play a role in cell proliferation; therefore, these results have important implications regarding the control of tumorigenesis.
Assuntos
Proteínas Proto-Oncogênicas c-myc/metabolismo , Ribossomos/metabolismo , Transativadores/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Camundongos , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fator de Processamento Associado a PTB , Polirribossomos/metabolismo , Ligação Proteica , Biossíntese de Proteínas/genética , Proteínas Proto-Oncogênicas c-myc/classificação , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transativadores/genéticaRESUMO
Snu13p is a Saccharomyces cerevisiae protein essential for pre-messenger RNA splicing and pre-ribosomal RNA processing. Snu13p binds U4 snRNA of the spliceosome and box C/D snoRNAs of the pre-ribosomal RNA processing machinery to induce assembly of each ribonucleoprotein complex. Here, we present structural and biochemical analysis of Snu13p. The crystal structure of Snu13p reveals a region of the protein which could be important for protein interaction during ribonucleoprotein assembly. Using the structure of Snu13p we have designed the first temperature-sensitive mutants in Snu13p, L67W and I102A. Wild-type and mutant Snu13p proteins were assayed for binding to U4 snRNA and U3 snoRNA. Both temperature-sensitive mutants displayed significantly reduced RNA binding compared to wild-type protein. As the temperature-sensitive mutations are not in the known RNA binding region of Snu13p this indicates that these mutants indirectly influence the RNA binding properties of Snu13p. This work provides insight into Snu13p function during ribonucleoprotein assembly.
Assuntos
Mutação , Precursores de RNA/genética , RNA Mensageiro/genética , RNA Ribossômico/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas de Saccharomyces cerevisiae/genética , Clonagem Molecular , Modelos Moleculares , Ribonucleoproteínas Nucleares Pequenas/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
During apoptosis there is a substantial reduction in the rate of protein synthesis, and yet some mRNAs avoid this translational inhibition. To determine the impact that receptor-mediated cell death has on the translational efficiency of a large number of mRNAs, translational profiling was performed on MCF7 cells treated with the apoptosis-inducing ligand TRAIL. Our data indicate that approximately 3% of mRNAs remain associated with the polysomes in apoptotic cells, and genes that are involved in transcription, chromatin modification/remodeling, and the Notch signaling pathway are particularly prevalent among the mRNAs that evade translational inhibition. Internal ribosome entry segments (IRESs) were identified in several of the mRNAs that remained associated with the polysomes during apoptosis, and, importantly, these IRESs functioned efficiently in apoptotic cells. Finally, the data showed that polypyrimidine tract binding protein (PTB, a known IRES trans-acting factor or ITAF) is pivotal in regulating the apoptotic process by controlling IRES function.
Assuntos
Apoptose/fisiologia , Regulação da Expressão Gênica , Proteína de Ligação a Regiões Ricas em Polipirimidinas/fisiologia , Sequências Reguladoras de Ácido Ribonucleico/genética , Regiões 5' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/genética , Proteínas Reguladoras de Apoptose/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Glicoproteínas de Membrana/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Pre-mRNA splicing is executed by the spliceosome, a complex of small nuclear RNAs (snRNAs) and numerous proteins. One such protein, 15.5K/Snu13p, is associated with the spliceosomal U4/U6.U5 tri-snRNP and box C/D small nucleolar ribonucleoprotein particles (snoRNPs), which act during preribosomal RNA (rRNA) processing. As such, it is the first splicing factor to be identified in two functionally distinct particles. 15.5K binds to an internal helix-bulge-helix (K-turn) structure in the U4 snRNA and two such structures in the U3 snoRNA. Previous work has concentrated on the structural basis of the interaction of 15.5K with the RNAs and has been carried out in vitro. Here we present a functional analysis of Snu13p in vivo, using a galactose inducible SNU13 strain to investigate the basis of three lethal mutations in Saccharomyces cerevisiae. Two are point mutations that map to the RNA-binding domain, and the third is a C-terminal deletion. These mutations result in accumulation of unspliced pre-mRNA, confirming a role for Snu13p in pre-mRNA splicing. In addition, these mutants also display rRNA processing defects that are variable in nature. Analysis of one mutant in the RNA-binding domain reveals a reduction in the levels of the U4 snRNA, U6 snRNA, and box C/D snoRNAs, but not H/ACA snoRNAs, supporting a role for Snu13p in accumulation and/or maintenance of specific RNAs. The mutations in the RNA-binding domain exhibit differential binding to the U4 snRNA and U3 snoRNA in vitro, suggesting that there are differences in the mode of interaction of Snu13p with these two RNAs.