RESUMO
We have derived a secondary structure model for the c-myc internal ribosome entry segment (IRES) by using information from chemical probing of the c-myc IRES RNA to constrain structure prediction programs. Our data suggest that the IRES is modular in nature, and can be divided into two structural domains linked by a long unstructured region. Both domains are required for full IRES function. Domain 1 is a complex element that contains a GNNRA apical loop and an overlapping double pseudoknot motif that is topologically unique amongst published RNA structures. Domain 2, the smaller of the two, contains an apical AUUU loop. We have located the ribosome landing site and have shown that ribosomes enter in a 16 nt region downstream of the pseudoknots in a situation similar to that observed in several viral IRESs. To test the structure, several key regions of the IRES were mutated and, interestingly, it appears that some of the structural elements that we have identified function to repress c-myc IRES function. This has profound implications for de-regulation of c-myc expression by mutations occurring in the IRES.
Assuntos
Genes myc/genética , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Ribossomos/metabolismo , Sequência de Bases , Sítios de Ligação , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estabilidade de RNA , RNA Mensageiro/genética , Alinhamento de Sequência , Deleção de Sequência/genética , Software , Termodinâmica , TransfecçãoRESUMO
The 5' UTR of c -myc mRNA contains an internal ribo-some entry segment (IRES) and consequently, c -myc mRNAs can be translated by the alternative mechanism of internal ribosome entry. However, there is also some evidence suggesting that c -myc mRNA translation can occur via the conventional cap-dependent scanning mechanism. Using both bicistronic and monocistronic mRNAs containing the c- myc 5' UTR, we demonstrate that both mechanisms can contribute to c- myc protein synthesis. A wide range of cell types are capable of initiating translation of c- myc by internal ribosome entry, albeit with different efficiencies. Moreover, our data suggest that the spectrum of efficiencies observed in these cell types is likely to be due to variation in the cellular concentration of non-canonical translation factors. Interestingly, the c -myc IRES is 7-fold more active than the human rhinovirus 2 (HRV2) IRES and 5-fold more active than the encephalomyocarditis virus (EMCV) IRES. However, the protein requirements for the c -myc IRES must differ significantly from these viral IRESs, since an unidentified nuclear event appears to be a pre-requisite for efficient c -myc IRES-driven initiation.
Assuntos
Regiões 5' não Traduzidas/genética , Núcleo Celular/metabolismo , Iniciação Traducional da Cadeia Peptídica/genética , Proteínas Proto-Oncogênicas c-myc/genética , Transativadores/metabolismo , Regiões 5' não Traduzidas/química , Regiões 5' não Traduzidas/metabolismo , Animais , Extratos Celulares , Linhagem Celular , Códon de Iniciação/genética , Vírus da Encefalomiocardite/genética , Genes/genética , Genes Virais/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Conformação de Ácido Nucleico , Especificidade de Órgãos , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Reticulócitos , Rhinovirus/genética , Ribossomos/fisiologia , TransfecçãoRESUMO
Translation in eukaryotic cells is generally initiated by ribosome scanning from the 5' end of the capped mRNA. However, initiation of translation may also occur by a mechanism which is independent of the cap structure and in this case ribosomes are directed to the start codon by an internal ribosome entry segment (IRES). Picornaviruses represent the paradigm for this mechanism, but only a few examples exist which show that this mechanism is used by eukaryotic cells. In this report we show data which demonstrate that the 5' UTR of the proto-oncogene c-myc contains an IRES. When a dicistronic reporter vector, with c-myc 5' UTR inserted between the two cistrons, was transfected into both HepG2 and HeLa cells, the translation of the downstream cistron was increased by 50-fold, demonstrating that translational regulation of c-myc is mediated through cap-independent mechanisms. This is the first example of a proto-oncogene regulated in this manner and suggests that aberrant translational regulation of c-myc is likely to play a role in tumorigenesis.
