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Internal-ribosome-entry-site functional activity of the 3'-untranslated region of the mRNA for the beta subunit of mitochondrial H+-ATP synthase.

ABSTRACT: Translation in vitro of the mammalian nucleus-encoded mRNA for the beta subunit of mitochondrial H(+)-ATP synthase (beta-mRNA) of oxidative phosphorylation is promoted by a 150 nt translational enhancer sequence in the 3'-untranslated region (3' UTR). Titration of the eukaryotic initiation factor eIF4E with cap analogue revealed that translation of capped beta-mRNA was pseudo-cap independent. The 3' UTR of beta-mRNA stimulates the translation of heterologous uncapped mRNA species, both when the 3' UTR is placed at the 3' end and at the 5' end of the transcripts. The 3' UTRs of the alpha subunit of mitochondrial H(+)-ATP synthase (alpha-F1-ATPase) and subunit IV of cytochrome c oxidase (COX IV) mRNA species, other nucleus-encoded transcripts of oxidative phosphorylation, do not have the same activity in translation as the 3' UTR of beta-mRNA. On dicistronic RNA species, the 3' UTR of beta-mRNA, and to a smaller extent that of COX IV mRNA, is able to promote the translation of the second cistron to a level comparable to the activity of internal ribosome entry sites (IRESs) described in picornavirus mRNA species. These results indicate that the 3' UTRs of certain mRNA species of oxidative phosphorylation have IRES-like functional activity. Riboprobes of the active 3' UTRs on dicistronic assays formed specific RNA-protein complexes when cross-linked by UV to proteins of the lysate, suggesting that cytoplasmic translation of the mRNA species bearing an active 3' UTR is assisted by specific RNA-protein interactions.

SUBMITTER: Izquierdo JM

PROVIDER: S-EPMC1220922 | biostudies-other | 2000 Mar

REPOSITORIES: biostudies-other

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Project description:In recent years, hepatitis C virus (HCV)-related viruses were identified in several species, including dogs, horses, bats, and rodents. In addition, a novel virus of the genus Hepacivirus has been discovered in bovine samples and was termed bovine hepacivirus (BovHepV). Prediction of the BovHepV internal ribosome entry site (IRES) structure revealed strong similarities to the HCV IRES structure comprising domains II, IIIabcde, pseudoknot IIIf, and IV with the initiation codon AUG. Unlike HCV, only one microRNA-122 (miR-122) binding site could be identified in the BovHepV 5' nontranslated region. In this study, we analyzed the necessity of BovHepV IRES domains to initiate translation and investigated possible interactions between the IRES and core coding sequences by using a dual luciferase reporter assay. Our results suggest that such long-range interactions within the viral genome can affect IRES-driven translation. Moreover, the significance of a possible miR-122 binding to the BovHepV IRES was investigated. When analyzing translation in human Huh-7 cells with large amounts of endogenous miR-122, introduction of point mutations to the miR-122 binding site resulted in reduced translation efficiency. Similar results were observed in HeLa cells after substitution of miR-122. Nevertheless, the absence of pronounced effects in a bovine hepatocyte cell line expressing hardly any miR-122 as well suggests additional functions of this host factor in virus replication.IMPORTANCE Several members of the family Flaviviridae, including HCV, have adapted cap-independent translation strategies to overcome canonical eukaryotic translation pathways and use cis-acting RNA-elements, designated viral internal ribosome entry sites (IRES), to initiate translation. Although novel hepaciviruses have been identified in different animal species, only limited information is available on their biology on molecular level. Therefore, our aim was a fundamental analysis of BovHepV IRES functions. The findings which show that functional IRES elements are also crucial for BovHepV translation expand our knowledge on molecular mechanism of hepacivirus propagation. We also studied the possible effects of one major host factor implicated in HCV pathogenesis, miR-122. The results of mutational analyses suggested that miR-122 enhances virus translation mediated by BovHepV IRES.

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Internal-ribosome-entry-site functional activity of the 3'-untranslated region of the mRNA for the beta subunit of mitochondrial H+-ATP synthase.

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