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The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition.

ABSTRACT: Most eukaryotic mRNAs are monocistronic and translated by cap-dependent initiation. LINE-1 RNA is exceptional because it is naturally dicistronic, encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Here, we show that sequences upstream of ORF1 and ORF2 in mouse L1 function as internal ribosome entry sites (IRESes). Deletion analysis of the ORF1 IRES indicates that RNA structure is critical for its function. Conversely, the ORF2 IRES localizes to 53 nt near the 3' end of ORF1, and appears to depend upon sequence rather than structure. The 40 nt intergenic region (IGR) is not essential for ORF2 IRES function or retrotransposition. Because of strong cis-preference for both proteins during L1 retrotransposition, correct stoichiometry of the two proteins can only be achieved post-transcriptionally. Although the precise stoichiometry is unknown, the retrotransposition intermediate likely contains hundreds of ORF1ps for every ORF2p, together with one L1 RNA. IRES-mediated translation initiation is a well-established mechanism of message-specific regulation, hence, unique mechanisms for the recognition and control of these two IRESes in the L1 RNA could explain differences in translational efficiency of ORF1 and ORF2. In addition, translational regulation may provide an additional layer of control on L1 retrotransposition efficiency, thereby protecting the integrity of the genome.

SUBMITTER: Li PW

PROVIDER: S-EPMC1361618 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition.

Li Patrick Wai-Lun PW Li Jinfang J Timmerman Stephanie L SL Krushel Les A LA Martin Sandra L SL

Nucleic acids research 20060206 3

Most eukaryotic mRNAs are monocistronic and translated by cap-dependent initiation. LINE-1 RNA is exceptional because it is naturally dicistronic, encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Here, we show that sequences upstream of ORF1 and ORF2 in mouse L1 function as internal ribosome entry sites (IRESes). Deletion analysis of the ORF1 IRES indicates that RNA structure is critical for its function. Conversely, the ORF2 IRES localizes to 53 nt near the 3' end of ORF ...[more]

PMID: 16464823

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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.

Dataset Information

The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition.

Publications

The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition.

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