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Eukaryotic ribosomal protein RPS25 interacts with the conserved loop region in a dicistroviral intergenic internal ribosome entry site.

ABSTRACT: The intergenic region-internal ribosome entry site (IGR-IRES) of dicistroviruses binds to 40S ribosomal subunits in the absence of eukaryotic initiation factors (eIFs). Although the conserved loop sequences in dicistroviral IGR-IRES elements are protected from chemical modifications in the presence of the 40S subunit, molecular components in the 40S subunit, which interacts with the loop sequences in the IRES, have not been identified. Here, a chemical crosslinking study using 4-thiouridine-labeled IGR-IRES revealed interactions of the IGR-IRES with several 40S proteins but not with the 18S rRNA. The strongest crosslinking signal was identified for ribosomal protein S25 (rpS25). rpS25 is known to be a neighbor of rpS5, which has been shown to interact with a related IGR-IRES by cryo-electron microscopy. Crosslinking analysis with site-directed mutants showed that nucleotides UU(6089-6090), which are located in the loop region in conserved domain 2b in the IRES, appear to interact with rpS25. rpS25 is specific to eukaryotes, which explains why there is no recognition of the IGR-IRES by prokaryotic ribosomes. Although the idea that the IGR-IRES element may be a relict of a primitive translation system has been postulated, our experimental data suggest that the IRES has adapted to eukaryotic ribosomal proteins.

SUBMITTER: Nishiyama T

PROVIDER: S-EPMC1865070 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Eukaryotic ribosomal protein RPS25 interacts with the conserved loop region in a dicistroviral intergenic internal ribosome entry site.

Nishiyama Takashi T Yamamoto Hiroshi H Uchiumi Toshio T Nakashima Nobuhiko N

Nucleic acids research 20070207 5

The intergenic region-internal ribosome entry site (IGR-IRES) of dicistroviruses binds to 40S ribosomal subunits in the absence of eukaryotic initiation factors (eIFs). Although the conserved loop sequences in dicistroviral IGR-IRES elements are protected from chemical modifications in the presence of the 40S subunit, molecular components in the 40S subunit, which interacts with the loop sequences in the IRES, have not been identified. Here, a chemical crosslinking study using 4-thiouridine-labe ...[more]

PMID: 17287295

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Project description:All viruses must usurp host ribosomes for viral protein synthesis. Dicistroviruses utilize an intergenic region internal ribosome entry site (IGR IRES) to directly recruit ribosomes and mediate translation initiation from a non-AUG start codon. The IGR IRES adopts a three-pseudoknot structure that comprises a ribosome binding domain of pseudoknot II and III (PKII and PKIII), and a tRNA-like anticodon domain (PKI) connected via a short, one to three nucleotide hinge region. Recent cryo-EM structural analysis of the dicistrovirus Taura syndrome virus (TSV) IGR IRES bound to the ribosome suggests that the hinge region may facilitate translocation of the IRES from the ribosomal A to P site. In this study, we provide mechanistic and functional insights into the role of the hinge region in IGR IRES translation. Using the honeybee dicistrovirus, Israeli acute paralysis virus (IAPV), as a model, we demonstrate that mutations of the hinge region resulted in decreased IRES-dependent translation in vitro. Toeprinting primer extension analysis of mutant IRESs bound to purified ribosomes and in rabbit reticulocyte lysates showed defects in the initial ribosome positioning on the IRES. Finally, using a hybrid dicistrovirus clone, mutations in the hinge region of the IAPV IRES resulted in decreased viral yield. Our work reveals an unexpected role of the hinge region of the dicistrovirus IGR IRES coordinating the two independently folded domains of the IRES to properly position the ribosome to start translation. IMPORTANCE Viruses must use the host cell machinery to direct viral protein expression for productive infection. One such mechanism is an internal ribosome entry site that can directly recruit host cell machinery. In this study, we have identified a novel sequence in an IRES that provides insight into the mechanism of viral gene expression. Specifically, this novel sequence promotes viral IRES activity by directly guiding the host cell machinery to start gene expression at a specific site.

Dataset Information

Eukaryotic ribosomal protein RPS25 interacts with the conserved loop region in a dicistroviral intergenic internal ribosome entry site.

Publications

Eukaryotic ribosomal protein RPS25 interacts with the conserved loop region in a dicistroviral intergenic internal ribosome entry site.

Similar Datasets

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