Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The association of mRNA and ribosomes with the cytoskeleton of eucaryotic cells may be important for protein synthesis and its regulation. HeLa cells were gently lysed with detergent, and soluble and cytoskeletal framework subfractions were prepared by centrifugation. We analyzed these fractions for ribosomes and confirmed earlier findings that polysomes are preferentially associated with the cytoskeletal fraction. The levels of initiation factors eIF-2, eIF-3, eIF-4A, and eIF-4B were quantitated by immunoblotting; all are enriched in the cytoskeletal fraction relative to the soluble fraction. Heat shock, fluoride, pactamycin , and cytochalasin caused the release of both ribosomes and initiation factors into the soluble fraction. However, treatment of the cytoskeletal fraction with EDTA or low levels of ribonuclease resulted in polysome degradation but no release. Therefore initiation factor association with the cytoskeletal framework correlates with the presence of ribosomes, whereas ribosome association does not require intact mRNA.
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PMID:Translational initiation factor and ribosome association with the cytoskeletal framework fraction from HeLa cells. 672 78

The virion host shutoff protein (vhs) of herpes simplex virus triggers accelerated degradation of cellular and viral mRNAs while sparing other cytoplasmic RNA species. Previous work has shown that vhs forms a complex with translation initiation factor eIF4H, which displays detectable RNase activity in the absence of other viral or host proteins. However, the contributions of eIF4H and other host factors to the activity and mRNA targeting properties of vhs have not yet been directly examined. An earlier report from our laboratory demonstrated that rabbit reticulocyte lysate (RRL) contains one or more factors that strongly stimulate the RNase activity of vhs produced in Saccharomyces cerevisiae. We report here that such yeast extracts display significant vhs-dependent RNase activity in the absence of mammalian factors. This activity differs from that displayed by vhs generated in RRL in that it is not targeted to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). Activity was strongly enhanced by the addition of RRL, eIF4H, or the related translation factor eIF4B. RRL also reconstituted strong targeting to the EMCV IRES, resulting in a major change in the RNA cleavage pattern. In contrast, eIF4H and eIF4B did not reconstitute IRES-directed targeting. These data indicate that eIF4B and 4H stimulate the nuclease activity of vhs, and they provide evidence that additional mammalian factors are required for targeting to the EMCV IRES.
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PMID:Herpes simplex virus virion host shutoff protein is stimulated by translation initiation factors eIF4B and eIF4H. 1507 51

During lytic infections, the virion host shutoff (Vhs) protein of herpes simplex virus accelerates the degradation of both host and viral mRNAs. In so doing, it helps redirect the cell from host to viral protein synthesis and facilitates the sequential expression of different viral genes. Vhs interacts with the cellular translation initiation factor eIF4H, and several point mutations that abolish its mRNA degradative activity also abrogate its ability to bind eIF4H. In addition, a complex containing bacterially expressed Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein has RNase activity. eIF4H shares a region of sequence homology with eIF4B, and it appears to be functionally similar in that both stimulate the RNA helicase activity of eIF4A, a component of the mRNA cap-binding complex eIF4F. We show that eIF4H interacts physically with eIF4A in the yeast two-hybrid system and in GST pull-down assays and that the two proteins can be coimmunoprecipitated from mammalian cells. Vhs also interacts with eIF4A in GST pull-down and coimmunoprecipitation assays. Site-directed mutagenesis of Vhs and eIF4H revealed residues of each that are important for their mutual interaction, but not for their interaction with eIF4A. Thus, Vhs, eIF4H, and eIF4A comprise a group of proteins, each of which is able to interact directly with the other two. Whether they interact simultaneously as a tripartite complex or sequentially is unclear. The data suggest a mechanism for linking the degradation of an mRNA to its translation and for targeting Vhs to mRNAs and to regions of translation initiation.
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PMID:mRNA decay during herpes simplex virus (HSV) infections: protein-protein interactions involving the HSV virion host shutoff protein and translation factors eIF4H and eIF4A. 1601 27

Ribonucleoprotein complexes (RNP) remodeling by DEAD-box proteins is required at all stages of cellular RNA metabolism. These proteins are composed of a core helicase domain lacking sequence specificity; flanking protein sequences or accessory proteins target and affect the core's activity. Here we examined the interaction of eukaryotic initiation factor 4AI (eIF4AI), the founding member of the DEAD-box family, with two accessory factors, eIF4B and eIF4H. We find that eIF4AI forms a stable complex with RNA in the presence of AMPPNP and that eIF4B or eIF4H can add to this complex, also dependent on AMPPNP. For both accessory factors, the minimal stable complex with eIF4AI appears to have 1:1 protein stoichiometry. However, because eIF4B and eIF4H share a common binding site on eIF4AI, their interactions are mutually exclusive. The eIF4AI:eIF4B and eIF4AI:eIF4H complexes have the same RNase resistant footprint as does eIF4AI alone (9-10 nucleotides [nt]). In contrast, in a selective RNA binding experiment, eIF4AI in complex with either eIF4B or eIF4H preferentially bound RNAs much longer than those bound by eIF4AI alone (30-33 versus 17 nt, respectively). The differences between the RNase resistant footprints and the preferred RNA binding site sizes are discussed, and a model is proposed in which eIF4B and eIF4H contribute to RNA affinity of the complex through weak interactions not detectable in structural assays. Our findings mirror and expand on recent biochemical and structural data regarding the interaction of eIF4AI's close relative eIF4AIII with its accessory protein MLN51.
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PMID:Interactions between eIF4AI and its accessory factors eIF4B and eIF4H. 1871 48

The virion host shutoff protein (vhs), encoded by the gene UL41, has RNase activity and is the key regulator of the early host shutoff response induced by type 1 herpes simplex virus. Despite low amino acid similarity, the vhs protein of the swine herpesvirus, pseudorabies virus (PrV), also exhibits RNase activity. However, the mechanism underlying the action of vhs remains undefined. Here, we report that the RNA degradation profile of PrV vhs is similar, but not identical, to that of type 1 herpes simplex virus vhs. Notably, the presence of a cap structure enhances both the degradation rate and the preferential targeting of the vhs protein towards the 3'-end of the encephalomyocarditis virus internal ribosome entry site (IRES). Furthermore, type 1 herpes simplex virus vhs produces a simple degradation pattern, but PrV vhs gives rise to multiple intermediates. The results of northern blotting using probes recognizing various regions of the RNA substrate found that PrV vhs also cleaves downstream of the IRES region and this vhs protein overall shows 5' to 3' RNase activity. Moreover, addition of the translation initiation factors eIF4H and eIF4B significantly increased the RNase activity of recombinant PrV vhs against capped RNA. Nonetheless, these proteins did not fully reconstitute the IRES-directed targeting pattern observed for vhs translated in a rabbit reticular lysate system. The interaction between PrV vhs and eIF4H/eIF4B implies that the translation initiation machinery within the cell is able to stimulate the nuclease activity of PrV vhs. However, this process remains inefficient in terms of the IRES-targeting pattern.
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PMID:The pseudorabies virus vhs protein cleaves RNA containing an IRES sequence. 2674 29