We have identified a cellular target for proteasomal endonuclease activity. Thus, 20 S proteasomes interact with the 3-untranslated region of certain cytoplasmic mRNAs in vivo, and 20 S proteasomes isolated from Friend leukemia virus-infected mouse spleen cells were found to be associated with a mRNA fragment showing great homology to the 3-untranslated region of tumor necrosis factor- mRNA that contains AUUUA sequences. We furthermore demonstrate that 20 S proteasomes destabilize oligoribonucleotides corresponding to the 3-untranslated region of tumor necrosis factor-␣, creating a specific cleavage pattern. The cleavage reaction is accelerated with increasing number of AUUUA motifs, and major cleavage sites are localized at the 5 side of the A residues. These results strongly suggest that 20 S proteasomes could be involved in the destabilization of cytokine mRNAs such as tumor necrosis factor mRNAs and other short-lived mRNAs containing AUUUA sequences.
We have identified and characterized a specific nuclease activity to be tightly associated with proteasomes. Using tobacco mosaic virus RNA (TMV-RNA) as substrate to analyze and quantify the cleavage reaction, we supply several lines of evidence that this nuclease activity is an integral part of proteasomes. Thus, RNase activity was coincident with the elution profiles of proteasomes at each stage of purification. Proteasomal nuclease activity was resistant to strong dissociation conditions using 480 mM KC1, 0.5% sodium lauroylsarcosinate, and 6 M urea. This nuclease activity remained associated with an urea-resistant subcomplex of the proteasome comprising a specific set of proteins. Finally the digestion of TMV-RNA led to a well defined pattern of RNA fragments while 5 S ribosomal RNA and globin mRNA were not degraded. These results provide further evidence that proteasomes are able to discriminate between different RNAs, and the possible involvement of proteasomes in translation control is discussed.
We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism.
Proteasomes (prosomes) are large multiprotein complexes. They are involved in protein degradation of ubiquitin-conjugated proteins and in the generation of MHC class I peptides. We gave further evidence that they interfere with in vitro protein synthesis. Proteasomes inhibit the translation of Tobacco mosaic virus RNA. Analysis of cell-free systems by sucrose gradient centrifugation revealed that they prevent the formation of 80S initiation complexes but not the early phase of initiation.
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