Evidence for a highly nuclease resistant RNA fragment in prosomes

Dineva, Bistra; Tomek, Wolfgang; Köhler, Kurt; Schmid, Hans‐Peter

doi:10.1007/bf00788172

Cited by 17 publications

(6 citation statements)

References 13 publications

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“…Isolation of the particle by different purification procedures systematically led to copurification of small RNA species with the prosome-MCP particle. Furthermore, Dineva and co-workers [15] showed that most of the prosomal RNA is digested by nucleases only after phenol extraction, i.e. after elimination of the prosomal proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Analysis of the protection of prosomal RNA against nuclease digestion by intact and Zn2+-dissociated prosomal particles To test stringently the controversial question about the presence of RNA in the particle, we took advantage ofthe dissociation of the particle on Zn2+ treatment. Dineva and co-workers [15] gave evidence for high nuclease resistance of 80-90 % of the RNA in the intact prosomes. Taking this result as a basis, we addressed the question as to whether the pRNA could be digested by nuclease after dissociation of the complex into its protein subunits by Zn2+.…”

Section: Analysis Of the Dissociated Protein Subunitsmentioning

confidence: 99%

“…Small RNA (pRNA) with a length of 80-120 nucleotides has been isolated from purified prosomes of many types of cells and tissues, such as mouse or duck erythroblasts, rat liver, HeLa cells, calf liver, Drosophila and plants [6,[11][12][13][14]. This RNA seemed to be an intrinsic part of prosomes, since nucleases can only digest it after the elimination by phenol extraction of the prosomal protein components [15]. However, often investigators have not found pRNA in particles that seemed to be identical with prosomes.…”

Section: Introductionmentioning

confidence: 99%

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Disruption of prosomes by some bivalent metal ions results in the loss of their multicatalytic proteinase activity and cancels the nuclease resistance of prosomal RNA

Nothwang

Coux

Bey

et al. 1992

Biochemical Journal

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Prosomes are ribonucleoprotein particles constituted by a variable set of about 20 proteins found associated with untranslated mRNA. In addition, they contain a small RNA, the presence of which has been an issue of controversy for a long time. The intact particles have a multicatalytic proteinase (MCP) activity and are very stable; we have never observed autodigestion of the particle by its intrinsic proteinase activity. Surprisingly it was found that Zn2+ and Cu2+ ions at concentrations of 0.1-1 mM disrupt the prosome particles isolated from HeLa cells and duck erythroblasts and abolish instantaneously its MCP activity, without altering the two-dimensional electrophoretic pattern of the constituent proteins. Fe2+, however, seems to induce autodegradation rather than dissociation of the prosome constituents. Most interestingly, protein or oligopeptide substrates protect the particle and its proteinase activity from disruption by Zn2+ or Cu2+. Nuclease-digestion assays reveal that the prosomal RNA, which is largely resistant in the intact particle, becomes digestible after dissociation of prosomes by Zn2+. These data give, for the first time, unambiguous proof of the presence of an RNA in the particle. Furthermore, they demonstrate a structure-function relationship between the complex and its enzyme activity, which seems to be based on the particle as an entity and not on the single constituent proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Analysis Of the Dissociated Protein Subunitsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Disruption of prosomes by some bivalent metal ions results in the loss of their multicatalytic proteinase activity and cancels the nuclease resistance of prosomal RNA

Nothwang

Coux

Bey

et al. 1992

Biochemical Journal

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“…The individual particle has a defined morphological and biochemical structure (12 -17 nm in the electron microscope, z 720 kDa) and is made up of about 15 proteins with total molecular mass of 19 -36 kDa (Coux et al, 1992). Prosomes also contain several small RNAs 80 -120 nucleotides long (Schmid et al, 1984;Martins de Sa et al, 1986); this RNA (pRNA) is an intrinsic part of the complex, as shown by nuclease digestion assays, where over 80% of the length of the RNA was found to be protected (Dineva et al, 1989). Furthermore, prosomal RNAs inhibit, similarily to intact particles, in vitro protein synthesis and hybridize stably to mRNA (Martins de Sa et al, 1986;Horsch et al, 1989).…”

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confidence: 99%

Prosomes and their multicatalytic proteinase activity

Nothwang

Coux

Bey

et al. 1992

European Journal of Biochemistry

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Prosomes were first described as being mRNA-associated RNP (ribonucleoprotein) particles and subcomponents of repressed mRNPs (messenger ribonucleoprotein). We show here that prosomes isolated from translationally inactive mRNP have a protease activity identical to that described by others for the multicatalytic proteinase complex (MCP, 'proteasome'). By RNase or non-ionic detergent treatment, the MCP activity associated with repressed non-globin mRNP from avian erythroblasts, sedimenting at 35 S, could be quantitatively shifted on sucrose gradients to the 19-S sedimentation zone characteristic of prosomes, which were identified by monoclonal antibodies. The presence of small RNA in the enzymatic complex was shown by immunoprecipitation of the protease activity out of dissociated mRNP using a mixture of anti-prosome monoclonal antibodies; a set of small RNAs 80-120 nucleotides long was isolated from the immunoprecipitate. Furthermore, on CsCl gradients, colocalisation of the MCP activity with prosomal proteins and prosomal RNA was found, and no difference in the prosomal RNA pattern was observed whether the particles were fixed or not prior to centrifugation. These data indicate that the MCP activity is a property of prosomes, shown to be in part RNP and subcomplexes of in vivo untranslated mRNP. A hypothesis for the role of the prosome-MCP particles in maintaining homeostasis of specific protein levels is proposed.

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“…Therefore, in the light of these observations, it was suggested that these RNA species, in conjunction with proteasomes, play a role in the processing of RNA (Arrigo et al, 1985). Since it was shown that the protein-bound RNA species are nuclease resistant and that their digestion is only possible by initially digesting the proteasomal protein components, it was concluded that these RNAs must be fully protected by the proteasome particle (Dineva et al, 1989).…”

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confidence: 99%

Proteasome‐associated RNAs are non‐specific

Pamnani¹,

Haas²,

Pühler³

et al. 1994

European Journal of Biochemistry

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The RNA isolated from RNase-treated proteasome preparations from human erythrocytes, HeLa cells, the archaeon Thermoplasma acidophilum and also from recombinant proteasomes of T acidophilum expressed in Escherichia coli was characterized. The RNA associated with structurally similar protein particles, namely with the two molecular chaperones, groEL from E. coli and with the thermosome from ir: acidophilum, served as controls. Electrophoretic analysis on polyacrylamide gels of the radioactively end-labelled RNA revealed a very similar size distribution pattern, irrespectively of the protein particles from which they had been isolated. The predominant RNA species were in the size ranges 80 nucleotides and 120 nucleotides, respectively. Partial sequencing of their terminal regions by mobility-shift analysis revealed that, of the proteasomes from human erythrocytes, the approximately 80-nucleotide-long RNA consists of a heterogenous population of mostly tRNA species because they carried the tRNA-specific 3'-terminal sequence motif 5'-CCA-3'. The RNA in the size range 120 nucleotides isolated from the proteasomes of human erythrocytes and of T. acidophilum was also heterogeneous and displayed, in the terminal regions, a remarkable sequence similarity to the corresponding regions of the 5s rRNA from the same and different organisms. The total content of RNA of all the protein particles was quantified and found to be consistently sub-stoichiometric. All these findings strongly suggest that RNA associated with the proteasomes and with the molecular chaperones originate from the abundant cellular pool of the tRNAs and 5s rRNAs which bind non-specifically to these large protein particles.

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Evidence for a highly nuclease resistant RNA fragment in prosomes

Cited by 17 publications

References 13 publications

Disruption of prosomes by some bivalent metal ions results in the loss of their multicatalytic proteinase activity and cancels the nuclease resistance of prosomal RNA

Disruption of prosomes by some bivalent metal ions results in the loss of their multicatalytic proteinase activity and cancels the nuclease resistance of prosomal RNA

Prosomes and their multicatalytic proteinase activity

Proteasome‐associated RNAs are non‐specific

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