Alterations in the Processing of Rat-Liver Ribosomal RNA Caused by Cycloheximide Inhibition of Protein Synthesis

Abstract

Cycloheximide given in vivo at low doses (2 -5 mg/kg body weight) causes within 30 min a complete inhibition of protein synthesis in rat liver. The labelling of nuclear proteins is also strongly inhibited. Under these conditions, the amount of nucleolar 45-S pre-rRNA and its ['"CIorotate labelling remain unaffected for at least 4 h. These results show that initially the rates of synthesis and processing of 45-S pre-rRNA are not appreciably altered. On the other hand, drastic alterations in the 45-S pre-rRNA processing pathways occur at the early stages of cycloheximide action. Formation of 18-S rRNA is abolished and that of 28-S rRNA is reduced to about half the level in control rats. This dichotomy in the production of the two ribosomal particles may be correlated with a block in the formation of 41-S and 21-S pre-rRNA. Generation of 36-S and 32-S pre-rRNA is still taking place, but the rate of their processing to nucleolar 28-S rRNA is decreased, thus causing the accumulation of these two pre-rRNA species. In parallel, processing of 45-S prerRNA to an aberrant 39-S rRNA species is markedly enhanced. The results obtained show that the channelling of nucleolar pre-rRNA along alternative processing pathways is under stringent control by the continuous supply of critical protein(s).The formation of ribosomes in animal cells is dependent on the continuous supply of proteins. However, the critical sites in the pathway leading to mature ribosomes, affected primarily by the lack of control structural or regulatory protein(s), are not yet identified [1,2]. Experiments with HeLa cells [3] and rat liver in vivo [4] showed that cycloheximide block of protein synthesis results in a rapid inhibition of 45-S pre-rRNA synthesis. This early inhibition of transcription of rRNA genes appears to be due to an altered interaction of RNA polymerase A with nucleolar template chromatin [5,6]. The shortage of some control protein(s), characterized by a rapid turnover, has been implicated to explain the observed alterations in transcription [5-81. On the other hand, it was reported that block of protein synthesis by low doses of cycloheximide does not alter the synthesis of total nuclear RNA in rat liver [9]. Therefore, the inhibition of transcription, observed with high doses of cycloheximide, could be due to some side effect of the drug or its metabolites rather than to the shortage of putative control proteins (see [2]).Inhibition of 45-S pre-rRNA processing and formation of mature ribosomes upon cycloheximide block of protein synthesis was observed with HeLa [3,10] and L [ l l , 121 cells, regenerating rat liver [13] and culAbbreviations. pre-rRNA, precursor to rRNA; hnRNA, heterogeneous nuclear RNA. tured lymphocytes [14]. The exact mechanisms of this inhibition of ribosome formation and its possible dependence on simultaneous alterations of transcription remain unclear (see [1,2]).In this work we studied the action of cycloheximide in vivo on 45-S pre-rRNA synthesis and processing in rat liver. It is shown that low doses of cycl...

Processing of truncated mouse or human rRNA transcribed from ribosomal minigenes transfected into mouse cells.

“…9A and reference 22) by (i) the accumulation of prerRNA processing products which appear to be coterminal with mature rRNA species and (ii) the identification of minor products corresponding to nuclease cuts within ITS 1 or ITS 2. The similarity of the processing of truncated pre-rRNA transcripts to the phenomena observed in vivo is supported further by preliminary results showing that inhibition of protein synthesis by a low dose of cycloheximide (10 ,ug/ml) does not affect appreciably the synthesis of the primary transcript for the human-mouse minigene pWH/S.I1.L'.12.Ld, while the level of its processing products is markedly reduced, an observation in line with findings in vivo (22,45).…”

Differentiation of rat myoblasts

“…Rat liver, for example, when treated with actinomycin D at a concentration which inhibits only rRNA synthesis, continues to synthesis r-proteins at pre-treatment levels with the excess being rapidly degraded [43]. Ribosome precursors, either the pre-rRNAs or the r-proteins, clearly do not directly regulate each other's levels in these systems, unlike the situation in lower eukaryotes such as ycast [3] and Tetrulzyn?enu [44, 451.…”

Depletion of Saccharomyces cerevisiae ribosomal protein L16 causes a decrease in 60S ribosomal subunits and formation of half-mer polyribosomes.

“…Such turnover of excess molecules produced during ribosome biogenesis also occurs under several other circumstances. When the synthesis of ribosomal proteins is blocked, rRNA is transcribed but is not processed and is instead degraded (Shulman and Warner 1978;Stoyanova and Hadjiolov 1979). When rRNA synthesis or processing is blocked, ribosomal proteins are synthesized but degraded (Craig and Perry 1971;Gorenstein and Wamer 1977;Tsurugi and Ogata 1977;Wamer 1977;]acobs et al 1985;Pierandrei-Amaldi et al 1985).…”