Alterations in the processing of rat-liver ribosomal RNA caused by cycloheximide inhibition of protein synthesis.

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 proteint is also strongly inhibited. Under these conditions, the amount of nucleolar 45-S pre-rRNA and its [14C]-orotate 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 28S 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 pre-rRNA 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).     

Functional defects in transfer RNAs lead to the accumulation of ribosomal RNA precursors

Normal expression and function of transfer RNA (tRNA) are of paramount importance for translation. In this study, we show that tRNA defects are also associated with increased levels of immature ribosomal RNA (rRNA). This association was first shown in detail for a mutant strain that underproduces tRNA(Arg2) in which unprocessed 16S and 23S rRNA levels were increased several-fold. Ribosome profiles indicated that unprocessed 23S rRNA in the mutant strain accumulates in ribosomal fractions that sediment with altered mobility. Underproduction of tRNA(Arg2) also resulted in growth defects under standard laboratory growth conditions. Interestingly, the growth and rRNA processing defects were attenuated when cells were grown in minimal medium or at low temperatures, indicating that the requirement for tRNA(Arg2) may be reduced under conditions of slower growth. Other tRNA defects were also studied, including a defect in RNase P, an enzyme involved in tRNA processing; a mutation in tRNA(Trp) that results in its degradation at elevated temperatures; and the titration of the tRNA that recognizes rare AGA codons. In all cases, the levels of unprocessed 16S and 23S rRNA were enhanced. Thus, a range of tRNA defects can indirectly influence translation via effects on the biogenesis of the translation apparatus.     

Down-regulation of RNA helicase II/Gu results in the depletion of 18 and 28 S rRNAs in Xenopus oocyte

Genetic manipulations have revealed the functions of RNA helicases in ribosomal RNA (rRNA) biogenesis in yeast. However, no report shows the role of an RNA helicase in rRNA formation in higher eukaryotes. This study reports the functional characterization of the frog homologue of nucleolar RNA helicase II/Gu (xGu or DDX21). Down-regulation of xGu in Xenopus laevis oocyte using an antisense oligodeoxynucleotide results in the depletion of 18 and 28 S rRNAs. The disappearance of 18 S rRNA is accompanied by an accumulation of 20 S, indicating that xGu is critical in the processing of 20 to 18 S rRNA. The degradation of 28 S rRNA into fragments smaller than 18 S is also associated with a specific decrease in the level of xGu protein. These effects are reversed in the presence of in vitro synthesized wild type xGu mRNA but not its helicase-deficient mutant form. Similar aberrant rRNA processing is observed when antibody against xGu is microinjected. The involvement of xGu in processing of rRNA is consistent with the localization of Gu protein to the granular and dense fibrillar components of PtK2 cell nucleoli by immunoelectron microscopy. Our results show that xGu is involved in the processing of 20 to 18 S rRNA and contributes to the stability of 28 S rRNA in Xenopus oocytes.     

RNase G of Escherichia coli exhibits only limited functional overlap with its essential homologue,RNase E

RNase G (rng) is an E. coli endoribonuclease that is homologous to the catalytic domain of RNase E (rne), an essential protein that is a major participant in tRNA maturation, mRNA decay, rRNA processing and M1 RNA processing. We demonstrate here that whereas RNase G inefficiently participates in the degradation of mRNAs and the processing of 9S rRNA, it is not involved in either tRNA or M1 RNA processing. This conclusion is supported by the fact that inactivation of RNase G alone does not affect 9S rRNA processing and only leads to minor changes in mRNA half-lives. However, in rng rne double mutants mRNA decay and 9S rRNA processing are more defective than in either single mutant. Conversely, increasing RNase G levels in an rne-1 rng::cat double mutant, proportionally increased the extent of 9S rRNA processing and decreased the half-lives of specific mRNAs. In contrast, variations in the amount of RNase G did not alter tRNA processing under any circumstances. Thus, the failure of RNase G to complement rne mutations, even when overproduced at high levels, apparently results from its inability to substitute for RNase E in the maturation of tRNAs.     

Cloning of a gene involved in rRNA precursor processing and 23S rRNA cleavage in Rhodobacter capsulatus.

In Rhodobacter capsulatus wild-type strains, the 23S rRNA is cleaved into [16S] and [14S] rRNA molecules. Our data show that a region predicted to form a hairpin-loop structure is removed from the 23S rRNA during this processing step. We have analyzed the processing of rRNA in the wild type and in the mutant strain Fm65, which does not cleave the 23S rRNA. In addition to the lack of 23S rRNA processing, strain Fm65 shows impeded processing of a larger 5.6-kb rRNA precursor and slow maturation of 23S and 16S rRNAs from pre-23S and pre-16S rRNA species. Similar effects have also been described previously for Escherichia coli RNase III mutants. Processing of the 5.6-kb precursor was independent of protein synthesis, while the cleavage of 23S rRNA to generate 16S and 14S rRNA required protein synthesis. We identified a DNA fragment of the wild-type R. capsulatus chromosome that conferred normal processing of 5.6-kb rRNA and 23S rRNA when it was expressed in strain Fm65.     

Structure, expression and products of the ribosomal RNA operons of Rhodopseudomonas palustris No. 7

Rhodopseudomonas palustris strains carry one or two ribosomal rRNA operons, and those with duplicated rrn operons grow faster. The two rrn operons in R. palustris No. 7 are virtually identical over a 54,70-bp stretch containing the genes for 16S rRNA, tRNAile, tRNAala, 23S rRNA and 5S rRNA, as well as the intergenic spacers and part of the extragenic spacer. In R. palustris, unlike most bacteria with multiple rrn operons, the putative promoter sequences of the two operons are highly diverged, suggesting possible functional differentiation. By simultaneous primer-extension analysis of both pre-rRNAs, we detected a two-fold higher level of expression from rrnA under photoautotrophic conditions. Alteration of the conditions of growth leads to changes in the relative levels of expression of the two operons. Within the 5,470-bp segment, only two sequence differences are found between the 23S rRNA genes; one is at the center of the 23S rRNA molecule and affects a site of unknown function, and the other is within or immediately adjacent to sequences involved in processing of the 5' 23S rRNA IVS. In vitro processing of 5' IVS-containing 23S rRNA precursors from each operon does not reveal any detectable difference between them. The 5' ends of the mature 16S, 23S, and 5S rRNAs were determined by primer-extension analysis, and the 3' end of 23S rRNA was determined by RNA linker ligation-mediated cDNA cloning. The 5' and 3' ends of the R. palustris 23S rRNA molecule are extensively processed, suggesting that, unlike the situation in the established eubacterial model, these ends cannot basepair.     

Divergent mechanisms of 5' 23S rRNA IVS processing in the alpha-proteobacteria

Widespread occurrence of a separate small RNA derived from the 5'-end of 23S rRNA and of an intervening sequence (IVS) which separates this domain from the main segment of 23S rRNA in the alpha-proteobacteria implies that processing reactions which act to excise the IVS are also maintained in this group. We previously characterized the first example of processing of this IVS in Rhodopseudomonas palustris, which is classified with the Bradyrhizobia In this case, IVS excision occurs by a multistep process and RNase III appears to act at an early step. Here, we characterize in vivo and in vitro IVS processing in two other related, but phenotypically distinct, Bradyrhizobia We also examine in vivo and in vitro processing of rRNA precursors from a more distantly related alpha-proteobacterium, Rhodobacter sphaeroides which produces a separate 5' 23S rRNA domain but has different sequences in the 5' 23S rRNA IVS. The details of the in vivo processing of all of the Bradyrhizobial rRNAs closely resemble the R. palustris example and in vitro studies suggest that all of the Bradyrhizobia utilize RNase III in the first step of IVS cleavage. Remarkably, in vivo and in vitro studies with R.sphaeroides indicate that initial IVS cleavage uses a different mechanism. While the mechanism of IVS cleavage differs among these alpha-proteobacteria, in all of these cases the limits of the internal segments processed in vivo are almost identical and occur far beyond the initial cleavage sites within the IVSs. We propose that these bacteria possess common secondary maturation pathways which enable them to generate similarly processed 23S rRNA 5'- and 3'-ends.