RNase III cleavage is obligate for maturation but not for function of Escherichia coli pre-23S rRNA.

Abstract

RNase III makes the initial cleavages that excise Escherichia coli precursor 16S and 23S rRNA from a single large primary transcript. In mutants deficient in RNase Ill, no species cleaved by RNase Ill are detected and the processing of 23S rRNA precursors to form mature 23S rRNA fails entirely. Instead, 50S ribosomes are formed with rRNAs up to several hundred nucleotides longer than mature 23S rRNA. Unexpectedly, these aberrant subunits function well enough to participate in protein synthesis and permit cell growth. Consistent with the inference that RNase III cleavages are absolutely required for 23S rRNA maturation, when 50S ribosomes from a strain deficient in RNase HI were incubated with a ribosome-free extract from a RNase III+ strain, rRNA species processed by RNase III and species with normal mature 23S rRNA termini were produced. rRNA in Escherichia coli is synthesized as one transcript containing 16S, 23S, and 5S rRNA as well as precursor sequences. This transcript is thought to be processed first by RNase III, which cleaves the precursor at double-stranded stems and separates the three rRNA species from one another before transcription is complete; other processing activities then remove the remaining precursor sequences during ribosome formation, generating the mature termini (1, 2). Because RNase III initiates processing, and processing has been generally considered indispensable to produce functional RNA molecules, it was unexpected that mutants deficient in the enzyme could survive. It was originally suggested that a low level of RNase III might remain in such mutants or that other RNases might form an alternative pathway to yield mature rRNA (3-5). These ideas have persisted in recent discussions, but both are disproven here in the case of 23S rRNA formation. Nuclease S1 mapping is used to show that the lesion in RNase III is complete in these mutants, but no backup pathway replaces the function of RNase III. The 16S rRNA is matured normally without any preliminary RNase III cleavage, but 23S rRNA exists only as unmatured species. Maturation of 23S rRNA is therefore absolutely dependent on RNase III action, but at least some of the unmatured 23S rRNA species detected must function in protein synthesis because these mutant strains are viable. The maturation of the unprocessed 23S rRNA in 50S ribosomes is shown to occur in subcellular extracts of a RNase III+ strain. MATERIALS AND METHODSThe termini of rRNA species in two RNase III-deficient strains, ABL1 and AB301-105 (6, 7), were assessed by their capacity to hybridize to DNA probes composed of complementary sequences of rDNA. ABL1 and AB301-105 cells were grown in Luria broth at 30°C to an optical density of 0.25 at 550 nm and then harvested over ice. Total RNA was prepared by extracting cells directly at 65TC with phenol in the presence of 1% sodium dodecyl sulfate. The RNA was precipitated once with LiCI (8) and twice with ethanol and was then suspended at a standard concentration. Singlestranded DNA hybridization probes for each termi...

Genetic analysis of small nuclear RNAs in Saccharomyces cerevisiae: viable sextuple mutant.

“…In this view, ribosomes with subtle differences in their rRNA termini, or modification patterns, could be generated, which might differ in their performance characteristics (22).…”

RNA Processing and Degradation inBacillus subtilis

“…In E. coli, the endonucleases RNase E and RNase G combine to produce the mature 5Ј end of 16S rRNA (128), which presumably accounts for the presence of significant quantities of mature 16S in cells completely lacking RNase III. 23S rRNA is not completely matured in E. coli rnc cells, however, but is nonetheless functional (113,114 age site at the 3Ј side of 23S rRNA to the end of the operon (43,215). No other enzyme appears to be capable of performing this cleavage reaction, since all 5S rRNA occurs in its precursor form in rnmV mutants in vivo.…”

Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3′ ETS but not the 5′ ETS

“…Yeast Rnt1p was identified by sequence homology to bacterial RNase III (Abou Elela et al+, 1996)+ Rnt1p is a double-strand specific ribonuclease that is required for the synthesis of several small nucleolar RNAs (snoRNAs) from large precursors (Chanfreau et al+, 1998a(Chanfreau et al+, , 1998bQu et al+, 1999) and for 39 end maturation of the U1, U2, and U5 small nuclear RNAs (snRNAs) (Chanfreau et al+, 1997;Abou Elela & Ares, 1998;Seipelt et al+, 1999)+ The initial functions reported for yeast Rnt1p were, however, in the processing of the prerRNA (Abou Elela et al+, 1996)+ The eukaryotic 18S, 5+8S, and 25S/28S rRNAs are transcribed as a single precursor molecule that undergoes complex posttranscriptional processing to remove the external transcribed spacers (59 ETS and 39 ETS) and internal transcribed spacers (ITS1 and ITS2)+ This process involves several exonucleolytic and endonucleolytic steps Fig+ 1B) and is largely carried out in the nucleolus+ The two earliest processing events, in the 39 ETS and at site A 0 in the 59 ETS, were reported to be inhibited in a temperature-sensitive rnt1-1 strain in vivo (Abou Elela et al+, 1996)+ In addition, model 39 ETS and 59 ETS substrates comprising stem-loop structures were specifically cleaved in vitro by the recombinant GST-Rnt1p fusion protein+ This strongly suggested that Rnt1p directly cleaved these two sites+ Since RNase III in Escherichia coli also participates in pre-rRNA processing (King et al+, 1984), this result greatly influenced models for the evolutionary origins of the eukaryotic pre-rRNA processing machinery+ Cleavage at site A 0 also requires base pairing between the U3 snoRNA and the 59 ETS (Beltrame & Tollervey, 1995)+ We therefore investigated the relationship between U3 and Rnt1p+ In the course of this work we realized that a strain completely lacking Rnt1p is, in fact, able to efficiently cleave site A 0 + In contrast, cleavage in the 39 ETS is inhibited, as previously reported (Abou Elela et al+, 1996), although the sites of in vivo Rnt1p cleavage do not match the previously reported site of in vitro cleavage+…”