Genetic studies of cleavage-initiated mRNA decay and processing of ribosomal 9S RNA show that the Escherichia coli ams and rne loci are the same

We show in the present paper that the cleavages initiating decay of the ompA mRNA are suppressed both in the Escherichia coli ams(ts) strain (originally defined by a prolonged bulk mRNA half-life) and in the me(ts) strain (originally defined by aberrant 9S RNA processing). The temperature-sensitive defects of both these strains are complemented by a recombinant lambda phage containing a genomic segment that carries the putative ams locus. A 5.8 kb fragment from this genomic DNA segment was cloned into a low-copy plasmid and used to transform the ams(ts) and rne(ts) strains. This resulted in growth at the non-permissive temperature and a reoccurrence of the cleavages initiating decay of the ompA mRNA. Deletion analyses of this 5.8 kb fragment indicated that the putative ams open reading frame could complement both the Ams(ts) and the Rne(ts) phenotype with regard to the ompA cleavages. In addition we showed that the ams(ts) strain suppresses 9S RNA processing to 5S RNA to the same extent as the rne(ts) strain, and that the rne(ts0 strain has a prolonged bulk mRNA half-life, as was reported for the ams(ts) strain. Therefore we suggest that ams and rne reflect the same gene locus; one which is involved both in mRNA decay and RNA processing. We discuss how this gene locus may related to the previously characterized endoribonucleolytic activities of RNase E and RNase K.     

Escherichia coli cafA gene encodes a novel RNase, designated as RNase G, involved in processing of the 5' end of 16S rRNA.

We found that the Escherichia coli cafA::cat mutant accumulated a precursor of 16S rRNA. This precursor migrated to the same position with 16.3S precursor found in the BUMMER strain that is known to be deficient in the 5' end processing of 16S rRNA. Accumulation of 16. 3S rRNA in the BUMMER mutant was complemented by introduction of a plasmid carrying the cafA gene. The mutant type cafA gene cloned from the BUMMER strain had a 11-bp deletion in its coding region. A small amount of the mature 16S rRNA was still formed in the cafA::cat mutant. This residual activity was found to be due to RNase E encoded by the rne/ams gene by rifampicin-chase experiments of the cafA::cat ams1 double mutant. These results indicated that the cafA gene encodes a novel RNase responsible for processing of the 5' end of 16S rRNA.     

The gene specifying RNase E (rne) and a gene affecting mRNA stability (ams) are the same gene

A DNA clone complementing the rne-3071 mutation has been expressed and localized in the physical map of Escherichia coli. The DNA fragment from this clone was localized to the region of the E. coli chromosome where the rne-3071 mutation has been mapped. The position of this DNA fragment in the E. coli chromosome, the size of the product directed by this DNA fragment (110,000 Da), the restriction map of this fragment, the fact that the same clone complements the ams mutation, and the observation that the rne-3071 and the ams mutations cause similar patterns of RNA synthesis, show that the rne gene--a gene specifying the processing endonuclease RNase E--and the ams gene--a gene that affects mRNA stability--are identical.     

Enhanced cleavage of RNA mediated by an interaction between substrates and the arginine-rich domain of E. coli ribonuclease E

Endonucleolytic cutting by the essential Escherichia coli ribonuclease RNaseE has a central role in both the processing and decay of RNA. Previously, it has been shown that an oligoribonucleotide corresponding in sequence to the single-stranded region at the 5' end of RNAI, the antisense regulator of ColE1-type plasmid replication, is efficiently cut by RNaseE. Combined with the knowledge that alteration of the structure of stem-loops within complex RNaseE substrates can either increase or decrease the rate of cleavage, this result has led to the notion that stem-loops do not serve as essential recognition motifs for RNaseE, but can affect the rate of cleavage indirectly by, for example, determining the single-strandedness of the site or its accessibility. We report here, however, that not all oligoribonucleotides corresponding to RNaseE-cleaved segments of complex substrates are sufficient to direct efficient RNaseE cleavage. We provide evidence using 9 S RNA, a precursor of 5 S rRNA, that binding of structured regions by the arginine-rich RNA- binding domain (ARRBD) of RNaseE can be required for efficient cleavage. Binding by the ARRBD appears to counteract the inhibitory effects of sub-optimal cleavage site sequence and overall substrate conformation. Furthermore, combined with the results from recent analyses of E. coli mutants in which the ARRBD of RNase E is deleted, our findings suggest that substrate binding by RNaseE is essential for the normal rapid decay of E. coli mRNA. The simplest interpretation of our results is that the ARRBD recruits RNaseE to structured RNAs, thereby increasing the localised concentration of the N-terminal catalytic domain, which in turn leads to an increase in the rate of cleavage.     

Cloning and analysis of the entire Escherichia coli ams gene. ams is identical to hmp1 and encodes a 114 kDa protein that migrates as a 180 kDa protein.

We have used an antibody to a previously identified 180 kDa (Hmp1) protein in Escherichia coli to clone the corresponding gene, which encodes a polypeptide of 114 kDa that has a mobility equivalent to 180 kDa in SDS/PAGE. We have demonstrated that the 180 kDa polypeptide is the primary gene product and not due to aggregation with other molecules. Moreover, our data indicate that the highly charged C-terminal region of the protein is responsible for its anomalous behaviour when analysed by SDS/PAGE. The hmp1 gene is in fact identical to ams (abnormal mRNA stability), also designated rne (RnaseE), and reported to have an ORF of 91 kDa. This discrepancy with the data in this paper can be ascribed to the omission of two bases in the previously reported sequence, generating an apparent stop codon. We previously demonstrated that the 180 kDa Hmp1/Ams protein cross reacted with both a polyclonal antibody and a monoclonal antibody raised against a yeast heavy chain myosin. However, we could detect no homology with myosin genes in the ams/hmp1 sequence. From the DNA sequence data, we identified a putative nucleotide binding site and a transmembrane domain in the N-terminal half of the molecule. In the C-terminal half, which appears to constitute a separate domain dominated by proline and charged amino acids, we also identified a region homologous to the highly conserved 70 kDa snRNP protein, involved in RNA splicing in eukaryotes. This feature would be consistent with reports that ams encodes RNaseE, an enzyme required for the processing of several stable RNAs in E. coli.     

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.     

Maturation of precursor 10Sa RNA in Escherichia coli is a two-step process: the first reaction is catalyzed by RNase III in presence of Mn2+.

A precursor to 10Sa RNA accumulates in an rne mutant. However, the present studies indicate that RNase III is the enzyme that processes this RNA. Cell extracts prepared from an rne mutant failed to cleave p10Sa RNA, whereas E coli wild type, rne and rnp cell extracts processed p10Sa RNA under specific assay conditions that require the presence of Mn2+ but not under the customary conditions used for assaying RNase III. That the p10Sa cleaving activity is solely RNase III was confirmed by comparing the increase in p10Sa and poly(A).poly(U) cleaving activities in a strain harboring a plasmid carrying an RNase III gene as compared to a normal E coli strain. It is of interest that these 2 substrates are cleaved by RNase III efficiently, but under 2 different assay conditions. In all strains tested, with normal or elevated levels of RNase III, RNase III fractionates predominantly with the membrane. Further characterization of the maturation of 10Sa RNA revealed that the processing of 10Sa RNA is a 2 step reaction involving 2 separate activities, both sensitive to heat and proteinase K treatment. The first step is catalyzed by RNase III, and results in the formation of a molecule, p10Sa', which is larger than the mature 10Sa RNA. The second activity catalyzes the conversion of p10S' to 10Sa RNA, and this step does not require a divalent cation. The second activity is not any of the known processing endoribonucleases, RNase III, E or P, but could be a new enzyme having no obligate requirement for a divalent cation.     

RNA processing: new mutants that affect endonucleolytic processing of RNA

A strain of Escherichia coli carrying the rne-3071 mutation that affects the RNA processing enzyme ribonuclease E, was mutagenized, and double mutants deficient in RNA processing were isolated. The isolation was based on the appearance of a particular RNA precursor molecule upon infection of an rne mutant with a specific bacteriophage T4 deletion strain. From one of the double mutants the rne mutation was removed, and the new single mutant, designated rng, was examined. In this mutant the maturation of host RNA as well as of bacteriophage T4 RNA is affected. The effect of the rng mutation on RNA synthesis is unique and can be distinguished from the effects of the other established mutations in RNA processing. The effects of the rng mutation can be recognized in vivo and in vitro.     

RNase E polypeptides lacking a carboxyl-terminal half suppress a mukB mutation in Escherichia coli.

We have isolated suppressor mutants that suppress temperature-sensitive colony formation and anucleate cell production of a mukB mutation. A linkage group (smbB) of the suppressor mutations is located in the rne/ams/hmp gene encoding the processing endoribonuclease RNase E. All of the rne (smbB) mutants code for truncated RNase E polypeptides lacking a carboxyl-terminal half. The amount of MukB protein was higher in these rne mutants than that in the rne+ strain. These rne mutants grew nearly normally in the mukB+ genetic background. The copy number of plasmid pBR322 in these rne mutants was lower than that in the rne+ isogenic strain. The results suggest that these rne mutations increase the half-lives of mukB mRNA and RNAI of pBR322, the antisense RNA regulating ColE1-type plasmid replication. We have demonstrated that the wild-type RNase E protein bound to polynucleotide phosphorylase (PNPase) but a truncated RNase E polypeptide lacking the C-terminal half did not. We conclude that the C-terminal half of RNase E is not essential for viability but plays an important role for binding with PNPase. RNase E and PNPase of the multiprotein complex presumably cooperate for effective processing and turnover of specific substrates, such as mRNAs and other RNAs in vivo.     

Degradation of Escherichia coli uncB mRNA by multiple endonucleolytic cleavages.

The mechanism of segmental decay of the uncB sequence near the 5' end of the 7-kb Escherichia coli unc operon mRNA was investigated. Northern (RNA) blots of mRNA expressed from a plasmid carrying the uncBE portion of the operon revealed that the uncB message was rapidly degraded by multiple internal cleavages which resulted in the formation of at least five discrete species having a common 3' end. Turnover studies indicated that processing rapidly converted all species to the smallest. Identification of the 5' ends by primer extension analysis revealed that the cleavages were made either in the uncB coding region or in the intercistronic region between uncB and uncE, the latter being the most 3' cleavage. An rne mutant strain contained much higher levels of the uncBE message, implying that RNase E, the product of the rne gene, is essential for the normal degradation of uncB, and a number of the 5' ends were not detected in the rne mutant. The cleavage sites in chromosomally encoded unc mRNA were also identified by primer extension. These studies reveal that the segmental decay of the uncB region of unc mRNA occurs rapidly through a series of endonucleolytic cleavages. The rapid decay of uncB is expected to play a role in limiting expression of this gene relative to that of the other genes of the operon.     

Processing of bacteriophage T4 tRNAs: a precursor of species 1 RNA

A precursor molecule of species 1 RNA, p2Sp1, that accumulates when an rne (RNase E-) mutant is infected with a T4 deletion mutant (delta 27) is also found after infection of an rne host mutant by different deletion mutants or wild type bacteriophage T4. Low levels of this molecule were also found in a wild-type host infected with a wild-type T4. This precursor molecule accumulates at higher concentrations at 43 degrees C as compared to 30 degrees C or 37 degrees C. Structural analysis of the precursor molecules from the different sources has shown a complete identity of p2Sp1 RNA isolated from the different sources. Therefore, we suggest that this precursor is a normal intermediate in processing of T4 tRNAs, and that it is unrelated to a particular T4 deletion strain. Since RNase E does not process this precursor, its accumulation in an rne mutant reflects an interaction between RNase E and the enzyme that processes this intermediate.     

Escherichia coli endoribonuclease RNase E: autoregulation of expression and site-specific cleavage of mRNA

Mutations in the Escherichia coli rne (ams) gene have a general effect on the rate of mRNA decay in vivo. Using antibodies we have shown that the product of the rne gene is a polypeptide of relative mobility 180kDa. However, proteolytic fragments as small as 70kDa, which can arise during purification, also exhibit RNase E activity, in vitro studies demonstrate that the rne gene product, RNase E, is an endoribonuclease that cleaves mRNA at specific sites. RNase E cleaves rne mRNA and autoregulates the expression of the rne gene. In addition we demonstrate RNase E-dependent endonucleolytic cleavage of ompA mRNA, at a site known to be rate-determining for degradation and reported to be cieaved by RNase K. Our data are consistent with RNase K being a proteolytic fragment of RNase E.