This review focuses on the enzymes and pathways of RNA processing and degradation in Bacillus subtilis, and compares them to those of its gram-negative counterpart, Escherichia coli. A comparison of the genomes from the two organisms reveals that B. subtilis has a very different selection of RNases available for RNA maturation. Of 17 characterized ribonuclease activities thus far identified in E. coli and B. subtilis, only 6 are shared, 3 exoribonucleases and 3 endoribonucleases. Some enzymes essential for cell viability in E. coli, such as RNase E and oligoribonuclease, do not have homologs in B. subtilis, and of those enzymes in common, some combinations are essential in one organism but not in the other. The degradation pathways and transcript half-lives have been examined to various degrees for a dozen or so B. subtilis mRNAs. The determinants of mRNA stability have been characterized for a number of these and point to a fundamentally different process in the initiation of mRNA decay. While RNase E binds to the 5' end and catalyzes the rate-limiting cleavage of the majority of E. coli RNAs by looping to internal sites, the equivalent nuclease in B. subtilis, although not yet identified, is predicted to scan or track from the 5' end. RNase E can also access cleavage sites directly, albeit less efficiently, while the enzyme responsible for initiating the decay of B. subtilis mRNAs appears incapable of direct entry. Thus, unlike E. coli, RNAs possessing stable secondary structures or sites for protein or ribosome binding near the 5' end can have very long half-lives even if the RNA is not protected by translation.
In recent years, it has become clear that the common assumption that Bacillus subtilis is essentially Escherichia coli with a cell wall and the capacity to sporulate could not be further from the truth. While both organisms respond similarly to environmental stimuli, as, indeed, do the vast majority of mesophilic bacteria, the mechanisms underlying these responses have proven to be very different in practically every case examined. While an understanding of the mechanisms of RNA processing and degradation in B. subtilis to the depth available for E. coli is perhaps several years away, a simple comparison of the genomes of the two organisms (Table 1) provides sufficient evidence that the same will hold true for this particular aspect of cellular metabolism. In E. coli, a key enzyme of RNA decay is RNase E (178). It is an essential enzyme, responsible for the initial rate-limiting cleavage in the decay of many mRNAs (reviewed in references 77 and 121), as well as playing an important role in 5S and 16S rRNA processing (70, 128). However, neither the gene for RNase E, rne, nor that for oligoribonuclease, orn, also essential for E. coli viability (6, 71, 178), is present in B. subtilis. Also missing are the genes encoding a battery of exonucleases involved in tRNA maturation, namely, RNase T, RNase BN, and RNase D (124, 126). In contrast, B. subtilis, and other low-G+C gram-positive bacteria, have at least three enzymes identified thus far that are not found in E. coli: RNase M5, RNase Bsn and YhaM. Thus, only 6 of a total of 17 characterized RNase activities are shared between B. subtilis and E. coli: 3 exoribonucleases, PNPase, RNase R, and RNase PH, and 3 endoribonucleases, RNase III, RNase P, and RNase H (44). It is thought that the evolutionary split between E. coli and B. subtilis occurred more than a billion years ago, even before the split between plants and animals (177, 183, 231). Thus, it is difficult to go much deeper than this in evolutionary terms to compare the enzymes and pathways of bacterial RNA degradation of two well-studied organisms. It seems likely that the RNases and pathways of degradation that have been identified, or remain to be identified, in these two organisms will account for the vast majority of degradation mechanisms to be found in the whole eubacterial kingdom. By the same token, the enzymes and pathways these organisms have in common will probably be generally conserved throughout the eubacteria (see arguments in reference 67).
The decay of most mRNAs in E. coli is thought to be initiated by endonucleolytic cleavage by RNase E (158), an enzyme with relatively loose substrate specificity (AU-rich single-stranded regions) (63, 130, 147). This first cleavage is rate limiting and is dependent on the phosphorylation state of the 5' end of the molecule; RNAs with 5'-triphosphates (primary transcripts) are cleaved much less efficiently than RNAs with 5'-monophosphates (resulting from a prior cleavage event) (137, 138, 233). Thus, once the initial cleavage event has occurred, RNAs are very rapidly cut into smaller pieces. While this process is generally cited as proceeding with an overall 5'-to-3' directionality, specific examples are not so abundant. Indeed, a recent paper has suggested that RNase E may have intrinsic 3'-to-5' directionality on short RNA fragments (66). In a limited number of cases, the initial endonucleolytic cleavage is catalyzed by RNase III (11, 143, 188, 194, 195), an enzyme with far more restricted specificity (double-stranded RNA hairpins with specific side bulges) (34, 118, 200, 212, 242). The RNA fragments generated by endonucleolytic cleavage are attacked by the exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase II (22, 57, 86), which progressively remove individual nucleotides from the 3' end of the molecules either phosphorolytically (PNPase) (115, 222) or hydrolytically (RNase II) (171). Both of these enzymes are significantly slowed by secondary structures (37, 38, 81, 148) and are thought to be allowed multiple attempts at degrading such structures by successive rounds of addition and degradation of poly(A) tails, which serve as "on-ramps" for the exonucleases, PNPase in particular (19, 39, 40, 42, 140, 175, 233). The association of RNase E, PNPase, enolase, and an RNA helicase (RhlB) for unwinding structured RNAs in a complex known as the degradosome (31, 41, 152, 190, 191) is thought to facilitate the coordination of the RNA decay process (133). The C-terminal half of RNase E provides the scaffolding for the assembly of this complex (223). There is also some evidence that poly(A) polymerase, the enzyme responsible for the addition of poly(A) tails, is associated with RNase E at substoichiometric levels (192), oiling the mechanism even further.
The absence of an obvious RNase E homolog in B. subtilis (119) suggests that the pathway of mRNA degradation in this organism is fundamentally different, if only from the standpoint of the degradosome. Although it is possible that some other protein acts as the scaffold for the formation of a complex between PNPase, enolase, and helicase, we have failed to demonstrate an association between these proteins in B. subtilis (D. Brechemier-Baey, H. Putzer, and C. Condon, unpublished results). In this review, I describe what is known about the different RNases found in B. subtilis, their substrates, and the different stability determinants identified thus far.
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