Analysis of the decay pattern of specific mRNAs in a variety of RNase mutant backgrounds has allowed some conclusions regarding the role of the four known B. subtilis
3′-to-5′ exoribonucleases in mRNA decay. In the analysis of veg
mRNA and rpsO
mRNA, the only RNA detected at significant levels in the wild type and in the triple mutant that contained only PNPase was the full-length mRNA (Fig. , lanes 1 and 3, and Fig. , lanes 1 and 3). Furthermore, analysis of the decay pattern of small, monocistronic mRNAs in wild-type and PNPase-deficient strains (Fig. ) showed that the absence of PNPase alone resulted in an accumulation of mRNA decay fragments that was nearly undetectable in the presence of PNPase. This suggested that PNPase was the major activity required for the complete turnover of mRNA. Yet, despite the dominance of PNPase in degrading mRNA, the half-life of full-length rpsO
mRNA in the wild-type strain was similar to that in the pnpA
mutant strain (Fig. ). This striking result is important in considering the mechanism of mRNA decay initiation in B. subtilis
. Studies on the effect of 5′-proximal elements on mRNA half-life (6
) point to the 5′ end as the major determinant in mRNA half-life. This suggests the existence of an as yet unidentified RNase E-like activity in B. subtilis
which would initiate decay by endonucleolytic cleavage, as reviewed in the introduction. Our results here further support this hypothesis. If 3′-to-5′ exoribonucleolytic activity from the native 3′ end is a major element in the initiation of decay, we would expect that knocking out PNPase, the dominant 3′-to-5′ exonuclease, would result in an increased concentration of full-length mRNA at steady state, as well as an increased mRNA half-life. The fact that this was not observed suggests that decay initiation by endonuclease cleavage was still occurring normally in the pnpA
strain, but that the resultant decay intermediates were not being degraded efficiently. (As we have not measured rpsO
mRNA half-life in the full set of RNase mutants, there is still the formal possibility that the initiation of decay does occur from the 3′ end, but by a 3′-to-5′ exoribonuclease other than PNPase. However, we consider this possibility unlikely, since it would be difficult to explain how other exoribonucleases that are blocked by upstream secondary structure would be able to degrade past the strong terminator structure.)
Another result that bears on the mechanism of decay initiation is the similarity between the steady-state decay pattern detected by a 215-nt riboprobe complementary to most of the veg coding sequence (Fig. ) and that detected by a 21-nt DNA oligomer complementary to positions 4 to 24 of the veg transcription unit (Fig. ). This similarity demonstrated that only 5′-proximal fragments were observed for veg mRNA. The smallest decay fragments detected by all seven gene probes were between 100 and 150 nt long. Not shown in Fig. is the bottom of the gel, in which a 50-nt marker band was present. No fragments were detected between 40 and 100 nt. This result is compatible with the initiation of decay by an endonucleolytic cleavage distal (more than 100 nt) to the 5′ end. It is also possible that the initial cleavage occurs closer to the 5′ end but that RNA structures that block 3′-to-5′ processivity, and give rise to decay intermediates (see below), are not present near the 5′ end.
We chose the rpsO
mRNA for further study based on the absence of detectable decay fragments in the wild type, the intensity of the 180-nt band, and the relatively uncomplicated decay pattern (Fig. ). (Although the decay of E. coli rpsO
mRNA has been studied thoroughly by Régnier’s group [reference 24
and references therein], this does not provide a model for B. subtilis
, since there is no conservation at the nucleic acid sequence level between the E. coli
and B. subtilis rpsO
genes.) Two of the three prominent decay intermediates with sizes of between 100 and 200 nt were mapped to the base of predicted local secondary structures (Fig. ). There was a qualitative correlation between the amount of accumulated fragments (as measured by intensities of the bands) and the predicted strength of the secondary structure, with the 180-nt set mapping at the base of a structure with a ΔG
° of −10.6 kcal/mol and the 133-nt set mapping at the base of a structure with a ΔG
° of −3.9 kcal/mol. The 5′ end of E. coli rpsO
mRNA has been shown to be a site of translational regulation based on an alternative stem-loop or pseudoknot structure (30
). A similar form of regulation involving pseudoknot formation has been predicted for the B. subtilis rpsO
). The pseudoknot structure for B. subtilis rpsO
mRNA is predicted to end at nucleotide 97, which fits well with the observed 102-nt set of decay fragments.
Thus, blocks to rpsO
mRNA decay in the 3′-to-5′ direction by ribonucleases other than PNPase correspond to the downstream sides of predicted secondary structures. The simplest model for rpsO
mRNA decay is that endonuclease cleavages at sites in the coding sequence are followed by 3′-to-5′ exonucleolytic decay by PNPase. The indicated secondary structures are not an obstacle to PNPase, so that decay intermediates do not accumulate in the wild type or in the triple mutant strain containing PNPase. In strains lacking PNPase, however, decay intermediates whose 3′ ends map to the downstream sides of secondary structures do accumulate. The difference between PNPase and the other 3′-to-5′ exonucleases may be due to the superior processivity of PNPase or an interaction between PNPase and a poly(A) polymerase activity that would enhance the degradation of stem-loop structures by cycles of polyadenylation and 3′-to-5′ degradation (5
). Further study, using 3′-proximal probes, will be required to determine how the extreme 3′ end of rpsO
mRNA is degraded, since the transcription terminator structure (ΔG
° = −14.6 kcal/mol) is more stable than even the predicted structure that gives rise to the 180-nt decay intermediate.
Results with double and triple RNase mutants were revealing in terms of the secondary roles of exoribonucleases other than PNPase. The data in Fig. , lanes 2, 4, and 5 (quantitation in Fig. ), showed that there was little difference in the amounts of the three decay intermediates in strains that were missing YhaM or RNase PH in addition to PNPase. On the other hand, the mutant that was missing PNPase and RNase R (Fig. , lane 3) showed a substantial increase in the amount of the 102-nt fragment, as well as significant increases in the levels of 180-nt and 133-nt fragments. These results suggested that, in the absence of PNPase, RNase R was capable of degrading past secondary structure. The absence of RNase R in the pnpA
background left little exoribonuclease processivity to degrade through the predicted structures. The reason for the disproportionately large increase in the 102-nt fragment set is not clear, although it is possible that the absence of RNase R could affect regulation of rpsO
expression, which is a function of the 5′-proximal pseudoknot formation. Since it has been shown that RNase R is required by E. coli
for the quality control of rRNA (3
), we speculate that expression of rpsO
(encoding a ribosomal protein) is down-regulated in a strain that has an imbalance in fully processed rRNA content, and thus formation of the inhibitory pseudoknot is enhanced, resulting in a greater block to RNase processivity.
The ability of RNase R to degrade past secondary structure was demonstrated in a positive way using the triple mutant strains. In the strain containing only RNase R, few decay intermediates were observed (Fig. , lane 4). Thus, RNase R is also capable of degrading mRNA in vivo.
Surprisingly, the amount of decay fragment that accumulated when only RNase R was present (Fig. , lane 4) was significantly less than that which accumulated when RNase R was present with RNase PH (Fig. , lane 4) or with YhaM (Fig. , lane 5). One might expect that the presence of an additional exoribonuclease would correlate with less, not more, decay intermediates. We hypothesize that the ability of RNase R to degrade past secondary structure may be compromised when RNase PH or YhaM is present. The less-processive RNase PH and YhaM might hydrolyze nucleotides at the downstream side of a stem structure, leaving few single-stranded nucleotides at which RNase R can bind. We showed previously in vitro that RNase R could degrade past a strong stem structure that was followed by a 40-nt single-stranded tail, but was unable to do so when the tail consisted of only 12 nt (28
). Interestingly, the growth rate for the triple mutant strain containing only RNase R was higher than those for all other strains containing more than one RNase mutation and was similar to the growth rate for the single pnpA
mutant (Table ). This result may also be a reflection of the superior ability of RNase R to degrade mRNA when other exoribonucleases are not present.
The involvement of YhaM in mRNA decay was indicated by the decreased accumulation of decay products for the strain containing YhaM alone, relative to that for the strain containing RNase PH alone (compare Fig. , lanes 5 and 6, and Fig. , lanes 2 and 4). The suggestion that any of the known exoribonucleases can participate in mRNA decay was evident as well in the result obtained from the quadruple mutant, which was lacking all four of the 3′-to-5′ exoribonucleases (Fig. , lane 5). In this case, decay intermediates accumulated to levels similar to that for the triple mutant strain containing RNase PH only (Fig. , lane 2), but the intermediates were larger than those of the triple mutant. Whatever activity is responsible for 3′-to-5′ degradation in the quadruple mutant apparently cannot approach stem structures as closely as the other ribonucleases can. The conspicuously low growth rate of the quadruple mutant (Table ) also indicates that the presence of any one of the four exoribonucleases is sufficient to support a growth rate that is closer to that of the wild type than to that of the quadruple mutant.
A considerable amount of mRNA decay fragments accumulated in the pnpA strain. In the case of rpsO, we found that 81% of the total RNA detected was shorter than full length (average of four experiments). While the results shown in Fig. suggest that this fraction might be lower for other genes, the burden of broken mRNAs is clearly substantial in the pnpA strain, and even greater in strains with additional RNase mutations. As expected, the results in Fig. show that the tmRNA system operates at a higher level in the pnpA strain than in the wild type. Somewhat surprisingly, though, we detected only a threefold increase in the level of tagged peptide in the pnpA strain relative to that in the wild type. We suggest that, due to the efficiency of transcription and of mRNA decay, mRNA fragments are relatively rare in a wild-type strain and that the tmRNA system is designed merely to avoid the slight depletion of free ribosomes that would result from the translation of such fragments. The level of accumulated mRNA decay fragments in a PNPase mutant, however, is so high that it would overwhelm the tmRNA system, and the level of peptide tagging would not reflect the level of mRNA fragment accumulation. Other components of the tmRNA system besides ssrA RNA, such as SmpB protein or the requirement for charging by alanyl-tRNA synthetase, may be limiting. It would also be of interest to measure the level of peptide tagging in the large-colony-type pnpA pspac-ssrA strain, which had almost fivefold more ssrA expression in the presence of IPTG than did the small-colony-type strain (Fig. ).
Although Muto et al. found previously that ssrA
expression is induced under various stress conditions—up to 10-fold during heat shock and 4- to 6-fold in the presence of ethanol or cadmium chloride (26
)—we could not detect a high level of induction in the pnpA
strain. By the use of Northern blot analysis of three independent RNA isolations, the levels of ssrA
RNA in wild-type and pnpA
mutant strains were compared (data not shown). In each case, there was a slight increase in the amount of ssrA
RNA (1.2- to 1.4-fold). Somewhat higher levels of ssrA
RNA were found in the multiply mutant strains (1.5- to 1.8-fold increases). The weak responses we observed may be similar to the approximately 1.5- to 2-fold inductions found in the presence of elevated sodium or sucrose (26
). Thus, it appears that there is no robust mechanism to induce ssrA
expression in response to an accumulation of mRNA decay fragments.
It has been shown for E. coli
that the tmRNA system facilitates the degradation of truncated crp
mRNA, presumably by releasing protective ribosomes at the 3′ end of the mRNA fragments (37
). In preliminary experiments, comparing pnpA
, and pnpA ssrA
mutant strains, we have found an increased accumulation of ermC
mRNA decay fragments in the pnpA ssrA
double mutant, relative to that in either single mutant. However, we have not observed significant differences in mRNA decay half-lives between these strains (D. H. Bechhofer, unpublished data). Based on the detailed information about rpsO
mRNA obtained in the present study, it may be informative to study rpsO
mRNA decay in strains that contain both RNase and ssrA
More work needs to be done to understand the individual functions of exoribonucleases that have similar activities. The apparent redundancy of exoribonuclease activities is even greater in E. coli
, where there are eight exoribonucleases (39
), than it is in B. subtilis
. On the other hand, Mycoplasma
appears to have only one 3′-to-5′ exoribonuclease, RNase R (39
). Our finding that RNase R can participate in mRNA turnover suggests that this enzyme is likely to do the same in organisms that do not have PNPase. Although the primary function of RNase R in B. subtilis
might be in the quality control of rRNA, it might also serve as the chief backup mRNA decay enzyme and perhaps play an important role during growth in phosphate-limiting conditions that might reduce the activity of the phosphorolytic PNPase. Further in vitro work with these enzymes will be required to understand how they differ in their abilities to degrade past RNA secondary structure. In addition to requiring the exoribonucleases noted in the present work, the degradation of mRNA in B. subtilis
requires the participation of a 5′-end-dependent endoribonuclease (6
) and likely that of a poly(A) polymerase and a helicase. Identification of the genes encoding such activities will be required to gain a better understanding of the B. subtilis
mRNA decay pathway.