It has been reported that the overproduction of wild-type SprE is toxic to cells (34
). We have exploited the growth defect caused by SprE overproduction to uncover a novel function of SprE. Our data indicate that in exponentially growing cells, SprE stimulates polyadenylation, thereby affecting the decay of specific mRNA species. Indeed, the SprE60 mutant protein increases levels of polyadenylation by at least twofold, and poly(A) levels are reduced by twofold in the absence of SprE (Fig. ). Thus, SprE has two distinct functions, both of which affect the transcriptome. SprE controls what is made by regulating the stability of RpoS, and it also controls which mRNAs are destroyed by stimulating polyadenylation.
While the exact changes in the polyadenylation profile of RNA substrates in the presence of SprE60, or in the absence of SprE, are not yet known, we assume that these changes reflect changes in the activity of PAP I. In support of this idea, removal of the SprE protein led to changes in mRNA half-lives that were almost identical to that obtained upon inactivation of PAP I (Table ). However, not all of the decay intermediates observed in the absence of PAP I were present in the SprE mutant (Fig. ). For example, out of the two yfiA
decay intermediates that accumulated in the ΔpcnB
strain, only the larger species was stable in the ΔsprE
mutant (Fig. ). This could indicate that the effect of SprE on mRNA decay is dependent on PAP I-mediated polyadenylation as well as some other PAP I-independent mechanism. Furthermore, we cannot rule out that the strong effect of both sprE
mutations on the ompA
mRNA half-life (Table ) is due to a combination of effects related to other factors such as growth rate, Hfq modulation, and the action of small RNA regulators that affect RNase E cleavage of this transcript (35
). Nonetheless, our data strongly suggest that SprE and PAP I function together during exponential phase to regulate polyadenylation and mRNA stability.
Interestingly, connections between SprE and PAP I have been previously reported. Sarkar et al. (43
) identified SprE as a multicopy suppressor of the pcnB
plasmid-copy-number defect. PAP I controls the levels of an antisense RNA (RNA I) that inhibits an RNA replication primer of ColE1-type plasmids (14
). In the absence of pcnB
, RNA I is stabilized, which prevents plasmid replication. Overproduction of SprE suppresses this defect. Given our results, it seems likely that the overproduction of SprE suppressed the pcnB
defect either by stimulating PAP I-independent polyadenylation or by stimulating the degradation of the nonpolyadenylated RNA I species. One possibility is that SprE stimulates the other known polyadenylation enzyme PNPase, which accounts for the residual polyadenylation in a pcnB
null strain in exponential-phase cells (31
). While it is true that we did not observe a significant increase in PNPase-dependent polyadenylation by SprE60 in the ΔpcnB
background (Fig. ), this result may simply be due to the fact that the heteropolymeric tails generated by PNPase (32
) cannot be efficiently detected with RNA-DNA dot blots using the oligo(dT)20
probes we employed.
Santos et al. (42
) noted that in the absence of PAP I, RpoS levels were reduced by threefold in stationary-phase cells. They also demonstrated that the effect seen was posttranscriptional and specifically affected protein stability. They hypothesized that PAP I controls the activity of SprE, and that is the cause for reduced RpoS levels. Taking into account our findings, it is plausible that the reduction of RpoS levels the authors reported was due to the lack of SprE's second function. Perhaps when there is no PAP I, higher levels of SprE are available to promote RpoS degradation. Indeed, the rssA2
allele, which increases SprE levels by fourfold, decreases RpoS levels in all growth phases (39
). What the authors perceived as PAP I regulation of SprE may actually be a titration of SprE into the polyadenylation pathway.
PAP I and SprE clearly function together during exponential phase, and we believe this is true during stationary phase as well. Polyadenylation and mRNA stability in stationary phase have not been extensively studied and remain poorly understood. However, we believe that SprE must play some role in regulating polyadenylation in stationary phase, since the localization of PAP I changes in a SprE-dependent manner when growth ceases. Moreover, this localization defect is associated with clear phenotypic changes.
In an otherwise wild-type background, PAP I-GFP was localized to the inner membrane during exponential phase and was released from the membrane as cells entered stationary phase (Fig. ). This release of PAP I-GFP from the membrane was SprE dependent; in cells lacking SprE, PAP I-GFP remained membrane associated. These SprE-dependent changes in the localization of PAP I-GFP provide evidence for a functional connection between PAP I and SprE during stationary phase. It should be noted that the hybrid gene producing PAP I-GFP was carried on a multicopy plasmid, and thus, the fusion protein is overproduced compared to the wild-type endogenous PAP I. This overproduction facilitated visualization of the fluorescent molecule. We assume, but have not proven, that this change in PAP I-GFP localization reflects what happens with endogenous PAP I. If this is true, then it seems likely that during exponential phase, efficient polyadenylation takes place at the inner membrane. It is also possible that the entire mRNA degradation process is compartmentalized. Indeed, it has been recently reported that the mRNA degradosome is anchored to the inner membrane by the N-terminal portion of RNase E (18
It is important to note that the cells overproducing PAP I-GFP (Fig. ) exhibit a defect in the exit from stationary phase that is similar to that caused by overproducing PAP I-His6 (Fig. ). We believe that this exit defect is caused by the elevated activity of PAP I, because removal of SprE suppresses this exit defect. We have shown that removal of SprE decreases PAP I activity in exponential-phase cells (Fig. ), and we assume its removal has similar effects on PAP I-GFP and PAP I-His6 in stationary phase as well. Strikingly, in reciprocal fashion, increased production of SprE also causes a defect in the exit from stationary phase, and removal of PAP I suppresses this defect. We assume that overproduction of SprE in stationary-phase cells also increases PAP I activity. Indeed, SprE60 increases polyadenylation in exponential-phase cells (Fig. ). However, we do not know if the elevated activity of PAP I that occurs in stationary-phase cells under conditions of either PAP I or SprE overproduction is due to changes in PAP I localization, activity, or both. Nonetheless, this reciprocal suppression provides additional evidence for a functional connection between SprE and PAP I.
As noted above, little is known about polyadenylation and mRNA stability in stationary-phase cells, and we do not currently understand why increasing PAP I activity, by increasing production of either PAP I or SprE, would cause a stationary-phase exit defect. It seems likely that this exit defect results from alterations in stationary-phase polyadenylation. It is possible that altered polyadenylation results in the depletion of an essential mRNA or noncoding RNA, or that the tRNA pool is damaged and therefore depleted. In wild-type cells, a reduction of PAP I activity during stationary phase may help change the polyadenylation profile of mRNAs and therefore greatly change which ones are degraded. The RNAs that are degraded during exponential phase may be quite different from those that are degraded during stationary phase. In fact, the stationary-phase-specific mRNAs generally contain polynucleotide tails (9
), which are most probably generated by PNPase (32
), and the tails generated by PNPase are poor substitutes for poly(A) tails (33
Upon entry into stationary phase, SprE levels are increased by two- to threefold (41
). It is somewhat paradoxical that SprE levels would increase when the protein is inactive with regard to RpoS degradation. One proposed explanation is that increasing SprE levels in stationary phase is necessary to promote the rapid destruction of RpoS when cells encounter nutrients and return to exponential phase. This explanation seems unlikely because cells that only mildly overproduced SprE exhibited a defect in the exit from stationary phase. Alternatively, we suggest that SprE levels increase because the protein has an additional function in stationary phase, the regulation of polyadenylation. While we currently have no evidence for a direct interaction between SprE and PAP I, SprE clearly plays a role in the PAP I-pathway in exponential phase, and we have documented numerous genetic interactions between sprE
that affect both exponential- and stationary-phase phenotypes. Further studies are required to elucidate the exact mechanism by which SprE contributes to the polyadenylation pathway.