SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Previous work showed that a 42-nucleotide sequence from an SP82 bacteriophage early RNA functions as a 5′ mRNA stabilizer in Bacillus subtilis. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis of decay of a model mRNA with alterations at the 5′-end was used to elucidate the mechanism of SP82-mediated stability. A predicted 5′-terminal stem–loop structure was essential for stabilization. Increasing the strength of the 5′-terminal structure above a minimum level did not result in increased stability. A thorough analysis of the context in which the stabilizing structure occurred included the effects of distance from 5′-end, translation of downstream coding sequence, and distance between the secondary structure and the ribosome binding site. Our data are consistent with the dominant mRNA decay pathway in B. subtilis being 5′-end dependent.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Messenger RNA stability has been shown to play an important role in the regulation of gene expression, and much needs to be learned about the elements that function as mRNA stability determinants. A large body of evidence from Escherichia coli studies suggests that RNase E, a 5′-end-dependent endoribonuclease, is responsible for the initiation of decay of most mRNAs. A general model for mRNA decay in E. coli has been developed (Coburn and Mackie, 1999; Grunberg-Manago, 1999; Kushner, 2002). In this model RNase E binds to an accessible 5′-end, followed by looping or tracking to an internal cleavage site. Following the initial rate-limiting endonucleolytic cleavage, an mRNA with a 5′ monophosphate end is created, to which RNase E has greater affinity than the 5′-triphosphate end of the initial transcribed product. Successive cleavage events result in mRNA fragments with accessible 3′-ends, which are rapidly degraded by 3′−5′-exoribonucleases (primarily RNase II and PNPase) to oligonucleotides (Donovan and Kushner, 1986; Régnier and Arraiano, 2000). Final turnover of mRNA oligonucleotides to mononucleotides is accomplished by oligoribonuclease (Ghosh and Deutscher, 1999). Interfering with the accessibility of the 5′-end of an mRNA results in mRNA stabilization. For example, stem–loop structures present at the 5′-end of ompA or papA mRNA confer stability (Emory et al., 1992; Bricker and Belasco, 1999). Disruption of the 5′ stem–loop, or the presence of three to five single-stranded nucleotides (nts) preceding the stem–loop structure, both of which create an accessible 5′-end, abolishes stabilization (Bouvet and Belasco, 1992; Emory et al., 1992). When rpsT mRNA was circularized in vivo, generating an mRNA without a 5′-end, the message half-life increased four- to sixfold (Mackie, 2000).

Much less is known about the mechanism of mRNA decay in Bacillus subtilis. How mRNA decay occurs in this organism is of particular interest, because the B. subtilis genome lacks sequence homologues of RNase E, RNase II and oligoribonuclease, which are the major mRNA decay ribonucleases in E. coli.

Experiments in B. subtilis have shown that 5′-proximal secondary structure or events such as ribosome stalling, regulatory protein binding, and ribosome binding can be important factors in determining mRNA stability. Erythromycin-induced ribosome stalling in the 5′ leader region of the ermA or ermC mRNAs leads to a 10- to 15-fold increase in mRNA half-life (Bechhofer and Dubnau, 1987; Bechhofer and Zen, 1989; Sandler and Weisblum, 1989). Binding of the regulatory protein, GlpP, in the 5′ untranslated region (UTR) of glpD mRNA results in an increase in mRNA stability (Glatz et al., 1996; 1998). A 5′ stem–loop and ribosome binding are important for the stability of aprE mRNA (Hambraeus et al., 2000; 2002). Results from our laboratory have shown that ribosome binding and ternary complex formation, but not actual translation of the coding sequence, are important factors for mRNA half-life in B. subtilis (Sharp and Bechhofer, 2003). These studies, which point to the 5′-end as the key element in determining mRNA stability, suggest the existence of a 5′-end-dependent, RNase E-like activity. Indirect evidence for such an activity comes from the study of Condon et al. (1997), who showed that endonucleolytic cleavage of B. subtilis thrS mRNA occurs at the same site in B. subtilis and E. coli, and this cleavage was RNase E-dependent in E. coli. However, the identity of a gene encoding such an activity in B. subtilis is unknown at this time.

In previous work, we found that insertion into ermC mRNA of a bacteriophage SP82 early RNA sequence, which contained a B. subtilis RNase III endonuclease cleavage site, resulted in a downstream RNA fragment that was extremely stable (DiMari and Bechhofer, 1993). Further study with this SP82 sequence showed that a 42 nt sequence downstream of the RNase III cleavage site could function by itself as a 5′ mRNA stabilizer (Hue et al., 1995). When the 42 nt SP82 sequence (Table 1, construct 2) was present at the 5′-end of ermC or lacZ mRNA, the mRNA half-life was increased by 15-fold. The SP82 5′ stabilizer sequence contains a polypurine sequence that likely acts as a strong Shine–Dalgarno (SD) sequence (ΔG0 for interaction with 16S rRNA-3′-end sequence is −11.2 kcal mol−1), and it was shown that this SD sequence was required for SP82-mediated stability (Hue et al., 1995). The initial goal of the present work was to elucidate the sequence element(s) necessary for SP82-mediated stability.

Table 1.  Effect of SD strength on mRNA stability. Thumbnail image of

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Model mRNAs and real-time reverse transcriptase polymerase chain reaction (RT-PCR)

Model mRNAs designed for this study were based on a 254 nt mRNA, which is a deletion derivative of ermC mRNA and is termed ‘ΔermC’ (Drider et al., 2002). A schematic diagram of the basic mRNA is shown in Fig. 1A.ΔermC is plasmid-borne, has a 32 nt 5′ UTR, followed by a 62-amino acid coding sequence and a strong 3′ transcriptional terminator stem–loop structure. The half-life of ΔermC mRNA, as measured in this study, is 6.0 ± 0.3 min (Table 1). This is more stable than the average mRNA half-life in B. subtilis of about 3 min, based on our earlier measurements of bulk mRNA half-life in cultures grown under similar conditions (Wang and Bechhofer, 1996). We have shown that ΔermC mRNA half-life is dependent on ribosome binding at the SD site (Sharp and Bechhofer, 2003).

image

Figure 1. Model mRNA and real-time RT-PCR analysis. A. Schematic diagram of model mRNA used in this study. Open rectangle represents the 5′ UTR in which sequence changes were made; arrows indicate location of RT and PCR primer binding. B. Standard curve for number of mRNA template molecules. Numbers to right of curves are the number of molecules of in vitro-transcribed ΔermC mRNA added to the RT-PCR reaction. C. Melting curve analysis of PCR products from B. D. Real-time RT-PCR results of in vivo-isolated ΔermC mRNA. Numbers are minutes after rifampicin addition. E. Melting curve analysis of PCR products from D.

Download figure to PowerPoint

The model mRNAs used in this study differ from each other only in the 5′ UTR sequence, indicated by the open rectangle in Fig. 1A. In this study, ‘SP82’ mRNA is identical with ΔermC mRNA except that the 5′ UTR has been replaced with the 42 nt SP82 sequence.

To aid in analysis of mRNA half-life, a quantitative real-time RT-PCR protocol was established, utilizing the Roche LightCycler and the Qiagen QuantiTect SYBR Green one-step RT-PCR kit. Primers were designed to amplify a 150 bp product downstream of the start codon; thus, one primer set could be used to analyse all of the constructs with altered 5′ UTRs (Fig. 1A). For quantitative measurements a standard curve was created, which is shown in Fig. 1B. The indicated number of in vitro-transcribed ΔermC mRNA molecules was added to a one-step RT-PCR reaction mix. As the PCR cycle at which fluorescence starts to increase exponentially (referred to as the ‘crossing point’) is proportional to the amount of starting template, a quantitative standard curve could be established. Figure 1C shows a melting curve analysis for the PCR products generated in each sample shown in Fig. 1B. The set of coincident peaks in Fig. 1C demonstrates that a single product is generated with the primer set and ΔermC template. A small peak, which is observed in the sample that had no ΔermC mRNA added, is likely resulting from primer-dimers. In the presence of template, primer-dimer product is minimized because of competition with the true template. Figure 1D shows the real-time RT-PCR data for RNA isolated at various times after rifampicin was added to a B. subtilis strain carrying wild-type ΔermC. The LightCycler data analysis program compares crossing points of the actual samples with those of the standard curve, and calculates the number of molecules of template mRNA present at each time point. Regression analysis of the LightCycler data was used to determine mRNA half-life. The melting curve analysis for ΔermC PCR products using in vivo-isolated RNA (Fig. 1E) shows the same profile as the curve generated using in vitro-transcribed ΔermC template (Fig. 1C). Similar results were obtained for all constructs used in this study.

Effect of predicted SD sequence strength

Replacement of the ΔermC 5′ UTR with the 42 nt SP82 sequence (to give ‘SP82’ mRNA) resulted in a threefold increase in mRNA stability (Table 1, construct 2). A major difference between the ΔermC 5′ UTR and the SP82 5′ UTR is the predicted free energy of the interaction between their respective SD sequences and the 16S rRNA-3′-end (Table 1). These were ΔG0 = −8.4 for ΔermC and ΔG0 = −11.2 for SP82. Derivatives were constructed to determine if the SP82 SD sequence alone was sufficient to function as a stabilizing element. The SD sequence of SP82 mRNA was changed to the SD sequence of ΔermC, giving an mRNA designated ‘SP82 w/ΔermC SD.’ This mRNA had a half-life of 20 min, which was not significantly different from the half-life of SP82 mRNA itself (Table 1, construct 3). In the converse experiment, the SD sequence of ΔermC was changed to the SD sequence of SP82, yielding ‘ΔermC w/SP82 SD’. The half-life of this mRNA increased by only 1 min compared with ΔermC, a significant but minor increase (Table 1, construct 4). These data suggested that the SP82 SD sequence may play a minor role in mRNA stabilization, but that it was not sufficient to account for the large increase in stability conferred by the SP82 5′ UTR.

Role of SP82 sequence upstream of SD

The role of the 26 nts upstream of the SP82 SD sequence was investigated. Derivatives were constructed that contained sequence changes of the first half or the entire 26 nt sequence (Table 1, constructs 5 and 6). In either case, the sequence changes resulted in decreases of 4–5 min in mRNA half-life. However, these mRNAs were still nearly 2.5-fold more stable than ΔermC mRNA. Thus, the stabilizing effect of the SP82 sequence could not be attributed entirely to a particular linear sequence (see below).

Distance from 5′-end to SD

As both the SP82 SD sequence and the sequence upstream of SD played only minor roles in mRNA stabilization, we proceeded to examine if stabilization was resulting from a particular distance between +1 (the start of transcription) and the start of the SD sequence. This distance is 18 nts for ΔermC mRNA (t1/2= 6 min) and 26 nts for SP82 (t1/2 = 18.6 min) (Table 2, constructs 1 and 2). Derivatives of ΔermC where made with insertion of random nucleotides into the 5′ UTR (Table 2, constructs 3–10). Insertions of 2 or 4 nts resulted in an increase of mRNA half-life of 1.5 and 0.8 min respectively (constructs 3 and 4). Insertion of 6 nts resulted in an increase of mRNA half-life of 4.8 min, a 75% increase in stability. Remarkably, insertion of 8 nts, which made the +1-to-SD distance equal to that of SP82, but with a completely different 5′ sequence (construct 6), resulted in a 19.6 min half-life, which was not significantly different from that of SP82 itself (P = 0.436). This result was also confirmed by Northern blot analysis (data not shown). When the +1-to-SD distance was increased to 27 nts (construct 7), just one more nucleotide than SP82, the half-life dropped by almost twofold to 9.1 min. As the distance was increased further to 28, 32 and 34 nts (constructs 8, 9 and 10), the mRNA half-life decreased to 7.3, 7.2 and 5.9 min respectively.

Table 2.  Effect of +1-to-SD distance on ΔermC mRNA half-life.
Construct5′ UTR Sequencea+1-to-SDHalf-lifeb P-valuedΔG0 (5′nts)d
  • a.

    SD sequences are in bold. Inserted nts are underlined. Italicized nts in constructs 11 and 12 indicate differences from ΔermC.

  • b.

    mRNA half-lives are given in minutes ± standard deviation.

  • c.

    P-values are comparing mutant mRNA half-life to ΔermC mRNA half-life.

  • d.

    Values are predicted free energies of secondary structure, in kcal mol−1. Values in parentheses are the number of unpaired nucleotides at the 5’ end. Predicted free energy value for the most stable structure of each construct is given.

 1. SP82GGAGCCGCUGAGCUACCACAGAUUGUGAAAGGAG...2618.6 ± 1.30.004−4.7 (0)
 2. ΔermCGGAGAUCUAAGCUUUUAUAAGGAGG...18 6.0 ± 0.3N/A+1.1 (0)
 3. ΔermC+2GGUCAGAUCUAAGCUUUUAUAAGGAGG...20 7.5 ± 0.20.004+1.6 (0)
 4. ΔermC+4GGUCGGAGAUCUAAGCUUUUAUAAGGAGG...22 6.8 ± 0.20.024−0.8 (0)
 5. ΔermC+6GGUCGGAUAGAUCUAAGCUUUUAUAAGGAGG...2410.8 ± 1.50.006−3.1 (0)
 6. ΔermC+8GGUCGGAUCCAGAUCUAAGCUUUUAUAAGGAGG...2619.6 ± 1.70.006−2.8 (0)
 7. ΔermC+9GGUCGGAAUCCAGAUCUAAGCUUUUAUAAGGAGG...27 9.1 ± 0.60.015−3.1 (0)
 8. ΔermC+10GGUCGGAUCCUGAGAUCUAAGCUUUUAUAAGGAGG...28 7.3 ± 0.20.009−3.9 (4)
 9. ΔermC+14GGUCGUUUUGAUCCUGAGAUCUAAGCUUUUAUAAGGAGG...32 7.2 ± 0.20.009−5.7 (4)
10. ΔermC+16GGUCGGUUUUUUAUCCUGAGAUCUAAGCUUUUAUAAGGAGG...34 5.9 ± 0.30.714−2.5 (4)
11. ΔermC+8 Ver.1 GGAGAUCUAAGCAUCUAAGCUUUUAUAAGGAGG...26 5.6 ± 0.30.151−2.3 (8)
12. ΔermC+8 Ver.2GGUCGGAGAUCUAUCUAAGCUUUUAUAAGGAGG...26 5.8 ± 0.020.274−0.8 (0)

Initially, these data suggested that SP82-mediated stability was a function of the distance between +1 and the SD sequence. A distance of 26 nts, irrespective of sequence, would be sufficient to confer SP82-like stability. To test this, two additional constructs were made that also contained 26 nts from +1 to the SD sequence, but with different nucleotides (constructs 11 and 12). The half-life of mRNA encoded by each of these constructs was similar to that of ΔermC mRNA, suggesting that some element(s) other than the +1-to-SD distance was necessary for SP82-mediated stabilization.

Effect of 5′-terminal secondary structure

An analysis of RNA secondary structure, using the mfold structure prediction program of Zuker (Zuker, 2003) was performed on the region from +1 up until, but not including, the SD sequence of the constructs shown in Table 2. The free energy of predicted secondary structures are shown in the rightmost column of Table 2 and diagrams of selected structures are shown in Fig. 2. ΔermC and ΔermC +2 (Table 2, constructs 2 and 3; Fig. 2A) had positive ΔG0 values, which indicate an unstructured 5’ end. On the other hand, analysis of SP82 5’ UTR predicted a 5′-terminal stem–loop structure with a ΔG0 value of −4.7 (Fig. 2B). ΔermC +8 mRNA, which had a half-life similar to SP82, also had a predicted 5′-terminal stem–loop, with a ΔG0 value of −2.8 (Table 2, construct 6; Fig. 2D). Constructs 8, 9 and 10 also had predicted 5′ structures; however, these were not located at the very 5′-end but were preceded by four unpaired nucleotides (not shown). Construct 11 contained eight unpaired nucleotides before a 5′-proximal stem–loop (Fig. 2E), and construct 12 had a weak 5′ secondary structure. The mRNA half-life of constructs 8–12 were very similar to that of ΔermC itself (Table 2). Finally, the versions of SP82 that were relatively stable and that had a different nucleotide sequence at the 5′-end from that of SP82 (Table 1, constructs 5 and 6) also had a 5′-terminal secondary structure (Fig. 2F). These data were consistent with the hypothesis that the presence of secondary structure could confer stability, provided that such a structure was located at the very 5′-end.

image

Figure 2. Predicted structures of 5′ UTRs of selected constructs. The names of the constructs and, in parentheses, the number of each construct from Table 2 are given (except for F, which is from Table 1, as indicated). Structures were calculated using nucleotides from + 1 up until, but not including, the SD sequence. ΔG0 values and mRNA half-lives are as in Tables 1 and 2. SD sequences are underlined. A and C residues with bold dots next to them in B–D indicate positions that were methylated in vivo.

Download figure to PowerPoint

Disruption of 5′-terminal stem–loop structures

To test whether 5′-terminal stem–loop structure was required for stability, constructs were made that were predicted to disrupt the SP82 stem–loop. Creation of a BamHI site (Fig. 3B) was predicted to result in a changed 5′-terminal structure, with an overall increase in free energy from −4.7 to −1.8. The half-life for the SP82(BamHI) construct was 2.6-fold lower than SP82 (18.6 min vs. 7.1 min). Creation of an NheI site (Fig. 3C) was predicted to give an mRNA that had six unpaired nucleotides preceding a stem–loop structure. The half-life of the SP82(NheI) construct was 2.3-fold lower than SP82 (18.6 min vs. 8.0 min). Similarly, the 5′-terminal stem loop structure in ΔermC +8 was disrupted by changing nucleotide 15 from C to G, which increased the ΔG0 to −1.1 (Fig. 3D). This resulted in a 2.6-fold decrease in mRNA half-life (19.6 min vs. 7.5 min). These data confirmed that disruption of 5′-terminal secondary structure significantly destabilized the mRNA.

image

Figure 3. Predicted structures of 5′ UTRs with disrupted 5′-proximal secondary structure. SD sequences are underlined. Boxed nucleotides in B were added to SP82 to give SP82(BamHI); BamHI nucleotides in italics. Boxed nucleotides in A were deleted, and boxed nucleotide in C was added, to give SP82(NheI); NheI nucleotides in italics. Vertical arrowhead in D indicates C to G change.

Download figure to PowerPoint

Effect of 5′-terminal stem–loop without ternary complex formation

Ribosome binding and formation of a ternary complex have been shown to be determinants of mRNA stability in B. subtilis (Sharp and Bechhofer, 2003). We asked whether the SP82 5′-terminal stem–loop was sufficient for stabilization in the absence of ternary complex formation. A construct was made in which the AUG start codon of SP82 was changed to the non-functional ACG (Table 3, construct 2), which would preclude ternary complex formation. The half-life of SP82 ACG mRNA was only 7.9 min, almost a 2.5-fold reduction compared with SP82. These data indicated that, in addition to a 5′-terminal stem–loop, formation of a ternary complex was necessary for stabilization by the SP82 sequence.

Table 3.  Effect of 5′ UTR mutations on SP82 mRNA half-life. Thumbnail image of

Strength of 5′-terminal stem–loop and distance from SD

The SP82 5′-terminal stem–loop structure had a moderately strong ΔG0 of −4.7. We tested whether a stem–loop structure with a substantially lower predicted free energy would result in a longer mRNA half-life. The SP82 stem structure has a single nucleotide bulge (Fig. 2B), and such bulges are destabilizing (Groebe and Uhlenbeck, 1989). A construct was made with an added U nucleotide that was predicted to base pair with the A bulge in the stem, reducing the predicted ΔG0 from −4.7 to −9.6 (Table 3, construct 3). Interestingly, the half-life of this construct was not significantly different from that of SP82.

We tested whether relocating the stem–loop further away from the SD sequence would decrease the ability of the stem–loop to act as a stabilizing element. Constructs were made in which the 5′-terminal stem–loop was located 12 or 24 nts further from the SD sequence (Table 3, constructs 4 and 5). We found no significant difference in the mRNA stability of these constructs, compared with SP82.

Length of unpaired sequence at the 5′-end

We tested whether unpaired 5′-terminal nucleotides would eliminate the stabilizing effect of the SP82 stem–loop. Constructs were made that added three, five or seven unpaired nucleotides to SP82 (Table 3, constructs 6, 7 and 8). These additions were predicted not to affect the SP82 secondary structure. While the presence of three or five unpaired nucleotides had no significant effect on SP82-mediated stabilization, the presence of seven unpaired nucleotides resulted in a large decrease of mRNA half-life, from 18.6 min to 8.7 min.

Proximity of secondary structure to SD sequence

As the presence of only five unpaired nucleotides had no effect on SP82 stability (Table 3, construct 7), we revisited the results obtained with the ΔermC +14 construct (Table 2, construct 9).ΔermC +14 has a predicted stem–loop structure that is significantly more stable than SP82 (ΔG0 = −5.7), and this structure is located only 4 nts away from the 5′-terminus (Fig. 4A). Surprisingly, the half-life of ΔermC +14 mRNA was only 7.2 min. Deletion of 3 nts at the 5′-terminus created a 5′-terminal stem–loop structure (ΔermC +14/5′T), but this did not result in stabilization (Fig. 4B). Even when three changes were made in the ΔermC+14 stem sequence, to give a perfectly base-paired stem (ΔermC +14/PS, Fig. 4C), the mRNA half-life remained remarkably unchanged, despite a large change in ΔG0 from −5.9 to −12.8. Thus, neither the unpaired nucleotides at the 5′-end, nor destabilizing features of the stem–loop itself, could explain why ΔermC +14 was not particularly stable. One other element that we considered was the proximity of the ΔermC+14 stem–loop to the SD sequence. Indeed, when an additional 7As were inserted downstream of the predicted stem–loop structure (ΔermC +14/7A), which made the distance from the stem–loop to the SD sequence 11 nts, like that found in ΔermC +8, stability increased dramatically to 21.6 min (Fig. 4D).

image

Figure 4. Predicted structure of 5′ UTRs of ΔermC +14 and derivatives. SD sequence is underlined. Changed nucleotides to give ΔermC+14/PS (C) are boxed. Added 7A residues to give ermC+14/7A (D) are italicized. A and C residues with bold dots next to them in D indicate positions that were methylated in vivo.

Download figure to PowerPoint

In vivo methylation

To test for the presence of predicted 5′-terminal secondary structure in selected cases, in vivo methylation studies were performed. Addition of dimethylsulphate to a cell culture results in the methylation of unpaired A and C residues. A and C residues engaged in base-pairing are not modified. A subsequent primer extension reaction reveals the sites of methylation, as methylation blocks the progression of RT. We chose to examine one RNA with no predicted secondary structure (ΔermC, Fig. 2A), three RNAs with weak or moderate predicted structure (SP82, ΔermC +6 and ΔermC +8, Fig. 2B–D), and one RNA with strong predicted structure (ΔermC +14/7A, Fig. 4D). The results of the primer extension analysis on untreated and methylated in vivo isolated RNA are shown in Fig. 5. In these exposures, numerous bands appear even in the untreated lanes, and these presumably represent sites of RT pausing. The relevant data are the presence of stronger bands in the methylated samples at positions of A and C residues, relative to these positions in the untreated samples. Such methylated As and Cs are indicated by bold dots Figs 2 and 4. The striking observation was that no A or C residues in predicted stem regions were methylated, and, with one exception, all A and C residues in predicted loop regions were methylated. Even the bulged A residue off the predicted stem in SP82 (Fig. 2B) was clearly methylated, while the neighbouring C residues that are in the stem were not. The one exception was the first C residue in the upper loop of SP82. The strong RT stop that could be predicted at the base of the ΔermC +14/7A stem, even in the untreated sample, was also quite evident in this analysis (Fig. 5). Thus, the results of in vivo structural analysis are largely consistent with the computer predictions.

image

Figure 5. Primer extension analysis of in vivo-methylated RNA. RNAs were isolated from untreated cultures (–) or cultures treated with dimethylsulphate (+). Names of constructs are indicated above each pair of lanes; ‘Δe’, ΔermC. Carats indicate methylated C residues; wavy lines indicate methylated A residues; asterisks indicate the base of predicted stem structures. Lanes A, C, G, T contain a DNA sequencing reaction, performed on an unrelated DNA, used to measure the length of primer extension fragments. Sizes in nucleotide are indicated on the left. ‘BG1’ indicates two control lanes (untreated and methylated) that contained primer extension products on RNA from a strain that did not contain a ΔermC plasmid.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Real-time RT-PCR was used to investigate the effect on mRNA stability of sequence changes in the 5′ UTR of ΔermC mRNA, consisting of either the native ermC 5′ UTR or the SP82 42 nt 5′ UTR. The ease with which mRNA half-life could be assayed using this system allowed us to measure the half-lives of many different mutants in a reasonable amount of time, with measurements made in triplicate (at least) and using only data from experiments in which linear regression of mRNA decay at time points after rifampicin addition gave an R2 value greater than 0.9. Interestingly, when measured by real-time RT-PCR, the base-line half-life of ΔermC mRNA was always around 6 min (this study), whereas ΔermC mRNA half-life measured by Northern blot analysis in multiple previous experiments was closer to 8 min (Sharp and Bechhofer, 2003). Many differences in the Experimental procedures might explain this discrepancy, but what was striking was the internal consistency of the observed half-lives in both types of experiments.

We can divide the present findings into five different elements of a 5′ UTR that may affect mRNA stability: (i) strength of SD sequence; (ii) distance from 5′-terminus to SD sequence; (iii) unpaired 5′ nucleotides; (iv) strength of 5′-terminal secondary structure; (v) distance of SD sequence from 5′-terminal secondary structure.

Strength of SD sequence

We found that the predicted SD sequence strength did not correlate with mRNA stability (Table 1, constructs 1–4). This is in agreement with the findings of Hambraeus et al. (2002), who concluded from a study of the stable aprE mRNA that the strength of a B. subtilis SD sequence is not predictive of mRNA stability. Our findings are also consistent with the results of Hambraeus et al. (2003), who examined decay of a large number of B. subtilis mRNAs using a microarray system and found no correlation between strength of SD−16S rRNA interaction and mRNA stability. On the other hand, it was found for Bacillus thuringiensis cryIIIA mRNA expressed in B. subtilis (Agaisse and Lereclus, 1996) and gsiB mRNA (Jürgen et al., 1998) that stability depended on a strong SD sequence. We note that in these cases, the SD sequence is located only four (cryIIIA) or nine (gsiB) nucleotides away from the 5′-end. Taken together with our earlier results regarding the effect of ternary complex formation on mRNA stability (Sharp and Bechhofer, 2003), we propose that SD sequence strength by itself will determine stability only when ribosome binding occurs in close proximity to the 5′-terminus. Ternary complex formation at such a site would sequester the 5′-end, and higher-affinity SD−16S rRNA interaction would enhance protection. When occurring further downstream, however, the strength of the SD sequence would not be a determining factor in mRNA stability.

Distance from 5′-terminus to SD sequence

The data in Table 2, regarding mRNA half-lives of a large set of ΔermC 5′ UTR insertions, are informative. We tested the hypothesis that distance between the 5′-end and the SD sequence could account for the difference in SP82 and ΔermC half-life. The basis for this hypothesis was our earlier study on the importance of ternary complex formation for mRNA stability (Sharp and Bechhofer, 2003). If ribosomes bound in a ternary complex could restrict access to the 5′-end, perhaps location of the SD sequence a particular distance from the 5′-end would allow, at once, optimal ribosome binding and optimal interaction of 5′-proximal nucleotides with the ribosome. This would provide maximal protection of the 5′-end. In fact, the data on constructs in Table 2 did not reveal a consistent relationship between distance from 5′-end to SD sequence and stability. Rather, the conclusion from these constructs was that 5′-terminal secondary structure above a minimal ΔG0, located less than 4 nts from the 5′-end, was the key element for stabilization. Thus, the most stable construct was ΔermC +8 (Table 2, construct 6; t1/2 = 19.6 min), which had a 5′-terminal secondary structure with a ΔG0 of −2.8 and no unpaired nucleotides. Constructs with weak 5′ secondary structure or 4 or more unpaired 5′ nucleotides had short half-lives similar to that of ΔermC. Apparent exceptions to this conclusion were ΔermC +6 and ΔermC +9 (Table 2, constructs 5 and 7), both of which had 5′-terminal secondary structures with ΔG0 of −3.1 and no unpaired nucleotides. The mRNAs of these constructs were significantly more stable than ΔermC (t1/2 = 10.8 and 9.1 min respectively, vs. 6.0 min for ΔermC) but not nearly as stable as ΔermC +8 (t1/2 = 19.6 min). Another exception was SP82 Ver. 1 (Fig. 2F), which had a stronger predicted 5′-terminal structure than SP82 itself, but still had only a 13.7 min half-life. In the case of ΔermC +9, we found that mfold predictions gave an alternative structure, with a slightly lower ΔG0 of −2.7 (not shown), and one might argue that the possibility of forming closely related alternative structures may be destabilizing. However, this would not explain the relative instability of ΔermC +6 and SP82 Ver. 1. A more likely explanation is that we cannot rely completely on predicted secondary structures. Such predictions are based on RNA sequence alone, and this may not be accurate for an mRNA in vivo with ribosomes binding in close proximity. The smaller the number of base–pairing interactions, the less reliable such predictions would be. However, the good correlation otherwise between predicted 5′-terminal structure and mRNA stability engenders confidence that such predictions are good approximations in general. The results of in vivo methylation studies (Fig. 5), which largely confirmed the RNA secondary structure predictions, also suggests that, in many cases, the computer-predicted structures do exist in vivo.

Unpaired 5′ nucleotides

The half-lives of constructs shown in Table 2 that have 5′ unpaired nucleotides suggested that the protective secondary structure needed to be less than 4 nts from the 5′-terminus. However, those constructs (e.g. 8–10) had different predicted structures, so it was difficult to make a conclusion about the length of unpaired nucleotides that could affect stability. To examine this more systematically, 3, 5 or 7 nts were added at the SP82 5′-end, and these were predicted to remain as single-stranded tails upstream of the identical secondary structure. The data (Table 3, constructs 6, 7 and 8) showed that the presence of three or five unpaired nucleotides gives a half-life not significantly different from that of SP82 itself, but the presence of seven unpaired nucleotides resulted in almost a complete loss of stabilization (t1/2 = 18.6 min for SP82 and 8.7 min for construct 8). This is consistent with the requirement for access to the 5′-end to initiate mRNA decay, which would be denied by 5′-terminal secondary structure but would be provided by upstream 5′ unpaired nucleotides.

These experiments speak to the proposed mechanism of a putative 5′-dependent endonuclease in B. subtilis. Based on a number of differences between the nature of 5′ stabilizers in E. coli and B. subtilis, Condon (2003) has suggested that 5′-end-dependent decay in B. subtilis requires 5′-end binding followed by tracking to an internal cleavage site, unlike the case in E. coli where the 5′-binding endonuclease (RNase E) may loop to an internal site. Our results suggest that providing an accessible 5′-end will result in a loss of stability, even though a strong secondary structure is present downstream. Either the 5′ binding activity can loop around this structure or its processivity in tracking from the 5′-end is not disturbed by RNA secondary structure.

Strength of 5′-terminal secondary structure

Once the results in Table 2 had demonstrated the importance of secondary structure at the 5′-end, the effect of the predicted strength of this structure on mRNA stability was analysed. The predicted strength of 5′-terminal structures of the stable SP82 and ΔermC +8 mRNAs were decreased by insertion of nucleotides, and these constructs gave mRNAs with much reduced half-lives (Fig. 3), as predicted. Interestingly, a single nucleotide addition to the 5′-terminal sequence of SP82, which resulted in a large increase in predicted stability of secondary structure, gave no additional increase in mRNA half-life (Table 3, construct 3). This latter result suggests that formation of a stable, 5′-terminal structure is sufficient to confer stability by blocking 5′-end-dependent decay initiation. Further increases in stability of such structures above a certain minimum would not result in increased mRNA stability. This was indeed the observation in comparing the half-lives of SP82 (t1/2 = 18.6 min) and ΔermC +8 (t1/2 = 19.6 min). Based on the significant difference in predicted free energy of formation for the respective stem–loop structures (−4.7 for SP82 and −2.8 for ΔermC +8), one might have expected that SP82 would be more stable than ΔermC +8. We suggest that the terminal secondary structure of ΔermC +8 is above the threshold required to block 5′-access. A similar conclusion was reached by Arnold et al., 1998) in their study of E. coli ompA mRNA.

The observation that abolishing ternary complex formation (by changing the AUG initiation codon to ACG; Table 3, construct 2) had a major effect on stability suggests that 5′ stem structure is not sufficient to confer stability. Perhaps both 5′-terminal structure and 5′-proximal ribosome binding contribute to protection against 5′-end-dependent initiation of decay, and the presence of both acts synergistically.

Distance of SD sequence from 5′-terminal secondary structure

Increasing the distance between the SP82 5′-terminal secondary structure and the SD sequence had a statistically insignificant effect on SP82 half-life (Table 3, constructs 4 and 5). This is consistent with the idea that the major determinant for mRNA stability is a blocked 5′-end. It would be of interest to determine whether locating the SD sequence even further from the 5′-end would cause a decrease in half-life. This might be attributed to endonucleolytic cleavage between the 5′-terminal secondary structure and the SD sequence. Insertion of only 12 or 24 nts, as we have done, may not have provided a sufficiently large target for such cleavages.

The experiments shown in Fig. 4 demonstrated that even a highly stable 5′-terminal structure could not confer stability without adequate distance between the 3′-end of the structure and the SD sequence. We propose that the binding of ribosomes at an SD sequence that is located too close to the 5′-terminal structure will result in perturbation of the structure. Based on crystallography studies (Yusupova et al., 2001), 15 nts upstream of the initiation codon are protected by the ribosome bound in a ternary complex. In the case of ΔermC +14/5′T or ΔermC +14/PS (Fig. 4B and C), there are 18 nts between the downstream base of the stem–loop structure and the initiation codon. We suggest that this distance is short enough to allow ribosome binding to perturb formation of the protective 5′-terminal structure. Once the distance is increased to 25 nts (ΔermC +14/7A; Fig. 4D), ribosome binding no longer affects secondary structure formation, resulting in a very stable mRNA. The reverse could also be true, i.e. the presence of the secondary structure could affect translation, which is also required for stability. It has been shown that secondary structure located immediately upstream of an SD sequence reduces translational efficiency (Coleman et al., 1985).

A recent article (Hambraeus et al., 2003) reported the use of microarray analysis to examine genome-wide mRNA stability in B. subtilis. Fifty-two mRNAs were identified that had half-lives ≥ 15 min, and seven of these (abnA, acoA, aprE, cotG, gsiB, sunA and yhxD) were confirmed by Northern blot or primer extension analysis. It was stated that no common features were found that could explain the stability of these mRNAs. Based on our conclusions from the current study, however, we can explain the stability of five of these seven. Analysis of the +1-to-SD sequences by mfold predicted the presence of 5′-terminal secondary structure for three of the seven stable mRNAs (sunA, cotG and aprE; Fig. 6). acoA mRNA lacks any predicted 5′-terminal structure but its SD sequence is only 13 nts from the 5′-end. Thus, it may be similar to the case of gsiB, which has a +1-to-SD distance of 9 nts, as discussed above. For the final two cases, abnA has a 116 nt 5′ UTR, which may assume structures that cannot be predicted or may be bound by protein. The yhxD gene function is unknown, and its transcription start site has not been mapped.

image

Figure 6. Predicted 5′-terminal secondary structures of stable B. subtilis mRNAs.

Download figure to PowerPoint

Our results that identified specific 5′ elements that can confer stability were based on ΔermC mRNA, which is a relatively small mRNA (254 nts). While we would like to believe that the elements found in this study to confer stability are applicable to B. subtilis mRNAs generally, it is possible that similar 5′ elements will not confer stability to longer mRNAs that have increased target size for an alternative decay pathway. In this respect, however, we note that the 42 nt SP82 5′ UTR could confer stability when fused to lacZ mRNA, the fusion mRNA being at least 10-fold longer than ΔermC (Hue et al., 1995). On the other hand, we should also note that, based on our observations from the 5′ UTRs described here, we have constructed mRNAs with other 5′-terminal stem–loop structures and measured their stability. In one particular case (data not shown), a ΔermC mRNA with a strong 5′-terminal stem structure (ΔG0 = −9.5) had a surprisingly short half-life of only 5.8 min. The sequence of nucleotides in the stem and loop of this construct, as well as the nucleotides between the stem–loop structure and the SD sequence, differed from those of the stable constructs found in this study. Thus, there must be other factors, of which we are not aware, that can affect initiation of mRNA decay.

Finally, we suppose that a 5′-end-dependent pathway of mRNA decay would be the result of an RNase E-like activity. However, as stated above, there is no RNase E sequence homologue in the B. subtilis genome. We and others have tried unsuccessfully to complement a temperature-sensitive E. coli RNase E mutant, using shotgun-cloned B. subtilis chromosomal DNA (D.H. Bechhofer, unpubl. results). Elucidation of the basis for 5′-end-dependent mRNA decay in B. subtilis remains a high priority.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bacterial strains

The B. subtilis host was BG1, which is trpC2 thr-5. E. coli strain DH5α (Grant et al., 1990) was the host for plasmid constructions.

Standard procedures

The preparation and transformation of B. subtilis competent cell cultures were as described previously (Dubnau and Davidoff-Abelson, 1971). For RNA isolation, B. subtilis cultures were grown to mid-logarithmic phase in minimal medium containing Spizizen salts with 0.5% glucose, 0.1% casamino acids, 0.001% yeast extract, 50 µg ml−1 tryptophan, 50 µg ml−1 threonine, and 1 mM MgSO4. RNA was isolated essentially as described (Köhrer and Domdey, 1991), except that the buffer for phenol extraction (ANE) was replaced with 50 mM sodium acetate/1 mM EDTA (pH 6.0). For unstable mRNAs, RNA isolation was carried out at 0, 3, 6 and 9 min after rifampicin addition. For stable mRNAs, RNA isolation was carried out at 0, 5, 10 and 20 min after rifampicin addition.

Plasmids

ΔermC mRNA was expressed from plasmid pYH196, an E. coli-B. subtilis shuttle plasmid that has been described elsewhere (Drider et al., 2002). To facilitate cloning of mutated DNA fragments, an EcoRI site was introduced downstream of the ΔermC transcription terminator, giving plasmid pYH250. NcoI, BglII and HindIII sites are present at the beginning of the ΔermC transcriptional unit. These sites were used in conjunction with the downstream EcoRI site to insert PCR fragments that were generated with mutagenic 5′ primers containing NcoI, BglII or HindIII sites.

Real-time RT-PCR

The Qiagen QuantiTect SYBR Green one-step RT-PCR kit was used to quantify the number of target mRNA molecules present at time points after rifampicin addition. Reactions were prepared in a 4°C cooling block. Each reaction contained the following: 8.3 µl H2O, 0.25 µl forward primer at 20 pmol ml−1, 0.25 µl reverse primer at 20 pmol ml−1, 0.2 µl QuantiTect RT Mix, and 10.0 µl 2× Quantitect SYBR Green RT-PCR Master Mix. The reaction mix was placed into a LightCycler capillary, and 1 µl of in vitro-transcribed RNA or 1 µl of a 1:100 dilution of RNA isolated in vivo was added. The capillary was centrifuged at 1300 g for 10 s and loaded into the LightCycler thermocycler.

The thermocycler conditions were as follows: RT step = 50°C for 30 min; Taq polymerase activation/RT inactivation step = 95°C for 15 min; 45 PCR cycles = 94°C for 15 s, 55°C for 25 s, 70°C for 15 s and 68°C for 5 s. SYBR Green fluorescence was measured at the end of each cycle. Melting curve analysis was performed after the PCR cycles as follows: the temperature was dropped to 60°C for 15 s followed by a 0.05°C s−1 increase in temperature until 92°C was reached. SYBR Green fluorescence was measured continually during the melting curve analysis.

For the RT and PCR reactions, the forward primer was the same sequence as nts 43–67 of ΔermC mRNA (5′-TAGTATTTTTGTAATCAGCACAGTT-3′) and the reverse primer was complementary to nts 168–192 of ΔermC mRNA (5′-AAAAGAGATAAGAATTGTTCAAAGC-3′).

In vivo methylation analysis

Methylation of ΔermC mRNAs in vivo was performed essentially as described by Mayford and Weisblum (1989), except that potassium borate was omitted from the annealing buffer. Dimethylsulphate (Sigma) was added to 2 µl per millilitre of culture. The products of AMV RT (Roche Diagnostics) primer extension were separated on an 8% denaturing, high-resolution polyacrylamide gel. The 5′-end labelled primer was complementary to nts 64–86 of the ΔermC transcript (5′-GTTAACTGGTTGATAATGAACTG-3′).

Data analysis and free energy calculations

Half-lives were determined by linear regression analysis plots of the number of target mRNA molecules remaining versus time. Half-life data are the average of at least three determinations from independent RNA isolations, using only those experiments in which the R2 value was greater than 0.9. A two-sample t-test comparing wild-type and mutant mRNA half-lives was used to calculate P-values. A P-value of less than 0.05 was considered significant. The free-energy values for SD−16S rRNA interactions were calculated using the program for RNA:RNA hybridization at the Zuker RNA website http://www.bioinfo.rpi.edu/applications/mfold, assuming a temperature of 37°C, 0.1 mM RNA concentration, and 10 mM Na+ concentration. The free energy of mRNA secondary structures was calculated using the mfold program at the Zuker RNA website.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This work was supported by Public Health Service Grant GM-48804 from the National Insitutes of Health. We thank Arsalan Shabbir for instruction on the use of the LightCycler thermocycler.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References