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Nucleic Acids Research 27:3891-3898 (1999)
© 1999 Oxford University Press

Article

In vivo oligo(A) insertions in phage MS2: role of Escherichia coli poly(A) polymerase

Dico van Meerten1, Marina Zelwer1,2, Philippe Régnier2,3 and Jan van Duin1,a

1Leiden Institute of Chemistry, Department of Biochemistry, Leiden University, Gorlaeus Laboratories, PO Box 9502, 2300 RA Leiden, The Netherlands, 2University Paris 7 and 3Institut de Biologie Physico Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously we introduced an RNase III site into the genome of RNA phage MS2 by extending a hairpin with a perfect 18 bp long stem. One way in which the phage escaped from being killed by RNase III cleavage was to incorporate uncoded A residues on either side of the stem. This oligo(A) stretch interrupts the perfect stem that forms the RNase III site and thus confers resistance. In this paper we have analyzed the origin of these uncoded adenosines. The data strongly suggest that they are added by the host enzyme poly(A) polymerase. Apparently the 3"-OH created by RNase III cleavage becomes a substrate for poly(A) polymerase. Subsequently, MS2 replicase makes one contiguous copy from the two parts of the genome RNA. The evolutionary conversion from RNase III sensitivity to resistance provides a large spectrum of solutions that could be an important tool to understand what essentially constitutes an RNase III site in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The single-stranded RNA phages like MS2 contain a genome of ~3500 nt (Fig. 1A). The RNA encodes four proteins necessary for phage multiplication and spreading. In addition, the sequence prescribes a specific RNA structure, optimized for translational control, replication and other viral functions. These structures thereby contribute substantially to the reproductive success of the organism. For example, amino acid neutral mutations that change critical aspects of RNA folding can lead to a dramatic loss in viral fitness (1,2). At the same time all these structures must function within the Escherichia coli cell and phage RNA must thus be resistant to the host’s nuclease machinery dedicated to the turnover of mRNA. Major components of this system are the endonuclease RNase E, poly(A) polymerase I (PAP I) and the 3"->5" exonucleases polynucleo­tidephosphorylase (PNPase) and RNase II (3–5). It is believed that mRNA degradation is triggered by endonucleolytic cleavage, presumably by RNase E (6). The new 3"-OH end is then poly­adenylated by PAP I, which facilitates degradation by PNPase (6,7). Furthermore, there is RNase III, cutting endonucleolytically on both sides of long duplexes of double-stranded RNA (8). This enzyme, though primarily involved in RNA maturation, can also be the trigger to mRNA degradation (9). RNase III, like RNase E, creates breaks ending in a 3"-OH group, which is the substrate for PAP I (4).



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  		Figure 1. (A) Genetic map of single-stranded RNA phage MS2. (B) On the left is shown the wild-type stem–loop structure including the stop codon of the maturation protein gene and the XbaI site (UCUAGA) used to insert the RNase III-sensitive stem shown on the right as R. The dashed line is at the boundary of the insert. RNase III is known to cleave on both sides in a staggered way to produce a 2 nt 3" overhang. Cleavage sites indicated by black arrows were determined previously (10). Open arrows are predicted cleavage sites.

 
In a previous study (10) we started to analyze the sensitivity and adaptation of phage RNA genomes to host nucleases by introducing an RNase III site (a long regular stem) in an intercistronic region (Fig. 1B). In rnc– hosts, which do not produce RNase III, the insertion of the long regular stem had no effect on the viability of the phage, as witnessed by its potential to form an unchanged number of plaques. However, in wild-type hosts the number of plaques was down by four orders of magnitude, indicating that the majority of the genomic RNA was inactivated by RNase III cleavage. However, adapted revertants that were no longer sensitive to RNase III and that produced wild-type quantities of plaques quickly appeared. There were four ways in which the phage acquired RNase III resistance: (i) it destroyed perfect base pairing in the long regular stem by introducing mismatches; (ii) it made big deletions that shortened the stem of the hairpin; (iii) it made small deletions leading to bulges in the stem; (iv) it introduced extra A or U residues at the RNase III cleavage site, again leading to bulges (10).

The mismatches under (i) are simply the result of replication errors. The deletions mentioned under (ii) and (iii) can be caused by a variety of events such as replicase jumps (copy choice) on the intact or already cleaved and perhaps exonucleo­lytically trimmed genome. It is even possible that the deletions arise by chemical religation of the RNase-cleaved hairpin as proposed by Chetverin et al. (11).

In this paper we analyze the origin of the non-templated A or U residues in the genome of MS2 as described under (iv). We envisage two possibilities. One is that the MS2 replicase, idling at the site where the genome is cleaved by RNase III, adds A residues to the nascent chain before it manages to complete complementary strand synthesis by jumping to the other side of the cut. The other possibility is that the extra residues are added by PAP I at the 3"-terminus created by RNase III cleavage. The prediction then is that in a strain devoid of PAP I, oligo(A) or oligo(U) insertions must be absent from revertant phages. For either mechanism the presence of uncoded uridines is the result of oligo(A) addition to the minus strand.

Here, we describe our findings that E.coli mutants lacking the PAP I enzyme yield MS2 revertants in which oligo(A) or oligo(U) insertions are absent.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and bacterial strains
Plasmid pMS.R, conferring kanamycin resistance, carries the entire cDNA of MS2 with the R hairpin inserted into the XbaI site (position 1303). The complete MS2 sequence is under control of the thermoinducible PL promoter. The bacterial strains used are described in Mikkelsen and Gerdes (12). As wild-type E.coli strain we used NDM5006 (F–, thr–, leu–). NDM5006{Delta}pcnB (F–, thr–, leu–, {Delta}pcnB) is PAP I deficient, NDM5004pnp7 (F–, thr–, leu–, {Delta}pcn7) is PNPase deficient and the double mutant is called NDM5004pnp7,{Delta}pcnB (F–, thr–, leu–, {Delta}pcn7, {Delta}pcnB). To obtain F+ derivatives of these strains the F factor pGP655 (Ftet) (13) was transferred from E.coli strain KA 1078 ({Delta}trp, bio–, strepr) to the above-mentioned F– strains by conjugation (14) as follows. The F– strains were grown in LC broth (containing per liter 10 g bactotryptone, 5 g yeast extract, 8 g NaCl, 2 g MgSO4, 140 mg thymine and 1 ml 1 M Tris–HCl (pH 7.6) at 37°C to OD650 0.2. Strain KA1078 harboring the F factor was also grown to OD650 0.2 and washed in the same volume of LC. F– cells were mixed with an equal volume of KA1078 cells and incubated at 37°C for 60 min without shaking. Cells were plated on minimal medium containing threonine, leucine and tetra­cycline (50 µg/ml). The F+ derivative of NDM5006 was called FNDM5006, and so on.

Infection of cells by the pMS.R plasmid
Competent F– cells were transformed with the infectious plasmid pMS.R by heat shock. After incubation at 28°C for 30 min 3 ml of LC containing 50 µg/ml kanamycin was added and the total mixture was incubated overnight at 28°C. The supernatants of the overnight cultures were plated on a lawn of the corresponding F+ strain to obtain separate plaques. Phages present in individual plaques were analyzed by sequencing.

Infection of cells by R phages
R phages were obtained from rnc– cells by transforming strain RCL1 (F+) with plasmid pMS.R (10). Appropriate dilutions were plated on lawns of FNDM5006 cells and phages present in individual plaques were analyzed by sequencing.

Phage evolution
Phages from plaques were amplified overnight at 37°C in 2 ml liquid cultures using the appropriate F+ cells. Thereafter phages were further analyzed by sequencing.

RT–PCR and sequence analysis
Phages from individual plaques or 1 µl of an overnight infected E.coli culture were dissolved in 6 µl H2O and heated at 92°C for 2 min. An aliquot of 1 µl was used for RT–PCR in a total of 50 µl according to standard procedures recommended by the suppliers (Promega and Eurogentec). The primers used were biotin-labeled DUI715 (complementary to nt 1182–1205) and unlabeled DUI59 (complementary to nt 1422–1409). Samples were analyzed on 2% agarose gels. PCR fragments were sequenced with DUI59 after separation and purification of the strands with Dynabeads (Dynal).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The experimental system
Some time ago we cloned the complete cDNA sequence of MS2 RNA in a plasmid (15). Escherichia coli cells transformed with this plasmid produce phages spontaneously. In this study we used three E.coli strains, mutant in loci involved in mRNA degradation. These are NDM5006{Delta}pcnB lacking the PAP I enzyme, NDM5004pnp7 lacking PNPase and the double mutant NDM5004pnp7,{Delta}pcnB. As a control the isogenic wild-type NDM5006 was used. From these four strains F+ derivatives were prepared to allow infection by MS2 phages.

In a typical experiment any of the four strains was transformed with the plasmid containing the mutant MS2 cDNA sequence pMS.R, which carries the RNase III site in hairpin R (Fig. 1B). The transformation mixture was left to grow overnight in liquid culture on medium selective for the plasmid-encoded resistance. Then the supernatant was plated on the F+ variant of the same strain in the absence of antibiotics. Plaques were taken and subjected to RT–PCR using primers around the RNase III site. The size of these PCR fragments was inspected on agarose gels.

Two classes of revertants can be distinguished by this technique. Those which have deleted most or all of the insert lead to an RT–PCR fragment of wild-type size. These revertants are not very interesting for the present study because they frequently turn out to be wild-type and only a small number were sequenced. The interesting revertants were those that had largely maintained the insert. They are the ones that potentially have acquired RNase III resistance by insertion of oligo(A) or oligo(U) stretches and the majority of these revertants have been sequenced. Some plaques gave both wild-type and insert size fragments and these have not been analyzed further.

To minimize the effects of chance we carried out three independent transformations with infectious pMS.R for each E.coli mutant. We began the experiments by repeating our previous work but now in host NDM5006, which is the isogenic parent for the PNPase and PAP I mutants. This experiment was necessary to confirm that the new host, like the old one (M5219), yields revertants carrying oligo(U) or oligo(A) insertions.

Oligo(A) insertions are also present in NDM5006
As summarized in Table 1, 18 plaques recovered from wild-type parent NDM5006 were analyzed by RT–PCR followed by electrophoresis. In six of these most or all of the insert had been removed as judged by the size of the band. These were not further analyzed. Eleven plaques had retained most of the insert and the sequences of eight of these are shown in Figure 2. Sometimes, the same sequence was found several times. For instance, WTR1 was obtained three times. As can be seen, all but one of these revertants carry oligo(A) or oligo(U) insertions, sometimes in combination with variously sized deletions. As expected, these results are basically the same as those obtained previously with host M5219 (10).


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  		Table 1. Analysis of revertants recovered from three independent transformations
 


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  		Figure 2. Revertants recovered from the isogenic wild-type strain NDM5006 after infection with plasmid pMS.R. Uncoded nucleotides are in black. In parentheses we show how many times the sequence was found.

 
Revertants from a PAP I-deficient strain
In the next experiment we infected the PAP I mutant with pMS.R. Three independent transformations were carried out and from each 18 plaques were analyzed by RT–PCR (Table 1). All of the 11 plaques that gave insert size RT–PCR fragments were sequenced. Of the 41 plaques that gave wild-type size RT–PCR fragments only 12 were sequenced, because these revertants frequently turned out to be wild-type that had removed the complete insert.

Figure 3 shows the sequences of the revertants in the context of the secondary structure. Indeed, six of the revertants that gave a wild-type size RT–PCR fragment turned out to be wild-type while the others, PAPR4–PAPR7, had deleted the bulk of the insert.



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  		Figure 3. Revertants recovered from the PAP I-deficient host NDM5006{Delta}pcnB, after infection with plasmid pMS.R. In parentheses we show how many times the sequence was found. For revertant PAPR1 we show 6+1. This means it was found six times in one transformation and once in a second transformation. In PAPR4–PAPR7 the sequence above the black line was deleted.

 
Most important, however, none of the 11 revertants that gave an insert size RT–PCR fragment contained oligo(A) or oligo(U) insertions. There were only small deletions creating a bulge. PAPR3 had an extra C, the origin of which is unknown. These results indicate that PAP I is responsible for the oligo(A/U) stretches found in revertants.

Revertants from a PNPase-deficient strain
Although there seem to be many ways to obtain deletions in hairpin R it was both attractive and feasible to examine a possible contribution of PNPase to this process. Accordingly, plasmid pMS.R was transformed to the PNPase-defective strain and the resulting phages were separated by plating. Three independent transformations were carried out and 63 plaques were analyzed by RT–PCR (Table 1). Of these, 16 were selected for sequencing and the results are shown in Figure 4.



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  		Figure 4. Revertants recovered from the PNPase-deficient host NDM5004 after infection with plasmid pMS.R. See legends to Figures 2 and 3 for further details.

 
It is clear that the absence of an active PNPase does not prevent the introduction of deletions in the R hairpin. As mentioned in the Introduction, it is possible that RNase II is responsible for nucleotide removal. Alternatively, a replicase jump or even chemical linkage across the gap can be considered as causing the deletion of nucleotides from the sequence.

Another important result from this experiment is that, as expected, a substantial number of the revertants again carry oligo(U/A) insertions [pnpR1 (8x) and pnpR3].

Revertants from the PNPase, PAP I double mutant
Three independent transformations were carried out and a total of 57 plaques were characterized by RT–PCR. Six contained a mixture of wild-type size and mutant size fragment(s) and these were not analyzed further. Forty-two plaques contained wild-type size fragments and the sequences of two were determined. One was wild-type, while the other is shown as DBLR6 (Fig. 5). Nine plaques gave an insert size RT–PCR fragment and their sequences are displayed in Figure 5. It is clear that none of them carries uncoded oligo(A/U) stretches, indicating again that PAP I is responsible for adding oligo(A/U) stretches to the genome of the revertants.



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  		Figure 5. Revertants recovered from the PNPase, PAP I double mutant NDM5004pnp7,{Delta}pcnB. See legends to Figures 2 and 3 for further details.

 
The other conclusion from Figure 5 is, as before, that PNPase is not needed for the appearance of deletions in the hairpin.

Revertants from mutant phages
So far the mutant phage had been introduced into the host as a cDNA sequence on a plasmid. We can also infect our strains directly with mutant phages carrying the RNase III-sensitive site (R phages). These phages can be harvested from F+ rnc– cells transformed with pMS.R.

Wild-type E.coli FNDM5006 was infected with R phages and 23 plaques analyzed by RT–PCR. Two plaques yielded wild-type size fragments and 21 insert size fragments. Of these, 15 were sequenced. The majority of the revertants had become RNase III resistant by the introduction of one or more mismatches in the R hairpin as found earlier (10). Only three revertants had acquired oligo(A/U) inserts (Fig. 6). Thus it seemed unrewarding to evolve the R phages in the {Delta}pcnB strain because it would be difficult to decide whether the absence of oligo(A/U) sequences in revertants was due to the absence of PAP I or to a sampling artefact unless one would sequence large numbers of plaques.



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  		Figure 6. Revertants recovered from the isogenic wild-type strain NDM5006 after infection with R phages. All revertants were found once except ØR2, which was found twice.

 
Nevertheless, some of the revertants we obtained are interesting from the viewpoint of what constitutes an RNase III site. Next to the many revertants that had acquired simple mismatches in the stem, like ØR1, ØR5 or ØR7, it was interesting to note that even a simple G-C->G·U change in the stem could relieve the threat of RNase III cleavage (ØR2). Still more striking is that the change of the last U-A pair of the RNase III-sensitive stem into C·A (ØR8) is sufficient to allow the phage to grow in a wild-type host.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The introduction of a long uninterrupted stem into the MS2 RNA genome makes this phage sensitive to RNase III. However, spontaneous pseudorevertants that have become resistant appear quickly. Basically, three pathways are used to turn the cleavage site into a structure that is no longer recognized by RNase III. One is the introduction of mismatches in the long uninterrupted stem. The second is the deletion of a stretch of nucleotides, creating bulges in the stem or shortening it so that it is no longer a substrate. The third escape route is the addition of uncoded A or U residues in the side of the stem, also creating bulges (10).

In this paper we have presented data suggesting that these new nucleotides are added to the genome by the host enzyme poly(A) polymerase I. These extra nucleotides are absent in revertants that evolved in two different hosts that were both mutant in the pcnB gene, while the extra A or U residues are present in two different wild-types and in pnp7 hosts.

In normal E.coli cells PAP I is believed to be a component of the RNA degrading machinery (4,7). The addition of A residues to the 3"-end of structured RNA is supposed to tag the RNA for destruction by the exonuclease PNPase. It is suggested that the poly(A) tract might serve as a binding site for this enzyme. Poly(A) addition requires a free 3"-OH on the terminal ribose and cleavage by RNase III produces this hydroxyl group.

We envisage the creation of our oligo(A) revertants as follows. The first step is a single cleavage event of the MS2 RNA by RNase III. This may either occur on the 5"- or 3"-side of the stem and either in the plus or the minus strand. (The minus strand is predicted to contain a nearly identical hairpin, which is probably also cleaved by RNase III.) Subsequently, the free 3"-terminus is polyadenylated. For the majority of the molecules (>99%) this will set off their complete degradation. However, a small fraction [estimated previously (10) to be <0.1%] is saved from destruction by the intervention of MS2 replicase. In fact, the cleaved molecule itself is not saved by the replicase but the enzyme succeeds in making a full-length copy of the two half-molecules. To do so the replicase, when arriving at the RNase III-cleaved position, must jump over to the poly(A) tail and continue the copying reaction (Fig. 7). This template switch would cause the loss of a variable number of A residues which would go unnoticed in the experiments. [The jump is an example of random or illegitimate RNA recombination, because there is no sequence identity whatsoever between the take off and the landing site of the replicase (16).]



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  		Figure 7. Schematic showing how untemplated A residues are introduced into MS2 revertants.

 
In accordance with this explanation we find that most poly(A/U) tails are positioned approximately at the sites where we have measured RNase III cleavage in the plus strand (Fig. 1) or where one would expect cleavage in the minus strand. Polyadenylation at extremities slightly nibbled by exonuclease could explain why poly(A/U) tracts are often associated with deletions (Figs 2, 4 and 6). The fact that the insertions are only found on one side of the helix at a time suggests that only those templates that are cut once can be rescued. Our model is also consistent with the notion that PAP I can polyadenylate most 3"-OH extremities, including those resulting from endonucleolytic cleavage and exonucleolytic degradation (17–19). Moreover, lack of substrate discrimination by PAP I is substantiated by in vitro data showing that tRNA and RNA fragments terminated by 3" single-stranded nucleotides or stable terminal hairpins can all be polyadenylated (20; E.Hajnsdorf and P.Régnier, unpublished data).

Meanwhile it is clear that polyadenylation is not required to save the cleaved genome. In {Delta}pcnB mutants the replicase also manages to produce a copy from a cleaved genome. It may be noted that the replicase jump is not necessarily to the other half of the same genome. It could also be to another genome, although this seems less likely.

As argued in the Introduction, it is not strictly necessary to invoke a copy choice event by the MS2 replicase. It seems possible that the 3"-terminal ribose hydroxyl group of the poly(A) tail spontaneously attacks a phosphodiester bond across the gap, thus re-establishing the link, though at the expense of a few nucleotides. This possibility to create RNA recombinants was recently suggested (11) but it seems that the efficiency of this reaction is too low to account for the number of recombinants obtained by us in our previous study (10).

Another aspect of the present evolutionary approach is that it can reveal a large array of changes that transform a structure from being a substrate for an enzyme to one that is no longer a substrate. For RNase III this has resulted in the finding that a stem of 18 uninterrupted base pairs is a good substrate for RNase III, while 17 continuous pairs show a large degree of resistance in vivo (ØR8). The system employed here may also be useful to further understand what the characteristics are of, for example, an RNase E site.

Finally, this study, like many of our previous ones, offers a glimpse of the concoctions involved in the creation of a new phage (21). MS2 RNA destined for destruction by RNase III cleavage and poly(A) tagging can sometimes be saved in an inimitable way by the seemingly unlimited recombinatorial potential of its replicase. ØR13 in Figure 6 is the example. This revertant contains the traces of at least four mutational events. The G->A substitution in the stem and the {Delta}CUAGAU deletion are, in a sense, normal, but that the poly(A) insert consists of two parts interrupted by a GGC sequence is unusual. Most peculiarly, however, the complete top section of the R hairpin (or only the four loop nucleotides) have a sequence complementary to the original R hairpin. The most likely explanation is that the top section of hairpin R has been taken from the minus strand by a double crossover.


   ACKNOWLEDGEMENTS
 
We thank Ken Gerdes for the strains used in this study, Nora Goossen for help in preparing the F+ derivatives and René Olsthoorn for his continued interest. Janis Klovins is acknow­ledged for essential, practical instructions. M.Z. was a participant in the Socrates student exchange program sponsored by the EC.


   FOOTNOTES
 
a To whom correspondence should be addressed. Tel: +31 71 527 4759; Fax: +31 71 527 4340; Email: j.duin{at}chem.leidenuniv.nl Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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  10. Klovins,J., van Duin,J. and Olsthoorn,R.C.L. (1997) Nucleic Acids Res., 25, 4201–4208.

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Received May 7, 1999. Revised July 12, 1999; Accepted August 16, 1999.


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