From the Nuffield Department of Clinical
Biochemistry, Institute of Molecular Medicine, John Radcliffe Hospital,
University of Oxford, Oxford OX3 9DS, United Kingdom, the
§ Laboratoire de Microbiologie et Génétique
Moléculaire, CNRS, 118 Route de Narbonne, 31062 Toulouse, France,
and the ¶ Medical Research Council Clinical Sciences Centre,
Imperial College School of Medicine, Hammersmith Hospital, Du Cane
Road, London W12 ONN, United Kingdom
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ABSTRACT |
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Polyadenylation contributes to the
destabilization of bacterial mRNA. We have investigated the role of
polyadenylation in the degradation of RNA by the purified
Escherichia coli degradosome in vitro. RNA
molecules with 3'-ends incorporated into a stable stem-loop structure
could not readily be degraded by purified polynucleotide phosphorylase
or by the degradosome, even though the degradosome contains active RhlB
helicase which normally facilitates degradation of structured RNA. The
exoribonucleolytic activity of the degradosome was due to
polynucleotide phosphorylase, rather than the recently reported
exonucleolytic activity exhibited by a purified fragment of RNase E
(Huang, H., Liao, J., and Cohen, S. N. (1998) Nature
391, 99-102). Addition of a 3'-poly(A) tail stimulated degradation by
the degradosome. As few as 5 adenosine residues were sufficient to
achieve this stimulation, and generic sequences were equally effective.
The data show that the degradosome requires a single-stranded
"toehold" 3' to a secondary structure to recognize and degrade the
RNA molecule efficiently; polyadenylation can provide this
single-stranded 3'-end. Significantly, oligo(G) and oligo(U) tails were
unable to stimulate degradation; for oligo(G), at least, this is
probably due to the formation of a G quartet structure which makes the
3'-end inaccessible. The inaccessibility of 3'-oligo(U) sequences is
likely to have a role in stabilization of RNA molecules generated by
Rho-independent terminators.
Polyadenylation of mRNA contributes to its stability and
maturation and to the initiation of translation in eukaryotic cells (2-4). In prokaryotes, poly(A) polymerase activity was first identified over 3 decades ago (for a review of polyadenylation in
bacteria, see Ref. 5). Nevertheless, only recently was polyadenylation considered to have a role in determining mRNA stability in
bacteria. Two poly(A) polymerases have been cloned and characterized
from Escherichia coli (6, 7), and several mRNAs have
been shown to possess poly(A) tails (6, 8-12). Post-transcriptional
addition of a poly(A) tail at the 3'-end of mRNA has been shown to
destabilize certain RNA molecules (13, 14). Furthermore, disruption of the pcnB gene, encoding poly(A) polymerase I, results in
increased stability of RNA I (12, 15) as well as lpp,
ompA, rpsO, and trxA mRNAs in a
pnp rnb rne background (9, 11). This is in contrast to the
situation in eukaryotes, where polyadenylation stabilizes RNA.
In E. coli, degradation of mRNA is mediated by the
concerted action of endo- and exoribonucleases. A large multienzyme
complex (the degradosome) includes two of the most important
ribonucleases, the endoribonuclease RNase E and the 3' Bacterial Strains, Growth Conditions, Plasmids, and
Enzymes--
The genotypes of the E. coli strains used in
this study were as follows: BL21 (F Oligonucleotides and DNA Fragments for in Vitro
Transcription--
For in vitro RNA degradation assays, RNA
was transcribed from DNA templates generated by the polymerase chain
reaction (PCR). DNA fragments from the intergenic region of the
malE-malF operon of E. coli were amplified using
pCH77 as template (22) with the same 5'-primer A
(5'-AAATTAATACGACTCACTATAGGG-3') and each of the three alternative
3'-primers: 3'-primer i, 5'-TGCCGGATGCGACGCTGACG-3'; 3'-primer ii,
5'-(T15)TGCCGGATGCGACGCTGACG-3'; and
3'-primer iii, 5'-TTATCCGACAACAACTGCCGGATGCGACGCTGACG-3'.
The PCR fragments generated (320, 335, and 335 bp, respectively)
contained a T7 promoter at the 5'-end and could therefore be used
directly for in vitro transcription (see below) to
synthesize RNA molecules of 297, 312, and 312 nucleotides,
respectively. All these RNAs also contained the A
Two DNA fragments in which the malE-malF intergenic region
was shortened at the 5'-end by 86 nucleotides, compared with the fragments described above, were amplified using 5'-primer B
(5'-AAATAATACGACTCACTATAGGGCCCGCAGATGTCCGCTTTC-3') together with each
of 3'-primers ii and 3'-iii to generate 3'-A15 or
3'-N15 tails downstream of the stem-loop, respectively.
These PCR products (250 bp) also contained a T7 promoter at the 5'-end such that in vitro transcription generated RNA molecules of
228 bp (named "sstemA15" and
"sstemN15").
A DNA fragment in which the stem-loop structure of the
malE-malF region was replaced by the terminator stem-loop of
the lpp gene (for the mRNA sequence, see Ref. 22) was
amplified using 5'-primer A (see above) and the 3'-primer
5'-AAAAAAATGGCGCACAATGTGCGCCATTTTTCATTTCACAGCATTACTTGGTGAT-3'. The PCR
product (266 bp) used as a template for in vitro
transcription gave a 243-nucleotide RNA molecule (named
"lpp-stem").
In Vitro Transcription--
Radiolabeled RNA was synthesized
in vitro from linearized, gel-purified DNA templates using
bacteriophage T7 RNA polymerase (Pharmacia) and
[ Polyadenylation of mRNA--
In vitro
transcribed, radiolabeled RNA was incubated with E. coli
poly(A) polymerase and 5 mM ATP in the buffer recommended by the manufacturer (Cambio) for 20 min at 37 °C. The reaction mixture was extracted with phenol/chloroform and further treated as
described above (see "In Vitro Transcription").
In Vitro RNA Degradation Assays--
In vitro RNA
degradation assays were performed as described (19). The reaction (100 µl) contained exonuclease assay buffer (20 mM Tris-HCl
(pH 7.5), 1 mM MgCl2, 20 mM KCl, 5 mM dithiothreitol, and 10 mM
K2HPO4), 1 mM ATP, and 80 units of
RNasin (Promega) and was incubated with enzyme at 37 °C. Unless
otherwise stated, 0.18 µg of His-tagged PNPase or 0.66 µg of the
degradosome was added to each reaction. Aliquots were taken at
appropriate time points, loaded onto a 4 or 6% sequencing gel, and
electrophoresed at 60 watts. The gel was dried on Whatman No. 3MM
paper, and radioactivity in each band was quantitated with a PhosphorImager.
Cloning of the Gene Encoding Polynucleotide
Phosphorylase--
The gene encoding PNPase was amplified by PCR from
E. coli HB101 genomic DNA using Vent polymerase (New England
Biolabs Inc.). Oligonucleotide primers
5'-ATATTAATCATATGCTTAATCCGATCGTT-3' and
5'-CCCCAAGCTTCTCGCCCTGTTCAGC-3' were based on the
pnp sequence (GenBankTM accession number U18996)
and amplified the pnp gene flanked by NdeI and
HindIII restriction sites (boldface) to facilitate subsequent cloning. After amplification, gel purification, and cleavage
with HindIII, the 2254-bp PCR product was cloned into pBluescript II KS(+) digested with SmaI and
HindIII. The pnp gene was excised from the
resulting plasmid with NdeI and HindIII and recloned into pET-20b (NdeI/HindIII). The
resulting plasmid, pPNPHIS, contained the pnp gene under
the control of the T7 promoter, allowing inducible overexpression of a
PNPase derivative with a C-terminal hexahistidine tag in E. coli strain BL21.
Purification of His-tagged PNPase--
His-tagged PNPase was
purified by metal-chelate affinity chromatography (24) as described
previously for His-tagged polyphosphate kinase (20) with the following
modifications. Buffer A for resuspending the cell pellet and for
washing steps consisted of 0.1 M
Na2HPO4/NaH2PO4 (pH
7.8), 10% glycerol, 1% Tween, 1 M NaCl, and 5 mM imidazole. The protein was eluted in steps using 5 ml of
buffer B (0.1 M Na2HPO4/NaH2PO4 (pH
7.8), 10% glycerol, and 1% Tween) containing 30, 50, 80, 120, and 500 mM imidazole, respectively. His-tagged PNPase was purified
to near homogeneity as judged by Coomassie Blue staining (see Fig.
4A). The fractions containing the protein peak were pooled
and stored in 50% glycerol (final concentration) at Effects of 3'-Adenylation on the Stability of Structured RNA in
Vitro--
To examine the influence of polyadenylation on RNA
degradation in vitro, various polyadenylated and
non-polyadenylated mRNAs were incubated with the degradosome in the
presence or absence of 1 mM ATP. As the primary substrate a
segment of malE-malF mRNA (19, 20) was used (named
stem). The 3'-end of this RNA corresponded to the last nucleotide of
the stable stem-loop structure of the malE-malF intergenic
region (Fig. 1A), such that
the 3'-end was fully incorporated into the secondary structure.
Derivatives of this mRNA were generated with an additional 15 adenosine residues (stemA15) or a poly(A) tail
("stem-poly(A)" containing 200-300 adenosine residues), providing
a single-stranded 3'-tail. These substrates were incubated with the
degradosome, samples were taken at defined time points, and the
products were separated on a denaturing gel. Fig.
2A shows an example of such a
gel. The rate of disappearance of the mRNA species was quantified
using a PhosphorImager (Fig. 2B).
In all reactions, the oligo(A) tail (when present) was rapidly removed
to generate a stable intermediate with its 3'-end close to the base of
the stem-loop structure (Fig. 2) (22). The curves are biphasic (Fig.
2B) because, for a small proportion of the RNA starting
material, it appears the degradosome falls off the RNA at the base of
the stem-loop (having removed the poly(A) tail), and this species is
then unable to be degraded further (see below). In the absence of ATP
this intermediate RNA species was only poorly degraded by the
degradosome, irrespective of the starting RNA from which it was
generated, because ATP is required for the RhlB helicase to unwind the
secondary structure (19). In the presence of ATP, however, this
intermediate was rapidly degraded, but only for the RNA molecules that
initially had a single-stranded A15 or poly(A) tail at
their 3'-ends (stemA15 and stem-poly(A)); the RNA lacking
any nucleotides added to the 3'-base of the stem-loop (stem) was still
resistant to degradation (Fig. 2). The degradation of RNAs containing 5 or 10 adenosine residues did not differ significantly from those
containing 15 adenosine residues or a poly(A) tail, demonstrating that
the length of the oligo(A) tail is relatively unimportant (Fig.
3).
As a control, we purified His-tagged PNPase (Fig.
4A). This C-terminally
His-tagged PNPase was active as a 3'-exoribonuclease (Fig.
4B), in contrast to N-terminally His-tagged PNPase which is
inactive (17). The stem and stemA15 RNA molecules were
incubated with the purified His-tagged PNPase. The purified
exoribonuclease did not degrade either stem RNA or stemA15
RNA, apart from removing the oligo(A) tail (Fig. 4C),
confirming that, under these conditions, the RhlB helicase of the
degradosome is required for the stem-loop structure to be degraded (19,
22).
Effect of Alternative Tails on the Degradation of Structured
RNA--
Since addition of a 3'-poly(A) tail stimulated degradation of
the stem-loop structure by the degradosome, we assessed whether there
was any specificity for the composition of the 3'-tail. The rate of
degradation of RNAs with a 3'-tail of 15 guanosines ("stemG15") or 15 uridines (stemU15) was
compared with that of the stem, stemA15, and
stemN15 substrates. The stemN15 substrate incorporated a 3'-tail consisting of the 15 nucleotides normally 3' of
the stem-loop in the malE-malF mRNA. Assays were in the presence of 1 mM ATP. The stemN15 RNA was
degraded as rapidly as the stemA15 RNA (Fig.
5). In contrast, the stemG15
and stemU15 RNAs were degraded slowly, at a rate similar to
that of the stem RNA. Furthermore, and in contrast to the
stemA15, stemN15, and stemU15 RNA
substrates, the oligo(G) tail of the stemG15 RNA was removed poorly by the degradosome (Fig.
6). Incubation of RNA substrates with
purified His-tagged PNPase showed that the purified enzyme was also
unable to degrade the oligo(G) tail (Fig. 6), demonstrating that the
oligo(G) tail is not accessible to either enzyme.
Degradation of Native malE-malF RNA Intermediates in
Vitro--
In vivo, transcription of the
malE-malF mRNA terminates several hundred base pairs
downstream of the large intergenic stem-loop. A stable RNA molecule
with the stem-loop near its 3'-end is then generated by processive
exoribonucleolytic degradation from the 3'-end (22, 26). To mimic the
in vivo situation in vitro, stemN15
RNA was incubated with His-tagged PNPase for 15 min; the reaction was
stopped; and the resulting RNA was purified. Fig. 7B shows that all the initial
substrate was converted to the stable intermediate, designated
"stem(N15)*." Aliquots of stem(N15)* RNA
were then polyadenylated and used as substrate for the degradosome in
the presence of ATP. Although the residual 3'-tail on
stem(N15)*, compared with stem RNA, permitted slightly
faster degradation of the stem-loop structure, the degradosome degraded
the polyadenylated RNA significantly faster than the non-polyadenylated
precursor (Fig. 7, A and C). Similar results were
obtained when the stem(N15)* RNA was generated using the
degradosome (in the absence of ATP) rather than His-tagged PNPase (data
not shown). Thus, polyadenylation enhances the rate of degradation of
intermediates equivalent to those generated in vivo.
E. coli PNPase Does Not Have a Preference for 3'-Poly(A) Tails
Compared with Other Single-stranded 3'-Tails--
To determine whether
PNPase has higher affinity for poly(A) tails than for generic
single-stranded 3'-ends, the rate of degradation of the N15
tail of stemN15 RNA was compared in the presence of different competing RNA substrates. 5'-Truncated malE-malF
competitor RNAs were constructed to distinguish them from the substrate
RNA following gel electrophoresis. These shorter RNAs
(sstemA15 and sstemN15) ended with an
A15 or N15 tail 3' to the stem-loop structures, respectively. In this assay, the concentration of enzyme was reduced to
amounts equimolar to the substrate RNA. The competitor RNAs were also
added in equimolar amounts. The rate of degradation of the
N15 tail of stemN15 in the presence of either
competitor (band a going to band b) calculated
from the data shown in Fig. 8 give the
time, in which 50% of the full-length stemN15 substrate is
shortened to the base of the stem-loop structure (td50), as 0.5 min for the sstemN15 substrate alone. Addition of
either of the competing substrates (sstemN15 or
sstemA15) resulted in a similar increase in
td50 to ~2 min. The removal of the 3'-tails from
sstemN15 and sstemA15 can also be seen to occur
at a similar rate (generated band d and f,
respectively). These data demonstrate that, as long as the 3'-tail is
single-stranded and accessible to the enzyme (unlike the
G15 tail; see above), it does not matter whether the
3'-tail is poly(A) or is a generic sequence.
Degradation of RNA Containing the lpp Terminator Stem-loop--
To
demonstrate the general relevance of polyadenylation, the degradation
of a primary transcript ending in a native terminator stem-loop was
characterized. The lpp terminator (Fig. 1B) was selected because the lpp mRNA is relatively stable and
polyadenylation has been reported in vivo (6, 27). The
native lpp terminator was used to replace the large
stem-loop of the malE-malF RNA, and the resulting mRNA
(lpp-stem) and its polyadenylated derivative were used as substrates
for the degradosome and for His-tagged PNPase. Equimolar amounts of
PNPase were used, whether His-tagged or in the degradosome (0.18 µg
of purified His-tagged PNPase is equivalent to 0.66 µg of degradosome
containing 28% PNPase (19)). In contrast to the malE-malF
stem-loop, ATP was not required for degradation of the lpp
stem-loop by the degradosome, presumably because the lower
thermodynamic stability of the stem-loop is such that the RhlB helicase
is not essential for unwinding (Fig. 9).
Therefore, addition of ATP promoted the degradation only slightly. Nevertheless, the presence of a 3'-poly(A) tail still markedly stimulated degradation of the RNA (whether or not ATP was present) both
by the degradosome and by purified PNPase (Fig. 9).
Polyadenylation of bacterial mRNA is increasingly recognized
as a potentially important destabilizing signal for the RNA degradation machinery. RNA molecules or fragments generated by endoribonuclease cleavage are degraded by one of the 3' The malE-malF intergenic region forms a very stable
stem-loop structure, and its degradation does not require RNase E
cleavage (19).2 The
degradosome and purified PNPase are stalled by the stem-loop structure
to give a very stable intermediate in vivo and in
vitro (19, 22, 26-29). A derivative of the malE-malF
message with its 3' terminus incorporated into the stem-loop structure
was degraded very poorly by the degradosome in vitro, even
when RhlB was active. In contrast, 3'-adenylated derivatives of the RNA were degraded rapidly. This demonstrates that the ability of the degradosome can be limited by the absence of a single-stranded 3'-RNA
end. Addition of an oligo(A) tail efficiently destabilized the message
in vitro, even when as few as 5 nucleotides were added. Fewer than 5 adenosine residues did not seem to be recognized (data not
shown). Although it has been suggested that as few as 2 adenosine
residues might target RNA for degradation (12), these studies were
carried out in vivo, and nucleases other than the
degradosome might be involved. Similarly, Coburn and Mackie (14) have
indicated that RNA with <6-10 unpaired 3'-residues is stable; these
were not adenosines, and the experiments were carried out with purified
nucleases, not the degradosome. Although the model malE-malF
transcript studied initially is not normally generated in
vivo, we also showed that polyadenylation stimulated degradation
of a malE-malF intermediate equivalent to that generated in vivo and of the lpp 3'-stem-loop generated
in vivo by transcription termination. Thus, polyadenylation
appears to stimulate degradation by the degradosome of any structured
RNA generated as a termination product or degradation intermediate
where the 3'-end has a limited single-stranded tail. Since an
N15 generic single-stranded sequence of the stem-loop was
as effective as the A15 sequence and since A15
and N15 sequences competed equally well for degradation,
the destabilizing effect appears be due to the single-stranded nature of the 3'-end rather than to a high affinity for poly(A) per
se.
Interestingly, a 3'-oligo(G) tail did not stimulate degradation of RNA
in vitro (Fig. 5). Furthermore, neither the degradosome nor
purified PNPase could even shorten the G15 tail, suggesting that the sequence is inaccessible to these enzymes. G-rich sequences are able to form intramolecular or intermolecular G quartets (30, 31),
which are very resistant to nucleases (32) and which presumably render
the 3'-end inaccessible to enzymatic attack.
A 3'-U15 tail also failed to destabilize the RNA. There is
no known structure an oligo(U) tail might adopt, although in contrast to the G15 tail, the U15 tail was shortened by
the degradosome and by PNPase in vitro (Fig. 6), showing
that it remains accessible. More important, every RNA generated from a
gene with a Rho-independent terminator ends in a 3'-oligo(U) tail.
Thus, apart from the terminator stem-loop itself, this oligo(U)
sequence of the terminator may contribute significantly to the
stability of such mRNAs, protecting against 3' Recently, a purified N-terminal fragment of RNase E was reported to
remove 3'-poly(A) and 3'-poly(U) tails (1). However, several lines of
evidence argue against the possibility that RNase E, rather than
PNPase, is responsible for the exoribonucleolytic activities of the
degradosome observed here. First, the exoribonucleolytic activity of
the degradosome containing a temperature-sensitive RNase E is still
efficient at the nonpermissive temperature (19). Since both the
endoribonucleolytic and poly(A) tail-shortening activities of RNase E
are in the same N-terminal region of the polypeptide (1), the poly(A)
shortening should also be inactive at the nonpermissive temperature.
Second, purified PNPase gave the same pattern of 3'-poly(A) tail
shortening as the degradosome. Third, characterization of the released
nucleotides by thin-layer chromatography allowed us to distinguish
which enzyme is involved.3
Thus, the reported exoribonucleolytic activity reported for a fragment
of RNase E is unlikely be significant in RNA degradation by the degradosome.
In conclusion, we have shown that polyadenylation can play an important
role in the degradation of mRNA molecules that have 3'-terminal
secondary structures, providing a single-stranded toehold recognized by
the degradosome. 3'-Structured RNAs are normally generated by
Rho-independent termination, by endonuclease cleavage, or by stalling
of 3'
INTRODUCTION
Top
Abstract
Introduction
References
5'
exoribonuclease PNPase1
(16-19), together with the DEAD box helicase RhlB, enolase, and a
non-stoichiometric amount of polyphosphate kinase (19, 20). The roles
of enolase and polyphosphate kinase in mRNA degradation are not yet
clear. However, RhlB has a central role in the degradation of mRNAs
with stable stem-loop structures (19). Purified PNPase is impeded by
stem-loop structures in RNA, whereas PNPase in the degradosome can
degrade structured RNA: in the degradosome, the RhlB helicase unwinds
such structures in an ATP-dependent fashion to permit the
passage of PNPase. Thus, the degradosome is generally efficient at
degrading structured RNA. However, in this study, we show that the
degradosome is inefficient in degrading RNA molecules with their
3'-ends incorporated into a stem-loop structure. Addition of a
single-stranded 3'-poly(A) tail facilitated degradation, providing a
"toehold" permitting the degradosome to initiate its attack on the
3'-end of the RNA.
EXPERIMENTAL PROCEDURES
ompT
r
Bm
B) and
HB101 (F
(gpt-proA)62 leuB6 supE44
ara-14 galK2 lacY1
(mcrC-mrr) rpsL20 (Strr) xyl-5 mtl-1 recA13). Cells were grown in
LB medium with antibiotics as appropriate (21). Plasmids pCH77 (22),
pET-20b (Novagen), and pBluescript II KS(+) (Stratagene) have been
described. DNA manipulations were performed as described (21), and
enzymes were used as recommended by the manufacturers. Purified
E. coli poly(A) polymerase was purchased from Cambio
(Cambridge, United Kingdom). The degradosome was purified following the
protocol described by Carpousis et al. (16) as modified by
Py et al. (19).
C change
indicated in Fig. 1. The 3' termini of these RNAs ended precisely at
the 3'-base of the malE-malF stem-loop structure
("stem") (3'-primer i), with a 3'-A15 tail ("stemA15") (3'-primer ii), or with 15 nucleotides of
the original malE-malF sequence downstream of the stem-loop
("stemN15") (3'-primer iii). To transcribe RNA
molecules with 5 or 10 adenosine residues or 15 guanosine or 15 uridine
residues downstream of the stem-loop, derivatives of 3'-primer ii were
used in which the T15 sequence (boldface) was exchanged for
the appropriate C or A nucleotides.
-32P]CTP (400 Ci/mmol; Amersham) as described
previously (22). The transcription reactions were diluted to 70 µl in
water, extracted with phenol/chloroform, and eluted through a Push
column (Stratagene) to separate unincorporated nucleotides. The RNA was
precipitated with ethanol and resuspended in water (MilliQ). The RNA
products stemA15, stemU15, and
sstemA15 migrated as broad bands (especially sstemA15 in Fig. 8, band e), probably because
runoff transcription does not stop precisely at the end of the DNA
template due to the weak deoxyribo(T)n-ribo(A)n or
deoxyribo(A)n-ribo(U)n interactions.
20 °C.
Protein concentrations were determined as described (25) with bovine
serum albumin as standard.
RESULTS
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Fig. 1.
RNA secondary structures. A,
sequence of the malE-malF stem-loop structure. In all
experiments described here, a derivative was used with an A C
change, as indicated, to correct the mismatch at the base of the stem
and to ensure that no single-stranded "tail" is exposed.
B, terminator of the lpp mRNA (23).
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Fig. 2.
Different rates of degradation of adenylated
and non-adenylated derivatives of 3'-structured malE-malF
RNA. A, example autoradiograph showing RNA degradation
as a function of time in the absence (upper panel) or
presence (lower panel) of ATP. B, graph showing
the rates of RNA degradation, determined by phosphoimaging of gels
similar to the gel illustrated in A, plotted as the
percentage of RNA remaining as a function of time. Derivatives of
malE-malF RNA with 3'-ends incorporated into the stem-loop
structure (stem; ,
) or with a 3'-A15 tail
(stemA15;
,
) or a 3'-poly(A) tail (stem-poly(A);
,
) downstream of the stem-loop structure were incubated with the
degradosome in the absence (open symbols) or presence
(closed symbols) of ATP.
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Fig. 3.
Rates of degradation of malE-malF
RNA with different length adenylated tails 3' to the stem-loop
structure. A, example autoradiograph showing a time course
of RNA degradation assayed in the presence of ATP. B, graph
showing the rates of RNA degradation, derived by phosphoimaging of gels
similar to the gel in A. As the "fuzzy" 0-min band was
difficult to accurately quantitate, the 1-min part was designated the
100% value. The RNA substrates used have no 3'-tail (stem ( )) or
have 3'-A tails of different lengths (stemA5 (
),
stemA10 (
), stemA15 (
), and stem-poly(A)
(
)).
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Fig. 4.
Purified C-terminally His-tagged PNPase is
active. A, Coomassie blue-stained protein gel loaded with
1.2 µg of purified His-tagged PNPase (Pnp-His6;
arrow). B and C, autoradiographs of
6% gels of RNA degradation assays as a function of time (in minutes).
B, 8 ng of purified His-tagged PNPase was incubated with
malE-malF mRNA (19) to demonstrate that the enzyme is
active. Because this study was designed to show that the His-tagged
PNPase was active, only a small amount of enzyme was used. Under these
conditions, the enzyme paused at small stem-loop structures (as does
native PNPase) (22) and finally stalled at the large stem-loop,
generating a stable degradation intermediate (arrow).
C, His-tagged PNPase did not degrade the stem RNA
(left panel). Only the A tail of the stemA15 RNA
substrate (right panel) was degraded by His-tagged PNPase,
again generating a stable intermediate (arrow). The
intermediate migrated slightly slower than the stem RNA, indicating
that PNPase does not remove the entire 3'-tail, but leaves a few
nucleotides (nt) of the base of the stem-loop structure (as
shown previously (22)).
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Fig. 5.
Degradation of malE-malF RNA
containing different 3'-tails. A, autoradiograph showing RNA
degradation as a function of time. Assays were in the presence of ATP.
The schematic stem-loop symbol indicates the position where
the stable intermediate migrated. Full-length RNA migrated only
slightly slower in this gel. The minor additional degradation product
appearing in the assay when stemU15 was used (*) has not
been identified. One explanation might be that the structure of the RNA
is changed by the oligo(U) sequence and that an RNase E site is
exposed. B, graph showing rates of RNA degradation, derived
by phosphoimaging of the gel in A. Derivatives of
malE-malF RNA containing an A15 ( ) or an
N15 (
) tail downstream of the stem-loop structure were
degraded by the degradosome faster than RNAs with a G15
(
) or an U15 (
) tail or the 3'-structured stem RNA
itself (
).
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Fig. 6.
Accessibility of RNAs with different
homopolymeric tails. Shown is autoradiograph of a denaturing gel.
The indicated malE-malF RNA derivatives were incubated with
the degradosome (D), with His-tagged PNPase (P),
or with no added enzyme ( ) for 10 min (stemA15) or for 30 min (stemG15 and stemU15) in the presence of
ATP.
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Fig. 7.
Degradation of the stable in vivo
degradation intermediate of malE-malF RNA
compared with its polyadenylated derivative. A,
autoradiograph showing RNA degradation as a function of time. Assays
were in the presence of ATP. The initial RNA substrate
(stemN15) was processed by His-tagged PNPase, and the
degradation intermediate was purified. The degradation intermediate
(stem(N15)*) and its polyadenylated derivative were then
used as substrates for the degradosome. B, the
stemN15 RNA ( ) is fully processed to a stable
intermediate by His-tagged PNPase (+ Pnp). C,
graph showing rates of RNA degradation. The polyadenylated degradation
intermediate (stem(N15)*-poly(A);
) was less stable than
its precursor (stem(N15)*;
), whereas the 3'-structured
stem RNA (
) was most resistant to degradation.
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Fig. 8.
Relative affinities of PNPase for
3'-N15 or 3'-A15
tails. An autoradiograph showing the processing of RNA with
a 3'-N15 tail (stemN15) by His-tagged PNPase
(100 fmol). Upper panel,100 fmol of stemN15
(band a) as the only substrate was processed to a stable
intermediate (band b). Middle and
lower panels,100 fmol of 5'-truncated malE-malF
RNAs were added as competitors to the stemN15 RNA.
Bands c and e indicate the full-length
sstemN15 and sstemA15 RNAs, respectively, and
bands d and f indicate the stable intermediates
generated.
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Fig. 9.
Degradation of RNA with the 3'-lpp
terminator stem-loop and its 3'-polyadenylated derivative.
A, autoradiograph showing RNA degradation as a function of
time. The polyadenylated and degradation intermediate RNAs are
indicated as schematic symbols. B, graphs showing
the rates of RNA degradation derived from the autoradiograph in
A. Left panel,the substrate RNA containing the
lpp terminator at the 3'-end (lpp-stem; ,
) and its
polyadenylated derivative (lpp-stem-poly(A):
,
) were incubated
with the degradosome in the presence (closed
symbols) or absence (open symbols) of ATP.
Right panel, the substrate RNAs were incubated with
His-tagged PNPase (lpp-stem (
) and lpp-stem-poly(A) (
)).
DISCUSSION
5' exoribonucleases: PNPase
or RNase II. These processive exoribonucleases are stalled by stem-loop
structures; temporarily at smaller stem-loop structures, but more
stable stem-loops (e.g. the malE-malF intergenic
stem-loop structure) (19, 22) or complex secondary structures (3'-end of the S20 mRNA) (14) act as a very effective block to these enzymes. In the cell, however, a proportion of PNPase is part of a
multiprotein complex, the degradosome (16-19). The
ATP-dependent helicase activity of RhlB in the degradosome
strongly promotes the degradation of structured RNA by unwinding
sequences and offering them as single-stranded substrates to PNPase
(19). Thus, the degradosome appears to be fully equipped to degrade
highly structured RNA molecules (19). Since polyadenylation is
important for destabilizing structured RNA molecules (9, 11, 13), we
set out to address the apparent paradox of how polyadenylation might
stimulate the activity of the degradosome. In particular, it has
previously been suggested that polyadenylation may provide a point of
access for ribonucleases (11); this idea was tested experimentally for
the degradosome.
5'
exoribonucleolytic attack.
5' exonucleases at an internal secondary structure. Poly(A)
polymerase can potentially pin a label "to be degraded" to
structured RNA generated by any of these processes that is otherwise
inaccessible to ribonucleases. Thus, "initiation of degradation" of
structured RNA by polyadenylation may play an important role not only
in the degradation of functional RNA molecules with inaccessible
(structured) 3'-ends, but also in recycling the small structured RNA
degradation intermediates generated during cellular RNA turnover.
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ACKNOWLEDGEMENTS |
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We thank Beatrice Py, Kenneth McDowall, and Chris Burns for helpful discussions.
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FOOTNOTES |
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* This work was supported by the Biotechnology and Biological Sciences Research Council, CNRS, the European Union, and the Imperial Cancer Research Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Howard Hughes International Research Scholar. To whom
correspondence should be addressed: MRC Clinical Sciences Centre,
Imperial College School of Medicine, Hammersmith Hospital, Du Cane Rd., London W12 ONN, UK. Tel.: 44-181-383-8335; Fax: 44-181-383-8337; E-mail:chiggins{at}rpms.ac.uk.
The abbreviations used are: PNPase, polynucleotide phosphorylase; PCR, polymerase chain reaction; bp, base pair(s).
2 G. S. C. Dance and C. F. Higgins, unpublished data.
3 C. Burns and C. F. Higgins, unpublished data.
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REFERENCES |
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