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J. Biol. Chem., Vol. 275, Issue 34, 26523-26529, August 25, 2000
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§,,
, and
From the Department of Genome Organization and
Group of Genes Chemical Synthesis, Engelhardt Institute of
Molecular Biology Russian Academy of Sciences, Vavilov str. 32, Moscow
117984, Russia and the ¶ Laboratory of Bacterial Genetics, State
Scientific Center of Russian Federation GNIIGENETICA, 1th Dorozhnii pr.
1, Box 825, Moscow 113545, Russia
Received for publication, April 4, 2000, and in revised form, June 6, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Gene-specific silencing refers to a phenomenon in
which expression of an individual gene can be specifically repressed by different mechanisms on the levels of transcription, RNA splicing, transport, degradation in nuclei or cytoplasm, or blocking of translation. In different species gene-specific silencing was observed
by expression or injections of antiparallel double-stranded RNA formed
by a fragment of mRNA and antisense RNA. Here we show a potent and
specific gene silencing in bacteria by expression of RNA, that is
complementary in a parallel orientation to Escherichia coli
lon mRNA. Moreover, the expression of parallel RNA is more effective at producing interference than expression of antisense RNA
corresponding to the same mRNA region. Both effects of interference mediated either by parallel RNA or antiparallel RNA gradually decrease
up to the 40th generation. Together with in vitro nuclease protection studies these results indicate that a parallel RNA duplex
might be formed in vivo and both types of duplexes,
antiparallel or parallel, can induce gene-specific silencing by similar mechanisms.
There has been dramatic recent progress in uncovering the
gene-specific silencing in a number of organisms (1-3). Several lines
of evidence suggest that
dsRNA1 is the effector
molecule responsible for RNA-mediated silencing or co-suppression.
dsRNA is formed by mRNA and antisense RNA, that corresponds to the
non-coding strand of the same gene. However, in experiments on
Caenorhabditis elegans it was demonstrated that injections
of gel-purified antisense RNA corresponding to an abundant transcript
is less active at producing interference than in vitro annealed dsRNA samples (1). The purification was performed to remove
the traces of dsRNA from in vitro synthesized RNA
preparations because of the nonspecific activity of RNA polymerases.
It was concluded that the observation of co-suppression and RNA
interference uncovered the existence of a novel cellular mechanisms for
regulation of gene expression (2, 4). The phenomenon has been described
in fungi, protozoa, plants, invertebrates, and vertebrates (5-10).
This suggests a evolutionary conservation of the physiological
mechanisms involved.
The mechanisms of RNAi remain largely unknown. It was concluded that
RNAi and co-suppression work by an equivalent core mechanism produces
decrease or elimination of a target mRNA transcript (2, 3, 4, 6).
In experiments on gene silencing ("quelling") in Neurospora
crassa it was shown that in the mutant defective in quelling the
gene specifying RNA-dependent RNA polymerase was affected
(5). The genes involved in RNA degradation also could be connected with
RNAi (12). It was considered that RNAi mechanisms might operate at the
level of transcription and involve proteins of the polycomb complex
(13). But recently it was clearly demonstrated that this
transcriptional cosuppression mechanism is distinct from RNAi and
involve homology recognition at the DNA level (14). The fact
demonstrates that although the phenomenology of gene-specific silencing
is similar in different taxa, the underlying molecular mechanisms are
not yet understood.
The physiological role of mechanisms leading to gene-specific silencing
remains mysterious. Genes regulated by antisense transcripts were
described in Eubacteria, Archaebacteria, and some primitive eukaryotes
(15). RNAi was not described in prokaryotes. However, antisense control
is bacteria is now recognized as an efficient and specific means of
gene (16). This control occurs at many levels, including premature
transcription termination, facilitated RNA decay, and direct or
indirect blocking of translation.
We had previously reported that RNA molecules are capable of forming
parallel RNA-RNA duplexes in vitro (17). To address the capacity of parallel double-stranded RNA to induce gene-specific silencing, we have used the construct expressing small RNA that is
complementary in a parallel orientation (pRNA) to the endogenous lon mRNA in Escherichia coli cells. To our
surprise, we found a potent and specific effect of interference on the
expression of the gene. In vitro experiments with pRNA and
corresponding sense RNA of the gene have revealed nuclease protection
bands after annealing. Our results indicate that parallel
double-stranded RNA might be formed in vivo and serve as
effector for some mechanisms of gene regulation similarly to regular
dsRNA.
Construction of the Plasmids for in Vivo Experiments--
For
construction of parlon and mirlon plasmids the chemically synthesized
95-bp DNA (see Fig. 4A) was inserted into the
SmaI site of the pUC12 vector in opposite orientations. For
preparation of the antilon construct, the
EcoRI-HindIII fragment from the pUC19AL1
construct, containing 341-bp HpaI-HindIII
fragment of the lon gene (18), was subcloned in the pUC12
vector. All the constructs are expressed under control of the
lac promoter of the vector and the in vivo
transcripts possess identical stretches of 229 and 300 bases in length
at their 5' and 3' ends, respectively, coming from the lac
gene, as well as the different sequences in the middle, corresponding
to the DNA inserts. The transcription termination signals are provided
by pBR322 origin flanking the lac operon fragment in the
pUC12 vector (19). All inserts interrupt the open reading frame of the
lacZ gene and only the N-part of the gene could be translated.
Degradation of Puromycin Peptides--
HB101 transformants were
grown for 14 h at 37 °C on plates. Separate colonies were
inoculated separately in M9 medium containing 80 µg/ml DL proline and
50 µg/ml ampicillin and grown at 37 °C to
A550 = 0.25-0.35.
Isopropyl-1-thio-
Direct isolation and in vitro testing of the Lon
preparations was performed. Fresh transformed HB101 cells were grown in
L broth with 100 µg/ml ampicillin at 37 °C to
A550 = 0.3. Then
isopropyl-1-thio-
To test the effect of KCN administration on degradation of puromycin
peptides the HB101 transformants were grown in the presence of
puromycin as described above in two tubes and KCN was added in one tube
to 5 mM 1 h before cells were precipitated and
degradation of 3H-peptides was tested. To test the
degradation of normal 3H-proteins the HB101 transformants
were grown and analyzed as described above, but no puromycin was added.
RNA Isolation and Northern Blot Analysis--
After
transformation the portions of HB101 cell suspension in L medium,
containing up to 100 transformed cells were incubated in 20 ml of L
medium containing 200 µg/ml ampicillin for 14 h at 37 °C. Up
to 105 of nontransformed cells of different strains were
grown in L medium during the same time without ampicillin. For RNA
isolation cells were precipitated, suspended in at 0 °C in solution
containing 25% sucrose, 50 mM Tris-HCl, pH 8, and treated
with 0.5 mg/ml lysozyme for 5 min and then 1 volume of the lysing
buffer containing 2% SDS, 1% Brij 58, 0.4% deoxycholate, 0.1 M EDTA, 20 mM Tris-HCl, pH 8, was added on ice
for 15 min. Then one-half volume of saturated NaCl solution was added.
After precipitation at 16,000 rpm at 4 °C the supernatant was
treated 5 times with phenol/chloroform mixture, pH 5, and ethanol
precipitated. To remove DNA the pellet was dissolved in 100 µl of
water and RNA was precipitated with 300 µl of 4 M sodium
acetate, pH 4.8, after incubation for 1 h at In Vivo Measurements of the Luciferase Activity--
The plasmid
pAC16, containing the lux regulon (16 kilobase
BamHI DNA segment from Vibrio fischeri) in
pACYC184 vector was introduced in lon+ AB1157
cells and lon
To study the effect of the lon gene inhibition up to 50 generations, the cells after transformation with constructs or pUC12 vector (100 transformants/ml) were grown as described above to A550 = 2.0 (4 × 108 cells/ml),
which corresponds to 20 generations (n = 20), and the
first measurement of the luminescence was performed. Then cells were
1000 times diluted in L medium and the growth was proceeded to
A550 = 2.0 (n = 30) and the
second measurement of the luminescence was performed. Similarly the
measurements were performed until the 50th generation.
Nuclease Protection Assay--
The pGEM constructs were
linearized completely. RNA transcription was performed in 20 µl of
solution containing 2-5 µg of DNA, 40 mM Tris-HCl, pH
7.5, 6 mM MgCl, 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol, 1 units/µl of
RNasin, ATP, GTP, CTP, UTP (500 µM each), 2.5 µM
About 4.5 ng of [33P]RNA parlon was annealed alone or
separately with 45 ng each of 3H-labeled RNAs in 6 µl of
solution containing 0.1 M NaCl, 10 mM Tris-HCl,
pH 7.5, 10 mM MgCl2, and 0.1% SDS under oil in
a wet chamber that was cooled from 37 to 0 °C or from 65 to 10 °C
for 16 h. The parlon and mirlon RNAs were annealed at 53 °C for
16 h. Then to some samples 10 µl of S1 nuclease in
concentration from 0.1 to 5 units/µl were added in a solution
containing 0.3 M NaCl, 0.03 M Na acetate, pH
4.5, 3 mM ZnSO4, 0.5% glycerol, and 25 mg/ml
bovine serum albumin and digestion was performed for 40 min at
10 °C. To the samples not treated with S1 nuclease 10 µl of the
same solution were added. Incubation was performed at 10 °C for 40 min. The antiparallel parlon/mirlon probe was incubated at 37 °C for
40 min. Then 25 µl of 90% formamide containing 50 mM
EDTA and dyes was added. 1-mm thick denaturing 4% polyacrylamide gels
were run in the "Macrophor LKB System" at 65 °C. The samples before loading were heated at 100 °C for 2-3 min. After
fractionation the gels were dried and autoradiographed to observe the
fractionation of [33P]RNA.
Software Used--
RNA secondary structures constructions (Fig.
4C) were performed on the Genebee server. The longest
antiparallel stretches formed by the lon mRNA and
different transcript were selected. The Tm values of
the stretches were determined on the Virtual Genome Center server.
Relations between the lon mRNA and Two RNAs Expressed on
Symmetrical DNA--
To study the potential of pRNA in gene-specific
silencing we selected the lon gene from E. coli.
The gene specifies the ATP-dependent Lon protease that
plays the important role in the selective degradation of abnormal
proteins and limits the time of availability of critical regulatory
proteins (21). Fig. 1 shows the
relationships between the lon mRNA and short RNAs
expressed under control of the lac promoter on three
constructs in the pUC12 vector. One RNA, antilon, corresponds to the
341-nucleotide antisense RNA synthesized on the fragment of the
lon gene (bases 247-587, numberings here and then as
indicated in GenBank sequence accession number J03896). Earlier this
fragment was successfully used for potent and selective inhibition of
the lon gene expression by production of antisense RNA (18).
That is why in our experiments we used the same region in the antilon
construct as a positive control of inhibition by antisense RNA. The
antilon RNA can form antiparallel duplex with the mRNA. The second
RNA, parlon, has 95 nucleotides and is complementary in the same
polarity to the lon mRNA fragment from 369 to 463 nucleotides, and potentially might bind in parallel orientation with
the mRNA. The third RNA, mirlon, has inverted sequence
corresponding to the same 95-nucleotide lon mRNA region
and can form neither antiparallel nor parallel duplexes with the
lon mRNA. The parlon and mirlon RNAs could be
synthesized only on the heterologous DNA sequence possessing the mirror
inversion of nucleotide sequence corresponding to the selected region
of the gene. Such chemically synthesized DNA was inserted separately in
different orientations in the pUC12 vector (Fig. 1A). All
constructs were checked by sequencing. The constructs were introduced
by transformation in E. coli cells.
Gene-specific Silencing Tested Biochemically as Inhibition of
Selective Energy-dependent Degradation of Puromycin
Peptides--
The expression of the lon gene in the
transformants is tested by different approaches. The first one was
based on the measurements of the degradation of abnormal proteins,
synthesized in the presence of puromycin (20). The drug acts as an
analog of aminoacyl-tRNA and terminates the polypeptide chain. HB101
transformants containing the pUC12 plasmid or the mirlon construct
released up to 9% of the labeled peptides for 1 h (Fig.
2A). In the presence of
antilon or parlon RNA the peptides were degraded appreciably slower.
The lon
In the first experiments we used the chemically synthesized DNA
(synlon), corresponding to the 95-bp region of the lon gene, for the in vivo production of regular antisense RNA (not
shown). The letter exactly corresponds to the same region of the
mRNA as parlon RNA does (see Fig. 1B). As far as we
observed that parlon reveals better inhibition of the lon
gene than synlon, we decided to use the 341-bp region of the
lon gene from the pUC19AL-1 construct as a positive control
of inhibition (18). This region, including the 95-bp fragment, was
described as the most effective in the selective inhibition of the gene
by expression of antisense RNA (18). We found that the antilon
construct, possessing this 341-bp fragment, and the synlon plasmid
demonstrate the same inhibition of the lon gene activity and
that both are less effective than the parlon construct.
The higher effectiveness of the parlon transcript was independently
confirmed by direct isolation of the Lon protease that could be readily
isolated on the phosphocellulose (18) from equal amounts of cells
(Table I, Footnote a). In these
experiments the inhibition of the expression of the
ATP-dependent Lon protease by antilon or parlon RNAs was
observed.
To inhibit the ATP-dependent proteolysis the administration
of KCN to the cell suspension was performed as described earlier (18,
20). It was found that parlon and antilon RNAs affect only an
energy-dependent proteolysis of abnormal
3H-peptides and practically have no effect on the
degradation of normal 3H-proteins (Table I, Footnotes b and
c). These findings strongly suggest that both parlon and antilon RNAs
are mediators of a potent and specific repression of the target gene
expression, and parlon RNA is more efficient in this process.
Gene-specific Silencing in E. coli HB101 Cells Does Not Reduce the
mRNA Content--
It was demonstrated earlier, that RNAi
establishes an intracellular state leading to a specific decrease or
elimination of the corresponding eukaryotic mRNA (4, 6). To
elucidate a mechanism of the detected inhibition of the lon
gene expression, we performed Northern blot analysis. The same amounts
of mRNA were observed in all HB101 transformants (Fig.
2B). The method clearly can check the physiological decrease
of the lon mRNA to 16 S RNA. In different nontransformed
cells, incubated during the same time but grown to stationary state in
the absence of ampicillin, an appreciable decrease of the
lon transcripts was observed. The mRNA was absent only
in the lon-146 mutant (22) that was used as a negative
control. Thus, in our experiments the gene-specific silencing does not
induce an evident mRNA degradation. The Northern blot analysis also
demonstrates that parlon, mirlon, and antilon constructs are expressed
at the same level in the transformed cells (Fig. 2B). Thus,
the differences in the effects of parlon, mirlon, and antilon
constructs on the lon gene expression are not due to
differences in amounts of the corresponding RNAs in the transformed cells.
pRNA Action Tested Genetically as Inhibition of the Effect of the
Gene on the lux Regulon--
The last method that we have used for the
study of gene-specific silencing established by expression of pRNA was
based on an genetic approach allowing the observation of the
lon
Plasmid pAC16, containing the lux regulon was introduced in
both lon+ and lon
Fig. 3C shows that activation of the luminescence in this
system gradually decreases to zero up to the 40 generations. In separate experiments we found that this is not due to changes in the
constructs. The constructs isolated from cells after 50 generations are
capable of activating the luminescence after a new transformation (data
not shown).
Partial Nuclease Protection after Annealing of Parlon RNA and Sense
RNA in Vitro--
The observation of the readily reproducible
inhibition of the lon gene induced by expression of pRNA
encouraged us to study the possibility of formation of a parallel RNA
duplex between the parlon RNA and sense RNA in vitro by
nuclease protection assay. The 95-bp sequence of the lon
gene and the corresponding stretches in the parlon and mirlon
constructs are shown on Fig.
4A. The inserts from the
parlon and antilon constructs in the pUC12 vector were subcloned into
the pGEM vectors in the orientation allowing synthesization in
vitro with T7 RNA polymerase, the parlon, mirlon, or sense RNA
products (Fig. 4B). The correctness of the constructs was
confirmed by sequencing. To purify the 33P-labeled parlon
transcript from contamination with products of unintended synthesis on
the opposite strand due to ectopic transcription, the electrophoresis
in low gelling temperature agarose was performed.
The gel-purified 33P-parlon RNA was annealed alone or with
a 10-fold excess of 3H-labeled sense RNA in a wet chamber
by the temperature shift from 37 to 0 °C or from 65 to 10 °C for
16 h. In separate experiments we selected the condition for the
complete digestion of the 33P-parlon RNA preparation by
adding S1 nuclease containing solution (0.5 units/µl) to the
substrate in the annealing buffer (Fig. 5, lane 2). Annealing with the
sense RNA protected 33P-parlon RNA because the 70-bp band
and two smaller bands appeared after both temperature shifts and the
same digestion conditions (lanes 7 and 10). This
was not simply due to an increase of RNA in the probes and as a result,
partial digestion of single stranded 33P-parlon RNA. After
adding the same amount of Drosophila Kruppel transcript, as
was prepared and annealed in the same condition, no protection was
observed (lanes 8 and 11). Similarly, adding of
lon sense RNA without annealing also did not interfere with the complete digestion of 33P-parlon RNA (lane
13). It should be stressed that no protection after annealing of
33P-parlon RNA with the lon sense RNA was
observed with 2 units/µl of enzyme concentration (lane 5),
while at 0.1 unit/µl concentration the 134-bp full-length
33P-parlon RNA still remained (lane 3). The
duplex formed does not survive adding of 2 units/µl of S1 nuclease
and incubation at 10 °C for 40 min, while regular antiparallel
duplex, formed by the parlon and mirlon transcrips, are stable after
adding of 5 units/µl of the enzyme and incubation at 37 °C
(lane 12). In the latter case the full-length 124-bp
antiparallel duplex, including the attached polylinker sequence, was
observed. The full-length complementary region between the sense RNA
and parlon transcript is 95 bp long, but we have reproducibly observed
only smaller protected bands. Nevertheless, the observation of partial
protection after annealing of RNA molecules possessing complementary
bases in the same polarity, suggests both the formation of
non-canonical parallel duplex and its lesser stability compared with
the regular antiparallel duplex.
Fig. 4C presents the longest possible antiparallel stretches
between the lon mRNA and the parlon or mirlon
transcripts. The intermolecular duplex formed by the lon
mRNA with itself is longer and more stable. Even The only possible explanation of the data on the gene-specific
silencing observed in this study is the assumption that a parallel duplex could be formed between the lon mRNA and the
parlon transcript. The transcripts coming from mirlon construct or
pUC12 vector have two characteristics in common. They do not possess
complementary nucleotide texts neither in antiparallel nor in parallel
orientation with respect to the mRNA. Also, they do not induce
gene-specific silencing. Although both parlon and mirlon RNAs
potentially could form 8- or 9-bp long antiparallel stretches with the
mRNA, only the parlon transcript demonstrates a potent silencing
ability, while mirlon RNA produces no effect at all. Moreover, the
antilon transcript can form much longer antiparallel duplex with the
lon mRNA (about 340 bp), but reproducibly induces less
prominent effect than parlon RNA.
The assumption concerning a possibility that the parallel RNA duplex
formation was independently confirmed by in vitro studies. Our data show that only after annealing the parlon transcript with the
sense lon RNA could partial protection be observed. It was
difficult to select the condition for S1 analysis, because in this
particular case the interval in which the complete digestion of
single-stranded parlon RNA on one hand, and incomplete digestion of
protected regions on the other, is narrow. We believe, that this is a
case when testing aimed to reveal an object could disrupt it at the
same time. Obviously, this is due to a low stability of the duplex
formed. However, using a similar in vitro approach recently,
we observed clearly more stable and longer duplex formed between a pRNA
and a corresponding Drosophila sense
RNA.2 There is one general
argument in favor of the capability of RNA to shape a parallel duplex.
It is known from the in vitro experiments that DNA molecules
are physically capable of forming such structures (25, 26). There is
evidence suggesting that versatile RNA molecules possess a higher
plasticity (27). This view on RNA is clearly consistent with our data
obtained in vivo and in vitro.
The first experimental data demonstrating the possibility of parallel
RNA-RNA duplex formation at acidic pH were provided about 40 years ago
(28). It was concluded that the poly(A) duplex is stabilized by
protonated symmetrical self-pairs formed by hydrogen bonds between
N6 and N7. One more independent argument in
favor of formation of parallel RNA duplexes came from EM observations of different viral molecules forming dimers in the physiological condition by joining their non-poly(A) sequences close to the 5' ends
(29). Recently the formation of parallel RNA duplexes between the
molecules possessing alternating stretches of A and U bases was shown
in vitro at neutral pH (17). The data suggest that these
duplexes could be stabilized either by A-U bonds or A-A and U-U
self-pairs, and that the symmetric A-A pair is formed by two hydrogen
bonds between N1 and N6.
The gene-specific silencing produced by expression of pRNA, described
in this paper, strongly suggests that parallel RNA-RNA duplex also
could serve as a potent and specific signal for similar factors and
mechanisms as the regular dsRNA. It follows that exclusively the
involvement of mRNA in any kind of duplex, antiparallel or parallel, is the effector of the interference. Independently the capability of pRNA to serve as a trigger for gene-specific silencing was observed in experiments on Drosophila Kruppel
gene.2
It was convincingly demonstrated in C. elegans and
Drosophila that single stranded molecules of aRNA or sense
RNA are substantially less effective at producing interference than
in vitro annealed and injected dsRNA. Clearly, sense RNA
could induce the interference only if a preparation is contaminated
with dsRNA (1). Interestingly, after injections of equal masses of
dsRNA or purified aRNA corresponding to abundant mRNA, aRNA
exhibits much lower interference than dsRNA. These data could be
interpreted as a strong demonstration of the fact that the amount of
dsRNA formed in vivo after injection of purified aRNA is
low, and that annealing of 0.5-1 kilobase long RNA molecules in
vivo is really hampered.
Parallel duplexes are less stable and energetically more favorable for
apparent regulatory cellular mechanisms then antiparallel ones (30).
Thereafter, parallel RNA duplexes could be considered as more probable
candidates for physiological interference mechanisms. The data
presented here suggest at least two reasons in favor of this
conclusion. First, parallel RNA could be more potent in inducing
gene-specific silencing. Also, unstable parallel RNA duplex could be
readily formed under in vivo conditions. It cannot be
excluded that the observed higher activity of parallel RNA at producing
interference is due to a higher sensitivity of some cellular factors to
parallel RNA duplex than to antiparallel one.
Whatever mechanism of gene-specific silencing in E. coli is
operating, it does not give rise to degradation of mRNA. This result was not expected essentially because mRNAs in prokaryotes exhibit short half-lives when compared with eukaryotic mRNAs (31). The results presented here indicate that the block of translation plays
a role in the interference described here. Our data are clearly
consistent with the view of the important role of RNA duplexes for
regulation of gene activity in prokaryotes (16). The data on the
gradual fading of both types of gene-specific silencing observed by
expression of the parlon or antilon RNAs suggest that common
physiological mechanisms might be involved. We speculate that fading
might reflect the existence of some mechanism of physiological
adaptation that gradually switches off a gene silencing. The search of
mutants resistant to the detected effect of pRNA and aRNA on the
lon expression or to the fading effect, that we perform now,
may answer some questions arisen by this study.
The data presented in this study raise a question about the possible
role of symmetrical sequences in the modulation of gene expression. The
analysis of the nucleotide data bases revealed that in different
genomes there are a lot of diverged sequences possessing this kind of
symmetry (11). The sequences are about 50 bp in length and possess up
to 80% symmetric positions of the same bases. These data may indicate
that transcripts from symmetric sequences (mirror inversions of
nucleotide sequences) could potentially serve as molecules for
regulation of gene expression by physiological mechanisms. In any case,
the data presented here suggest that mirror inversions of nucleotide
sequences do possess biological activity.
Acknowlegments--
We thank Prof. A. A. Krayevsky for
discussions; Prof. G. P. Georgiev and Prof. V. A. Gvozdev for
support and encouragement; N. A. Ponomarenko for help in
preliminary experiments and K. S. Pustovoit for technical assistance.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside and puromycin were
added to 0.5 mM and 100 µg/ml, respectively, and after
incubation for 20 min [3H]leucine (120-190 Ci/mmol) was
added to 1 µCi/ml for 5 min. Then cells were centrifuged, washed, and
suspended in the same medium but containing 300 µg/ml unlabeled
leucine and a number of 0.5-ml aliquots were taken immediately (zero
point) and after 15, 30, 45, and 60 min of incubation. The samples were
precipitated by 7% trichloroacetic acid with 10% bovine serum albumin
for 30 min to determine the label of acid-soluble fraction. Protein
degradation was calculated as a percentage of acid-soluble fraction to
the total label incorporated in cells.
-D-galactopyranoside was added to 1 mM and incubation was continued to
A550 = 1.0. Then suspensions of cells were
diluted to the equal A550 and equal amounts of
cells were peletted, and isolation of the Lon preparations on PC
columns was performed as described earlier (18). Protein fractions
enriched with the Lon protease were used for testing of
ATP-dependent proteolysis and results were calculated as % of hydrolysis of [14C]acetylcasein for 1 h at
37 °C with 1 mg of the Lon preparations in the presence of 3 mM ATP (18).
20 °C. About 20 µg of RNA was run in 1.2% agarose gel. Hybridization experiments
were performed on identical blots separately with different
32P-labeled RNA probes (specific activities about
109 cpm/µg): with antisense RNA synthesized with T7 RNA
polymerase in vitro on the lon gene
PstI-HindIII fragment (583-1036 bp, GenBank accession number J03896) cloned in the pGEM-1 vector; with probe,
prepared by extension of the primer (5'-CCGGGTCGACTTAACGCGTTAGCTCC-3') by avian myeloblastosis virus reverse transcriptase on 16 S ribosomal RNA; with RNAs complementary to antilon, parlon, or mirlon transcripts synthesized with T7 RNA polymerase in vitro on the inserts
cloned in the pGEM-1 or pGEM-2 vectors. Hybridizations were performed in 5 ml of solution containing 50% formamide, 5 × SSC, Ficoll, polyvinylpyrolidone, bovine serum albumin (0.1% each) 0.1% SDS, denatured salmon DNA (50 µg/ml), denatured pUC12 DNA (20 µg/ml), tRNA (50 µg/ml), and 106 cpm of a probe. After
hybridization for 24 h at 43 °C the filter was washed 2 times
(20 min each) in 2 × SSC, 0.1% SDS solution at 43 °C, then 3 times at 65 °C in the same solution and finally 2 times at 65 °C
in 0.2 × SSC, 0.1% SDS.
AB1899 cells. The obtained
lon+ cells were transformed by the parlon
or antilon expressing constructs or by pUC12 vector and immediately
grown in L medium with 200 µg/ml ampicillin (100 transformed
cells/ml) at 28 °C. 200 µl of suspension was mixed with 4 µl of
0.001% n-decanal in ethanol for 0.5-1 min before measuring
bioluminescence. Then the samples were transferred to the luminometer
(consisting of a FEU-85 photomultiplier and a V2-15 microvoltmeter) in
the special cells and the luminescence was measured in µv (1 µv = 107 quants/s).
-33P-labeled UTP (4000 Ci/mmol), or 50 µM 3H-labeled ATP
(27 Ci/mmol), and 20-30 units of T7 RNA polymerase. Specific
activities of 33P- and 3H-labeled RNAs were
about 4.2 × l07/µg and 1.6 × l06
cpm/µg, respectively. For purification of
[33P]RNA the electrophoresis in low melting temperature
agarose gels containing methylmercuric hydroxide was performed followed
by dithiothreitol treatment, short exposition, and isolation of RNA from a gel slice by heating on 0.5 M ammonium acetate,
phenol/chloroform extraction, and ethanol precipitation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
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Fig. 1.
The relations between the lon
mRNA and RNAs expressed by the constructs. A,
the relations between the lon gene and synthesized DNA,
inserted in both orientations into the pUC12 vector under control of
the lac promoter. B, the sizes of RNA molecules
are not shown to scale. The sequences with gaps around
triplets refer to complete texts of the parlon and antilon RNAs
corresponding to the mRNA. In the lon mRNA the
corresponding region possesses the 5' non-coding sequence of 44 bases
in length, initiation codon (shown as triplet with the gaps around) and
the coding region specifying the following 16 amino acids. Texts under
the mRNA and antilon RNA are restricted only to sequences
corresponding to the parlon and mirlon RNAs. The axis shows
the symmetry of nucleotide ordering between the fragment of mRNA
and mirlon RNA.
cells served as a negative control,
demonstrating the effect of other proteases. The relative % of the Lon
activity in different transformants were calculated. Up to 28 and 10%
of the Lon activity were observed upon expression of the antilon and
parlon RNAs, respectively (Fig. 2A). Surprisingly, parlon
RNA was more effective at producing the interference that antilon
RNA.
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Fig. 2.
Analysis of the lon gene
expression in HB101 transformants. A, degradation of
puromycin 3H-peptides. The cells were treated with
[3H]leucine for 5 min in the presence of puromycin. The
label was removed and protein degradation was measured in aliquots as
percent of the acid-soluble fraction to the total label incorporated in
cells as described earlier (18, 20). B, analysis of the
lon mRNA content in HB101 transformants and in some
nontransformed cells by Northern blotting and hybridization with the
lon antisense [32P]RNA probe. Relative levels
of the 16 S RNA, antilon, parlon, and mirlon transcripts were
determined by hybridization with different probes (see "Experimental
Procedures"). The numbers on the left indicate
the lengths in kilobase.
Testing of the energy dependence of proteolysis
phenotype easily. It was found earlier
that the Lon protease is a negative regulator of the lux regulon,
introduced in E. coli cells from Vibrio fischeri
(23, 24). Inhibition of the lon gene expression considerably
increases the bioluminescence provided by luciferase since the regulon
activator, LuxR, is not attacked by the protease (Fig.
3A).
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Fig. 3.
Effect of the constructs on the
lon expression in E. coli cells
possessing the lux regulon. A, scheme
showing the regulon genes (not in scale). Promoters
pr and pl are located on
different strands. Genes I, C, D, A, B, and E
specify proteins essential for the luminescence. B,
activation of luminescence in the lon+ AB1157
cells by constructs expressing the parlon or antilon RNAs (presented as
luminometer data in µv against A550, see
"Experimental Procedures"); the lon AB1899
strain was used as a control. C, fading of the luminescence
in the lon+ AB1157 cells up to 40 generations.
cells. The resultant lon+ cells were
transformed separately by the constructs expressing parlon RNA, antilon
RNA, and pUC12 vector. The biochemical approach for testing the
lon expression by the degradation of puromycin peptides
allows one to measure the Lon activity only after 20-23 generations
after transformation. The sensitive bioluminescence method allows one
to check the inhibition of the lon activity much earlier,
after 12-15 generations. We observed that the luminescence of the
lon+ cells expressing the parlon or antilon RNAs
was increased 20 and 350 times, respectively, in comparison with the
luminescence of the pUC12 containing lon+ cells
(Fig. 3B). The luminescence of the
lon cells was much higher. Nevertheless, the
data strongly suggest the repression of the lon gene
triggered by the parlon or antilon RNAs. The AB1157
lon+ transformants expressing the parlon RNA
after growth on plates at 26 °C reveal the characteristic property
of the lon cells and form rather mucous
colonies. Thus, parlon RNA expression specifically induces changes in
the phenotype of the lon+ host cells. These
results are in good agreement with the data obtained by the biochemical approaches.
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Fig. 4.
Texts of artificial sequences corresponding
to the fragment of the lon gene and the
pGEM-constructs used for in vitro experiments.
A, relations between the fragment of the lon
gene, parlon, and mirlon sequences used in the constructs. The
numbers above the fragment of the gene refer to numbers in
GeneBank accession number J03896. B, the schematic
presentation of the constructs used for in vitro synthesis
of the 33P-labeled parlon transcript or the
3H-labeled mirlon and sense RNAs. The constructs were
linearized by restriction enzymes indicated on the scheme and used for
RNA synthesis by T7 RNA polymerase. The parlon and mirlon RNAs possess
95-nucleotide sequences symmetric to the lon gene sequence
and the attached sequences coming from the vectors. C, the
longest antiparallel stretches that could be formed between the
lon RNA and the transcripts derived from the constructs
in vivo or in vitro.
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Fig. 5.
Nuclease protection assay.
33P-Parlon RNA was annealed alone (lanes 1-3)
or after adding of the sense RNA (lanes 4-7, 9, and 10) in 6 µl of the solution by temperature shifts
(37-0 °C or 65-10 °C) for 16 h. Then 10 µl of the S1
solution were added in the concentrations as indicated. After
incubation the probes were denatured and run on denaturing 4%
polyacrylamide gel. In the probes shown on lanes 8 and
11, the sense RNA corresponding to the fragment of
Kruppel cDNA was added instead of the lon
RNA. Lane 12 presents the result of annealing antiparallel
parlon and mirlon transcripts. Lane 13 shows the digestion
of the mixed parlon and sense lon transcripts at 0 °C
without incubation followed by S1 digestion. The numbers
show marker (M) positions of pBR322-HinfI DNA
fragments (left) or protected RNA bands (right).
The 124-bp band corresponds to the full-length antiparallel region
formed by parlon and mirlon RNAs containing the 95-bp stretch of the
construct and the adjacent regions from the pGEM vectors.
-lactamase
mRNA, that confers the resistance to ampicillin to the vector,
could form the 9-bp antiparallel stretch with the lon
mRNA. It follows that short antiparallel duplexes should not
interfere in both the in vivo and in vitro studies described in this work.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
FOOTNOTES |
|---|
* This work was supported by Russian State Program "Frontiers in Genetics" Grant 99-1-085 and Russian Foundation for Basic Research Grants 97-04-49897 and 99-04-48065).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) J03896.
§ To whom all correspondence should be addressed. Tel.: 7-095-1359753; Fax: 7-095-1351405; E-mail: tchur@imb.ac.ru.
Published, JBC Papers in Press, June 9, 2000, DOI 10.1074/jbc.M002833200
2 N. A. Tchurikov, N. G. Shostak, O. V. Okladnova, and B. K. Chernov, manuscript in preparation.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: dsRNA, antiparallel double-stranded RNA; pRNA, RNA possessing complementary bases in the same polarity to mRNA; parlon, construct or its transcript possessing complementary bases in parallel orientation to a fragment of the lon mRNA; mirlon, construct or its transcript possessing mirror ordering of bases in respect to a fragment of the lon mRNA; antilon, construct or its transcript comprising the lon antisense RNA; RNAi, RNA interference; bp, base pair(s).
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
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