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J Biol Chem, Vol. 274, Issue 29, 20688-20692, July 16, 1999
From the Rat liver perchloric acid-soluble protein (L-PSP)
is a potent inhibitor of cell-free protein synthesis; however, its
mechanism of action is not known. Here we show that the protein is a
unique ribonuclease and that this activity is responsible for the
inhibition of translation. The addition of perchloric acid-soluble
protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 µM led to an almost complete inhibition of
protein synthesis. The kinetics are unlike those of hemin-controlled
inhibitor, a protein that acts at the initiation step. The inhibition
appears to be due to an endoribonucleolytic activity of perchloric
acid-soluble protein because L-PSP directly affects mRNA template
activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by
thin-layer chromatography of [ Rat liver perchloric acid-soluble protein
(L-PSP)1 is a 136-amino acid
protein that inhibits protein synthesis (1). Oka et al. (1)
demonstrated that L-PSP, when added to a rabbit reticulocyte cell-free
system, causes inhibition of a biphasic kinetic nature and also leads
to the disaggregation of polysomes. This would be similar to the mode
of inhibition of translation by the heme-regulated eukaryotic
initiation factor 2 A 14-kDa translational inhibitor protein remarkably similar to L-PSP
has been characterized in human monocytes and mouse liver (3-5). A
homology search revealed that these proteins belong to a new group of
small proteins named the YER057c/YJGF family (3), which is of unknown
physiological function. The protein sequences of these family members
are highly conserved in prokaryotes (including cyanobacteria), fungi,
and eukaryotes, suggesting that the proteins may be involved in a basic
cellular process. Indeed, mRNA of the translational inhibitor
p14.5, the human homologue of L-PSP, becomes significantly up-regulated
with the induction of differentiation to macrophages (3), and the
synthesis of PSP from rat kidney increases from the 17th fetal day to
the fourth postnatal week and then enters a steady-state level (6). In contrast, the expression of PSP from rat kidney in renal tumor cells
was down-regulated (6).
Recently, Schmiedeknecht et al. (7) have identified the
functional promoter of the human p14.5 translational inhibitor. They
reported a head-to-head orientation of p14.5 with the gene for the
protein subunit hPOP1 of RNase P and with RNase MRP ribonucleoproteins; the promoter region between p14.5 and hPOP1 acts as a bidirectional promoter. Because bidirectional transcription units commonly encode proteins that are different in structure but have similar biological function (8, 9), the authors suggested that the p14.5-hPOP1 cluster may
encode functionally related proteins as well.
As the first step in our endeavor to understand the physiological role
of the PSP proteins, we studied the mechanism of action of the
translational inhibitor L-PSP.
General--
The following procedures were either described or
cited previously (10-12): preparation of rabbit reticulocyte lysate,
sucrose density gradient analysis of polysomes, preparation of
ribosomes, extraction of RNA with phenol and SDS, preparation of
plasmid pBR322, analysis of the nucleic acids by polyacrylamide gel
electrophoresis, separation of nucleotides by thin-layer
chromatography, the methods used for the sequencing of 5 S rRNA, and
the source of materials such as
L-[U-14C]leucine, human placental
ribonuclease inhibitor (133 units/ml), [ Preparation of Authentic and RecombinantL-PSP--
Both forms
of L-PSP were purified as described previously (1, 13). Briefly,
the 5% perchloric acid-soluble fraction of a rat liver lysate was
loaded onto CM-Sephadex C-25, and pure L-PSP was recovered in the
flow-through fraction. Recombinant PSP was expressed in
Escherichia coli as a fusion protein with glutathione
S-transferase using plasmid pGEX-PSP. The fusion protein was
purified from the extract with glutathione-Sepharose 4B and then
incubated with thrombin, and recombinant L-PSP was isolated using the
same affinity column as described above. The recombinant protein was
confirmed to be L-PSP by its mobility on SDS-polyacrylamide gels and by
direct sequencing of the eight N-terminal amino acids. Furthermore, the
recombinant protein and authentic L-PSP were immunologically identical.
Protein Synthesis Inhibition Assay--
The standard cell-free
protein synthesis system derived from rabbit reticulocyte lysate (10)
contained the following in a final volume of 30 µl: 15 µl of
micrococcal nuclease-treated lysate (containing 25 µM
hemin), 25 mM Hepes (pH 7.6), 2 mM
dithiothreitol, 1 mM ATP, 20 µM GTP, 8 mM creatine phosphate, 1.2 µg of creatinine phosphokinase, 25 µM of each of the 20 amino acids, 0.1 µCi of L-[U-14C]leucine (13.3 mCi/mmol), 90 mM potassium acetate, 1 mM magnesium acetate,
0.6 mM spermidine, and 2 µg of mRNA. Endogenous
globin synthesis was done under the same conditions as described above using lysate that was not treated with micrococcal nuclease.
Synthesis of
[32P]mRNA--
mRNA having the cap
structure was synthesized with [ L-PSP inhibits protein synthesis at the elongation step rather
than the initiation step. We started with experiments to confirm that
L-PSP inhibits protein synthesis with a similar mode of action as
heme-regulated eukaryotic initiation factor 2 An interesting and probably important observation here is that the
kinetics of the inhibition is monophasic in both systems, rather than
biphasic, as reported previously (1). In fact, careful examination of
the earlier data does not reveal an inhibition lag, and the discrepancy
may stem from an incorrect explanation of the kinetic data. Inhibition
of translation initiation by proteins such as heme-regulated eukaryotic
initiation factor 2 In experiments that used higher amounts of mRNA than the standard
reaction, the inhibitory effect on protein synthesis tended to be less.
For instance, when mRNA was 20 µg instead of 2 µg in a 30 µl
system, 20 nM L-PSP did not show significant inhibition (data not shown). To gain more insight into the mode of action of this
small protein, we examined the effect of preincubation of mRNA with
L-PSP. For these experiments, mRNA was first incubated with a low
amount of L-PSP for a prolonged time, and then the activity of the
mRNA of a small portion of the preincubation reaction mixture was
tested in the standard translation system. This strategy minimizes the
effect of L-PSP on the translation system because the concentration of
L-PSP in the translation reaction mixture is very low. As shown in Fig.
1C, preincubation of mRNA with L-PSP results in a
significant decrease of protein synthesis. Recombinant L-PSP affected
the template quality of mRNA in a similar manner, making it
unlikely that the observed inactivation of mRNA was due to
contaminants in the L-PSP preparation. However, the activity on
mRNA of authentic L-PSP differed significantly from that of recombinant PSP, as had been found previously (13). Although we cannot
be certain, the difference is likely to be due to a loss of activity
because of the expression of recombinant PSP in a prokaryotic system. A
similar but even larger discrepancy has been reported for the human
homologue of PSP, p14.5: the authentic form is more active than the
recombinant form by 3 orders of magnitude (3).
Nevertheless, the results indicate that the main target of the protein
is mRNA rather than the translation system. There are at least two
alternative mechanisms for mRNA inactivation: 1) L-PSP might
specifically modify mRNA at its 5'-untranslated region, including
the cap structure (m7GpppG) that is nearly essential for
the initiation reaction, or 2) the protein might degrade mRNA nucleolytically.
To test the alternative possibilities, we performed experiments using
sucrose density gradient centrifugation to investigate polysome
profiles in the presence of cycloheximide. Incubation of the globin
synthesizing lysate system with L-PSP resulted in a disaggregation of
polysomes into 80 S ribosomes (Fig.
2A), supporting the earlier
result (1). The addition of the ribonuclease inhibitor at the start of
the incubation did not prevent polysome disaggregation. An important
feature of this experiment was that both the incubation and the
analysis were performed in the presence of a low concentration of
cycloheximide at which the antibiotic freezes the elongation reaction
but not initiation. Polysome disaggregation in the presence of
L-PSP demonstrates that the protein does not affect the initiation process. Rather, the results strongly suggested that L-PSP
disintegrates polysomes through fragmentation of polysomal mRNA
because of ribonuclease activity.
Ribonuclease Activity of Rat Liver Perchloric Acid-soluble
Protein, a Potent Inhibitor of Protein Synthesis*
,,,,,¶
Department of Applied Chemistry, Faculty of
Engineering, Ehime University, Matsuyama 790-8577, Japan and
§ Department of Veterinary Physiology, Faculty of
Agriculture, Kagoshima University, Kagoshima 890-0065, Japan
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES
-32P]UTP-labeled
mRNA incubated with the protein showed production of the
ribonucleoside 3'-monophosphates Ap, Gp, Up, and Cp, providing direct
evidence that the protein is an endoribonuclease. When either 5'- or
3'-32P-labeled 5 S rRNA was the substrate, L-PSP cleaved
phosphodiester bonds only in the single-stranded regions of the molecule.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
kinase (2) (however, see our results below).
Based on these data, it was suggested that the protein inhibits the
initiation step rather than the elongation step (1).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]UTP, and
SP6 RNA polymerase.
-32P]UTP and the four
nucleotide triphosphates using phage SP6 RNA polymerase and linearized
plasmid pSP65 containing the gene coding for E. coli form 1 dihydrofolate reductase as template (14). The transcript is 1079 nucleotides long and consists of a coding region of 477 nucleotides
close to the 5' end and a 3' noncoding region of 565 nucleotides.
RESULTS AND DISCUSSION
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kinase, as reported previously (1). Incubation of micrococcal nuclease-treated rabbit
reticulocyte lysate containing exogenous capped mRNA encoding dihydrofolate reductase and all other components necessary for protein
synthesis at 30 °C led to the incorporation of
[14C]leucine into newly synthesized proteins. In
agreement with the previous report (1), the addition of L-PSP to the
system resulted in inhibition of protein synthesis in a
concentration-dependent manner (Fig.
1A). The estimated
IC50 (51 nM) was in the same range as the
previously reported value (1). A L-PSP concentration of 1 µM almost completely abolished the capacity of the
cell-free system to support protein synthesis. However, when similar
experiments were done using endogenous globin mRNA, inhibition by
L-PSP appeared to be about 10 times less efficient (IC50,
0.68 µM) (Fig. 1B). The observed difference in
the IC50 can be ascribed to the character of the two
translation systems: in the latter system, a large population of
ribosomes is already engaged with globin mRNA (as polysomes) at the
start of incubation, whereas practically no mRNA is associated with
ribosomes in the former system. Thus, the results suggested that the
lysate containing a higher proportion of polysomes was less susceptible
to L-PSP.
View larger version (16K):
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Fig. 1.
Inhibition of cell-free protein synthesis by
L-PSP. Exogenous mRNA programmed lysate (A) or
globin synthesizing lysate (B) was incubated without or with
the indicated concentration of L-PSP. In C, 30 µg of
mRNA were incubated in a 30-µl reaction volume with 20 nM L-PSP or recombinant L-PSP for 3 h, and aliquots
containing the same amounts of mRNA were then added to the
transcription reaction. In these reactions, the final concentration of
L-PSP was about 1.3 nM. Reactions were done at 30 °C,
and 5-µl aliquots were removed at the specified times, spotted on
paper filters, and assayed for [14C]leucine incorporation
into hot trichloroacetic acid-insoluble material.
kinase shows biphasic kinetics (2). However,
this typical biphasic shape, in which protein synthesis proceeds at the
initial rate for several minutes before an abrupt decline occurs, can
only be expected when the globin-synthesizing system is used because only this system has an initial rate (run-off of polysomes). Obviously, inhibitors of initiation show monophasic kinetics of translation inhibition in the exogenous mRNA programmed system, which has no
initial rate because there is no initial protein synthesis. The
inhibition of protein synthesis by L-PSP was not prevented by the
ribonuclease inhibitor from human placenta that tightly binds and
inhibits the activity of the ribonuclease A family (data not shown)
(15).
View larger version (41K):
[in a new window]
Fig. 2.
Effect of L-PSP on polysome profiles and
degradation of mRNA. A, translation mixtures (30 µl) prepared with endogenous globin synthesizing lysate were
incubated in the presence of cycloheximide (0.25 µM) at
30 °C for 15 min without ( 
) or with L-PSP (2.1 µM)
(
) or with L-PSP together with human ribonuclease inhibitor (266 units) (
). Samples were analyzed on a linear 10-45% sucrose
gradient, and 20 drops were collected from the bottom of the tube and
used for absorbancy measurement at 254 nm. B, translation
mixtures (30 µl) composed of micrococcal nuclease-treated lysate,
[32P]mRNA (0.5 µg; 10,000 cpm), and 266 units of
human ribonuclease inhibitor were incubated in the absence (lanes
1-4) or in the presence (lanes 5-8) of L-PSP (2.1 µM) at 30 °C for the indicated periods of time. RNA
was extracted and separated on a 4% polyacryamide gel containing 7 M urea, and a radioautograph of the gel was prepared.
Ribonucleolytic Activity of L-PSP-- To obtain direct evidence, micrococcal nuclease-treated rabbit reticulocyte lysate was incubated as described above with 32P-labeled dihydrofolate reductase mRNA in the presence of both the ribonuclease inhibitor and L-PSP. After incubation for various periods of time (Fig. 2B), RNA was extracted and separated on polyacrylamide gels. Consistent with our hypothesis, the radioautograph shows intensive digestion of mRNA into small fragments in the presence of L-PSP, whereas the mRNA was fairly stable in the control sample. A similar digestion pattern of the mRNA was observed when the incubation was carried out in the absence of the ribonuclease inhibitor (data not shown). The amount of the ribonuclease inhibitor (266 units) used in Fig. 2, A and B, inhibits the activity of 1.33 µg of ribonuclease A by 50%, whereas the amount of L-PSP was 0.89 µg in a 30-µl reaction mixture. The result excludes the possibility that the inhibition by L-PSP is due to a contamination with ribonuclease A or other A-like ribonuleases, RNases known to be abundant in both animal tissues and E. coli. All of these results show that L-PSP is a ribonuclease that hydrolyzes phosphodiester bonds of RNA in an endonucleolytic fashion.
We next determined the substrate specificity of the enzyme, and the
results are shown in Fig. 3A.
The [32P]mRNA was incubated with L-PSP,
and then the sample was separated by thin-layer chromatography.
Subsequent autoradiography showed the production of the four 3'
nucleotide monophosphates as identified by comparison with standard
nucleotides obtained from ribonuclease T2 digests. The
results are conclusive: L-PSP cleaves the phosphodiester bonds of all
four nucleotides, yielding 3'-AMP, 3'-GMP, 3'-UMP, and 3'-CMP. In
addition to the four discrete spots, some streaks are also seen in Fig.
3A that may represent residual small oligomers accumulating
under the digestion conditions. Practically the same digestion pattern
of the RNA was obtained using recombinant L-PSP (data not shown). By
measuring the radioactivity of the spots, we determined the relative
catalytic activity of L-PSP for the various substrates:
Np/A32p/U = Np/G32p/U = Np/U32p/U > Np/C32p/U (slashes indicate
the sites of hydrolysis). The mechanism of cleavage probably involves
2',3'-cyclic phosphate intermediates.
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Because there are nucleases that hydrolyze both RNA and DNA (16), we determined whether L-PSP has any deoxyribonuclease activity. We chose pBR322 as the substrate for the test because a single nick in the circular supercoiled DNA of the plasmid produces a change in conformation that alters its mobility in agarose gels. Prolonged incubation of the plasmid with L-PSP (2.1 µM) under conditions similar to those used for the RNA substrate did not result in any significant effect on the integrity of the plasmid (data not shown). Thus, we conclude that the protein does not have deoxyribonuclease activity.
We next determined whether L-PSP is specific for single- or
double-stranded regions of RNA. We chose rat liver 5 S rRNA as substrate for the test because 1) the secondary structure is known, and
2) the RNA has a highly ordered structure (17). 5 S rRNA was labeled at
its 3' end with ([5'-32P]pCp or at its 5' end
with [
-32P]ATP, and the sample was treated under
nondenaturing conditions with either authentic PSP from rat liver or
recombinant PSP. An alkaline digest and T1 digests were
analyzed together with the products of the protein treatment on a 10%
polyacrylamide sequencing gel (Fig. 4).
Both L-PSP preparations cleaved only in single-stranded regions of the
molecule. Once again, recombinant L-PSP was about four times less
active than authentic L-PSP. The addition of the ribonuclease inhibitor
to the reaction mixtures did not alter the digestion pattern (data not
shown). The results clearly indicated that L-PSP itself is a
ribonuclease with characteristics unique among liver RNases.
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Effect of L-PSP on other RNAs--
Other ribonucleases with unique
properties are known: the cytotoxin -sarcin and related proteins
(12), colicin E3 and related bacteriocins (18), and angiogenin, which
is important for angiogenesis (19). -Sarcin is a basic 17-kDa
protein produced by the mold Aspergillus giganteus. The
basis of the action of the protein is an inhibition of protein
synthesis caused by the inactivation of ribosomes. Although there are
5000-7000 nucleotides in ribosomes, -sarcin hydrolyzes only a
single phosphodiester bond between G4325 and A4326 of 28 S (or 23 S)
rRNA. This cleavage inactivates the ribosome and is entirely
responsible for the toxicity of the protein (11, 12, 20). Having
established that L-PSP acts on mRNA, we further examined whether
the protein attacks other RNA molecules. A micrococcal
nuclease-untreated cell-free system was incubated with L-PSP as
described for Fig. 2A, and total RNA was extracted. Samples
were separated on agarose and polyacrylamide gels, and RNA bands were
visualized with ethidium bromide (data not shown). Despite a careful
examination of the bands, we did not observe any significant
degradation of rRNA nor tRNA at 1.5 µM of the authentic
protein (which has an IC50 of 0.68 µM).
However, when similar experiments were done with higher concentrations (>2 µM) of the protein, significant fragmentation of
rRNA could be observed, which supports the notion that L-PSP is a
ribonuclease. These results eliminate the possibility that L-PSP
inhibits protein synthesis by cleaving rRNA or tRNA. We thus conclude
that L-PSP inhibits cell-free protein synthesis by cleaving mRNA.
These results may have an important bearing on the physiological role
of L-PSP and its related proteins.
Finally, it may be worthwhile to mention here that besides the lack of
significant sequence homology with other ribonucleases, L-PSP also
lacks histidine residues (1). Histidine is known to be the
indispensable general acid in the catalytic activity of other
ribonucleases (21, 22). Recently, another ribonuclease that lacks
histidine has been reported (23): the C-terminal domain of the
bacteriocin colicin E5 inhibits bacterial protein synthesis in
vitro by cleaving several tRNAs at a specific site, the 3' side of
the queosine nucleotide in the anticodon loop. The cytotoxin recognizes
the same site even if unmodified tRNA is the substrate, in which case,
cleavage occurs 5' side of the guanosine nucleotide that is at the
queosine position. The proposed mechanism of cleavage involves a
2',3'-cyclic phosphate intermediate, but the exact enzymatic mechanism
is unknown. A computer-aided homology search between L-PSP and the
colicin E5 peptide revealed a short sequence shared by both enzymes,
NDFGTV (amino acids 87-92) in L-PSP and NDPATV (positions 84-89 of
the 115-amino acid C-terminal domain) in E5, but the functional
significance of these hexapeptides remains to be seen.
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FOOTNOTES |
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* This work was supported by The Japan Society for the Promotion of Science Grant JSPS-RFTF 96100305 (to Y. E.).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.
¶ To whom correspondence should be addressed. Tel.: 81-89-927-9936; Fax: 81-89-927-9941.
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ABBREVIATIONS |
|---|
The abbreviations used are: L-PSP, rat liver perchloric acid-soluble protein; PSP, perchloric acid-soluble protein.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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