From Natural Sciences/Biology, York College of the City University
of New York and the City University Graduate School,
Jamaica, New York 11451
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ABSTRACT |
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Drosophila RNase P and 3-tRNase
endonucleolytically process the 5
and 3
ends of tRNA precursors. We
examined the processing kinetics of normal substrates and the
inhibitory effect of the tRNA product on both processing reactions. The
product is not a good RNase P inhibitor, with a KI
approximately 7 times greater than the substrate KM
of ~200 nM and is a better inhibitor of 3
-tRNase, with a
KI approximately two times the
KM of ~80 nM.
We generated matrices of substitutions at positions
G18/U55 and G19/C56
(two contiguous universally conserved D/T loop base pairs) in Drosophila tRNAHis precursors. More than half
the variants display a significant reduction in their ability to be
processed by RNase P and 3-tRNase. Minimal substrates with deleted D
and anticodon stems could be processed by RNase P and 3
-tRNase much
like full-length substrates, indicating that D/T loop contacts and D
arm/enzyme contacts are not required by either enzyme.
Selected tRNAs that were poor substrates for one or both enzymes were further analyzed using Michaelis-Menten kinetics and by structure probing. Processing reductions arise principally due to an increase in KM with relatively little change in Vmax, consistent with the remote location of the sequence and structure changes from the processing site for both enzymes. Local changes in variant tRNA susceptibility to RNase T1 and RNase A did not coincide with processing disabilities.
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INTRODUCTION |
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tRNAs are small (~76
nt)1 molecules that possess a
conserved cloverleaf secondary structure (1) and a compact L-shaped
tertiary structure stabilized by numerous contacts (2). tRNA precursors undergo endonucleolytic end processing and modification before their
aminoacylation and use in translation (for reviews, see Ref. 3). An
additional G is added to the 5 end of tRNAHis (4), and CCA
is added to the 3
end of all eukaryotic tRNAs (5) following the RNase
P and 3
-tRNase reactions in vivo.
Several post-transcriptional tRNA processing reactions have been used
to evaluate the relation between primary, secondary, and tertiary
structure in recognition and catalysis. RNase P, a ribozyme (6),
recognizes internal tRNA tertiary structure; enzyme-substrate base
pairs which are important in RNase P catalysis have also been
demonstrated between tRNA nucleotides C74 and
C75 and Escherichia coli M1 RNA nucleotides
G293 and G292 (7). Although not directly
relevant to eukaryotic tRNA end processing reactions (eukaryotic tRNA
genes lack a transcriptionally encoded 3-CCA), the observation of base
pairing between enzyme and substrate nonetheless raises the possibility
of direct readout of internal substrate sequence, either by RNA or
protein in the processing enzymes.
None of the positions where tRNA sequence varies (8), however, could be
sequence-specific substrate determinants for recognition or catalysis
by general tRNA processing enzymes such as RNase P and 3-tRNase. The
universally conserved, paired nucleotides G18/
55, G19/C56 in
the tRNA D and T loops are good candidates for direct readout. These
base pairs also stabilize the tRNA tertiary fold. Herein, we examine
the effects of substitutions in these D and T loop nucleotides.
tRNA tertiary contacts have recently been analyzed using substitutions
in studies of aminoacylation (9-11) and of suppressor tRNA function
(12). Interestingly, the first C of the 3-CCA pairs with conserved
G2252 in the peptidyltransferase center of 23 S ribosomal
RNA, as proven by partial matrix analysis (13).
We have studied the 5 and 3
end endonucleolytic processing of
Drosophila tRNAHis precursors (14). In the
present report, we investigate processing kinetics and the inhibitory
effect of the tRNA product on the RNase P and 3
-tRNase reactions. We
generated matrices of substitutions in the conserved nucleotides that
stabilize the D/T loop tertiary interaction; most of these variants
display reduced processing by RNase P and/or 3
-tRNase. Mini-tRNAs with
deleted D and anticodon arms could be processed much like wild type
tRNAs by both RNase P and 3
-tRNase, demonstrating the absence of
required substrate contacts by either enzyme within these regions. By
structure probing, we establish that substrate defects do not generally
coincide with the disruption of tertiary contacts. Structure modeling
the D and T loop substitutions suggests that local perturbations may have occurred in the variants.
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EXPERIMENTAL PROCEDURES |
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Cell Culture, Extract Preparation, and Fractionation of RNase P
from 3-tRNase--
Drosophila Kc0 cells
adapted to serumless growth were cultured and harvested, and S100
extracts were prepared as described previously (15). RNase P and
3
-tRNase in the S100 were separated by S-Sepharose column
chromatography (14). The 50 mM KCl S-Sepharose flow-through
containing the 3
-tRNase and the 200 mM KCl fraction containing RNase P were stored in aliquots at
70 °C and used in processing reactions for up to 6 months without loss of
activity.
Substrate Construction and Mutagenesis--
The initial
Drosophila tRNAHis template linked to a T7 RNA
polymerase promoter was constructed using overlapping oligonucleotides (14) and subcloned into the small high copy number vector PHC624 (16)
for propagation and into SK (Stratagene) for mismatch
mutagenesis (17). The template for preparation of RNase P substrates
has an inserted 16-nt 5
end leader and an NsiI runoff site
at A73 (Fig. 1B). For preparation of 3
-tRNase
substrates, T7 transcription initiates at +1 and runs off at a
DraI site, including a 36-nt 3
end trailer (Fig.
1C). To investigate further possible inhibitors, we prepared
the 72-nt product of both tRNA end processing reactions by introducing
an NsiI runoff site at +73 into the template for the
3
-tRNase substrate.
Preparation of RNAs for Processing and Structure
Probing--
Internally labeled RNAs were prepared by transcription
with T7 RNA polymerase using [-32P]UTP and three
unlabeled NTPs (15). Templates for transcription were obtained by
digestion with NsiI (New England Biolabs) to obtain an RNase
P substrate or with DraI (Amersham Corp.) to obtain a
3
-tRNase substrate. Runoff transcripts were gel-purified, extracted by
a cut-and-crush procedure, and recovered for processing reactions.
Processing Reactions--
RNase P reactions were performed using
a 1/10 dilution of the S-Sepharose 200 mM KCl fraction
incubated with the internally labeled RNase P substrate (Fig.
1B) in a volume of 60 µl containing 30 mM
K-Hepes, pH 8, 175 mM KCl, 3 mM
MgCl2, 3 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, 0.1% Tween 20, 10% glycerol, and 1 µl of RNasin (40 units; Promega) at 28 °C. Reactions were sampled
after 0, 15, 30, 60, and 120 min, recovered, and analyzed on 6%
denaturing polyacrylamide gels. Results were obtained by autoradiography. 3-tRNase reactions were performed similarly except
that the S-Sepharose flow-through was the source of the enzyme; the
3
-tRNase substrate (Fig. 1C) was processed, and the reaction buffer contained 50 mM KCl.
Quantitation--
Microdensitometry was performed on all lanes
of the autoradiograms using an LKB XL laser microdensitometer. The
processing product of both RNase P and 3-tRNase is a 72-nt tRNA
which runs from G1 to A73 (Figs. 1-4). Bands
above and below the 72-nt product were not scored. These quantitation
procedures using a limiting amount of labeled substrate (14) yielded
first-order processing rate constants (Vmax/KM) relative to wild
type (bottom of Figs. 3 and 4); processing reactions with
variants and wild type were performed in parallel on the same day to
correct for possible day to day differences in enzyme activity or in
the preparation of processing buffer. The lower limit for detection of
processing using the gel assay is less than 1% of the wild type
rate.
Structure Probing--
Recovered end-labeled tRNAs were heated
to 70 °C for 5 min in distilled H2O and refolded by
cooling to room temperature for 10 min in 15 mM K-MOPS, pH
7, 175 mM KCl, and 3 mM MgCl2
containing 1.25 µg of unlabeled tRNA per 5-µl reaction. Samples
were incubated for 5 min at room temperature with RNase T1 at 0.4 or 1 unit/ml or with RNase A at 1.5 or 4 × 103 units/ml,
deproteinized, recovered, and analyzed on 10% polyacrylamide denaturing gels.
Structure Modeling-- BioSym software was used on a Silicon Graphics Indigo2 Extreme to obtain wild type and variant tRNA structure models. A yeast tRNAPhe structure from the Brookhaven Protein Data Bank was stripped of modifications and Mg2+ so that potentials could be recognized by the software (following instructions provided by J. Stuart, with technical support by E. VanRiper). The structures were minimized by 100 iterations of steepest descent.
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RESULTS |
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The Numbering and Sequence Conservation of Drosophila tRNAHis-- We have renumbered Drosophila tRNAHis to match yeast tRNAPhe (Fig. 1; 20) by omitting nt 17 and including nt 20a in the D loop and by omitting nt 46 in the V loop. Five of the eight tertiary contacts in yeast tRNAPhe (8-14, 15-48, 18-55, 19-56, 22-46, 23-9, 26-44, and 54-58; see Ref. 21), are conserved in Drosophila tRNAHis (bold), based on its sequence and presumed secondary structure (dashed lines in Fig. 1A; green and blue bases in Fig. 1E).
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Unlinking the Substrates for RNase P and 3-tRNase--
The
Drosophila tRNAHis precursor has a 5
end leader
and a 3
end trailer which are endonucleolytically removed by RNase P
and 3
-tRNase, respectively (Fig. 1A; Refs. 14 and 22); both
enzymes share the same internal substrate. We simplified the substrates from the in vivo precursor to minimize possible interference
between the 5
end leader and 3
end trailer in tRNA refolding and to avoid any problem with reaction order, which appears to be first 5
end, then 3
end, processing in vitro (22, 23). The RNase P
substrate has a 16-nt 5
end leader and a 3
end at A73
(Fig. 1B). The 3
-tRNase substrate has a mature 5
end and a 36-nt 3
end trailer (Fig. 1C). The product of both
reactions is thus a 72-nt tRNA (nt 1-73). We have used this 72-nt tRNA
to determine product KI values for both RNase P and
3
-tRNase (Fig. 2).
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Drosophila in Vitro tRNA End Processing Reactions-- Prokaryotic RNase P is subject to product inhibition, especially when its catalytic RNA component is used for processing (24-26). Fierke and co-workers (27) have found that under physiological conditions, however, the affinity of RNase P RNA for substrate and product decreases significantly due to an increase in the dissociation rate constant, thereby also lessening inhibition by product.
We investigated the kinetics of the fly RNase P and 3Purity of the Enzymes--
Before examining the effects of
internal substrate sequence on RNase P and 3-tRNase processing, we
consider the purity of enzymes used for the analysis and the possible
effects of endogenous inhibitors. Fractions from early stages of enzyme
purification might have at least two types of endogenous inhibitors of
tRNA processing reactions as follows: tRNAs and other RNAs, and tRNA binding proteins. To examine the possibility that further purification of the enzymes might remove endogenous inhibitors and thereby reduce
KM, we repeated the kinetic analysis using wild type
substrates and enzyme fractions that had been further fractionated by
anion exchange chromatography (MonoQ; data not shown), which greatly
reduces the concentration of endogenous RNA. We found no decrease in
KM, and in the case of 3
-tRNase,
KM increased with purification.
Matrix Analysis--
The D/T loop tertiary contacts are
illustrated schematically in Fig. 1D, and in
green in the tRNAPhe tertiary structure (Fig.
1E). We made all 15 possible pairwise substitutions at 18/55
and 19/56 (Fig. 1D) in both RNase P and 3-tRNase substrates
(Fig. 1, B and C) and tested their ability to be
processed (Figs. 3 and 4). Relative
processing rates are presented in the 16-place matrices (below the
processing autoradiograms in Figs. 3 and 4), in which the rows
represent the indicated D loop and the columns represent the T loop
substitutions. A row in the matrices with reduced processing values
suggests that a D loop substitution disables processing; similarly, a
column of reduced values implies a processing defect caused by the T
loop substitution.
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U55A Severely Reduces Processing by Both RNase P and 3-tRNase,
Regardless of the Substitution at nt 18--
There are four
combinations in the matrices with the substitution U55A as follows:
U55A which is wild type at G18 (Fig. 3, C parts
of panels 2 and 7), G18C/U55A (D parts
of panels 2 and 7), G18A/U55A (B parts
of panels 5 and 10), and G18U/U55A (C
parts of panels 5 and 10). The RNase P
substrates are poorer than the 3
-tRNase substrates in the cases of
G18A/U55A and G18C/U55A. The left-most columns in the
matrices at the bottom of Fig. 3 (enclosed in
dashed vertical rectangles) show that the inhibitory effect
of the U55A substitution on both RNase P (left) and
3
-tRNase (right) cannot be compensated by changes at nt
18.
G18C Substitutions Reduce Processing--
The four G18C
substitutions, which include G18C with a wild type U at nt 55 (Fig. 3,
panel 2B), G18C/U55A (panel 2D), G18C/U55C (panel 4B), and G18C/U55G (panel 4C), all
severely inhibit RNase P. 3-tRNase, on the other hand, is not
inhibited by G18C (Fig. 3, panel 7B) and G18C/U55G
(panel 9C). The quantitative results are enclosed in
dashed horizontal rectangles in the third row of
the matrices at the bottom of Fig. 3.
Some 18/55 Substitutions Inhibit 3-tRNase More Than RNase
P--
In contrast to the above and our previous observation (14) that
RNase P tends to be more sensitive to substrate changes than 3
-tRNase,
we found three instances in the 18/55 matrix in which 3
-tRNase is more
severely inhibited than RNase P (indicated by the dashed
circles in the matrices at the bottom of Fig. 3): G18A/U55C (Fig. 3, D parts of panels 3 and
8), U55C (C parts of panels 3 and
8), and U55G (C parts of panels 1 and
6).
Substitutions at G19 and C56 Interfere
Differently with Processing by RNase P and
3-tRNase--
G19/C56 is the only
Watson-Crick base pair among the tRNA tertiary contacts. There are four
C56A substitutions as follows: C56A (Fig.
4, part C of panels
3 and 8), G19U/C56A (part D of panels 3 and 8), G19A/C56A (part B of panels
4 and 9), and G19C/C56A (part D of
panels 4 and 9). Three of the C56A substitutions
inhibit processing by both RNase P and 3
-tRNase (vertical
rectangle on the left of the matrices at the
bottom of Fig. 4), whereas C56A with a wild type
G19 (enclosed by a dashed square in the matrix
at the bottom left of Fig. 4) inhibits RNase P processing
less severely.
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Processing Kinetics of Selected Variants--
We prepared
unlabeled selected variant RNAs (G18C, U55A, and C56A), mixed them in
varying amounts with a fixed amount of internally labeled substrate,
and performed Michaelis-Menten experiments to determine
KM and Vmax for both RNase P
and 3-tRNase reactions (Table I). These
variants were selected for kinetic analysis because each one was a poor
substrate for either RNase P (G18C) or 3
-tRNase (C56A) or both (U55A).
Reduced processing was due principally to increases in
KM, with relatively little effect on
Vmax, perhaps due to impaired substrate
recognition and binding. Using the formula
G0 =
RT ln(1/Kd) and setting
KM equivalent to Kd, we can
convert the ~75-fold increase in KM for U55A
processed either with 3
-tRNase to a
G0 of
2.6, approximately equivalent to breaking 1-2 H bonds that could be
involved in stabilizing the E-S complex (Ref. 29; see "Discussion").
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Deleted Substrates for RNase P and 3-tRNase Processing--
The D
arm and anticodon arm were established to be dispensible for RNase P
processing by M1 RNA in E. coli (30) and by RNase P
holoenzyme in toad (31) and human (32). On the other hand, all four
G18C substitutions are poor substrates for RNase P (Fig. 3) and the D
stem substitution U11A is a poor substrate for both RNase P and
3
-tRNase (14). We thus investigated the possible functional importance
of the D arm for the RNase P and/or the 3
-tRNase reaction by making
fly mini-tRNAs consisting of the acceptor stem, T arm, and variable
loop (Fig. 5). Both RNase P and 3
-tRNase
can efficiently process the mini-tRNAs (
11-42), as indicated by the
40-nt labeled internal product (I) at the predicted position
on the gel, as well as the 16-nt 5
end leader (5
EL, Fig. 5A) and 36 nt 3
end
trailer (3
ET, Fig. 5B) characteristic of these reactions.
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Structure Changes Revealed by Nuclease Susceptibility Do Not
Coincide with Processing Reductions--
In the case of variants that
gave different processing results for RNase P and 3-tRNase, the
possibility of differential misfolding arising from the internal
substitutions was addressed by structure probing both 3
-end-labeled
3
-tRNase substrates and 5
-end-labeled RNase P substrates (Figs.
6 and 7). These variants include G18U
(RNase P worse than 3
-tRNase), U55C (RNase P better than 3
-tRNase),
G18C/U55G (RNase P worse than 3
-tRNase), illustrated in Fig. 3, and
C56A and G19A/C56U (RNase P better than 3
-tRNase in both cases; Fig.
4). The most prominent RNase T1 site in wild type tRNA is at
G34 in the anticodon loop; D and T loop nucleotides can
become susceptible to T1 as a result of D/T loop substitutions
(arrowheads in the structure diagrams at left in Fig. 6 and
Ref. 14).
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DISCUSSION |
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Some Processing Determinants Are Shared between RNase P and
3-tRNase, While Others Are Different--
Eukaryotic 3
-tRNase has
been little studied, and the contacts this processing endonuclease
makes with tRNA have not been extensively mapped. Castaño
et al. (23) suggested that Xenopus 3
-tRNase is a
polypeptide enzyme. Zenarro et al. (33) found a
temperature-sensitive lethal substitution (C61U) at the T stem-loop boundary in yeast mitochondrial tRNAAsp which abolished
3
-tRNase catalysis. T arm sequence may thus be important for
3
-tRNase, as it is for RNase P. Nashimoto (34, 35) determined that
annealed half-tRNAs without an intact anticodon loop could be good
3
-tRNase substrates and that tRNA with CCA at its 3
end is a poor
substrate for 3
-tRNase.
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Influence of the D Arm on Processing Endonuclease
Recognition--
The D arm contributes to Drosophila RNase
P processing, based on the following results. First, U11A, a single
substitution in a D stem base pair, abolishes processing (14). Second,
D loop substitutions, especially G18C (Fig. 3), severely inhibit RNase
P. Human RNase P is significantly inhibited by deletion of the tRNA D
arm but not the anticodon arm (36). The tRNA contact surface with RNase
P observed in prokaryotes, toad, and human (30-32) may thus be
stabilized by a wild type D arm, perhaps due to an indirect effect on
the spatial arrangement of the stacked acceptor stem and T arm.
Conversely, the required substrate conformation could be destabilized
by unfavorable D arm substitutions. By deleting the entire D and
anticodon arms (Fig. 5), we produced RNase P and 3-tRNase substrates
that are processed much like full-length tRNAs. This approach is
fundamentally different from the analysis of single or double
substitutions; D arm substitutions might interfere with T loop
structure and acceptor stem-T loop stacking in ways that a short linker
would not.
Variant Processing Kinetics--
The processing changes that arise
from D/T loop substitutions are due largely to increases in
KM (Table I), with relatively little change in
Vmax. The reduced processing rate arising from
D/T loop substitutions would not be expected to affect catalysis, these
positions being remote from the processing sites at the end of the
acceptor stem. They might, on the other hand, be expected to affect
substrate recognition and binding, since the T arm has important RNase
P recognition elements (37, 38). Others (e.g. Refs. 7, 29,
39, and 40) have examined the effects of substrate changes in the
neighborhood of the mature tRNA 5 and 3
ends on RNase P kinetics and,
in some cases, found significant reductions in
Vmax (due to effects on the chemistry of
catalysis).
Tertiary Folding-- Gs at nts 14, 18, and 19 in the D loop and at 57 in the T loop are protected from RNase T1 in wild type tRNA and can become exposed as a result of D/T loop substitutions (Figs. 6 and 7 and Ref. 14). In our previous report, based on analysis of three D/T loop substitutions (G19C, C56G, and G19C/C56G), we suggested that disruption of the D/T contact in C56G (revealed by structure probing) causes the inability of the tRNA to be processed. Herein, by analyzing a larger set of D/T loop substitutions, we find that the disruption of contacts revealed by structure probing does not generally coincide with reduced ability of the substrate to be processed.
Structure Modeling-- We modeled the G18C, U55A, and C56A substitutions (Fig. 8). Interestingly, energy minimization buckles the substituted base in the G18C (Fig. 8C) and C56A (Fig. 8D) variants, perhaps to reduce steric clash. Another possibility is that the phosphate on U54 which stabilizes the T loop U-turn could be pushed aside by the U55A substitution. The modeling tools we used (Fig. 8) are not powerful enough, however, to elucidate the substrate structure changes responsible for reduced processing.
Matrix methods do not necessarily map the processing effects of substrate sequence changes to the level of a single functional group or nucleotide. Some processing defects could result from intraloop propagation (U55A being the most probable example; Fig. 3), and others could result from loop-loop tertiary contacts (G18C being the most probable example; Fig. 3). The T loop is stabilized by a tertiary base pair, a U-turn nucleotide-phosphate contact, a 2 ![]() |
ACKNOWLEDGEMENTS |
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We thank C. Francklyn, K. Musier-Forsyth, J. Puglisi, and J. R. Williamson for helpful conversations; R. Gumport for advice on 3-end-labeling and a gift of T4 RNA ligase; T. Shrader for the plasmid and a procedure for purifying His-Tag T7 RNA
polymerase; J. Stuart for instruction in the use of BioSym software; E. Westhof for suggesting the significance of the T stem U-turn; D. Engelke and R. Hartmann for a critical reading of the manuscript; and A. Birk, J. Cunniffe, V. Greene, A. Oladipo, E. VanRiper, and S. Whyte
for technical assistance.
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FOOTNOTES |
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* This work was supported by Academic Research Enhancement Award R15GM52654, Minority Biomedical Research Support Grant SO6GM08153, and Minority Access to Research Careers Grant T34GM08498 from the National Institutes of Health and by awards from the York College Faculty Development Fund and from the Professional Staff Congress of the City University of New York.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.: 718-262-2704; Fax: 718-262-2652; E-mail: louie{at}mbrs.york.cuny.edu.
¶ Present address: Howard University School of Medicine, Washington, D. C. 20059.
Present address: SUNY Health Sciences Center at Brooklyn
School of Medicine, Brooklyn, NY 11203.
** Present address: Jefferson Medical College, Philadelphia, PA 19107.
1 The abbreviations used are: nt, nucleotide(s); MOPS, 4-morpholinepropanesulfonic acid.
2 T. Shrader, unpublished observations.
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REFERENCES |
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