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Transfer RNA synthesis in HeLa cells
J. Mol. Bid. (1970) 50, 143-151
Transfer RNA Synthesis in HeLa Cells II. t Formation
of tRNA from a Precursor in vitro and Formation of Pseudouridine
DEBORAH BERNIXA~DT MOW~HO~ITZ Departments of Bioch.em&y and Cell Bidog~ Albert Einstein College of Medicine, Bronx, N.Y. 10461, U.S.A. (Received 3 September 1969, and in revised form 3 December 1969) Pre-tRNA, a presumed precursor to tRNA,
has been converted to tRNA by incubation in vitro with a crude cytoplasmic extract. The formation of pseudouridine has been examined during the conversion of pre-tRNA to tRNA ilz viwo. It has been found that pm-tRNA contains considerable pseudouridine and that newly synthesized tRNA or tRNA formed from pretRNA in the absence of net RNA synthesis contains more pseudouridine than pre-tRNA but less than mature tRNA. It is concluded from these and from previous results that pre-tRNA is a precursor to tRNA which is a longer polynucleotide than tRNA and which contains some minor bases, and that pre-tRNA is converted to the size of tRNA before modification of bases is complete.
1. Introduction A class of low molecular weight RNA molecules which has been called pre-tRNA has been found in Krebs II ascites cells (Burdon, Martin & Lal, 1967) and HeLa cells (Bernhardt & Damnell, 1969). I’re-tRNA has been tentatively identified as a precursor to tRNA on the basis of (1) the relative rates of synthesis and accumulation of pretRNA and tRNA and (2) the parallel disappearance of pre-tRNA and appearance of tRNA in the absence of net RNA synthesis. All of these experiments were carried out in intact cells, so that the apparent precursor-product relationship of pre-tRNA and tRNA could have been the fortuitous result of the simultaneous degradation of pre-tRNA and conversion of some other RNA molecule into tRNA. Experiments described in the first part of this report demonstrate that pre-tRNA can be converted in vitro to molecules that migrate during electrophoresis like tRNA. The second series of experiments explores the relationship of this conversion to the formation of paeudouridine. It has been shown previously that the conversion can occur without the completion of all base methyl&ion and thus appears to represent a ohange in molecular size and not primarily a change in minor base content (Bernhardt t Darnell, 1969; Burdon & &son, 1969). It has been found that the formation of pseudouridine, like the formation of methylated bases, does not coincide completely with the conversion of pre-tRNA to tRNA; but that pre-tRNA is converted to the size of tRNA before formation of pseudouridine is complete. t Paper I in this series is Bembardt & Darneli, 1969. 143
144
D. B. MOWSHOWITZ 2. Materials and Methods (a) Cell g+rwthand labelilagprocedures
The details of the growth, labeling and harvesting of HeLa cells have been described previously (Eagle, 1969; Warner, So&o, Birnboim, Girard & Darnell, 1900). It has been shown (Bernhardt & Darn&, 1969) that the incorporation of radioactive uridine into the tRNA of growing HeLa cells represents predominantly synthesis of new chains and not turnover of the terminal CMP of the -CCA ends. Methionine-free medium was used in some of the experiments in order to slow down the synthesis and processing of low molecular weight RNA (Bemhardt t Darnell, 1969). An “actinomycin chase” wss used in oertain experiments to stop further RNA synthesis. The final concentrations of the materials added to the growth medium were : aotinomycin D 10 pg/ml.; unlabeled &dine 10-*x; cytidine 6 x IO-% and thymidine 5 x 10e5~. Cytoplasmic extracts were prepared from HeLa cells by the technique of hypotonic swelling and homogenization as previously described (Penman, Scherrer, Becker & Darnell, 1963). Soluble cytoplasm was prepared from the cytoplasmic extracts by centrifugation for 90 min at 180,000 g at 4°C (Spinco no. 60 rotor, 45K) except that dithiothreitol was added to the hypotonic buffer to a final concentration of 0.25 mg/ml. when the extracts were used for in vitro incubations. The oytoplasmic extracts or fractions to be analyzed were made 0.6 to 1 y0 in sodium dodecyl sulfate to relertse the RNA. If removal of protein was necessary, the sodium dodecyl sulfate-treated fraction was extracted at 60°C with phenol aa previously described (Warner et al., 1966). The RNA was precipitated by addition of NaCl to 0.2 IVI and 2.6 vol. of ethanol. After overnight storage at -20°C the precipitate wu collected by centrifugation and dissolved in gel sample buffer (running buffer diluted 1:lO and made 0.5% in sodium dodecyl sulfate). (e) Electrophoretic analyses of RNA TheRNAwas analyzed by electrophoresis on 10% polyacrylamide gels, 5 mm x 180 mm, 25”C, 10 mA/gel) as previously described (Summers, Maize1 & Darnell, 1966; Bernhardt & Darnell, 1969). The gels were fractionated by the method of Maize1 (1966). Samples containing only 1% were collected on planchets, and the radioactivity was measured in a gas flow counter. Samples containing 3H or both SH and 14C were collected into vials. 10 ml. of Bray’s solution was added to each l-ml. sample and the radioactivity was determined in a liquid scintillation counter. (d) In vitro imubatiom The incubation conditions used to convert pre-tRNA to tRNA in vitro were patterned after the methylation assay of Rodeh, Feldman t Littauer (1967). The standard incubation mixture contained: Tris pH 9.0, O-5 M Dithiothreitol, 2.5 mg/ml. Ammonium acetate 4.0 M Cytoplasmic extract in hypotonic buffer plus dithiotbreitol Purified RNA as indicated
Per 3.0 ml. 0.5 ml. 0.1 ml. 0.2 ml.
Final concentration 83 rnM 0.08 mg/ml. 0.27 M
2.2 ml.
Hypotonic buffer plus dithiothreitol consisted of: MgCI, 1.5 mM; NaCl, 10 IIIM; Tris, pH 7.4, 10 maa; dithiothreitol, 0.26 mg/ml. The incubations were carried out under sterile conditions to avoid bacterial contamination, which had been suspected in earlier experiments (not reported here) involving sucrose-containing cytoplasmic fractions. The reaction mixture was incubated at 37% for the length of time given in each experiment and then sodium dodecyl sulfate was added to a fmal concentration of 0.5%
TRANSFER
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SYNTHESIS
IN
HELA
CELLS
to stop the reaction and release the RNA. The RNA waa then precipitated by gel electrophoresie ae described above. (e) Dt&mnhathn
145
and analyzed
of pseudoddine content of RNA
The method used was a modification of that of Attsrdi, Parnas, Hwang & Attardi (1966). Gel fractions containing labeled RNA were frozen, thawed and allowed to settle. Samples of the super-n&ant f&&ion were counted in a liquid scintillation counter. The appropriate fractions were pooled and the supernatant layer wse removed and concentrated in Garbowax. Yeast RNA was added as a carrier and the RNA precipitated with ethanol and N&l. The RNA wae redissolved in O-3 to O-6 ml. of O-3 N-KOH and hydrolyzed for 16 to 18 hr at 37°C. The hydrolysate wee diluted IO-fold with H,O and adjusted to pH 8.0 with formic acid. The neutralized hydrolysate wee applied to a column of Dowex-l-for-mate x 8, 0.56 x 6 cm prepared in a Pasteur pipette. The column was eluted first with 0.15 N-HCOOH at 0.7 ml./min to remove nucleosides, CMP and AMP; then eluted with O-01 N-HCOOH + O-05 N-ammonium formate at 0.1 to O-2 ml./min to remove YMP and UMP, respectively. The component which elutes before UMP, and which is well resolved from it under the present conditions, has been investigated and identified ea YMP by Amaldi & Attardi (1968).
3. Results (a) In vitro expetiments When HeLa cells are labeled for less than 30 minutes, the majority of the labeled low molecular weight RNA migrates on gel electrophoresis between 5 s RNA and tRNA (Bernhardt & DarnelI, 1969). This material has been called pre-tRNA. In intact cells it appears to be a precursor to tRNA, i.e. it is synthesized more rapidly than tRNA but does not accumulate, and in the absence of net RNA synthesis it disappears st the same rate that tRNA appears (Bernhardt & Darnell, 1969). The following in vitro experiments were designed to demonstrate that pre-tRNA can be converted into tRNA. In the first series of in vitro experiments, cytoplasmic extracts were prepared from cells briefly labeled with [“HI- or [14C]uridine and the extracts were used as sources of both pre-tRNA and of the potential enzymes (and other factors) required for the conversion. The reaction mixtures containing cytoplsamic extract were incubated for 0,20,40 or 60 minutes and then the RNA was examined by gel electrophoresis as described in Materials and Methods. The electrophoretic analysis of such an experiment is shown in Figure 1. As indicated in the Figure, pre-tRNA disappeared, an equivalent amount of tRNA appeared during the incubation and no non-specific degradation occurred on prolonged incubstion. This experiment indicated that 8 oytoplasmic polynucleotide was converted into tRNA in vitro. To demonstrate that this polynucleotide was pre-tRNA, the experiment was repeated, except that soluble cytoplasm prepared from briefly labeled cells (previously labeled overnight with [14C]uridine) was used as the only source of labeled RNA; additional cytoplasm, prepared from unlabeled cells, was used as the source of enzymes, etc. Soluble cytoplasm was used because it contained no significant amounts of labeled RNA larger than 5 s which entered a 2.5% gel as shown in Figure 2 and therefore it was assumed that it contained no larger molecular weight RNA at all, since RNA of molecular weight up to 7 to 8 x lo6 would enter a 2.5% gel. The results
of this experiment
are shown in
Table 1, Experiment 1. Again pre-tRNA disappeared and an equivalent amount of tRNA appeared. Since in this case no other 3H-labeled RNA was present except that shown in Figure 2 it appeared that pre-tRNA was actually converted into tRNA. 10
146
D. B. MOWSHOWITZ I
I
3
No incubation
sb ncubation
1
'O-
li
II 9
i0 :
10:
O, L
I IO
I
I 30
1
I
IO Fraction
I
30 no.
11
I
10
I
I
I
30
FIG. 1. Incubation of pulse-labeled cytoplasm in vitro. 6 x 10’ cells were harvested, resuspended at 4 X 10s cells/ml. and labeled for 15 min with 0.5 +/ml. of [1*C]uridine. A cytoplasmic extract was prepared and used as a source of labeled RNA and enzymes, in a 3.0-ml. incubation mixture as described in Materials and Methods but supplemented with 260 mpoles of S-adenosyl-methionine. The mixture was divided into 4 parts and and each portion was incubated for 0, 20, 40 or 60 min. The RNA WBB released, precipitated an&lyzed as described. S-Adenosyl-methionine was added because it was thought at the time that the conversion of pre-tRNA to tRNA might be dependent on methyl&ion. This does not appear to be correct (Bernherdt & Darnell, 1969). In any case the compound has no effect in this crude system and was subsequently omitted. I
I
Pulse-lob&d
Fraction no. FIG. 2. Analysis of soluble cytoplasm on a 2.5%/10% gel to detect moleoules of high molecular weight. 2.5~ lo* cells were hervested, resuspended in methionine-free medium at 5 x lo6 cells/ml., incubated for 3 hr and labeled for 15 min with 20 pc/ml. [zH]uridine. RNA was prepared from the soluble cytoplasm, deproteinized, precipitated and redissolved in hypotonic buffer. A sample was diluted, mixed with long-term [1*CJuridine-18beled tRNA also obtained from the soluble cytoplasm and the mixture was examined on a polyacrylamide gel which consisted of a 3-cm 2.5% gel overlying an l&cm 10% gel. The entire gel is shown in the Figure. 1’C radioactivity; --o--o--, 3H radioactivity. -0-O-9
TRANSFER
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SYNTHESIS
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147
To confirm this result, RNA was prepared from the soluble cytoplasm of briefly 1abeIed cells, deproteinized with phenol, precipitated twice to remove sodium dodecyl sulfate, redissolved in hypotonic buffer and used as the only source of labeled RNA in an in vitro incubation with unlabeled cytoplasm. The gel patterns before and after incubation are compared in Figure 3. The results of a similar experiment are shown in Table 1, Experiment 2. Pre-tRNA was apparently converted to tRNA as before.
150--300
Fraction
no.
FIG. 3. Incubation of purified RNA from soluble cytoplrssm in Z.&O. 6 X 10’ cells were harvested, resuspended at 5 X IO6 cells/ml. in methionine-free medium, incubated for 2 hr and labeled for 16 min with 20 &ml. [3H]uridine. RNA was prepared from the soluble oytoplasm, deproteinized, precipitated twice to remove sodium dodecyl sulfate and redissolved in sterile hypotonic buffer. Cytoplasm was prepared from 2 X 10’ cells and used as a source of enzymes. The purified 3H-labeled RNA was mixed with the unlabeled cytoplasm and incubated in a total of 0.5 ml. for 0 of 90 min. RNA was released, precipitated and analyzed 8~ described. -•-•--, 14C redioaotivity; --O---O--, 3H radioactivity.
To rule out the possibility that generalized degradation of RNA was occurring during the incubations in the absence of new tRNA formation, this experiment was repeated with the addition to the reaction mixture of 14C-labeled tRNA. The results indicated that net conversion of pre-tRNA to tRNA occurred in the absence of extensive degradation, since (1) the electrophoretic mobility of the 14C-labeled rRNA was unaffected by the incubation and (2) most of the 3H-pulse-labeled RNA was recovered after the incubation, as shown in Table 1, Experiment 3. It was concluded that the apparent conversion of pre-tRNA to tRNA observed both in vivo and in vitro represented an actual conversion of pre-tRNA molecules into tRNA. (b) Pseudouridine formation When pre-tRNA was first observed it was suspected that it might be “unmodified tRNA”, and that its conversion to tRNA might involve the modification of individual
2
3
1 2
1
1
2 2
Incubated without cytoplasm Incubeted with cytoplesm Not incubated
Not incubated Incubeted 90 min
Incubated without cytoplaem 60 min Incubated with cytoplasm 60 min Incubeted with cytoplasm 6 min
Description
of Total
0.72 0.38 0.70
0.12 o-11
0.35
O-18 0.17
0.59
068
0.54
0.72
pre-t-RNA
Freotion
0.21
O-18
0.15
0.17
6sRNA
0.49 0.19
0.11
0.47
0.20
0.14
o-31
0.21
tRNA
4975 6725
8126
3511
4272
9998
8098
10,588
Total 3H cts/min
4967 5665
6714
8720
8740
8930
Normmlized “Ht (cts/min)
t 3H cts/min/lOOO r4C cts/min recovered. Experiment 1 lo* cells were lebeled overnight with 0.5 PO [W]uridine (spec. a& 0.1 pc/qole), harvested, resuspended in methionine-free medium at 5 x 10s cells/ml,, incubated for 2 hr and labeled for 15 min with 20 PC of [3H]uridine/ml. Soluble cytoplssm (2.0 ml.) was prepsred and used 8s a source of RNA. A cytoplesmic extract (0.6 ml.) wa8 prepared from 2.5 x 10’ growing cells end used 8s a souroe of enzymes, feotors, etc. One-third of the double-labeled soluble CytoplasIll ~8s mixed with Tris, etc. and incubated with sdditionel hyptonic buffer + dithiothreitol for 60 min (Sample 1). The remsinder was mixed with cytoplasm (as well w Tris, etc.) and incub8ted for 60 or 5 min (samples 2 and 3, respectively). In e8oh c8se I.0 ml. incubation mixtures were used. The RNA was released, precipitated, and enalyzed es described. The radioactivity in each species ~8s est.¬ed from the gel electropherogrems by adding together all of the counts under each peak plus h8lf the counts in the troughs (of one or two freotions) on either side. Ezperimmta 2 and 3 The W-lsbeled tRNA ~8s prepared from 2.5 x 10s cells labeled overnight with 6 ~c[14C]uridine. The 3H-labeled low molecular weight RNA w8s prepared from 2.6 x lo* cells incubated in methionine-free medium at 5 x 10s cells/ml. for 3 hr and then labeled with 20 pc/ml. of [3H]uridine for 15 mm. The soluble cytoplasm was prepared from each sample. The RNA w8s released with sodium dodecyl sulfete, precipitated, and deproteinized with phenol. The purified RNA ~8s precipitated 3 times to remove sodium dodecyl sulfete end redissolved in 1-O ml. sterile hypotonic buffer. For experiment 2, each sample contained only 3H-18beled RNA and was divided into 3 tubes of 0.5 ml. each which were pooled after the incubation. For experiment 3, each semple consisted of O-5 ml. conteining sH-labeled low molecular weight RNA and 14C-18beled tRNA. Samples 2 and 3 each contained cytoplasm from 2 x 10s cells; samples 1 and 2 were incubsted for 90 min et 37°C.
1
Sample
1
Experiment
1
In vitro incubations of low nw2ecular weight RNA
TABLE
TRANSFER
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SYNTHESIS
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CELLS
149
bases. It was found, however, that the conversion probably involved a reduction in the size of the pre-tRNA molecule (Bernhardt & Darnell, 1969) and that methylation, the only modification examined, could be completed after pre-tRNA was converted to the size of tRNA. Therefore it was considered likely that formation of minor bases was a separate process not necessarily related to the conversion. To test this, the formation of pseudouridine was examined, because pseudouridine is the most frequently occurring minor base. The experiments were designed to determine: (1) if pre-tRNA contained pseudouridine; (2) if pseudouridine were formed during and/or after the conversion (in the absence of net RNA synthesis). All of the RNA used in these experiments was prepared from cells lebeled with [14C]uridine or [6-3H]uridine; the exact labeling conditions are given in the legends to the Tables. The RNA was fractionated on gels and the appropriate fractions were pooled. After short labels (less than 10 minutes in regular medium or less than 20 minutes in methionine-free medium) all of the RNA smaller than 5 s was considered to be pre-tRNA; after overnight labels it was considered to be entirely tRNA and after labels of intermediate length the pre-tRNA and tRNA were pooled separately. The pseudouridine content of each fraction was determined as described in Materials and Methods. The results are expressed in the Tables as the percentage of the total amount of uridine mononucleotidea (UMP + ?KMP) recovered as YMP. Table 2 gives the pseudouridine content of pre-tRNA, pulse-labeled tRNA and equilibrium-labeled tRNA. Clearly pre-tRNA contained a significant amount of pseudouridine, but less than the tRNA made during the same period of time. Both newly synthesized species contained less pseudouridine than mature tRNA. This is the result expected if pseudouridine were formed after (and possibly also during) the conversion. To attack this directly, the pseudouridine content of pre-tRNA was compared with that of the tRNA formed from it in the absence of net RNA synthesis. Cells were labeled briefly and then further RNA synthesis was inhibited with an TABLE
2
Pseudouridine content of pre-tRNA
Experiment
1 1 2 2
Labeling conditions
[l’C]uridine overnight [6-3H]uridine overnight [6-3H]uridine pulse [5-3H]uridine pulse
and tRNA
Tottll UMPfYMP cts/min 1700 3405 10640 12290
% YMP
23-l 24.3 10.7 15.5
Species of RNA
tRNA tRNA pm-tRNA tRNA
The method used for the determination of the YMP content is described in Materials section (e). The RNA used in etlch experiment ww prepared aa described below.
snd Methoda
Experiment 1 Psrallel cultures were labeled overnight with 0.5 &ml. [Wluridine ( lo8 cells) or 2 &ml. [6-3H]uridine (6 x 10” cells). RNA w= precipitated from the soluble cytoplasm of each oulture.
Experimnt
2
2~5 x lo8 cells at 5 x IO8 cells/ml. were labeled for 10 min with proteinized RNA was prepared from the cytoplasmic extract.
30 PC/ml. of [6-3H]uridine.
De-
150
D.
B.
MOWSHOWITZ
actinomycin chase. The pre-tRNA and tRNA were pooled in samples taken two minutes after the addition of the actinomycin and after 30 minutes more of chase. Before the chase most of the label was in pre-tRNA; afterwards the label was primarily in tRNA. The pseudouridine content of the chased and unchased samples from two experiments are shown in Table 3. In each case the pseudouridine content of the pooled tRNA and pre-tRNA increased significantly during the chase but not to the level found in equilibrium-labeled tRNA. It appears that formation of pseudouridine overlaps the conversion of pre-tRNA to tRNA but is probably a separate process which is completed after the conversion of pre-tRNA to the electrophoretio mobility of t-RNA (see the diagram following the discussion). TABLE 3
Pseudo&dine
Experiment
1 1 2 2
content of pulse-labeled low molecular weight RNA before and after chase Total UMP+?FMP (cts/min)
Labeling Conditions
[6-3H]uridine [6-3H]uridine [6-SH]uridine [6-3H]uridine
pulse pulse; chased pulse pulse; chased
3484 3092 6555 8871
%YMP
10.2 18.4 10.4 16.7
Species of RNA
pre-tRNA pre-tRNA pm-tRNA pre-tRNA
The method used for the determination of the YMP content is described in Materials section (e). The RNA used in each experiment was prepared as described below.
+ + + +
tRNA tRNA tRNA tRNA
and Methods
Experimwat 1 2.5 x lOa cells were incubated at 5 x lo6 cells/ml. in methionine-free medium for 4.5 hr at 37°C. They were labeled for 10 min with 20 &ml. of [6-3H]uridine, and then an actinomycin chase was added. One-half of the culture ws8 stopped 2 min after the addition of the chase. The other half was chased for 30 min longer in the presence of methionine and then stopped. RNA was precipitated from the soluble cytoplasm of each sample. Experiment 2 2.5 x lo* cells were incubated as in the preceding experiment.
for 3 hr in methionine-free medium, labeled for 16 min and chased Deproteinized RNA was prepared from cytoplasmic extracts.
4. Discussion Experiments described in this paper have demonstrated that purified pre-tRNA can be converted to tRNA by incubation with a crude cytoplasmic extract in vitro. Since it has been shown previously that pre-tRNA and tRNA exhibit a precursorproduct relationship in intact cells (Bernhardt & Darnell, 1969), it ia concluded that pre-tRNA is a bonafide intermediate in tRNA synthesis. Previous results have indicated that the conversion of pre-tRNA to a molecule with the electrophoretic mobility of tRNA does not require complete methylation (Bernhardt t Darnell, 1969). Experiments described in this report have shown that complete formation of pseudouridine is not required either. Since there is no direct evidence that modification of bases occurs during (as opposed to after) the conversion and since there is evidence that pre-tRNA and tRNA differ in molecular weight (Burdon t Clason, 1969; Bernhardt & Darnell, 1969), it is unnecessary to invoke a
TRANSFER
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SYNTHESIS
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151
role for modification of bases in the conversion of pre-tRNA to the electrophoretic mobility of tRNA. However, modification is involved at some point(s) in the conversion of pre-tRNA to mature tRNA. The apparent relationship between modification and conversion is shown diagrammatically below: Hypothetical primary gene product 4 base alterations pre-tRNA
1
1 peak on gel (pre-tRNA)
4 size change modification-deficient tRNA J- base alterations mature tRNA
1 peak on gel (tRNA) i
I thank Dr James E. Darnell for many invaluable suggestions and discussions. This work was supported by grants from the National Institutes of Health (CA 07861-05), the National Science Foundation (G.B. 4565) and the Health Research Council of the City of New York. REFERENCES Amaldi, F. & Attardi, G. (1968). J. Mol. Biol. 33, 737. Attardi, G., Parr-me, H., Hwang, M-I BEAttardi, B. (1966). J. Mol. Biol. 20, 145. Bernhard& D. t Darnell, J. E. (1969). J. Mol. Biol. 42, 43. Burdon, R. H. & Clason, A. E. (1969). J. Mol. BioZ. 39, 113. Burdon, R. H., Martin, B. T. & Lal, B. M. (1967). J. Mol. BioZ. 28, 357. Eagle, H. (1959). Science, 130, 432. Maizel, J. V. (1966). Science, 151, 988. Penman, S., Scherrer, K., Becker, Y. & Darnell, J. E. (1963). Proc. Nat. Ad. Sk., Wash. 49, 654. Rodeh, R., Feldman, M. & Littauer, U. (1967). Biochembt~g, 6, 451. Summers, D. F., Maizel, J. V. & Darnell, J. E. (1965). Proc. Nat. Acad. Sk., Wash. 54, 505. Warner, J. R., Soeiro, R., Birnboim, H. C., Girard, M. & Darnell, J. E. (1966). J. Mol. BioZ. 19, 349.
Transfer RNA Synthesis in HeLa Cells II. t Formation
of tRNA from a Precursor in vitro and Formation of Pseudouridine
DEBORAH BERNIXA~DT MOW~HO~ITZ Departments of Bioch.em&y and Cell Bidog~ Albert Einstein College of Medicine, Bronx, N.Y. 10461, U.S.A. (Received 3 September 1969, and in revised form 3 December 1969) Pre-tRNA, a presumed precursor to tRNA,
has been converted to tRNA by incubation in vitro with a crude cytoplasmic extract. The formation of pseudouridine has been examined during the conversion of pre-tRNA to tRNA ilz viwo. It has been found that pm-tRNA contains considerable pseudouridine and that newly synthesized tRNA or tRNA formed from pretRNA in the absence of net RNA synthesis contains more pseudouridine than pre-tRNA but less than mature tRNA. It is concluded from these and from previous results that pre-tRNA is a precursor to tRNA which is a longer polynucleotide than tRNA and which contains some minor bases, and that pre-tRNA is converted to the size of tRNA before modification of bases is complete.
1. Introduction A class of low molecular weight RNA molecules which has been called pre-tRNA has been found in Krebs II ascites cells (Burdon, Martin & Lal, 1967) and HeLa cells (Bernhardt & Damnell, 1969). I’re-tRNA has been tentatively identified as a precursor to tRNA on the basis of (1) the relative rates of synthesis and accumulation of pretRNA and tRNA and (2) the parallel disappearance of pre-tRNA and appearance of tRNA in the absence of net RNA synthesis. All of these experiments were carried out in intact cells, so that the apparent precursor-product relationship of pre-tRNA and tRNA could have been the fortuitous result of the simultaneous degradation of pre-tRNA and conversion of some other RNA molecule into tRNA. Experiments described in the first part of this report demonstrate that pre-tRNA can be converted in vitro to molecules that migrate during electrophoresis like tRNA. The second series of experiments explores the relationship of this conversion to the formation of paeudouridine. It has been shown previously that the conversion can occur without the completion of all base methyl&ion and thus appears to represent a ohange in molecular size and not primarily a change in minor base content (Bernhardt t Darnell, 1969; Burdon & &son, 1969). It has been found that the formation of pseudouridine, like the formation of methylated bases, does not coincide completely with the conversion of pre-tRNA to tRNA; but that pre-tRNA is converted to the size of tRNA before formation of pseudouridine is complete. t Paper I in this series is Bembardt & Darneli, 1969. 143
144
D. B. MOWSHOWITZ 2. Materials and Methods (a) Cell g+rwthand labelilagprocedures
The details of the growth, labeling and harvesting of HeLa cells have been described previously (Eagle, 1969; Warner, So&o, Birnboim, Girard & Darnell, 1900). It has been shown (Bernhardt & Darn&, 1969) that the incorporation of radioactive uridine into the tRNA of growing HeLa cells represents predominantly synthesis of new chains and not turnover of the terminal CMP of the -CCA ends. Methionine-free medium was used in some of the experiments in order to slow down the synthesis and processing of low molecular weight RNA (Bemhardt t Darnell, 1969). An “actinomycin chase” wss used in oertain experiments to stop further RNA synthesis. The final concentrations of the materials added to the growth medium were : aotinomycin D 10 pg/ml.; unlabeled &dine 10-*x; cytidine 6 x IO-% and thymidine 5 x 10e5~. Cytoplasmic extracts were prepared from HeLa cells by the technique of hypotonic swelling and homogenization as previously described (Penman, Scherrer, Becker & Darnell, 1963). Soluble cytoplasm was prepared from the cytoplasmic extracts by centrifugation for 90 min at 180,000 g at 4°C (Spinco no. 60 rotor, 45K) except that dithiothreitol was added to the hypotonic buffer to a final concentration of 0.25 mg/ml. when the extracts were used for in vitro incubations. The oytoplasmic extracts or fractions to be analyzed were made 0.6 to 1 y0 in sodium dodecyl sulfate to relertse the RNA. If removal of protein was necessary, the sodium dodecyl sulfate-treated fraction was extracted at 60°C with phenol aa previously described (Warner et al., 1966). The RNA was precipitated by addition of NaCl to 0.2 IVI and 2.6 vol. of ethanol. After overnight storage at -20°C the precipitate wu collected by centrifugation and dissolved in gel sample buffer (running buffer diluted 1:lO and made 0.5% in sodium dodecyl sulfate). (e) Electrophoretic analyses of RNA TheRNAwas analyzed by electrophoresis on 10% polyacrylamide gels, 5 mm x 180 mm, 25”C, 10 mA/gel) as previously described (Summers, Maize1 & Darnell, 1966; Bernhardt & Darnell, 1969). The gels were fractionated by the method of Maize1 (1966). Samples containing only 1% were collected on planchets, and the radioactivity was measured in a gas flow counter. Samples containing 3H or both SH and 14C were collected into vials. 10 ml. of Bray’s solution was added to each l-ml. sample and the radioactivity was determined in a liquid scintillation counter. (d) In vitro imubatiom The incubation conditions used to convert pre-tRNA to tRNA in vitro were patterned after the methylation assay of Rodeh, Feldman t Littauer (1967). The standard incubation mixture contained: Tris pH 9.0, O-5 M Dithiothreitol, 2.5 mg/ml. Ammonium acetate 4.0 M Cytoplasmic extract in hypotonic buffer plus dithiotbreitol Purified RNA as indicated
Per 3.0 ml. 0.5 ml. 0.1 ml. 0.2 ml.
Final concentration 83 rnM 0.08 mg/ml. 0.27 M
2.2 ml.
Hypotonic buffer plus dithiothreitol consisted of: MgCI, 1.5 mM; NaCl, 10 IIIM; Tris, pH 7.4, 10 maa; dithiothreitol, 0.26 mg/ml. The incubations were carried out under sterile conditions to avoid bacterial contamination, which had been suspected in earlier experiments (not reported here) involving sucrose-containing cytoplasmic fractions. The reaction mixture was incubated at 37% for the length of time given in each experiment and then sodium dodecyl sulfate was added to a fmal concentration of 0.5%
TRANSFER
RNA
SYNTHESIS
IN
HELA
CELLS
to stop the reaction and release the RNA. The RNA waa then precipitated by gel electrophoresie ae described above. (e) Dt&mnhathn
145
and analyzed
of pseudoddine content of RNA
The method used was a modification of that of Attsrdi, Parnas, Hwang & Attardi (1966). Gel fractions containing labeled RNA were frozen, thawed and allowed to settle. Samples of the super-n&ant f&&ion were counted in a liquid scintillation counter. The appropriate fractions were pooled and the supernatant layer wse removed and concentrated in Garbowax. Yeast RNA was added as a carrier and the RNA precipitated with ethanol and N&l. The RNA wae redissolved in O-3 to O-6 ml. of O-3 N-KOH and hydrolyzed for 16 to 18 hr at 37°C. The hydrolysate wee diluted IO-fold with H,O and adjusted to pH 8.0 with formic acid. The neutralized hydrolysate wee applied to a column of Dowex-l-for-mate x 8, 0.56 x 6 cm prepared in a Pasteur pipette. The column was eluted first with 0.15 N-HCOOH at 0.7 ml./min to remove nucleosides, CMP and AMP; then eluted with O-01 N-HCOOH + O-05 N-ammonium formate at 0.1 to O-2 ml./min to remove YMP and UMP, respectively. The component which elutes before UMP, and which is well resolved from it under the present conditions, has been investigated and identified ea YMP by Amaldi & Attardi (1968).
3. Results (a) In vitro expetiments When HeLa cells are labeled for less than 30 minutes, the majority of the labeled low molecular weight RNA migrates on gel electrophoresis between 5 s RNA and tRNA (Bernhardt & DarnelI, 1969). This material has been called pre-tRNA. In intact cells it appears to be a precursor to tRNA, i.e. it is synthesized more rapidly than tRNA but does not accumulate, and in the absence of net RNA synthesis it disappears st the same rate that tRNA appears (Bernhardt & Darnell, 1969). The following in vitro experiments were designed to demonstrate that pre-tRNA can be converted into tRNA. In the first series of in vitro experiments, cytoplasmic extracts were prepared from cells briefly labeled with [“HI- or [14C]uridine and the extracts were used as sources of both pre-tRNA and of the potential enzymes (and other factors) required for the conversion. The reaction mixtures containing cytoplsamic extract were incubated for 0,20,40 or 60 minutes and then the RNA was examined by gel electrophoresis as described in Materials and Methods. The electrophoretic analysis of such an experiment is shown in Figure 1. As indicated in the Figure, pre-tRNA disappeared, an equivalent amount of tRNA appeared during the incubation and no non-specific degradation occurred on prolonged incubstion. This experiment indicated that 8 oytoplasmic polynucleotide was converted into tRNA in vitro. To demonstrate that this polynucleotide was pre-tRNA, the experiment was repeated, except that soluble cytoplasm prepared from briefly labeled cells (previously labeled overnight with [14C]uridine) was used as the only source of labeled RNA; additional cytoplasm, prepared from unlabeled cells, was used as the source of enzymes, etc. Soluble cytoplasm was used because it contained no significant amounts of labeled RNA larger than 5 s which entered a 2.5% gel as shown in Figure 2 and therefore it was assumed that it contained no larger molecular weight RNA at all, since RNA of molecular weight up to 7 to 8 x lo6 would enter a 2.5% gel. The results
of this experiment
are shown in
Table 1, Experiment 1. Again pre-tRNA disappeared and an equivalent amount of tRNA appeared. Since in this case no other 3H-labeled RNA was present except that shown in Figure 2 it appeared that pre-tRNA was actually converted into tRNA. 10
146
D. B. MOWSHOWITZ I
I
3
No incubation
sb ncubation
1
'O-
li
II 9
i0 :
10:
O, L
I IO
I
I 30
1
I
IO Fraction
I
30 no.
11
I
10
I
I
I
30
FIG. 1. Incubation of pulse-labeled cytoplasm in vitro. 6 x 10’ cells were harvested, resuspended at 4 X 10s cells/ml. and labeled for 15 min with 0.5 +/ml. of [1*C]uridine. A cytoplasmic extract was prepared and used as a source of labeled RNA and enzymes, in a 3.0-ml. incubation mixture as described in Materials and Methods but supplemented with 260 mpoles of S-adenosyl-methionine. The mixture was divided into 4 parts and and each portion was incubated for 0, 20, 40 or 60 min. The RNA WBB released, precipitated an&lyzed as described. S-Adenosyl-methionine was added because it was thought at the time that the conversion of pre-tRNA to tRNA might be dependent on methyl&ion. This does not appear to be correct (Bernherdt & Darnell, 1969). In any case the compound has no effect in this crude system and was subsequently omitted. I
I
Pulse-lob&d
Fraction no. FIG. 2. Analysis of soluble cytoplasm on a 2.5%/10% gel to detect moleoules of high molecular weight. 2.5~ lo* cells were hervested, resuspended in methionine-free medium at 5 x lo6 cells/ml., incubated for 3 hr and labeled for 15 min with 20 pc/ml. [zH]uridine. RNA was prepared from the soluble cytoplasm, deproteinized, precipitated and redissolved in hypotonic buffer. A sample was diluted, mixed with long-term [1*CJuridine-18beled tRNA also obtained from the soluble cytoplasm and the mixture was examined on a polyacrylamide gel which consisted of a 3-cm 2.5% gel overlying an l&cm 10% gel. The entire gel is shown in the Figure. 1’C radioactivity; --o--o--, 3H radioactivity. -0-O-9
TRANSFER
RNA
SYNTHESIS
IN
HELA
CELLS
147
To confirm this result, RNA was prepared from the soluble cytoplasm of briefly 1abeIed cells, deproteinized with phenol, precipitated twice to remove sodium dodecyl sulfate, redissolved in hypotonic buffer and used as the only source of labeled RNA in an in vitro incubation with unlabeled cytoplasm. The gel patterns before and after incubation are compared in Figure 3. The results of a similar experiment are shown in Table 1, Experiment 2. Pre-tRNA was apparently converted to tRNA as before.
150--300
Fraction
no.
FIG. 3. Incubation of purified RNA from soluble cytoplrssm in Z.&O. 6 X 10’ cells were harvested, resuspended at 5 X IO6 cells/ml. in methionine-free medium, incubated for 2 hr and labeled for 16 min with 20 &ml. [3H]uridine. RNA was prepared from the soluble oytoplasm, deproteinized, precipitated twice to remove sodium dodecyl sulfate and redissolved in sterile hypotonic buffer. Cytoplasm was prepared from 2 X 10’ cells and used as a source of enzymes. The purified 3H-labeled RNA was mixed with the unlabeled cytoplasm and incubated in a total of 0.5 ml. for 0 of 90 min. RNA was released, precipitated and analyzed 8~ described. -•-•--, 14C redioaotivity; --O---O--, 3H radioactivity.
To rule out the possibility that generalized degradation of RNA was occurring during the incubations in the absence of new tRNA formation, this experiment was repeated with the addition to the reaction mixture of 14C-labeled tRNA. The results indicated that net conversion of pre-tRNA to tRNA occurred in the absence of extensive degradation, since (1) the electrophoretic mobility of the 14C-labeled rRNA was unaffected by the incubation and (2) most of the 3H-pulse-labeled RNA was recovered after the incubation, as shown in Table 1, Experiment 3. It was concluded that the apparent conversion of pre-tRNA to tRNA observed both in vivo and in vitro represented an actual conversion of pre-tRNA molecules into tRNA. (b) Pseudouridine formation When pre-tRNA was first observed it was suspected that it might be “unmodified tRNA”, and that its conversion to tRNA might involve the modification of individual
2
3
1 2
1
1
2 2
Incubated without cytoplasm Incubeted with cytoplesm Not incubated
Not incubated Incubeted 90 min
Incubated without cytoplaem 60 min Incubated with cytoplasm 60 min Incubeted with cytoplasm 6 min
Description
of Total
0.72 0.38 0.70
0.12 o-11
0.35
O-18 0.17
0.59
068
0.54
0.72
pre-t-RNA
Freotion
0.21
O-18
0.15
0.17
6sRNA
0.49 0.19
0.11
0.47
0.20
0.14
o-31
0.21
tRNA
4975 6725
8126
3511
4272
9998
8098
10,588
Total 3H cts/min
4967 5665
6714
8720
8740
8930
Normmlized “Ht (cts/min)
t 3H cts/min/lOOO r4C cts/min recovered. Experiment 1 lo* cells were lebeled overnight with 0.5 PO [W]uridine (spec. a& 0.1 pc/qole), harvested, resuspended in methionine-free medium at 5 x 10s cells/ml,, incubated for 2 hr and labeled for 15 min with 20 PC of [3H]uridine/ml. Soluble cytoplssm (2.0 ml.) was prepsred and used 8s a source of RNA. A cytoplesmic extract (0.6 ml.) wa8 prepared from 2.5 x 10’ growing cells end used 8s a souroe of enzymes, feotors, etc. One-third of the double-labeled soluble CytoplasIll ~8s mixed with Tris, etc. and incubated with sdditionel hyptonic buffer + dithiothreitol for 60 min (Sample 1). The remsinder was mixed with cytoplasm (as well w Tris, etc.) and incub8ted for 60 or 5 min (samples 2 and 3, respectively). In e8oh c8se I.0 ml. incubation mixtures were used. The RNA was released, precipitated, and enalyzed es described. The radioactivity in each species ~8s est.¬ed from the gel electropherogrems by adding together all of the counts under each peak plus h8lf the counts in the troughs (of one or two freotions) on either side. Ezperimmta 2 and 3 The W-lsbeled tRNA ~8s prepared from 2.5 x 10s cells labeled overnight with 6 ~c[14C]uridine. The 3H-labeled low molecular weight RNA w8s prepared from 2.6 x lo* cells incubated in methionine-free medium at 5 x 10s cells/ml. for 3 hr and then labeled with 20 pc/ml. of [3H]uridine for 15 mm. The soluble cytoplasm was prepared from each sample. The RNA w8s released with sodium dodecyl sulfete, precipitated, and deproteinized with phenol. The purified RNA ~8s precipitated 3 times to remove sodium dodecyl sulfete end redissolved in 1-O ml. sterile hypotonic buffer. For experiment 2, each sample contained only 3H-18beled RNA and was divided into 3 tubes of 0.5 ml. each which were pooled after the incubation. For experiment 3, each semple consisted of O-5 ml. conteining sH-labeled low molecular weight RNA and 14C-18beled tRNA. Samples 2 and 3 each contained cytoplasm from 2 x 10s cells; samples 1 and 2 were incubsted for 90 min et 37°C.
1
Sample
1
Experiment
1
In vitro incubations of low nw2ecular weight RNA
TABLE
TRANSFER
RNA
SYNTHESIS
IN
HELA
CELLS
149
bases. It was found, however, that the conversion probably involved a reduction in the size of the pre-tRNA molecule (Bernhardt & Darnell, 1969) and that methylation, the only modification examined, could be completed after pre-tRNA was converted to the size of tRNA. Therefore it was considered likely that formation of minor bases was a separate process not necessarily related to the conversion. To test this, the formation of pseudouridine was examined, because pseudouridine is the most frequently occurring minor base. The experiments were designed to determine: (1) if pre-tRNA contained pseudouridine; (2) if pseudouridine were formed during and/or after the conversion (in the absence of net RNA synthesis). All of the RNA used in these experiments was prepared from cells lebeled with [14C]uridine or [6-3H]uridine; the exact labeling conditions are given in the legends to the Tables. The RNA was fractionated on gels and the appropriate fractions were pooled. After short labels (less than 10 minutes in regular medium or less than 20 minutes in methionine-free medium) all of the RNA smaller than 5 s was considered to be pre-tRNA; after overnight labels it was considered to be entirely tRNA and after labels of intermediate length the pre-tRNA and tRNA were pooled separately. The pseudouridine content of each fraction was determined as described in Materials and Methods. The results are expressed in the Tables as the percentage of the total amount of uridine mononucleotidea (UMP + ?KMP) recovered as YMP. Table 2 gives the pseudouridine content of pre-tRNA, pulse-labeled tRNA and equilibrium-labeled tRNA. Clearly pre-tRNA contained a significant amount of pseudouridine, but less than the tRNA made during the same period of time. Both newly synthesized species contained less pseudouridine than mature tRNA. This is the result expected if pseudouridine were formed after (and possibly also during) the conversion. To attack this directly, the pseudouridine content of pre-tRNA was compared with that of the tRNA formed from it in the absence of net RNA synthesis. Cells were labeled briefly and then further RNA synthesis was inhibited with an TABLE
2
Pseudouridine content of pre-tRNA
Experiment
1 1 2 2
Labeling conditions
[l’C]uridine overnight [6-3H]uridine overnight [6-3H]uridine pulse [5-3H]uridine pulse
and tRNA
Tottll UMPfYMP cts/min 1700 3405 10640 12290
% YMP
23-l 24.3 10.7 15.5
Species of RNA
tRNA tRNA pm-tRNA tRNA
The method used for the determination of the YMP content is described in Materials section (e). The RNA used in etlch experiment ww prepared aa described below.
snd Methoda
Experiment 1 Psrallel cultures were labeled overnight with 0.5 &ml. [Wluridine ( lo8 cells) or 2 &ml. [6-3H]uridine (6 x 10” cells). RNA w= precipitated from the soluble cytoplasm of each oulture.
Experimnt
2
2~5 x lo8 cells at 5 x IO8 cells/ml. were labeled for 10 min with proteinized RNA was prepared from the cytoplasmic extract.
30 PC/ml. of [6-3H]uridine.
De-
150
D.
B.
MOWSHOWITZ
actinomycin chase. The pre-tRNA and tRNA were pooled in samples taken two minutes after the addition of the actinomycin and after 30 minutes more of chase. Before the chase most of the label was in pre-tRNA; afterwards the label was primarily in tRNA. The pseudouridine content of the chased and unchased samples from two experiments are shown in Table 3. In each case the pseudouridine content of the pooled tRNA and pre-tRNA increased significantly during the chase but not to the level found in equilibrium-labeled tRNA. It appears that formation of pseudouridine overlaps the conversion of pre-tRNA to tRNA but is probably a separate process which is completed after the conversion of pre-tRNA to the electrophoretio mobility of t-RNA (see the diagram following the discussion). TABLE 3
Pseudo&dine
Experiment
1 1 2 2
content of pulse-labeled low molecular weight RNA before and after chase Total UMP+?FMP (cts/min)
Labeling Conditions
[6-3H]uridine [6-3H]uridine [6-SH]uridine [6-3H]uridine
pulse pulse; chased pulse pulse; chased
3484 3092 6555 8871
%YMP
10.2 18.4 10.4 16.7
Species of RNA
pre-tRNA pre-tRNA pm-tRNA pre-tRNA
The method used for the determination of the YMP content is described in Materials section (e). The RNA used in each experiment was prepared as described below.
+ + + +
tRNA tRNA tRNA tRNA
and Methods
Experimwat 1 2.5 x lOa cells were incubated at 5 x lo6 cells/ml. in methionine-free medium for 4.5 hr at 37°C. They were labeled for 10 min with 20 &ml. of [6-3H]uridine, and then an actinomycin chase was added. One-half of the culture ws8 stopped 2 min after the addition of the chase. The other half was chased for 30 min longer in the presence of methionine and then stopped. RNA was precipitated from the soluble cytoplasm of each sample. Experiment 2 2.5 x lo* cells were incubated as in the preceding experiment.
for 3 hr in methionine-free medium, labeled for 16 min and chased Deproteinized RNA was prepared from cytoplasmic extracts.
4. Discussion Experiments described in this paper have demonstrated that purified pre-tRNA can be converted to tRNA by incubation with a crude cytoplasmic extract in vitro. Since it has been shown previously that pre-tRNA and tRNA exhibit a precursorproduct relationship in intact cells (Bernhardt & Darnell, 1969), it ia concluded that pre-tRNA is a bonafide intermediate in tRNA synthesis. Previous results have indicated that the conversion of pre-tRNA to a molecule with the electrophoretic mobility of tRNA does not require complete methylation (Bernhardt t Darnell, 1969). Experiments described in this report have shown that complete formation of pseudouridine is not required either. Since there is no direct evidence that modification of bases occurs during (as opposed to after) the conversion and since there is evidence that pre-tRNA and tRNA differ in molecular weight (Burdon t Clason, 1969; Bernhardt & Darnell, 1969), it is unnecessary to invoke a
TRANSFER
RNA
SYNTHESIS
IN
HELA
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151
role for modification of bases in the conversion of pre-tRNA to the electrophoretic mobility of tRNA. However, modification is involved at some point(s) in the conversion of pre-tRNA to mature tRNA. The apparent relationship between modification and conversion is shown diagrammatically below: Hypothetical primary gene product 4 base alterations pre-tRNA
1
1 peak on gel (pre-tRNA)
4 size change modification-deficient tRNA J- base alterations mature tRNA
1 peak on gel (tRNA) i
I thank Dr James E. Darnell for many invaluable suggestions and discussions. This work was supported by grants from the National Institutes of Health (CA 07861-05), the National Science Foundation (G.B. 4565) and the Health Research Council of the City of New York. REFERENCES Amaldi, F. & Attardi, G. (1968). J. Mol. Biol. 33, 737. Attardi, G., Parr-me, H., Hwang, M-I BEAttardi, B. (1966). J. Mol. Biol. 20, 145. Bernhard& D. t Darnell, J. E. (1969). J. Mol. Biol. 42, 43. Burdon, R. H. & Clason, A. E. (1969). J. Mol. BioZ. 39, 113. Burdon, R. H., Martin, B. T. & Lal, B. M. (1967). J. Mol. BioZ. 28, 357. Eagle, H. (1959). Science, 130, 432. Maizel, J. V. (1966). Science, 151, 988. Penman, S., Scherrer, K., Becker, Y. & Darnell, J. E. (1963). Proc. Nat. Ad. Sk., Wash. 49, 654. Rodeh, R., Feldman, M. & Littauer, U. (1967). Biochembt~g, 6, 451. Summers, D. F., Maizel, J. V. & Darnell, J. E. (1965). Proc. Nat. Acad. Sk., Wash. 54, 505. Warner, J. R., Soeiro, R., Birnboim, H. C., Girard, M. & Darnell, J. E. (1966). J. Mol. BioZ. 19, 349.