Transfer ribonucleic acid populations in concanavalin-A-stimulated bovine lymphocytes

Biochimica et Biophysica Acta 868 (1986) 91-99

91

Elsevier BBA 91640

T r a n s f e r ribonucleic acid p o p u l a t i o n s in c o n c a n a v a l i n - A - s t i m u l a t e d b o v i n e l y m p h o c y t e s Karl-Heinz Derwenskus and Mathias Sprinzl Laboratorium fh'r Biochemie der Universitiit Bayreuth and Bayreuther lnstitut fh'r makromolekulare Forschung, Bayreuth (F. R. G.)

(Received22 May 1986)

Key words: tRNA; Modifiednucleotide; Protein synthesis; Affinitychromatography;Mitogen; (Bovinelymphocyte)

Transfer RNA isolated from lymphocytes stimulated by concanavalin A and that from resting cells were compared with respect to amino-acid acceptance, integrity of the CCA-terminus, extent of modification and isoacceptor distribution. Following growth stimulation the overall amino-acid acceptance of the tRNA is elevated, in particular the relative acceptor activities for threonine and arginine are increased. The reduced acceptor activity of the tRNA from the quiescent cells is not due to a preferential degradation of the CCA-end, since it persits even in the presence of ATP(CTP):tRNA nucleotidyltransferase. We therefore conclude that this reduced activity is caused by structural differences of the tRNAs. The content of modified nucleotides in newly synthesized tRNA from lymphocytes cultured in the presence and absence of concanavalin A was determined, tRNA from resting cells was found to be undermodified with respect to pseudouridine and dihydrouridine. Upon monitoring the tRNA isoacceptor distribution by affinity chromatography on immobilized elongation factor Tu and subsequent two-dimensional gel electrophoresis, a preferential synthesis of particular lysine- and threonine-accepting tRNAs was observed upon mitogenic stimulation. Evidently, a specific tRNA population is needed by the proliferating cells. These results are discussed in view of the hypothesis that the commitment of iymphocytes to proliferation is at least in part under translational control.

Introduction Fluctuations in the tRN'A population have been observed as a result of changing a variety of conditions in microorganisms, plants, animals and cell cultures [1]. In many of these biological systems the conditions resulting in an altered tRNA distribution were artificial or pathological. With primary lymphocyte cultures we have the oppor-

Abbreviation: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid. Correspondence: Dr. M, Sprinzl, Laboratorium for Biochemie, Universit~itBayreuth, Universit~itsstrasse30, D-8580 Bayreuth, F.R.G.

tunity to study an inducible proliferation, which parallels a natural process. Lymphocytes are isolated as resting cells, locked in the GO-phase of the cell cycle, which can be stimulated in vitro by a variety of mitogenic substances, e.g., the plant lectin concanavahn A, They then enter the cell cycle, leading to proliferation, D N A synthesis and finally cell division. This process is believed to resemble the events caused by an antigen-triggered immune response. After the mitogenic stimulation of lymphocytes, a rapid increase in protein biosynthetic activity preceding any RNA synthesis is observed [2]. Even in the presence of actinomycin D an early onset of protein biosynthesis, at least for

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92 some proteins [3,4], was reported. These observations led to the postulation of posttranscriptional control mechanisms, resulting in a more efficient translation of pre-synthesized mRNA after lymphocyte stimulation. Increased transport of polyadenylated mRNA from the nucleus to the cytoplasm [2] and more efficient formation of the initiation complex due to a higher level of methionyl-tRNAiMet [5] contribute to the enhancement of the translation rate. Furthermore an increase in the overall methylation rate of nucleotides from cytoplasmic RNA [6] was reported and Cooper and Braverman [7] postulated the presence of a small, non-messenger RNA as being a regulatory signal in lymphocytes. In the past decade it has become clear that a well-balanced tRNA population is necessary for efficient translation [8,9]. Furthermore it has been demonstrated that in eukaryotic cells tRNA could be involved in regulatory processes. [10] and that not only the initiation of translation is a point of post-transcriptional control, but that also the elongation rates could differ under various conditions [11-13]. Since in the lymphocyte system, where a translational control is hypothesized, only few attempts have been made at examining the role of tRNAs [5,14], we decided to fill this gap and present a characterization of the tRNAs from resting and concanavalin-A-stimulated lymphocytes. Materials and Methods

Isolation and culture of lymphocytes. Lymphocytes were isolated from bovine retropharyngeal lymph nodes as described by Peters [15], including lysis of erythrocytes by osmotic shock in water. The cells were cultured in a humified atmosphere with 8% CO 2 in RPMI 1640 medium supplemented with 10% heat-inactivated newborn calf serum, 100 units/ml penicillin, 100 # g / m l streptomycin, 2 mM L-glutamine, (all obtained from Gibco) and 10 mM Hepes. The initial cell density was adjusted to (5-7)- 106 cells/ml. For triggering the stimulation, concanavalin A (BoehringerMannheim) was added to a final concentration of 5 ~tg/ml. Determination of RNA and DNA synthesis. The rates of RNA and DNA synthesis were measured

by [5-3H]uridine (5 Ci/mmol) or [methyl-3H] thymidine (5 Ci/mmol) incorporation into acidprecipitable material as described by Hauser et al. [2]. In parallel, cell viability was determined with a hemocytometer by the exclusion of Trypan blue dye. The amount of radioactivity incorporated was normalized to 1 • 106 viable cells.

Labelling of RNA with inorganic [3ep]phosphate. Cells were concentrated by centrifugation to a cell density of about 20.106 cells/ml in a phosphate-free RPMI 1640 medium with the same additives as described above. Incubation in the presence of 15 /~Ci/ml inorganic [32p]phosphate was performed for 18 h under the conditions described above. To stimulated cell cultures concanavalin A was added again to a final concentration of 5 ~g/ml. Isolation of tRNA from lymphocytes. Cells were harvested and broken essentially as described by Kecskemethy and Sch~ifer [16], except that heparin was replaced by ribonucleoside-vanadyl complexes as RNAase inhibitor [17]. tRNA was isolated by extracting three times with phenol and subsequent DEAE-cellulose chromatography [18]. Deacylation of endogenous aminoacyl-tRNA was achieved by CuSO4 treatment as described elsewhere [19]. For the comparison of amino-acid acceptance of tRNA from resting and stimulated lymphocytes, the tRNAs were separated from high-molecular-weight impurities by gel-filtration on Ultrogel AcA 54 columns (29 × 2 cm) with 20 mM sodium acetate (pH 4.5)/100 mM NaCI/10 mM MgCI 2 as elution buffer, t R N A was then precipitated with ethanol and resuspended in water. Alternatively, for the determination of nucleotide composition, [32p]tRNA was purified by gel electrophoresis in 10% polyacrylamide gels and eluted with the buffer system described in Ref. 20. Enzymatic aminoacylation, tRNA was aminoacylated in buffer containing 50 mM Hepes (pH 7.6), 160 mM KC1, 1 mM ATP, 200 /xM spermine, 2 m M MgCI 2, 50/~g/ml bovine serum albumin, 100 ~tM dithioerythritol, 7.5 A260 units/ ml tRNA, 18-24 /tM of the appropriate amino acid and, when indicated, 250 # g / m l ATP (CTP):tRNA nucleotidyltransferase from yeast (EC 2.7.7.21). (One A260 unit is the quantity of material contained in 1 ml of a solution with an

93

absorbance of I at 260 nm, when measured in a 1 cm pathlength cell.) After preincubation for 5 min at 37°C the aminoacylation reaction was started by the addition of aminoacyl-tRNA synthetase sufficient to achieve complete charging within 20 rain. Samples totalled 20 #1 from which 18 #1 were precipitated in 5% trichloroacetic acid and the radioactivity retained measured by scintillation counting. The aminoacyl°tRNA synthetase was prepared as described elsewhere [19]. tRNA isoacceptor patterns, tRNA isoacceptors were isolated by affinity chromatography on immobilized elongation factor Tu of Thermus thermophilus as reported recently [19] with one modification: the aminoacylation reaction was performed in the buffer described above. In this buffer, which is adjusted to physiological conditions [21], the extent of misaminoacylation is negligible. For the aminoacylation with lysine the pH was adjusted to 8.6, which was found to be optimal for the bovine lysyl-tRNA synthetase. The tRNA isoacceptors isolated by affinity chromatography were analyzed by two-dimensional polyacrylamide gel electrophoresis [22]. RNA was visualized by 'Stains All' (Serva, Heidelberg, F.R.G.) [23] or by autoradiography. Analysis of nucleotide composition. The tRNA was boiled in water for 2 min, chilled on ice and then subjected to enzymatic digestion by nuclease P1 (EC 3.1.30.1) for 2.5 h as outlined by Gehrke et al. [24]. Alkaline phosphatase was omitted from the reaction mixture in order to obtain nucleoside 5'-phosphates. The digests were analysed by twodimensional thin-layer chromatography as described by Silberklang et al. [25]. The positions of the nucleotides were determined by autoradiography and identified by comparison with standards. Relative amounts of each nucleotide were determined after detachment of the [32p]nucleotidecontaining areas from the thin-layer plates according to Turchinsky and Shershneva [26] and measurement of the radioactivity by scintillation counting.

Results Stimulation of bovine iymphocytes The mitogenic response of lymphocytes was measured by their capacity to incorporate [3H]-

8 15

b x

E 8t-

.o

10

5" c:

5 r-1

l

i

2s

so

7's

60

Time after concanavalin A addition (h)

Fig. 1. Effect of concanavalin A on the incorporation of [3 H]uridine by lymphocytes. Bovine lymphocytes were isolated and cultured in 400 ml batches. Following the addition of concanavalin A, 1 ml aliquots were removed after the time intervals indicated and the cells tested for their ability to incorporate [3H]uridine into acid-precipitable material within 30 rain (D). The corresponding values for control cultures without mitogen are represented by the dark symbols.

uridine or [3H]thymidine into RNA or DNA, respectively. In Fig. 1 the RNA synthesis during a 112 h culture period is shown. An increase in RNA synthesis 16 h after concanavalin A addition, reaching a maximal value at 65 h, was observed. The corresponding curve for DNA synthesis lags about 10-15 h behind that of RNA synthesis (data not shown). After 43 h, when ceils usually were harvested for tRNA isolation (see below), the stimulated lymphocytes showed on average a 12fold increase in RNA and a 20-fold increase in DNA synthesis relative to the resting cells. As an additional criterion for successfull stimulation a higher content of lymphoblasts was observed in the mitogen-treated cultures. If not indicated otherwise, tRNA was isolated from lymphocytes after 43 h of incubation, that is in the early S-phase of the cell cycle. At this time no mixed-lymphocyte reaction was detectable in the non-stimulated control cultures (Fig. 1), which otherwise could change the tRNA distribution in these cells. The content of bulk tRNA in proliferating lymphocytes was 2.3 + 0.2 A26o units/ 106 cells, about 1.4-times the amount found in resting cells (1.7 + 0.2 A26o units/106 cells). This

94 observation indicates that the higher content of total cytoplasmic RNAs reported for stimulated lymphocytes [16] also includes the tRNAs of the cells. Since cell viability was found to be higher than 75% in both resting and stimulated lymphocyte cultures, there is only little contamination of the tRNAs characteristic for proliferating or for resting lymphocytes by the RNAs of dead cells.

Amino-acid acceptance of the tRNA The ability to enzymatically aminoacylate tRNA preparations from resting and stimulated lymphocytes with six different amino acids was compared (Table I). In all experiments, bulk tRNA from proliferating cells was aminoacylated to a higher extent than the corresponding preparations from the untreated cultures. In particular, the relative acceptance for arginine and threonine rose significantly upon stimulation. Hence, this elevation is not a uniform process encompassing the whole tRNA population, and therefore cannot be due to a different t R N A content between the preparations from resting and proliferating ceils. Resting lymphocytes contain high nucleolytic activity. Although we used potential RNAase inhibitors during the tRNA isolation, a partial degradation of the tRNA could not be excluded entirely. On the other hand, a R N A a s e / R N A a s e inhibitor system could have physiological signifi.cance in regulating the level of functional tRNA. In order to examine the integrity of the most vulnerable part of the tRNA, the invariant CCA-

terminus, we repeated the aminoacylation experiments in the presence of A T P ( C T P ) : t R N A nucleotidyltransferase, the enzyme regenerating CCA-termini. The overall amino-acid acceptance was increased by the addition of this enzyme in all tRNA preparations, showing that about 15-25% of the tRNA from resting as well as from stimulated cells was devoid of the Y-terminal adenosine. But even in the presence of ATP(CTP) : tRNA nucleotidyltransferase, the tRNA from quiescent lymphocytes was less aminoacylated then that from stimulated cells and the arginine and threonine acceptance again was reduced significantly. Thus we concluded that structural requirements other then the CCA-end must be responsible for these differences.

tRNA isoacceptor distribution Bulk tRNA from stimulated and resting lymphocytes, harvested 43 h after concanavalin A addition, were compared by two-dimensional gel electrophoresis (Fig. 2). By this technique, 45 tRNA species could be resolved. This number is in good agreement with investigations of tRNAs from other vertebrate sources [27]. No significant differences between the tRNA patterns from proliferating and quiescent cells were detected. The corresponding tRNA patterns obtained from lymphocytes, which were analyzed immediately after their preparation from the lymph nodes without having been cultured, were virtually identical with that shown in Fig. 2. Thus, there also were no detectable changes of the tRNA distribution caused by the culture conditions.

TABLE I AMINOACYLATION OF BULK tRNA FROM RESTING AND STIMULATEDLYMPHOCYTES Data given in pmol/A26o unit tRNA are expressed as the means+ S.E. for triplicate determinations. Amino - N u c l e o t i d y l t r a n s f e r a s e acid + Con A - Con A Arg Leu Lys Ser Thr Val

63.9+ 9.9 38.1+ 1.7 42.3-+ 4.3 54.2_+ 6.5 51.7_+ 5.1 34.9-+ 1.7

41.7_+ 8.2 41.2-+ 6.2 36.6-+10.8 47.4___6.5 32.4-+ 3.4 35.85:0.9

Nucleotidyltransferase + Con A - Con A

+

86.9_+ 5.1 45.7-+ 0.9 49.4-+ 2.8 55.1+ 6.8 58.2_+ 5.1 38.1-+ 5.7

63.0_+16.2 50.3+ 9.4 40.0-+15.1 66.5-+ 9.7 45.7+ 2.0 38.6-+ 5.7

285.1+28.4 235.1_+33.5 333.4_+24.7 304.1_+54.5

Fig. 2. Distribution of tRNA isoacceptors in bovine lymphocytes. One A260unit of bulk tRNA from resting (a) or mitogen stimulated lymphocytes(b) was analyzed by two-dimensional gel electrophoresis(see Materials and Methods).

95 About 40% of the cell population isolated from bovine lymph nodes consists of T-lymphocytes [16]. Since only these cells are the primary targets for the interleukin-2-dependent concanavalin A stimulation, there is more than half the cell population in a quiescent state even after mitogen addition. This background can mask minor variations of the tRNA distribution. In order to circumvent this problem, we cultured the cells for different time intervals in the presence of inorganic [32p]phosphate. Thus, we were able to isolate [32p]tRNA synthesized de novo in resting and proliferating lymphocytes. From these [32p]_ tRNA preparations as well as from unlabelled tRNA the distribution of tRNA isoacceptors was

1. Dimension

1

02

Fig. 3. Distribution of tRNAT M isoacceptors in bovine lymphocytes. Unlabelled bulk tRNA was isolated from resting (a) or stimulated lymphocytes (b). 32p-labelled tRNA was prepared from stimulated cells which had been grown in the presence of inorganic [32p]phosphate for 18 h starting 31 h after mitogen addition (c). The threonine specific tRNAs were isolated by affinity chromatographyon immobilizedelongation factor Tu. Two-dimensional gel-electrophoreticanalysis was performed with 70 pmol threonyl-tRNAT M (a, b) or, in the case of [32P]tRNA~r, with an amount correspondingto 20000 cpm (c). Gels were stained (a, b) or autoradiographed (c). Because of the shrinkage due to freezingthe gel during autoradiography (c) the distance between the spots is shortened as compared to the stained gels.

monitored. Because of the difficulties in obtaining enough tRNA, particularly from the resting cells, we restricted our investigations to groups of certain isoaccepting tRNAs. Two major tRNA T M species were isolated by affinity chromatography from unlabelled tRNA of resting and stimulated lymphocytes. Their relative distribution was found to be equal in proliferating lymphocytes, whereas in the resting cells the isoacceptor corresponding to the lower spot on the gel was predominant (Fig. 3a, b). The 32p-labelled tRNA revealed clearly a preponderant synthesis of the tRNA isoacceptor corresponding to the upper spot (Fig. 3c). These results could be rationalized as follows: that an enhancement of synthesis of this particular tRNA T M isoacceptor upon stimulation leads to an equal distribution of the two tRNA T M species observed in the unlabelled tRNA from proliferating cells. Upon analysis, no differences in the distribution of the tRNA Lys isoacceptors in unlabelled tRNA from resting and stimulated lymphocytes were detectable (data not shown). The lysinespecific tRNAs were resolved into three major isoacceptors. The analysis of 32P-labelled tRNA Lys from resting lymphocytes revealed a predominant synthesis of the isoacceptors 1 and 2 as compared to tRNA Lys species 3 (Fig. 4a). In the early phase after concanavalin A addition an elevated synthesis of isoacceptor 1 and, in a later period, of isoacceptor 2 was observed (Fig. 4b, c). Therefore the equal distribution of the isoacceptors 1 and 2 seen in unlabelled tRNA isolated from stimulated cells after 43 h is obviously the result of a preferential synthesis of the particular isoacceptors at different culture periods. Since variations of the tRNA isoacceptor distribution were also detected for the serine-specific species, we conclude that an adaptation of the tRNA isoacceptor distribution is required by lymphocytes in different states of the cell cycle.

Analysis of nucleotide composition To determine whether the changes observed in the isoacceptor distribution were of transcriptional nature or due to post-transcriptional modification, we analyzed the base composition of the tRNA from resting and stimulated lymphocytes. In order to monitor only the new tRNA synthe-

96

b 0 t IP

1. Dimension

0

1

02 Ib3

Fig. 4. Distribution of tRNA Lys isoacceptors in bovine lymphocytes. Bulk tRNA was isolated from resting (a) or stimulated lymphocytes (b, c) which had been cultured in the presence of inorganic [32P]phosphate from the 31st until the 49th h (a, c) or from the 4th until the 22nd h (b) after concanavalin A addition. The isoacceptor patterns were obtained as described in the legend to Fig. 3. The amounts of [32p]tRNA used for electrophoreticai analysis corresponded to 10h0-8000 cpm; spots were detected by autoradiography.

TABLE II NUCLEOTIDE COMPOSITION OF BULK tRNA FROM RESTING AND STIMULATED LYMPHOCYTES Bulk tRNA was isolated from resting a mitogen treated lympbocytes cultured in the presence of inorganic [32p]phosphate for 18 h (31st until 49th h after mitogen addition). [32P]tRNA purified by polyacrylamide gel electrophoresis was digested and analyzed by two-dimensional thin-layer chromatography. Percentages are based on total radioactivity obtained by counting all spots removed from the particular plate (100% corresponds to 22300-47000 cpm). Data are given as the means4- S.E. with n = 3. Nucleotide

+ Con A

- Con A

pA pU pG pC pxO pD pmSC pm7G pT pm1G pm2G pm2G

17.49 4-0.87 15.584-0.61 27.68 4- 0.19 26.04 + 0.32 3.75 4- 0.04 2.89 + 0.10 2.575:0.14 0.48 4-0.07 0.57 + 0.02 1.20 + 0.14 0.84+0.15 0.79 + 0.08

18.67 + 0.73 14.804-0.59 27.40 4- 0.68 26.30 4- 0.40 3.29 4-0.24 2.42 4- 0.04 2.544-0.08 0.58 4- 0.02 0.50 4- 0.03 1.18 4- 0.08 1.01 4-0.09 0.69 4- 0.02

difference was reproducibly found in all experiments.

Discussion sized in response to the mitogenic stimulation, we labelled lymphocyte tRNA in vitro with inorganic [32p]phosphate. After enzymatic digestion and separation by two-dimensional thin-layer chromatography, 20 nucleoside 5'-[32p]phosphates were resolved. No qualitative differences were detected between the samples from resting and stimulated lymphocytes. From the 12 most abundant nucleotides the quantitative distribution was measured by liquid scintillation counting. The resuits for [32p]tRNA labelled for 18 h from mitogen-treated lymphocytes and from control cultures are shown in Table II. The content of modified nucleotides comprises 13.1% in the tRNA from stimulated and 12.2% in the tRNA from resting lymphocytes. The main difference concerning individual nucleotides between the tRNA preparations, is the higher degree of modification of the tRNA from the stimulated lymphocytes with respect to pseudouridine and dihydrouridine. This

The abbility of tRNA to accept the cognate amino acid is obviously the most important prerequisite for its function in protein biosynthesis. Griffin et al. [14] reported no significant differences in the extent of aminoacylation between tRNA from resting or phytohemagglutinin-stimulated human lymphocytes when tyrosine, phenylalanine or aspartic acid charging were compared. On the other hand, Cooper and Braverman [5] mentioned a complex pattern of alterations in the relative levels of tRNAs. Following growth stimulation, some accepting activities (glutamic acid, glycine) showed relative increases, other remained essentially constant (proline, valine), while again others decreased (aspartic acid, serine, phenylalanine) [5]. Our finding of an elevated amino-acid acceptance of the tRNA from stimulated as compared to resting cells could indicate a strong activity of

97 these tRNAs in protein biosynthesis. Furthermore, it has been suggested that the charging level of tRNA plays a key role in controlling metabolic changes in eukaryotic cells [28,29]. In particular, DNA-polymerase activity in lymphocytes [30], selective protein degradation by the ubiquitin- and ATP-dependent proteolytic system [10] and the initiation of translation [31,32] were thought to be controlled by the charging of tRNA species. These ideas, along with the belief that the availability of certain amino acids are important control mechanisms for protein synthesis in lymphocytes [33], it is conceivable that variations of chargeability with particular amino acids (e.g., threonine, arginine) may have profound implications on the mitogenic response. One potential target for controlling the level of aminoacylation is the invariant CCA-terminus common to all tRNAs. It is known that mammalian cell cytoplasm contains ribonucleases and ribonuclease inhibitors and that the rate of cytoplasmic degradation of RNA results from the balance between nuclease and inhibitor levels. Additionally, after mitogen application, a rise in the inhibitor level has been observed leading to nearly complete inhibition of ribonuclease activity [34]. Therefore we expected that the most sensitive part of the tRNA molecules, the CCA-end, may be affected by this potential control mechanism. However, our results reveal no significant differences in the integrity of the tRNAs' CCAtermini between resting and stimulated lymphocytes. Hence, the reduced level of amino-acid acceptance of the tRNA from the resting lymphocytes is, at least for the six amino acids under investigation, not due to incomplete CCA-ends. A unique feature of tRNA is the high degree of modification. In tRNA of eukaryotic origin up to 16.4% of the nucleotides are modified [39]. The content of modified nucleotides determined for tRNA from resting and stimulated lymphocytes, 12.2% and 13.1%, respectively, is in the same range as those found in other eukaryotic cells [35-37]. But it is less than the sequence-based value cited above, because not all rare nucleotides can be detected by any methodology applied. In particular 1-methyladenosine, which we found by HPLC analysis to comprise 0.6-0.9% of tRNA nucleotides, could not be detected by thin-layer chro-

matography for unknown reasons. Although no qualitative differences in the nucleotide composition of tRNA from resting and stimulated lymphocytes were detected, the relative amounts of dihydrouridine and pseudouridine were significantly elevated upon stimulation. These results are partly in contrast to those reported by Griffin et al. [14], who found no difference with respect to pseudouridine modification between resting and stimulated lymphocytes. The reason may reside in the different way, tRNA was labelled in each case. The sodium [14C]bicarbonate labelling used by Griffin et al. might be less sensitive than 32p labelling used in this work. It is, however, also possible that human lymphocytes stimulated with phytohemagglutinin behave differently than bovine lymphocytes after concanavalin A treatment. The observation of increased pseudouridine and dihydrouridine modification is of particular interest, not only because these are the most abundant tRNA modifications [38]. Beside the invariant pseudouridine in position 55, this nucleoside is very frequently located in the anticodon region, i.e., in the positions 32, 38 and 39 (numbering system according to Ref. 39). Since we investigated only cytoplasmic tRNA, which already should contain pseudouridine in position 55, because this modification is introduced in the nucleus [40], and since the enzymes responsible for pseudouridine formation in the anticodon region have been found also in the cytoplasm [41], the differences observed in pseudouridine modification are probably exclusively due to those in the anticodon region. It has been shown that the ability of histidyl,tRNA r~is from Salmonella typhimurium to recognize the histidine codons in the leader sequence of the mRNA of the His-operon is dependent on pseudouridine modifications in positions 38 and 39 [42,43]. Beside this direct evidence for the involvement of the pseudouridine modifications in codon recognition, it is generally believed that modifications in the anticodon region could affect the codon-anticodon interaction [44]. Therefore we conclude that lymphocytes may produce tRNAs capable of reading mRNAs more efficiently by introducing pseudouridine residues in the appropriate positions.

9~

The conversion of uridine to dihydrouridine leads to its inability to participate in stacking interactions and could result in a loop-out of the nucleotide [45]. These looped-out nucleotides are implicated as being potential enzyme-recognition sites [46]. According to this model there exists a simple and rapid opportunity for the cells to convert inactive tRNAs into substrates which could be recognized by enzymes, e.g., the appropriate aminoacyl-tRNA synthetases. The higher content of dihydrouridine in stimulated lymphocytes as compared to the resting cells could therefore reflect a switch of inactive to actively translating tRNAs. Beside the study already cited, only one report concerned with the modification of RNA following lymphocyte stimulation has been published [6]. Therein, an increased RNA methylation rate upon mitogenic stimulation was observed. Although total cellular RNA was investigated, it was assumed that the results are also valid for the tRNA moiety, since tRNAs are the main targets for methylation reactions. These observations are not in disagreement with our finding that the portion of most methylated nucleotides did not increase significantly, because in the work cited, the methylation activity rather than the nucleotide composition of the RNA was measured. Preliminary results obtained by HPLC analysis of tRNA nucleotides in our laboratory support the view that methylation also of tRNA is an early event after mitogenic stimulation, occurring before the onset of RNA synthesis [6]. Changes in the distribution of individual tRNA species after mitogenic stimulation were observed only when affinity chromatographically purified isoacceptors were analyzed. The pattern of tRNA~r isoacceptors clearly revealed a preferential synthesis of one tRNA species after stimulation. Although variations in tRNAThr isoacceptor distribution are not very frequently found upon changing physiological conditions, such changes were reported in plasmacytomas [47] and upon dimethylsulfoxide-induced differentiation of Friend's leukemia cells [48]. In contrast to these cells, which are specialized to synthesis of only one protein, in stimulated lymphocytes an increase of the synthesis of most cellular proteins is observed. Obviously, not only

for an efficient translation of particular mRNAs, an adaptation of tRNATh~ isoacceptors is necessary. The distribution of lysine isoaccepting tRNAs is of particular interest because one specific tRNALys isoacceptor was reported to correlate directly with the proliferation rate of cells [49]. This particular "isoacceptor" designated as tRNALys in the RPC-5 profile consists of two distinct tRNA species which are separable by polyacrylamide gel electrophoresis [50] and could be assigned to the tRNALys isoacceptors 1 and 3 in Fig. 4. Since we observe that only tRNA Lys isoacceptor 1 is synthesized predominantly in the early time interval after mitogenic stimulation, it is possible that only this particular isoacceptor in the 'mixture' of tRNA Lys species is the one correlated with cellular growth. Although we have no direct sequence information yet, we may assume in the light of the work by others [50-52] that the changes observed in the tRNALys isoacceptor distribution are not exclusively due to modification events but may also reflect differential gene expression. To prove this hypothesis and for the correlation of tRNA isoacceptor changes with different codon usage, additional investigations are under work.

Acknowledgements We wish to thank Drs. K.P. Schiller and N. Kecskemethy (Ruhr-Universit~it, Bochum) for the introduction of K.-H.D. into the technique of lymphocyte culture and to acknowledge the technical assistance of Ms. B. Wagner. The work was supported by Fonds der Chemischen Industrie and by Institut ftir makromolekulare Forschung, Universit~it Bayreuth.

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