Double-stranded ribonucleic acid in human lymphocytes

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Experimental Cell Research 88 (1974) 188-l 92

DOUBLE-STRANDED RIBONUCLEIC IN HUMAN LYMPHOCYTES U. TORELLI,

G. TORELLI

ACID

and R. CADOSSI

Institute of Medical Pathology, University of Modena, 41100 Modena, Italy

SUMMARY Labelled RNA extracted from human peripheral lymphocytes was digested with DNase and RNase and cbromatographed on cellulose columns. The results show that a significant proportion of the RNA synthesized in both unstimulated and PHA-stimulated human lymphocytes is in an RNase-resistant form with the properties of double-stranded RNA (ds-RNA). The proportion of total labelled cell RNA which appears in double-stranded form in unstimulated lymphocytes is much greater than in PI-IA-stimulated cells, presumably due to the much larger synthesis of ribosomal RNA in the latter cells.

Human small lymphocytes synthesize RNA molecules mostly confined to the nucleus whose size is larger than that of 28s ribosomal RNA [l, 21. These molecules show a very low degree of methylation and a high efficiency of DNA-RNA hydribization, and up to 20% of them are bound to polyadenylit sequences[l, 21. PHA stimulation is followed by a marked increase in the rate of both ribosomal RNA [l] and informational poly(A)-rich RNA [3], but methods used so far failed to reveal differences in RNA sequences synthesized after PHA stimulation

11,41. Since it has been recently demonstrated that a significant proportion of nuclear RNA of the heterogeneous type in animal cells is in an RNase-resistant form with properties of double-stranded RNA (ds-RNA), we have examined whether the RNA synthesized in both unstimulated and PHA-stimulated human lymphocytes includes double-stranded segments. Exptl Cell Res 88 (1974)

MATERIAL

AND METHODS

Lymphocyte cultures Normal small lymphocytes were obtained from the peripheral blood of two normal donors. Lymphocytes were separated from granulocytee by filtration through commercial nylon-fibres, according to the technique already described [l], and cultures were set up in Eagle’s medium containing 20% autologous plasma. The average cell concentration was 3 x 10’ -cells/ml. Incubation of unstimulated lymphocytes with labelled precursors ($H-uridine and SH-adenosine; Radiochemical Centre, Amersham) started 12 h after the cultures were set up, whereas incubation of PHA-stimulated cells started 48 h after the addition of PHA (Wellcome, reagent grade) to the cultures, at the concentration of 0.02 ml/ml.

RNA extraction Small pellets of cells washed twice with saline were resuspended in 20 ml of buffer (Na acetate 0.01 M, Na EDTA 0.01 M and bentonite 0.05 %). One ml of 10% sodium dodecyl sulphate was then’added, followed 30 set later bv 20 ml of a 90% solution of phenol-m-cresol (7.9:-l v/v) containing 0.1% hydroxyquinoline. The suspension was shaken vigorously for 3 min at 60°C in a water bath. The aqueous phase was separated by centrifuging for 10 min at 15 000 rpm and the extraction was repeated twice. The final aqueous phase was brought to 2 % potassium acetate and 75 % in ethanol and stored overnight at + 20°C.

Double-stranded RNA in human lymphocytes

for a sufficiently long time to obtain a statistical aaxracy of_ .k 6 % or better. To verify that the cellulose column was indeed separating ds-RNA, further experiments were performed in which RNase-resistant material was adsorbed to hydroxyapatite in low salt concentration (0.2 M NaCl, 0.05 M phosphate buffer) and eluted from the column in high salt concentration (0.2 M NaCl, 0.5 M phosphate buffer) [8]

Isolation of double-stranded RNA The precipitated nucleic acid was collected by centrifugation for 15 min at 2500 rpm at 4”C, rinsed with ethanol containing 2 % potassium acetate and washed twice with 10 ml of 3 M Na acetate. The pellet was then dissolved in 5 ml of buffer (50 mM NaCl, 2 mM MgCL 10 mM Tris-HCl, pH 7.4) and DNase (Worthington) was added to the final cont. of 2Opg/ml. After 30 min of incubation at room temperature, the solution was brouaht to 0.25 M NaCl bv adding 5 M NaCl. Thirty ,ug/&l of pancreatic RNaseand ;?O\nits/ ml of Tl RNase (Worthinaton) were then added and the solution was incubated for 30 min at 37°C. The reaction was terminated by extracting twice with phenol-m-cresol and the nucleic acid was precipitated as above. In order to isolate and purify double-stranded RNA from single-stranded RNA, transfer RNA and DNA, we used chromatography on cellulose columns, according to the method of Franklin [7]. Nucleic acids were dissolved in 0.5 ml of buffer (0.1 M NaCl, 0.05 M Tris HCl, 0.001 M NaEDTA, pH 6.9) and brought to 35 % with ethanol. The sample was applied to a cellulose column (1.5 x 10 cm) and the column was washed with the same solution. Under these conditions DNA and transfer RNA fail to remain on the column ana elute directly with buffer with 35 % ethanol. Single-stranded RNA remains on the column but can be eluted with buffer containing 15 % ethanol. Double-stranded RNA is finally removed with buffer alone. Five ml fractions were collected from the columns, for a total of 75 ml of each washing. Acid-precipitable radioactivity was determined for each fraction, Samples were counted lflhanol

15%

189

RESULTS From 5 to 15 % of the lymphocyte labelled RNA remained TCA-precipitable after nuclease treatment. Most of this RNA could be eluted from the column with buffer containing 35 or 15 % ethanol. However, a significant amount of labeled material left on the column was eluted with buffer containing no ethanol, as expected for double-stranded RNA (fig. 1). The material eluted in the third chromatographic peak was hydrolyzed completely in 0.3 % M NaOH, but was approx. 9.5% resistant to pancreatic RNase. Chromatographic samples were heated 3 min at lOO”C, chilled quickly to O”C, brought to

I Elulfer

Fig. 1. Abscissa: fraction no.; ordinate: cpm x lo-%. Isolation of labelled double-stranded RNAfrom unstimulated small lymphocytes. After treatment with DNase and RNase the total vield of re-extraction of dearaded nucleic acids was applied to a cellulose column. The radioactivity profile indicates that a definite peak of labelled material was obtained by elution with buffer alone. Background radioactivity has been subtracted. Blank vials had an average 8 cpm. 5

10

15

20

25

30

35

40

45

Exptl Cell Res 88 (1974

190

Torelli, Torelli and Cadossi

Table 1. Isolation of ds-RNA from human small lymphocytes Duration of incubation with 3H-5-uridinea 3 hours I Total RNA radioactivityb treated with nucleases II RNA radioactivity after digestion placed on column III RNA radioactivity in third chromatographic peak as % of I

6 hours

9 hours

6 255

8 950

10 730

15 240

13 870

21 380’

1 330

1 820

2 150

3 170

1700

2 530

127

192

206

282

242

384

2.03

2.15

1.92

1.85

1.80

1.80

o The two columns in each group show results obtained with cells from two different donors. b Acid-insoluble radioactivity, cpm. ’ In this experiment, 9 h of incubation with 3H-5-uridine was followed by 3 h of ‘chase’ with unlabelled uridine.

0.25 M NaCl and digested 30 min with 20 lug/ml of pancreatic RNase at 37°C. The 3H-5-uridine-labelled material was then made completely sensitive to RNase, whereas the 3H-adenosine-labelled RNA remained resisttant to pancreatic RNase to an extent up to 20%. These results were obtained with RNA from both unstimulated and PHA-stimulated lymphocytes. Experiments with hydroxyapatite chromatography also showed that 1.55 and 0.35 Y0 of the total RNA radioactivity extracted respectively from unstimulated and PHA-stimulated lymphocytes was eluted in high salt buffer, as expected from doublestranded RNA.

The difference between unstimulated and stimulated lymphocytes and the changes in the proportion of labelled ds-RNA observed by increasing the perio$ of incubation with labelled precursors are shown in tables 1 and 2. It is apparent that the proportion of labelled RNA with double-stranded characteristics found in PHA-stimulated lymphocytes is much lower than that found in unstimulated cells. Furthermore, in stimulated cells a decrease in this proportion was observed by prolonging the incubation and by ‘chasing’ the cells with unlabelled uridine; whereas no significant changewas observed in small lymphocytes under the sameconditions.

Table 2. Isolation of ds-RNA from PHA-stimulated

lymphocytes

Duration of incubation with 3H-5-uridinea

I Total RNA radioactivityb treated with nucleases II RNA radioactivity after digestion placed on column III RNA radioactivity in third chromatographic peak as % of I

9 hours

3 hours

6 hours

398.75 x 103 580.35 x 103

1108~10s

1350x103

2195x103

1830~10~’

24.5 x 10s

51.8 x 103

75.7 x 103

89.8 x 103

105 x 103

85x103

2 235

2960

4 575

5 130

4 390

732

0.55

0.51

0.41

0.38

0.2

0.04

a The two columns in each grsntp show results with cells from two different donors. b Acid-insoluble radioactivity, cpm. ’ In this experiment, 9 h of incubation with 3H-5-uridine were followed by 3 h of ‘chase’ with unlabelled uridine. Exptl Cell Res 88 (1974)

Double-stranded RNA in human lymphocytes

191

of ds-RNA apparently increases following PHA stimulation. In fact, the decrease in The results of our experiments show that a proportion of labelled ds-RNA observed by significant proportion of the RNA synthe- prolonging the incubation suggeststhat the acsized in both unstimulated and PHA-stimu- cumulation of ribosomal RNA is not parallated human lymphocytes in a short labelling leled by any accumulation of metabolically period is in an RNase resistant form with the stable ds-RNA. This is confirmed by the properties of ds-RNA. The proportion of further decreasein the proportion of labelled total labelled cell RNA which appears in ds-RNA caused by cold uridine chase of double-stranded form in small lymphocytes PHA-stimulated lymphocytes. The functions of ds-RNA in cellular meis considerably higher than that reported in other cell types [5, 9, lo], and is close to that tabolism are so far unknown. However, the found in isolated heterogenous nuclear RNA ability of very small amounts of cellular dsof HeLa cells [6]. This supports previous RNA to inhibit initiation of protein syntheconclusions that small lymphocytes synthe- sis in cell-free extracts has led to the hyposize mainly nuclear RNA of the hetero- thesis that these molecules play a physiological role in the regulation of translation of geneous type [l, 21. The large increase in the rate of synthesis mRNA in eukaryotic cells [ll]. of ribosomal RNA which follows PHA stiThe study of the pattern of ribosomal RNA mulation of small lymphocytes [l, 21justifies formation in small lymphocytes has shown the observation of a much lower proportion that in these cells, processing of the 45s riboof labelled ds-RNA in PHA-stimulated cells. somal precursor RNA is largely abortive, perThese results cannot therefore lead unambi- haps as a consequenceof an inadequate rate guously to the conclusion that the amount of protein synthesis [12, 13, 141.The demonof ds-RNA in unstimulated lymphocytes is stration that small lymphocytes synthesize higher than that in stimulated ones. How- ds-RNA segmentswhose life span is considerever, the above-reported data suggest that ably longer than in stimulated cells justifies the metabolic characteristics of the double- the hypothesis that the increase in protein stranded sequencesare markedly different in synthesis rate which occurs after PHA stimuthe two types of cells. The overall turnover lation may be related to the shortened life rate of nuclear RNA in small lymphocytes span of ds-RNA molecules. In this case, is markedly low, as shown by the association control of ds-RNA metabolism might repreof the majority of a labelled precursor with sent an important level of regulation of lymrapidly sedimenting molecules even after phocyte growth. several hours of actinomycin chase [l]. Since no significant changes in the proportion of This research was supported by a grant from the Consiglio Nazionale della Ricerche. labelled ds-RNA were observed in small lymphocytes during a 9 h period, this sugREFERENCES gests that the turnover rate of ds-RNA is DISCUSSION

also very low and similar to that of total nuclear RNA. This conclusion is also supported by the observation that cold uridine chase does not significantly alter the proportion of labelled ds-RNA. The turnover rate 13 - 741818

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192 Tovelli, Torelli and Cadossi 4. Berke, G, Sarid, S & Feldman, N, Biochim biophys acta 254 (1971) 440. 5. Kronenberg, L H & Humphreys, T, Biochemistry 11 (1972) 2020. 6. Jelinek, W & Darnell, J E, Proc natl acad sci US 69 (1972) 2537. 7. Franklin, R M, Proc natl acad sci US 55 (1966) 1504. 8. Bernardi, G, Nature 205 (1965) 779. 9. Stern, R & Friedman, R M, Nature 226 (1970) 612.

Exptl Cell Res 88 (1974)

10. Hare& L & Montaignier, L, Nature new biol 229 (1971) 106. 11. Robertson, H D & Mathews, M B, Proc natl sci US 70 (1973) 225. 12. Cooper, H L, Biochemistry of cell division (ed R Baserga) p. 91. Charles C Thomas, Springfield, Ill. (1969). 13. Cooper, H L, Nature 227 (1970) 1105. 14. Rubin, D, Blood 35 (1970) 708. Received April 8, 1974