Umberto Torelli, Giuseppe Torelli, and Ruggero Cadossi

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

Labelled double-stranded RNA (dsRNA) has been extracted from human leukemic blast cells incubated in-vitro with radioactive precursors. Purification of dsRNA has been obtained using selective nuclease digestion and chromatography on cellulose columns.The dsRNA molecules appear to be confined to the nucleus and carry, at least in part, polyadenylate sequences. Time course experiments show that the proportion of labelled dsRNA increases during in-vitro incubation of leukemic blast cells.

Introduction:

Double-stranded RNA (dsRNA) has been observed recently in several types of animal cells [1-5]. So far, the function of these molecules in cellular RNA metabolism is unknown. However, the ability of very small amounts of dsRNA to inhibit initiation of protein synthesis in cell-free extracts [6] has led to the hypothesis that these molecules play a role in regulation of of translation of messenger RNA in eukaryotic cells [6].

At least in some types of cells, dsRNA is bound to polyadenylate (poly (A)) sequences [4], is associated with large nuclear RNA molecules, and has metabolic properties characteristic of heterogenous nuclear RNA  [4, 5].

The occurrence in leukemic blast cells of a proportion poly(A)-bound nuclear RNA molecules larger than that observed in normal lymphocytes [7], led us to extend our investigations of the characteristics of nuclear RNA in human leukemic blast cells. We report here evidence that these cells do synthesize dsRNA. Some structural and metabolic characteristics are also reported.

Material and Methods:

Cells and Labelling Conditions:

Leukemic blast cells were obtained from the peripheral blood of four patients with acute myeloid leukemia having a high number of circulating leukocytes. Heparinized blood was sedimented by gravity at 37^oC and the supernatant was diluted to a final concentration of 2 x 10⁶ cells per ml. in Eagle's medium with 20% autologous plasma. [³H-5]-uridine and [³H]-adenine (Radiochemical Centre, Amersham) were added at the final concentration of 10UCi/ml and the cells incubated for periods up to 9 hr.

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 millilitre of 10% sodium dodecyl sulfate was then added, followed 30 sec later by 20 ml of 90% solution of phenol-m-cresol (7.9:1 v/v) containing 0.1% hydroxy-quinoline. The suspension was shaken vigorously for 3 min at 60^oC in a water bath. The aqueous phase was separated by centrifuging for 10 min at 15,000 rev.min and the extraction was repeated twice. The final aqueous phase was brought to 2% in potassium acetate and 75% in ethanol and stored overnight at -20^oC.

Isolation of Double-Stranded RNA:

The precipitated nucleic acid was collected by centrifugation for 15 min at 2500 rev/min at 4^oC, rinsed with ethanol 70% containing 2% potassium acetate and washed twice with 5 ml of 3 M sodium acetate. The pellet was then dissolved in 5 ml of buffer (50 mM NaCl, 2 mM MgCl₂, 10 mM Tris CCl, pH 7.4) and DNase (Worthington) was added at the final concentration of 20 ug/ml. After 30 min of incubation at room temperature, the solution was brought to 0.25 M NaCl by adding 5 M NaCl. Thirty microgrammes per millilitre of pancreatic RNase (Sigma) and 20 units/ml of T1 RNase (Worthington) were then added and the solution was incubated 30 min at 37^oC. Fifty microgrammes of subtilisn were then added and the reaction was terminated 30 min later by extracting twice with phenol-m-cresol and precipitating the nucleic acid as described 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 [8]. Nucleic acid was dissolved in 0.5 ml of buffer (0.1 M NaCl, 0.05 M Tris-HCl, 0.001 M Na EDTA, pH 6.9), 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 remian on the column but can be eluted 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. Six millilitre fractions were collected for a total of 72 ml of each washing. For each fraction, acid precipitable radioactivity was determined, and representative fractions of the third peak were pooled and further characterized.

Assay of Poly(A)-bound Double-Stranded RNA Sequences:

The proportion of dsRNA molecules bound to poly(A) sequences was evaluated by measuring the proportion of labelled dsRNA bound to glass fiber filters on which poly(U) had been previously immobilized by the technique of Sheldon et al [9].

To each fiberglass filter (Whatman GF.C, 2.4 cm), was added 0.15 ml of poly(U) solution (1 mg/ml in distilled water). The filters were then dried at 37^oC and irradiated for 2.5 min on each side at a distance of 22 cm from a 30 W Sylvania germicidal lamp. Each filter was rinsed with 50 ml of distilled water to remove unbound poly(U). Four fractions from the third peak were pooled and filtered through each filter at 2 ml/min. The filters were washed with 20 ml of buffer, followed by 20 ml of ice cold 5% trichloroactic acid and 10 ml of 95% ethanol, then dried and counted. Input radioactivity was evaluated by drying an filters 0.5 ml of the pooled fractions.

Nuclei Isolation:

To isolate nuclei of leukemic blast cells, the cells were suspended in cold hypotonic solution (0.01 M Tris, 0.08 M sucrose, 0.003 M CaCl₂, pH 7.4) and gently shaken by hand for 15 min. Triton X100 was then added at the final concentration of 0.2% and the suspension shaken again for 15 min and centrifuged at 2000 rev/min to separate nuclei.

Results:

As shown in Table 1:
 

Table 1. Synthesis of double-stranded RNA in leukemic blast cells incubated with [³H]-uridine for 3 hr.

Case	1	2	3	4
I. Total  RNA radioactivity treated with nuclease *	4.17 x 105	2.10 x 105	1.04 x 106	2.81 x 105
II.    RNA radioactivity after digestion placed on column	2.58 x 104	5.50 x 103	1.06 x 105	2.73 x 104
III.   RNA radioactivity in third chromatographic peak	4.76 x 103	1.44 x 103	8.40 x 103	1.70 x 103
As percentage of I.	1.0	0.7	0.8	0.6

*Acid insoluble radioactivity, counts/min

Fig. 1. Isolation of labelled double-stranded RNA from acute leukemia blast cells incubated with [³H]-uridine for 3 hr. After treatment with DNase and RNase, the total yield of reextraction of degraded nucleic acids was applied to a cellulose column. The radio-activity profile shows that a faily large amount of labelled material eluted in buffer alone.

Table 2. Effect of denaturation on ribonuclease reistance of labelled double-stranded RNA.

	[3H]-uridine labelled RNA	[3H]-adenosine labelled RNA
I.  Acid insoluble radioactivity from third chromatographic peak	1100             1860	550               2060
II.  Acid insoluble radioactivity after denaturation plus RNase	40                 32	155                  520
As percentage of I.	3.6                1.7	28                     25

heating for 3 min at 100^oC followed by rapid chilling made the [³H]-uridine labelled material completely sensitive to RNase, whereas the RNA labelled with [³H]-adenosine after denaturation by heating, was still resistant to RNase in a proportion up to 28 %. The proportion of double-stranded RNA from leukemic cells carrying poly(A) sequences is shown in Table 3.
 

Table 3. [³H]-uridine labelled ds-RNA bound to poly(U)-impregnated fiberglass filters.

Case	1	2
I.  Total RNA radioactivity from third chromatographic peak put on filter	1550	2470
II. RNA radioactivity bound to filter	350	665
As percentage of I.	23	27

Twenty-three per cent and twenty-seven per cent respectively of third peak radioactivity from two RNA samples labelled with [³H]-uridine was found bound to poly(U)-impregnated fiberglass filters.

A marked increase in the proportion of labelled RNA in double-stranded form was observed in some cases by increasing the period of incubation with the labelled precursor. As shown in Table 4, 
 

Table 4. Changes in proportion of labelled dsRNA after increasing periods of incubation with [³H]-uridine.

Case\Hours of Incubation
		3 hrs	6 hrs	9 hrs
1.	I. Total RNA radioactivity  treated with nucleases	2.10 x 105	2.10 x 105	3.925 x 104
	II. RNA radioactivity in  third peak	1.44 x 103	2.73 x 103	6.67 x 102
	As percentage of I.	0.7	1.3	1.7
2.	I. Total RNA radioactivity treated with nucleases	2.81 x 105	3.29 x 105	1.511 x 105
	II. RNA radioactivity in third peak	1.73 x 103	6.25 x 103	3.47 x 103
	As percentage of I.	0.6	1.9	2.3

in one of our cases this proportion did not reach 1 % after 3 hr, but was up to 2.3 % after 9 hr of incubation.

Experiments with cell fractionation showed that dsRNA in leukemic blast cells is almost exclusively confined to the nucleus. As shown in Table 5, 
 

Table 5. Occurrence of double-stranded RNA in nuclei of leukemic blast cells. A suspension of blast cells incubated for 2 hr with [³H]-adenosine was divided into two equal parts. One of these parts was immediately extracted with phenol, while the other was treated to fractionate the cells and separate the nuclei before RNA extraction.

	Whole cells	Nuclei
I.  Total RNA radioactivity treated with nucleases	2.737 x 105         3.865 x 105	9.528 x 104          8.36 x 104
II.  RNA radioactivity  in third peak	1.99 x 103            1.93 x 103	1.65 x 103           1.75 x 103
As percentage of I.	0.73                       0.5	1.73                        2.1

83 % and 91 % respectively of the total dsRNA of a certain amount of cells comes from the correspondent nuclear fractions.

Discussion:

The results of our experiments show that a significant proportion of the RNA synthesized in leukemic blast cells is in an RNase-resistant form with the properties of dsRNA. Poly-adenylate sequences would not be digested by pancreatic and T1 ribonucleases, but they do not co-chromatograph with dsRNA. This has been shown by Kronenberg and Humphrey [4] and confirmed in experiments which have shown that the material eluting with 15 % ethanol includes a large amount of RNase-resistant substance with poly(A) characteristics. It seems unlikely that the third chromatographic peak includes RNA-DNA hybrids since Nygard and Hall [10] have shown that such hybrids do not survive DNase digestion employed in our experiments. Subtilisin digestion makes also unlikely the presence in the third peak of RNA-protein complexes.

The whole of our results suggests that the substance eluting in the third peak is a duplex polyribonucleotide associated with polyadenylate sequences. The significance of this association, which has also been observed in sea urchins [4], is unknown. Polyadenylation had been considered originally an integral part of the processing mechanism by which the primary transcription products are converted to functional messenger RNA molecules [11, 12]. However, evidence has been presented recently to show that much of the nuclear polyadenylate sequences are degraded within the nucleus [13]. This is consistent with the view of Kronenberg and Humphrey [4] that poly(A)-bound dsRNA represents a portion of heterogenous nuclear RNA which is degraded in the nucleus. This conclusion is also supported by their kinetic experiments, showing that the proportion of total labelled RNA with double-stranded characteristics decreases with increasing the period of exposure to the label, as would be expected for a component of heterogenous nuclear RNA.

Although our results are at variance with those of the latter authors, one should not exclude the possibility that the dsRNA molecules which have been isolated in our experiments are part of of the heterogenous nuclear RNA of leukemic blast cells. We have shown previously that labelled heterogenous, rapidly sedimenting RNA molecules accumulate in leukemic blast cells after several hr of labelling [14, 15]. This suggests that heterogenous nuclear RNA of these cells does not behave kinetically as that of rapidly proliferating cells. A decrease in ribosomal RNA accumulation during the incubation period, accompanied by a reduced turnover rate of heterogenous nuclear RNA, might well explain our results. It must also be pointed out that an increase in the life span of dsRNA molecules, due to the ability of these molecules to inhibit initiation of protein synthesis, might reduce markedly the protein synthesis rate in leukemic blast cells.

Research supported by a grant from the Consiglio Nazionale delle Ricerche.

References:

1. C. Colby and P. H. Duesberg, "Double-Stranded RNA in Vaccinia Virus Infected Cells", Nature 222: 940 (1969).

2. R. Stern and R. Friedman, "Double-Stranded RNA Synthesized in Animal Cells in the Presence of Actinomycin D", Nature 222: 612 (1970).

3. L. Montagnier and L. Harel "Homology of Double-Stranded RNA from Rat Liver Cells with Cellular Genome", Nature, New Biology 229: 106 (1971).

4. L. H. Kronenberg and T. Humphreys, "Double-Stranded Ribonucleic Acid in Sea Urchin Embryos", Biochemistry 111: 2020 (1972).

5. W. Jelinek and J. E. Darnell, "Double-Stranded Regions in Heterogenous Nuclear RNA from HeLa Cells", Proc. Natl. Acad. Sci. USA 69: 2537 (1972).

6. H. D. Robertson and M. B. Mathews, "Double-Stranded RNA as an Inhibitor of Protein Synthesis and a Substrate for a Nuclease in Extracts of Krebs II Ascites Cells", Proc. Natl. Acad. Sci. USA 70: 225 (1973).

7. U. Torelli and G. Torelli, "Poly(A)-Containing Molecules in Heterogenous Nuclear RNA of Normal PHA-Stimulated Lymphocytes and Acute Leukemia Blast Cells", Nature, New Biology 244: 134 (1973).

8. R. M. Franklin, "Purification and Properties of the Replicative Intermediate of the RNA Bacteriophage R17", Proc. Natl. Acad. Sci. USA 55: 1504 (1966).

9. R. Sheldon, C. Jurale, and J. Kates, "Detection of Polyadenylic Acid Sequences in Viral and Eukaryotic RNA", Proc. Natl. Acad. Sci. USA, 69: 417 (1972).

10. H.P. Nygard, and B.D. Hall, "A Method for the Detection of RNA-DNA Complexes", Biochem. Biophys. Res. Comm., 12: 98 (1963).

11. J.E. Darnell, L. Philipson, R. Wall, and M. Adesnik, "Polyadenylic Acid Sequences: Role in Conversion of Nuclear RNA into Messenger RNA", Science 174: 507 (1971).

12. M. Edmonds, M.H. Vaughan, and H. Nakazato, "Polyadenylic Acid Sequences in the Heterogenous Nuclear RNA and Rapidly-Labelled Polyribosomal RNA of HeLa Cells: Posible Evidence for a Precursor Relationship", Proc. Natl. Acad. Sci. U.S.A., 68: 136 (1971).

13. R.P. Perry, D.E. Kelly, and J. La Torre, "Synthesis and Turnover of Nuclear and Cytoplasmic Polyadenylic Acid in Mouse L Cells", J. Molec. Biol. 82: 315 (1974).

14. U. Torelli, G. Torelli, and C. Mauri, "Competition Hybridization Studies on Rapidly Sedimenting RNA of Acute Leukemia Blast Cells", Europ. J. Cancer 8: 653 (1972).

15. U. Torelli, and G. Torelli, "Patterns of Macromolecular RNA Metabolism in Normal Human Lymphocytes and in Acute Leukemia Blast Cells", in: "Erythrocytes, Thrombocytes and Leukocytes", (ed. E. Gerlach, K. Moser, E. Deutsch, and W. Wilmanns), p. 372, Georg Thieme, Stuttgart, (1973).

Additional References:

1.  "Evidence for Inductive Properties of the Micromere-RNA in Sea-urchin Embryos".

2. "Nuclear Polyanions as De-Repressors of Synthesis of Ribonucleic Acid".

3. "Mated Models of Gene Regulation in Eukaryotes".

4. "Oncogenes as Molecular targets within Active Chromatin".

5. "Selective Gene De-Repression by De-Repressor RNA".