IN V I T R O C L E A V A G E O F 45S P R E - R I B O S O M A L RNA AND OF GIANT HETEROGENEOUS RNA EXTRACTED FROM HUMAN LEUKEMIC CELLS
U.L. TORELLI, ST. F E R R A R I , G . M . T O R E L L I , R. CADOSSI, SE. F E R R A R I , G. M O N T A G N A N I , and F. NARNI
Laboratory of Molecular Hematology, Institute of Medical Pathology, University of Modena, Italy
(Received 20 May; in revised form 9 July, 1977) Abstract.45S ribosomal precursor RNA and large heterogeneous RNA molecules (>45S) extracted from human leukemic cells were incubated in vitro with purified RNase II I, which specifically attacks double-helical RNA regions. About 50% of the ribosomal precursor was cleaved into two major fragments sedimenting at 28S and 32S respectively. A limited number of cleavages was also introduced in about 40% of heterogeneous RNA molecules sedimenting faster than 45S, causing a partial 'shift' to a polydisperse distribution in the 10S-45S range.
I.
INTRODUCTION
In eukaryotic cells, both the 45S preribosomal RNA and the larger heterogeneous molecules which some authors regard as 'nascent' messenger precursors [ 1] include segments with double-helical characteristics [2, 3, 4, 5, 6]. By analogy to what has been observed in bacterial cellsl some authors have suggested that in eukaryotes the double-helical regions may function as signals for cleavage of the large ribosomal and messagerial precursors to smaller final products [7, 8, 9]. As a matter of fact, evidence has been presented that 45S preribosomal RNA is cleaved in vitro by RNase III to fragments which are indistinguishable from those observed in vivo [7]. Large heterogeneous nuclear RNA molecules appear also susceptible to endonucleolytic cleavage by RNase III [7, 9]. Experiments of this type so far reported have been performed with large RNA precursor molecules from HeLa cells. In these cells, the rate of metabolic breakdown of those molecules is very high, so that their average life span is only a few minutes [ 10, 11]. On the other hand, Westphal and Crouch [ 12] have visualized in mammalian 28S and 18S ribosomal RNA a gradient of susceptibility of RNA sites to RNase III cleavage, with the primary sequence and steric conformation determining the various degrees of exposure to enzymatic attack. We have previously shown that both the 45S preribosomal RNA and the larger unmethy403 Molecular Biology Reports 3 (1977) 403-411. All Rights Reserved. Copyright 9 1977 by D. Reidel Publishing Company, Dordrecht-Holland.
lated heterogeneous RNA molecules in leukemic blast cells are characterized by a very low rate of cleavage, and that molecules of the latter type accumulate in the cells [ 13]. These observations prompted us to test the susceptibility of sites of the large ribosomal and messagerial RNA precursors extracted from leukemic cells to digestion by RNase III.
II.
MATERIALS AND METHODS
1. Cells and labeling conditions The methods used to maintain leukemic cells in culture and to label RNA with [5 -3H]uridine or [methyl -3H]methionine have been described previously [ 13]. 2. RNA extraction and separation Whole cell RNA was extracted by the hot phenol-m-cresol technique [ 13]. The extraction was repeated 4 times. The RNA was centrifuged through a 5-20% sucrose gradient, and fractions corresponding to 45S preribosomal RNA and to heterogeneous RNA sedimenting faster than 45S were separated and digested with RNase III as described in detail in the legends to the figures. 3. Isolation of double-helical RN,4 Double-helical segments of whole cell RNA extracted from leukemic cells were isolated b y the Franklin procedure [ 14] as described previously [ 15]. The RNase-resistant molecules eluted in a purified form from the cellulose column by buffer only were precipitated by ethanol in the presence of poly(C) as a carrier. 4. Assay of poly(A) +RNA molecules The assay of poly(A) + RNA molecules was carried out by the Dubroff and Nemer [ 16] modification of the method of Sheldon et al. [ 17], as described elsewhere [ 13]. 5. Preparation of RNase III E. Coli (Strain M RE 600) were purchased from Microbiological Research Establishment, Porton, England. Fifty g of frozen paste was ground in a cold mortar with 2.5 parts (w/w) of Alcoa A-305 (Serva, Heidelberg). All further operations were performed at 4 o C. The lysate was resuspended in 1.5 parts (w:v) of 0.02 M Tris buffer, pH 7.8, containing 0.03 M NH4CI, 0.01 M MgCI2, 0.005 M 2-mercapto-ethanol, 0.001 M NaEDTA and 10% glycerol. DNase (Worthington) was added at the final concentration of 5 ~g ml -j. When the viscosity vanished the suspension was centrifuged at 8000• for 15 min. The supernatant was further centrifuged at 30000• for 20 min. to obtain the $30 extract. To separate the ribosomal pellet the $30 extract was centrifuged at 105000• for 4 hr in a SW36 rotor of a Beckman ultracentrifuge. The supernatant was discarded and the pellet was rinsed with Tris buffer, pH 7.8. The pellet was then thoroughly resuspended in 50 ml of 0.02 M Tris buffer, pH 7.6, containing 0.22 M NH4C1, 0.01 M MgC12, 0.005 M 2-mercaptoethanol and 10% glycerol, stirred for 1 hr and centrifuged again at 105 000Xg for 4 hr. The supernatant was precipitated by slowly
404
adding (NH4)2SO4(22 g per 100 ml). The suspension was then centrifuged for 30 min at 15000 rpm in a SW 25.1 rotor. The sediment was discarded and (NH4)2SO4 ( 14 g per 100 ml) was again slowly added to the supernatant. The suspension was centrifuged for 30 min at 15000 rpm. The pH was controlled throughout the procedure and, when necessary, was maintained around 5.5 by adding a few drops ofa I M Tris solution. The precipitate was dissolved in 1/10 of the initial volume with 0.02 M Tris buffer, pH 7.6, containing 0.02 M NH4C1, 0.01 M MgC1z, 0.005 M 2-mercaptoethanol and 10% glycerol and applied to a column ( 1.5)
U.L. TORELLI, ST. F E R R A R I , G . M . T O R E L L I , R. CADOSSI, SE. F E R R A R I , G. M O N T A G N A N I , and F. NARNI
Laboratory of Molecular Hematology, Institute of Medical Pathology, University of Modena, Italy
(Received 20 May; in revised form 9 July, 1977) Abstract.45S ribosomal precursor RNA and large heterogeneous RNA molecules (>45S) extracted from human leukemic cells were incubated in vitro with purified RNase II I, which specifically attacks double-helical RNA regions. About 50% of the ribosomal precursor was cleaved into two major fragments sedimenting at 28S and 32S respectively. A limited number of cleavages was also introduced in about 40% of heterogeneous RNA molecules sedimenting faster than 45S, causing a partial 'shift' to a polydisperse distribution in the 10S-45S range.
I.
INTRODUCTION
In eukaryotic cells, both the 45S preribosomal RNA and the larger heterogeneous molecules which some authors regard as 'nascent' messenger precursors [ 1] include segments with double-helical characteristics [2, 3, 4, 5, 6]. By analogy to what has been observed in bacterial cellsl some authors have suggested that in eukaryotes the double-helical regions may function as signals for cleavage of the large ribosomal and messagerial precursors to smaller final products [7, 8, 9]. As a matter of fact, evidence has been presented that 45S preribosomal RNA is cleaved in vitro by RNase III to fragments which are indistinguishable from those observed in vivo [7]. Large heterogeneous nuclear RNA molecules appear also susceptible to endonucleolytic cleavage by RNase III [7, 9]. Experiments of this type so far reported have been performed with large RNA precursor molecules from HeLa cells. In these cells, the rate of metabolic breakdown of those molecules is very high, so that their average life span is only a few minutes [ 10, 11]. On the other hand, Westphal and Crouch [ 12] have visualized in mammalian 28S and 18S ribosomal RNA a gradient of susceptibility of RNA sites to RNase III cleavage, with the primary sequence and steric conformation determining the various degrees of exposure to enzymatic attack. We have previously shown that both the 45S preribosomal RNA and the larger unmethy403 Molecular Biology Reports 3 (1977) 403-411. All Rights Reserved. Copyright 9 1977 by D. Reidel Publishing Company, Dordrecht-Holland.
lated heterogeneous RNA molecules in leukemic blast cells are characterized by a very low rate of cleavage, and that molecules of the latter type accumulate in the cells [ 13]. These observations prompted us to test the susceptibility of sites of the large ribosomal and messagerial RNA precursors extracted from leukemic cells to digestion by RNase III.
II.
MATERIALS AND METHODS
1. Cells and labeling conditions The methods used to maintain leukemic cells in culture and to label RNA with [5 -3H]uridine or [methyl -3H]methionine have been described previously [ 13]. 2. RNA extraction and separation Whole cell RNA was extracted by the hot phenol-m-cresol technique [ 13]. The extraction was repeated 4 times. The RNA was centrifuged through a 5-20% sucrose gradient, and fractions corresponding to 45S preribosomal RNA and to heterogeneous RNA sedimenting faster than 45S were separated and digested with RNase III as described in detail in the legends to the figures. 3. Isolation of double-helical RN,4 Double-helical segments of whole cell RNA extracted from leukemic cells were isolated b y the Franklin procedure [ 14] as described previously [ 15]. The RNase-resistant molecules eluted in a purified form from the cellulose column by buffer only were precipitated by ethanol in the presence of poly(C) as a carrier. 4. Assay of poly(A) +RNA molecules The assay of poly(A) + RNA molecules was carried out by the Dubroff and Nemer [ 16] modification of the method of Sheldon et al. [ 17], as described elsewhere [ 13]. 5. Preparation of RNase III E. Coli (Strain M RE 600) were purchased from Microbiological Research Establishment, Porton, England. Fifty g of frozen paste was ground in a cold mortar with 2.5 parts (w/w) of Alcoa A-305 (Serva, Heidelberg). All further operations were performed at 4 o C. The lysate was resuspended in 1.5 parts (w:v) of 0.02 M Tris buffer, pH 7.8, containing 0.03 M NH4CI, 0.01 M MgCI2, 0.005 M 2-mercapto-ethanol, 0.001 M NaEDTA and 10% glycerol. DNase (Worthington) was added at the final concentration of 5 ~g ml -j. When the viscosity vanished the suspension was centrifuged at 8000• for 15 min. The supernatant was further centrifuged at 30000• for 20 min. to obtain the $30 extract. To separate the ribosomal pellet the $30 extract was centrifuged at 105000• for 4 hr in a SW36 rotor of a Beckman ultracentrifuge. The supernatant was discarded and the pellet was rinsed with Tris buffer, pH 7.8. The pellet was then thoroughly resuspended in 50 ml of 0.02 M Tris buffer, pH 7.6, containing 0.22 M NH4C1, 0.01 M MgC12, 0.005 M 2-mercaptoethanol and 10% glycerol, stirred for 1 hr and centrifuged again at 105 000Xg for 4 hr. The supernatant was precipitated by slowly
404
adding (NH4)2SO4(22 g per 100 ml). The suspension was then centrifuged for 30 min at 15000 rpm in a SW 25.1 rotor. The sediment was discarded and (NH4)2SO4 ( 14 g per 100 ml) was again slowly added to the supernatant. The suspension was centrifuged for 30 min at 15000 rpm. The pH was controlled throughout the procedure and, when necessary, was maintained around 5.5 by adding a few drops ofa I M Tris solution. The precipitate was dissolved in 1/10 of the initial volume with 0.02 M Tris buffer, pH 7.6, containing 0.02 M NH4C1, 0.01 M MgC1z, 0.005 M 2-mercaptoethanol and 10% glycerol and applied to a column ( 1.5)
