Cell, Vol . 4, 1 5 7 -165, February 1975, Copyright '© 1975 by MIT
Messenger Ribonucleoprotein Complexes Isolated With Oligo(dT)-Cellulose Chromatography from Kidney Polysomes David Irwin, Ajit Kumar, and Ronald A. Malt Surgical Services Massachusetts General Hospital and Shriners Burns Institute and the Department of Surgery Harvard Medical School Boston, Massachusetts 02114
The present study defines some of the characteristics of the mRNP complexes in normal mouse kidney as a base for later evaluating the contribution of mRNP and mRNP proteins to translational regulation during renal hypertrophy . Isolation of mRNP complexes was made possible by their binding to oligo(dT)-cellulose . Results
Summary As an initial step towards understanding the role of mRNP complexes in translational regulation during compensatory renal hypertrophy, characteristics of polysome-associated mRNP Isolated by affinity chromatography were studied . Renal mRNP contained 15-30% of the counts after a 1 hr pulse with 3 H-orotic acid ; it sedimented mainly between 10S and 100S and had a buoyant density of 1 .421 .44 g/cm 3 . RNA derived from the mRNP sedimented between 5S and 40S on sucrose density gradients, with the greatest radioactivity in the region of 15S . After labeling with 3 H-adenine for 1 hr, up to 17% of the radioactivity present in the mRNP-associated RNA was resistant to digestion by pancreatic and T1 ribonucleases . The mRNP protein moiety contained six polypeptides with molecular weights 69,000, 75,000, 80,000, 100,000, 109,000, and 118,000 daltons, which were undetected in the material not binding to ol igo(dT)-cel I u lose .
Isolation of mRNP Stable hybrids of poly(A) with oligo(dT) are completely disrupted at zero salt concentrations (Aviv and Leder, 1972) . However, Lindberg and Sund-
~tI 5,338 cpm 25% formamide
4
1
I
Introduction Messenger RNA (mRNA) of eucaryotes is associated with specific proteins in messenger ribonucleoprotein complexes (mRNP) (Hoagland and Askonas, 1963 ; Spirin and Nemer, 1965 ; Perry and Kelley, 1968 ; Henshaw, 1968) . Although the precise function of the proteins in mRNP is unclear, one of their roles may be to stabilize inactive precursor mRNA against degradation during transport from the nucleus and storage in the cytoplasm (Spohr et al ., 1970) . A reversible equilibrium between polysomes and free mRNP with ribosomal subunits might then form part of a translational control mechanism (Schochetman and Perry, 1972) . Previous studies on the nature of mRNP complexes have examined either continuously growing or terminally differentiated cells . A system of growing cells in mammals is provided by cortical cells of the remaining kidney following contralateral nephrectomy . The predominant response of these cells is hypertrophy, with a rapid accumulation of RNA and protein (Bucher and Malt, 1971) . There is little hyperplasia.
0.
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FRAC T/ON NUMBER Figure 1 . Fractionation of EDTA-Dissociated Polysomes on Oligo(dT)-Cellulose EDTA-dissociated polysomes were prepared from 8 kidneys labeled with 3 H-orotic acid . The dissociated polysomes (4 ml) were chromatographed on an oligo(dT)-cellulose (T-2) column, eluted successively with 20 ml of Tris elution buffer (0 .25 M NaCI), 10 ml of elution buffer containing 25% formamide, and finally 10 ml of elution buffer containing 50% formamide . Fractions of 0 .8-0 .9 ml were collected, from which 100 µl aliquots were counted directly in TX8 (o-o) . Counting efficiency was 30% .
Cell 1 58
quist (1974) showed that the mRNA-protein complexes from polysomes that are retained on ol igo(dT)-cel I u lose at high salt concentrations are not recovered by removal of salt alone unless the elution buffer is supplemented with 25-50% formamide . The binding of the mRNP is therefore not merely classical A-T hybridization : the mRNP proteins appear to contribute to hybrid stability . Figure 1 shows that 15-20% of the total polysome radioactivity from normal mouse kidney bound to oligo(dT)-cellulose (Type T-2) after pulse labeling with 3 H-orotic acid for 1 hr . The bound material was released with formamide elution buffer . Type T-3 oligo(dT)-celIulose bound 20-30% of the total polysome radioactivity under similar conditions . Approximately 60-65% of the bound material eluted with 25% formamide ; the remaining 35-40% eluted with 50% formamide . Sedimentation Coefficient of mRNP Previous experiments used sucrose gradients to prepare a fraction from EDTA-dissociated kidney polysomes sedimenting in the 90-170S region as shown in Figure 2a ; Figure 2b shows the isopycnic centrifugation of this fraction on CsCI gradients . The mRNP from the polysomes had a buoyant density of 1 .41-1 .45 g/cm 3 ; some material that had a buoyant density of 1 .54-1 .57 g/cm 3 was presumed to be contamination by large ribosomal subunits . Preparation of mRNP with oligo(dT)-cellulose presumably allows the isolation of poly(A)-contain-
ing complexes of widely differing sizes, including sizes similar to ribosomal subunits ; this kind of separation is not possible with sucrose gradients. The size distribution of mRNP prepared by the oligo(dT)-cellulose procedure therefore was examined on sucrose gradients as described in Figure 3 . The mRNP sedimented predominantly in the 10100S range, with a small amount sedimenting faster than 100S . The higher radioactivity in the 60S and 30S regions may be due to contamination by ribosomal subunits . Buoyant Density of mRNP To investigate whether the heterogeneously sedimenting mRNP included complexes with different protein to RNA ratios, mRNP was examined by isopycnic centrifugation on CsCI gradients . Figure 4a shows the oligo(dT) elution profile from EDTAtreated polysomes from 8 kidneys . The mRNP fractions indicated by bars were dialyzed, fixed, and banded on CsCI gradients . From Figure 4b it can be seen that the mRNP banded mainly at a density of 1 .42-1 .44 g/cm 3 , with
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Figure 2a . Sucrose Density Gradient Analysis of EDTA-Dissociated Polysomes
Figure 2b . Buoyant Density of Material in Fractions 10-18 from the Sucrose Gradients
Three mice were injected with 500 pCi of 3H-uridine (uridine 5,6- 3H, 40 .4 Ci/mmole) and the kidneys were removed after 1 hr . EDTAdissociated polysomes were prepared and resuspended in 3 ml of RSB. Aliquots of 1 ml were loaded on to 37 ml, 5-45% sucrose in NEB .25 gradients and centrifuged at 26,000 rpm for 3 hr in a Spinco SW27 rotor. A260 , was monitored and 1 .3-1 .4 ml fractions were collected . TCA insoluble radioactivity was determined in aliquots of 0 .5 ml from each fraction . (-) A260- ; (o-o) TCA insoluble radioactivity .
The remaining 0 .8-0 .9 ml of fractions 10-18 indicated by bars in Figure 2a were pooled and mixed with fractions 10-18 from a duplicate gradient . The fractions were precipitated with 2 vol of ethanol at-25°C for 20 hr . The precipitate was sedimented at 12,100 x g for 20 min, the supernatant was discarded, and the precipitate was dried under vacuum . Buoyant density of the dry residue dissolved in 1 .0 ml of HSB .25 was measured on a CsCI gradient . (x-A) density calculated from refractive index measurements . (o-o) radioactivity of filter discs .
mRNP Complexes of Kidney Polysomes 159
a small amount banding at 1 .39 g/cm 3 . The small amount of higher density material may result from contamination with ribosomal subunits or mRNP with a protein complement less than that normally seen in the formamide eluates. Under identical conditions, fixed EDTA-derived large ribosomal subunits and fixed polysomes exhibited buoyant densities of 1 .58 g/cm 3 and 1 .53-1 .56 g/cm3 , respectively . Less than 4% of the total radioactivity on the CsCI gradients sedimented to the bottom of the tube . The mRNP density of 1 .42-1 .44 g/cm 3 suggests a composition of approximately 20% RNA and 80% protein from the formula of Perry and Kelley (1966), calculated with values of 1 .87 g/cm 3 and 1 .35 g/cm 3 for the densities of RNA and protein, respectively . We recognize that it may not be possible to calculate . exact compositions from simple additive expressions (McConkey, 1974) . 4
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Figure 3 . Sedimentation Coefficient Distribution of mRNP Prepared on Oligo(dT)-Cellulose Columns Fractions 28-30 and 37-41 in Figure 1 were pooled and dialyzed against RSB for 2 hr and layered over a 1 ml cushion of 2 M sucrose . The dialysate was centrifuged at 45,000 rpm for 16 hr in a Spinco 65 rotor, the supernatant was discarded, and the sucrose cushion was diluted to 10 ml with NEB .25 . Aliquots of 2 ml were layered on to 36 ml 15-30% sucrose in NEB .25 gradients with 4 absorbance units of unlabeled HeLa large ribosomal subunits as marker and centrifuged at 25,000 rpm for 15 hr in a Spinco SW27 rotor . 1 .5 ml fractions were collected and precipitated . (o--o) TCA insoluble radioactivity.
The heterogeneity in the mRNP apparent from the distribution of sedimentation coefficient thus was not reflected in the protein-RNA composition . Size Distribution of RNA from mRNP Figure 5 shows the examination of RNA derived from mRNP on 15-30% sucrose in NETS gradients using unlabeled 28S ribosomal RNA as an internal marker . Activity was associated with RNA sedimenting between approximately 5S and 40S ; the greatest activity was in the region of 15S . A similar heterogeneous distribution was observed when mRNA prepared from mouse kidney polysomes by the method of Aviv and Leder (1972) was examined on sucrose density gradients (Ouellette, Kumar, and Malt, 1974) . We cannot rule out the possibility that some of the larger RNA and RNP (Figure 3) is nucleus derived . The amount of radioactivity binding to oligo(dT)-cellulose after a 10 min labeling period with 3 H-orotic acid was 15% of that which bound after a 1 hr labeling period . Although the mRNP eluted in two fractions-one eluting with 25% formamide and the other with 50% formamide-no difference in RNA size distribution was detected . A difference is suggested, however, in the proportion of poly(A) that the two mRNP fractions contain . When RNA from 3 H-adenine-labeled mRNP fractions was digested with T1 and pancreatic ribonucleases, the amount of radioactivity which would still bind to oligo(dT)-cellu lose in 0 .5 M NaCl was 9% and 17%, respectively, for the 25% formamide-eluted mRNP and 50% formamide-eluted mRNP . When RNA from the fraction which passes straight through the column during the isolation of mRNP was digested under the same conditions, 5% of the radioactivity bound to oligo(dT)-cellulose . This RNAase-resistant material from the column run-through fraction may derive from poly(A)-containing RNA that passes through the column with the ribosomal subunits . Polypeptides Present in Run-through and Formamide-eluted Fractions The polypeptides present in the 25% and 50% formamide-eluted mRNP and the column run-through fraction were characterized on SDS-polyacrylamide gels . The electrophoretic patterns are shown in the photograph in Figure 6 together with a schematic representation of the bands . The protein complement of the run-through material was similar to that of EDTA-derived large and small ribosomal subunits (unpublished results using kidney ribosomes) . In addition, the run-through material contained two polypeptides of 84,000 and 90,000 daltons . The 25% formamide eluate contained the 84,000 and 90,000 dalton polypeptides together with four polypeptides not seen in the run-
Cell 1 60
through material, 69,000, 75,000, 80,000 and 118,000 daltons . Two minor polypeptides with molecular weights of 100,000 and 109,000 daltons were also detected . Bands corresponding to molecular weights below 62,000 daltons appeared to have corresponding bands in the run-through material and probably represent contamination by ribosomal subunit proteins . Only one polypeptide, the 84,000 dalton polypeptide, was detected in the 50% formamide-eluted mRNP . The 25% formamide-eluted mRNP thus contains at least six distinct nonribosomal polypeptides . In addition, the 84,000 dalton polypeptide found in the three different eluates, and the 90,000 dalton polypeptide found in the run-through and the 25% formamide eluate are probably specific mRNP polypeptides . These two polypeptides could be associated with the poly(A)-containing RNA that does not bind to oligo(dT)-cellulose and is found in the ribosomal subunit run-through material . Proteins associated with nonpoly(A)-containing mRNA would also be expected to be found in the column run-through fraction .
Differences In Buoyant Density of the Formamide-eluted mRNP Fractions Although no difference in the size distribution of RNA from the 25% and 50% formamide eluates was apparent, differences in the protein composition and percentage of poly(A) were observed . These differences may have been detectable in the densities of the mRNP fractions had they been fixed and examined separately. Figure 7 shows the oligo(dT)-cellulose elution profile for EDTA-dissociated polysomes from 16 kidneys and the measurement of the densities of 25% and 50% formamide-eluted mRNP fractions separately . There was, indeed, a 4% difference in density of the two mRNP fractions . The 25% formamide-eluted mRNP had a major density of 1 .48-1 .50 g/cm3, and the 50% formamide-eluted mRNP had a major density of 1 .42-1 .43 g/cm3 . The average density that would be produced by these two values should be somewhat higher than the value of 1 .42-1 .44 g/cm3 shown in Figure 4b . However, the use of 0 .4 M NaCI (Figure 7) rather than 0 .25 M NaCl in the elution buffer may have removed some loosely attached surface proteins and caused a slight increase in the buoyant density .
f 54,812 cpa
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FRACT/ON NUMBER FRACT/ON NUMBER Figure 4 . Buoyant Density of Polysome-Derived mRNP (a, left) EDTA-dissociated polysomes were prepared from 8 kidneys and fractionated on an oligo(dT)-cellulose (T-2) column . (o-o) total radioactivity of 100 µl aliquots from 1 .1 ml fractions . (b, right) Fractions 25-30 and 32-25 were pooled and dialyzed against RSB for 3 hr . After the dialysate was fixed in 6% glutaraldehyde, 2 ml aliquots were examined on 5 ml CsCI gradients . (A-0) density calculated from refractive indices . (o-o) TCA insoluble radioactivity.
mRNP 161
Complexes
of Kidney
Polysomes
The 25% and 50% formamide eluates contained 37% and 20% RNA, respectively (Perry and Kelley, 1966). Discussion These results are an initial characterization of some of the polysome-associated mRNP complexes in mouse kidney. The oligo(dT)-cellulose method appears to select not only complexes containing mRNA with terminal poly(A)s attached, but also configurations permitting hybridization of the poly(A) to oligo(dT). Thus, histone mRNP complexes would not be expected to bind, and complexes in which the poly(A) regions are not exposed -for example those masked by proteins-may be unable to interact with oligo(dT). The RNAase-resistant poly(A) in the ribosomal run-through fraction therefore may derive from mRNP in which the poly(A) is shielded. The 84,000 and 90,000 dalton polypeptides present in the run-through fraction,
which are probably mRNP proteins, could be associated with this nonbinding mRNP. The heterogeneous distribution of the mRNP isolated using oligo(dT)-cellulose had a similar size range to that seen in several other mammalian systems listed in Table 1, the bulk of the mRNP in kidney sedimenting between 10s and 80s. To avoid excessive contamination by ribosomal subunits, the sucrose gradient procedure selects only the material sedimenting between 90s and 170s. It would appear that either the fast sedimenting mRNP complexes are not able to hybridize to oligo(dT), or that during oligo(dT)-cellulose chromatography the complexes are damaged, thereby producing smaller mRNPs. mRNP prepared by both oligo(dT)-cellulose and by sucrose gradients contained some ribosomal
8
12,400
B
I
25,000
67,000
III1
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/\ 64,000
90,000
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/
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1I 66,000’/ 75,000 I 64000
ii6,OOO
\
100S
1 .35-1 .55
Henshaw and Loebenstein (1970)
Rat liver
30->90S
1 .35-1 .45
Perry and Kelley (1968)
L cells (polysomes) (free)
12-60S 25-60S
1 .45 1 .38-1 .44
Blobel (1973)
L cells and Rat liver
After RNAase digestion 4-16S
2
52, 78
Spohr et al . (1970)
HeLa
10-70S
Kwan and Brawerman (1972)
Mouse ascites
12-15S for poly(A) region only
Lee et al . (1971)
Mouse ascites
25->160S
Blobel (1972)
Rabbit reticulocytes
20S
2
52, 78
Lebleu et al . (1971)
Rabbit reticulocytes
Morel at al . (1971)
Duck erythroblasts
15S
2
68, 130
20S
2
Morel et al . (1973)
49, 73
Duck erythroblasts
15S
2 major 6 minor
49, 73 52-64,86-120
Gander et al . (1973)
Duck erythroblasts (free)
12S 20S
7 10
7-65 15-51 all 17 different
Bryan and Hayashi (1973)
Chick embryo brain
3 different size ranges
2 common to all
54, 92 or 48 .4, 78 .5 by discontinuous polyacrylamide gel electrophoresis
4 Normal 4 + 1
56, 68, 78, 125 110
1 .40-1 .48
1 .40-1 .45 1 .40-1 .45
Kumar and Lindberg (1972)
KB Cells (isotonic) (0 .5 M NaCl)
up to 200S
1 .35-1 .47 1 .57-1 .60
Lindberg and Sundquist (1974)
KB Cells (normal) (Adeno viruss infected)
10-100S
1 .38
mRNP Complexes of Kidney Polysomes 163
a 5
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FRACTION NUMBER FRACTION NUMBER Figure 7 . Differences in Buoyant Densities of 25% and 50% Formamide-Eluted mRNP EDTA-dissociated polysomes from 16 kidneys were fractionated on oligo(dT)-cellulose, except that T-3 cellulose was used ; fractions were eluted with 25 ml of elution buffer (0 .4 M NaCl) followed by 15 ml of 25% formamide in elution buffer and 15 ml of 50% formamide in elution buffer . (a) Column elution ; (0 0) total radioactivity of 100 µI aliquots from 1 .6 ml fractions . Fractions 23 and 29 were dialyzed separately against RSB for 90 min and fixed in 6% glutaraldehyde, (volumes of fixed dialysates, 2 .3 ml and 2.8 ml, respectively) . Buoyant densities were measured using sample loads of 1 .5 ml . (b) 1 .5 ml of 25% formamide eluate . (c) 1 .5 ml of 50% formamide eluate . (d) 0.5 ml of 25% + 1 .0 ml of 50% formamide eluate mixed . (L-4) density . (0--o) TCA insoluble radioactivity .
Cell 1 64
than the 25% formamide eluate . Another difference between the two mRNP fractions therefore may be that the 50% formamide eluate contains mRNA with longer poly(A) sequences than those of the 25% formamide eluate . An alternative explanation would be that some nonpoly(A)-containing RNA (for example, ribosomal RNA) is present in the 25% formamide eluate . The sizes of the poly(A) sequences have yet to be determined by direct measurement . Experimental Procedures Solutions and Reagents RSB : 0 .01 M Tris, 0 .01 M NaCl, 0.0015 M MgCI2 pH 7.4 at 20°C . HSB .25 : 0 .01 M Tris, 0 .25 M NaCl, 0 .05 M MgCl2 pH 7 .4 at 20°C . NEB .25 : 0 .01 M Tris, 0 .25 M NaCl, 0 .01 M EDTA pH 7 .4 at 20°C. NETS : 0 .01 M Tris, 0 .1 M NaCl, 0 .01 M EDTA, 0.2% SDS . Elution buffer, 0 .01 M Tris or 0 .01 M P043- and 0 .25 M NaCl or 0.4 M NaCl pH 7 .4 at 20°C . TX8 : 2 I Triton X-100, 1 I xylene, 8 g Omnifluor (New England Nuclear Corp ., Boston, Mass .) SUP : 0 .5% SDS, 0 .5 M urea, 0 .01 M P043- pH 7 .0 at 20°C . Formamide (Fisher Scientific Co ., N .J .) was purified according to the procedure of Tibbets, Johansson, and Philipson (1973) except that the ether drying stage was omitted . Preparation of Polysomes Male Charles River mice (50-60 days old) were injected intraperitoneally with either 100 yCi of 3H-orotic acid (orotic-5-3H acid, 11 .1 Ci/mmole, New England Nuclear Corp .) or420tLCi of 3H-adenine (adenine 3H, 675 Ci/mole, New England Nuclear Corp .) . After 1 hr, kidneys were removed and disrupted in 1 ml of RSB per 2 kidneys using a glass Dounce homogenizer (10 strokes of the loose pestle followed by 4 strokes of the tight pestle) . The homogenate was centrifuged at 12,100 x g for 10 min, and the supernatant was layered on discontinuous sucrose gradients of 3 ml 2 M sucrose, 1 ml 1 .5 M sucrose and 1 ml 0 .5 M sucrose in HSB .25 . The gradients were centrifuged at 55,000 rpm for 4 hr in a Spinco 65 rotor at 2-4°C . The supernatant layers were discarded, and the clear polysome pellets were rinsed twice with 2 ml aliquots of RSB .
70% ethanol, dried and counted in 0 .4% Omnifluor in toluene with counting efficiencies greater than 30% . Isopycnic Centrifugation on CsCI Gradients mRNP preparations were fixed in 6% glutaraldehyde (Eastman Kodak Co ., Rochester, N .Y .) neutralized with NaHCO3 just before use . Densities were measured on CsCI gradients containing 0.8% Brij 35 (Fisher Scientific Co.) . Linear gradients were preformed from 2.4 ml of CsCI solution, density 1 .55 g/cm3, and 2 .6 ml of CsCI solution, density 1 .41 g/cm3 . Sample volumes of 1 .0-2 .0 ml were overlaid, and the gradients were centrifuged at 35,000 rpm for 20 hr at 2-4°C in a Spinco SW41 rotor, allowed to decelerate without braking . Twelve-drop fractions were collected from the bottom of the tubes directly onto Whatman 3MM filter discs . Following the first fraction and every sixth fraction thereafter, two drops were collected for density determinations from refractive index measurements . The discs were dried and rinsed in 10% trichloroacetic acid, 5% trichloroacetic acid, and 70% ethanol at 0°C, dried, and counted directly in 0 .4% Omnifluor in toluene. Sucrose Density Gradient Analysis of RNA from mRNP Ethanol was added to the mRNP preparations to a concentration of 70% . The precipitated mRNP was centrifuged at 12,100 x g for 30 min, and the supernatant was discarded . The precipitate was dried under vacuum and redissolved in NETS buffer including unlabeled 28S ribosomal RNA as an internal marker . Aliquots of 2 ml were run on 36 ml 15-30% sucrose (w/w) in NETS gradients at 21,000 rpm for 17 hr in a Spinco SW27 rotor at 23°C. SDS Polyacrylamide Gel Electrophoresis mRNP preparations were dialyzed against SUP and examined on 7.5% acrylamide gels according to Bhorjee and Pederson (1973), except that 6 mm x 10 cm gels were used and acrylamide (Fisher Scientific Co .) was not deionized, but twice recrystallized from chloroform . Bovine serum albumin, chymotrypsin A, and cytochrome c were used as reference standards in parallel gels . Molecular weights were calculated by the method of Weber, Pringle, and Osborn (1972) . Acknowledgment This work was supported by grants from the National Institutes of Health and from the National Science Foundation .
Preparation of mRNP by Affinity Chromotography Polysomes were resuspended in either 10 mM phosphate or 10 mM Tris buffer (pH 7 .4), and 10 mM EDTA . The ribosomal subunitmRNP mixture was centrifuged at 12,100 x g for 10 min to remove undissolved debris . 5 M NaCl was added to a final concentration of either 0 .25 M or 0.4 M NaCl . mRNP was then isolated using oligo(dT)-cellulose based on the method of Lindberg and Sundquist (1974) . A column 7 .5 mm x90 mm was formed in a disposable pipette with 0.8-1 .0 g of oligo(dT)-cellulose (Types T-2 or T-3, Collaborative Research, Inc ., Waltham, Mass .) . The ribosomal subunit-mRNP mixture was loaded on to a column and eluted with elution buffer, followed by 25% formamide in elution buffer, and finally, 50% formamide in elution buffer. The mRNP eluted by the formamide was dialyzed against either 0 .01 M Tris or 0 .01 M P04 3to remove the formamide . All operations were performed at 0-4°C .
Received September 18, 1974 ; revised December 2, 1974
Sucrose Density Gradient Analysis of mRNP mRNP was prepared as described and examined on either 15-30% or 5-45% linear density gradients of sucrose (w/w) in NEB .25 . The A 26p °m was monitored and the approximate S value range was estimated by reference to an unlabeled marker of 60S ribosomal subunits. Fractions with 2% bovine serum albumin coprecipitant were precipitated with equal volumes of 20% trichloroacetic acid . The fractions collected cold on glass fiber discs were rinsed successively with 10% trichloroacetic acid, 5% trichloroacetic acid,
Henshaw, E . C ., and Loebenstein, J . (1970) . Biochim . Biophys . Acta 199, 405 .
References Aviv, H ., and Leder, P . (1972) . Proc. Nat . Acad . Sci . USA 69, 1408 . Bhorjee, J . S ., and Pederson, T. (1973) . Biochemistry 12, 2766 . Blobel, G . (1972) . Biochem . Biophys. Res . Commun . 47, 88 . Blobel, G . (1973) . Proc. Nat. Acad . Sci . USA 70, 924 . Bryan, R . N ., and Hayashi, M . (1973) . Nature New Biol . 244, 271 . Bucher, N . L . R ., and Malt, R . A . (1971) . Regeneration of Liver and Kidney (Boston: Little Brown), p . 179 . Gander, E . S ., Stewart, A . G ., Morel, C. M ., and Scherrer, K . (1973). Eur . J . Biochem . 38, 443 . Henshaw, E . C . (1968) . J . Mol . Biol . 36, 401 .
Hoagland, M . B ., and Askonas, B . A. (1963) . Proc . Nat. Acad . Sci. USA 49, 130 . Kumar, A., and Lindberg, U . (1972) . Proc . Nat. Acad . Sci. USA 69, 681 . Kwan, S- . W ., and Brawerman, G . (1972) . Proc. Nat . Acad . Sci . USA 69, 3247 .
mRNP Complexes of Kidney Polysomes 165
Lebleu, B ., Marbaix, G ., Huez, G., Timmerman, J ., Burny, A ., and Chantrenne, H. (1971) . Eur. J . Biochem . 19, 264 . Lee, S . Y ., Krsmanovic, V ., and Brawerman, G . (1971) . Biochemistry 10, 895 . Lindberg, U ., and Sundquist, B . (1974) . J . Mol . Biol . 86, 451 . McConkey, E . H . (1974) . Proc . Nat . Acad . Sci . USA 71, 1379 . Morel, C ., Kayibanda, B ., and Scherrer, K . (1971) . FEBS Letters 18, 84 . Morel, C ., Gander, E . S ., Herzberg, M ., Dubochet, J ., and Scherrer, K . (1973) . Eur . J . Biochem . 36, 455 . Ouellette, A . J ., Kumar, A ., and Malt, R . A . (1974) . Federation Proceedings 33, 635 . Perry, R . P ., and Kelley, D . E . (1966) . J . Mol . Biol . 16, 255 . Perry, R . P., and Kelley, D . E . (1968) . J . Mol . Biol . 35, 37 . Schochetman, G ., and Perry, R . P . (1972) . J . Mol . Biol . 63, 577 . Spirin, A . S ., and Nemer, M . (1965) . Science 150, 214 . Spohr, G ., Granboulan, N ., Morel, C., and Scherrer, K . (1970) . Eur . J . Biochem . 17, 296 . Tibbetts, C ., Johansson, K ., and Philipson, L . (1973) . J . Virol . 12, 218 . Weber, K ., Pringle, J . R ., and Osborn, M . (1972) . Methods in Enzymology, 26, D . H . W . Hirs and S . N . Timasheff, eds . (New York and London : Academic Press), p. 3 .
Messenger Ribonucleoprotein Complexes Isolated With Oligo(dT)-Cellulose Chromatography from Kidney Polysomes David Irwin, Ajit Kumar, and Ronald A. Malt Surgical Services Massachusetts General Hospital and Shriners Burns Institute and the Department of Surgery Harvard Medical School Boston, Massachusetts 02114
The present study defines some of the characteristics of the mRNP complexes in normal mouse kidney as a base for later evaluating the contribution of mRNP and mRNP proteins to translational regulation during renal hypertrophy . Isolation of mRNP complexes was made possible by their binding to oligo(dT)-cellulose . Results
Summary As an initial step towards understanding the role of mRNP complexes in translational regulation during compensatory renal hypertrophy, characteristics of polysome-associated mRNP Isolated by affinity chromatography were studied . Renal mRNP contained 15-30% of the counts after a 1 hr pulse with 3 H-orotic acid ; it sedimented mainly between 10S and 100S and had a buoyant density of 1 .421 .44 g/cm 3 . RNA derived from the mRNP sedimented between 5S and 40S on sucrose density gradients, with the greatest radioactivity in the region of 15S . After labeling with 3 H-adenine for 1 hr, up to 17% of the radioactivity present in the mRNP-associated RNA was resistant to digestion by pancreatic and T1 ribonucleases . The mRNP protein moiety contained six polypeptides with molecular weights 69,000, 75,000, 80,000, 100,000, 109,000, and 118,000 daltons, which were undetected in the material not binding to ol igo(dT)-cel I u lose .
Isolation of mRNP Stable hybrids of poly(A) with oligo(dT) are completely disrupted at zero salt concentrations (Aviv and Leder, 1972) . However, Lindberg and Sund-
~tI 5,338 cpm 25% formamide
4
1
I
Introduction Messenger RNA (mRNA) of eucaryotes is associated with specific proteins in messenger ribonucleoprotein complexes (mRNP) (Hoagland and Askonas, 1963 ; Spirin and Nemer, 1965 ; Perry and Kelley, 1968 ; Henshaw, 1968) . Although the precise function of the proteins in mRNP is unclear, one of their roles may be to stabilize inactive precursor mRNA against degradation during transport from the nucleus and storage in the cytoplasm (Spohr et al ., 1970) . A reversible equilibrium between polysomes and free mRNP with ribosomal subunits might then form part of a translational control mechanism (Schochetman and Perry, 1972) . Previous studies on the nature of mRNP complexes have examined either continuously growing or terminally differentiated cells . A system of growing cells in mammals is provided by cortical cells of the remaining kidney following contralateral nephrectomy . The predominant response of these cells is hypertrophy, with a rapid accumulation of RNA and protein (Bucher and Malt, 1971) . There is little hyperplasia.
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40
50 TOP
FRAC T/ON NUMBER Figure 1 . Fractionation of EDTA-Dissociated Polysomes on Oligo(dT)-Cellulose EDTA-dissociated polysomes were prepared from 8 kidneys labeled with 3 H-orotic acid . The dissociated polysomes (4 ml) were chromatographed on an oligo(dT)-cellulose (T-2) column, eluted successively with 20 ml of Tris elution buffer (0 .25 M NaCI), 10 ml of elution buffer containing 25% formamide, and finally 10 ml of elution buffer containing 50% formamide . Fractions of 0 .8-0 .9 ml were collected, from which 100 µl aliquots were counted directly in TX8 (o-o) . Counting efficiency was 30% .
Cell 1 58
quist (1974) showed that the mRNA-protein complexes from polysomes that are retained on ol igo(dT)-cel I u lose at high salt concentrations are not recovered by removal of salt alone unless the elution buffer is supplemented with 25-50% formamide . The binding of the mRNP is therefore not merely classical A-T hybridization : the mRNP proteins appear to contribute to hybrid stability . Figure 1 shows that 15-20% of the total polysome radioactivity from normal mouse kidney bound to oligo(dT)-cellulose (Type T-2) after pulse labeling with 3 H-orotic acid for 1 hr . The bound material was released with formamide elution buffer . Type T-3 oligo(dT)-celIulose bound 20-30% of the total polysome radioactivity under similar conditions . Approximately 60-65% of the bound material eluted with 25% formamide ; the remaining 35-40% eluted with 50% formamide . Sedimentation Coefficient of mRNP Previous experiments used sucrose gradients to prepare a fraction from EDTA-dissociated kidney polysomes sedimenting in the 90-170S region as shown in Figure 2a ; Figure 2b shows the isopycnic centrifugation of this fraction on CsCI gradients . The mRNP from the polysomes had a buoyant density of 1 .41-1 .45 g/cm 3 ; some material that had a buoyant density of 1 .54-1 .57 g/cm 3 was presumed to be contamination by large ribosomal subunits . Preparation of mRNP with oligo(dT)-cellulose presumably allows the isolation of poly(A)-contain-
ing complexes of widely differing sizes, including sizes similar to ribosomal subunits ; this kind of separation is not possible with sucrose gradients. The size distribution of mRNP prepared by the oligo(dT)-cellulose procedure therefore was examined on sucrose gradients as described in Figure 3 . The mRNP sedimented predominantly in the 10100S range, with a small amount sedimenting faster than 100S . The higher radioactivity in the 60S and 30S regions may be due to contamination by ribosomal subunits . Buoyant Density of mRNP To investigate whether the heterogeneously sedimenting mRNP included complexes with different protein to RNA ratios, mRNP was examined by isopycnic centrifugation on CsCI gradients . Figure 4a shows the oligo(dT) elution profile from EDTAtreated polysomes from 8 kidneys . The mRNP fractions indicated by bars were dialyzed, fixed, and banded on CsCI gradients . From Figure 4b it can be seen that the mRNP banded mainly at a density of 1 .42-1 .44 g/cm 3 , with
10
1 .7
8
a
O 6 31.
E 4 v
1 .5 4
h W O
1 .4 2
1 .3
FRACTION NUMBER
FRACTION NUMBER
Figure 2a . Sucrose Density Gradient Analysis of EDTA-Dissociated Polysomes
Figure 2b . Buoyant Density of Material in Fractions 10-18 from the Sucrose Gradients
Three mice were injected with 500 pCi of 3H-uridine (uridine 5,6- 3H, 40 .4 Ci/mmole) and the kidneys were removed after 1 hr . EDTAdissociated polysomes were prepared and resuspended in 3 ml of RSB. Aliquots of 1 ml were loaded on to 37 ml, 5-45% sucrose in NEB .25 gradients and centrifuged at 26,000 rpm for 3 hr in a Spinco SW27 rotor. A260 , was monitored and 1 .3-1 .4 ml fractions were collected . TCA insoluble radioactivity was determined in aliquots of 0 .5 ml from each fraction . (-) A260- ; (o-o) TCA insoluble radioactivity .
The remaining 0 .8-0 .9 ml of fractions 10-18 indicated by bars in Figure 2a were pooled and mixed with fractions 10-18 from a duplicate gradient . The fractions were precipitated with 2 vol of ethanol at-25°C for 20 hr . The precipitate was sedimented at 12,100 x g for 20 min, the supernatant was discarded, and the precipitate was dried under vacuum . Buoyant density of the dry residue dissolved in 1 .0 ml of HSB .25 was measured on a CsCI gradient . (x-A) density calculated from refractive index measurements . (o-o) radioactivity of filter discs .
mRNP Complexes of Kidney Polysomes 159
a small amount banding at 1 .39 g/cm 3 . The small amount of higher density material may result from contamination with ribosomal subunits or mRNP with a protein complement less than that normally seen in the formamide eluates. Under identical conditions, fixed EDTA-derived large ribosomal subunits and fixed polysomes exhibited buoyant densities of 1 .58 g/cm 3 and 1 .53-1 .56 g/cm3 , respectively . Less than 4% of the total radioactivity on the CsCI gradients sedimented to the bottom of the tube . The mRNP density of 1 .42-1 .44 g/cm 3 suggests a composition of approximately 20% RNA and 80% protein from the formula of Perry and Kelley (1966), calculated with values of 1 .87 g/cm 3 and 1 .35 g/cm 3 for the densities of RNA and protein, respectively . We recognize that it may not be possible to calculate . exact compositions from simple additive expressions (McConkey, 1974) . 4
3
2
1
TOP FRACTION
NUMBER
Figure 3 . Sedimentation Coefficient Distribution of mRNP Prepared on Oligo(dT)-Cellulose Columns Fractions 28-30 and 37-41 in Figure 1 were pooled and dialyzed against RSB for 2 hr and layered over a 1 ml cushion of 2 M sucrose . The dialysate was centrifuged at 45,000 rpm for 16 hr in a Spinco 65 rotor, the supernatant was discarded, and the sucrose cushion was diluted to 10 ml with NEB .25 . Aliquots of 2 ml were layered on to 36 ml 15-30% sucrose in NEB .25 gradients with 4 absorbance units of unlabeled HeLa large ribosomal subunits as marker and centrifuged at 25,000 rpm for 15 hr in a Spinco SW27 rotor . 1 .5 ml fractions were collected and precipitated . (o--o) TCA insoluble radioactivity.
The heterogeneity in the mRNP apparent from the distribution of sedimentation coefficient thus was not reflected in the protein-RNA composition . Size Distribution of RNA from mRNP Figure 5 shows the examination of RNA derived from mRNP on 15-30% sucrose in NETS gradients using unlabeled 28S ribosomal RNA as an internal marker . Activity was associated with RNA sedimenting between approximately 5S and 40S ; the greatest activity was in the region of 15S . A similar heterogeneous distribution was observed when mRNA prepared from mouse kidney polysomes by the method of Aviv and Leder (1972) was examined on sucrose density gradients (Ouellette, Kumar, and Malt, 1974) . We cannot rule out the possibility that some of the larger RNA and RNP (Figure 3) is nucleus derived . The amount of radioactivity binding to oligo(dT)-cellulose after a 10 min labeling period with 3 H-orotic acid was 15% of that which bound after a 1 hr labeling period . Although the mRNP eluted in two fractions-one eluting with 25% formamide and the other with 50% formamide-no difference in RNA size distribution was detected . A difference is suggested, however, in the proportion of poly(A) that the two mRNP fractions contain . When RNA from 3 H-adenine-labeled mRNP fractions was digested with T1 and pancreatic ribonucleases, the amount of radioactivity which would still bind to oligo(dT)-cellu lose in 0 .5 M NaCl was 9% and 17%, respectively, for the 25% formamide-eluted mRNP and 50% formamide-eluted mRNP . When RNA from the fraction which passes straight through the column during the isolation of mRNP was digested under the same conditions, 5% of the radioactivity bound to oligo(dT)-cellulose . This RNAase-resistant material from the column run-through fraction may derive from poly(A)-containing RNA that passes through the column with the ribosomal subunits . Polypeptides Present in Run-through and Formamide-eluted Fractions The polypeptides present in the 25% and 50% formamide-eluted mRNP and the column run-through fraction were characterized on SDS-polyacrylamide gels . The electrophoretic patterns are shown in the photograph in Figure 6 together with a schematic representation of the bands . The protein complement of the run-through material was similar to that of EDTA-derived large and small ribosomal subunits (unpublished results using kidney ribosomes) . In addition, the run-through material contained two polypeptides of 84,000 and 90,000 daltons . The 25% formamide eluate contained the 84,000 and 90,000 dalton polypeptides together with four polypeptides not seen in the run-
Cell 1 60
through material, 69,000, 75,000, 80,000 and 118,000 daltons . Two minor polypeptides with molecular weights of 100,000 and 109,000 daltons were also detected . Bands corresponding to molecular weights below 62,000 daltons appeared to have corresponding bands in the run-through material and probably represent contamination by ribosomal subunit proteins . Only one polypeptide, the 84,000 dalton polypeptide, was detected in the 50% formamide-eluted mRNP . The 25% formamide-eluted mRNP thus contains at least six distinct nonribosomal polypeptides . In addition, the 84,000 dalton polypeptide found in the three different eluates, and the 90,000 dalton polypeptide found in the run-through and the 25% formamide eluate are probably specific mRNP polypeptides . These two polypeptides could be associated with the poly(A)-containing RNA that does not bind to oligo(dT)-cellulose and is found in the ribosomal subunit run-through material . Proteins associated with nonpoly(A)-containing mRNA would also be expected to be found in the column run-through fraction .
Differences In Buoyant Density of the Formamide-eluted mRNP Fractions Although no difference in the size distribution of RNA from the 25% and 50% formamide eluates was apparent, differences in the protein composition and percentage of poly(A) were observed . These differences may have been detectable in the densities of the mRNP fractions had they been fixed and examined separately. Figure 7 shows the oligo(dT)-cellulose elution profile for EDTA-dissociated polysomes from 16 kidneys and the measurement of the densities of 25% and 50% formamide-eluted mRNP fractions separately . There was, indeed, a 4% difference in density of the two mRNP fractions . The 25% formamide-eluted mRNP had a major density of 1 .48-1 .50 g/cm3, and the 50% formamide-eluted mRNP had a major density of 1 .42-1 .43 g/cm3 . The average density that would be produced by these two values should be somewhat higher than the value of 1 .42-1 .44 g/cm3 shown in Figure 4b . However, the use of 0 .4 M NaCI (Figure 7) rather than 0 .25 M NaCl in the elution buffer may have removed some loosely attached surface proteins and caused a slight increase in the buoyant density .
f 54,812 cpa
( a--0 )
(S---~) 4 .42-1 .44 g/cm3
4
10 4 1 .6 8 N1 O >
1 .5 6
v 4
1 .4
1 2 1 .3 Y I 10
20
I 30
40 TOP
I I I 10 20 30 40 50 TOP
FRACT/ON NUMBER FRACT/ON NUMBER Figure 4 . Buoyant Density of Polysome-Derived mRNP (a, left) EDTA-dissociated polysomes were prepared from 8 kidneys and fractionated on an oligo(dT)-cellulose (T-2) column . (o-o) total radioactivity of 100 µl aliquots from 1 .1 ml fractions . (b, right) Fractions 25-30 and 32-25 were pooled and dialyzed against RSB for 3 hr . After the dialysate was fixed in 6% glutaraldehyde, 2 ml aliquots were examined on 5 ml CsCI gradients . (A-0) density calculated from refractive indices . (o-o) TCA insoluble radioactivity.
mRNP 161
Complexes
of Kidney
Polysomes
The 25% and 50% formamide eluates contained 37% and 20% RNA, respectively (Perry and Kelley, 1966). Discussion These results are an initial characterization of some of the polysome-associated mRNP complexes in mouse kidney. The oligo(dT)-cellulose method appears to select not only complexes containing mRNA with terminal poly(A)s attached, but also configurations permitting hybridization of the poly(A) to oligo(dT). Thus, histone mRNP complexes would not be expected to bind, and complexes in which the poly(A) regions are not exposed -for example those masked by proteins-may be unable to interact with oligo(dT). The RNAase-resistant poly(A) in the ribosomal run-through fraction therefore may derive from mRNP in which the poly(A) is shielded. The 84,000 and 90,000 dalton polypeptides present in the run-through fraction,
which are probably mRNP proteins, could be associated with this nonbinding mRNP. The heterogeneous distribution of the mRNP isolated using oligo(dT)-cellulose had a similar size range to that seen in several other mammalian systems listed in Table 1, the bulk of the mRNP in kidney sedimenting between 10s and 80s. To avoid excessive contamination by ribosomal subunits, the sucrose gradient procedure selects only the material sedimenting between 90s and 170s. It would appear that either the fast sedimenting mRNP complexes are not able to hybridize to oligo(dT), or that during oligo(dT)-cellulose chromatography the complexes are damaged, thereby producing smaller mRNPs. mRNP prepared by both oligo(dT)-cellulose and by sucrose gradients contained some ribosomal
8
12,400
B
I
25,000
67,000
III1
I
II
/\ 64,000
90,000
\/J
C
/
#I#
1I 66,000’/ 75,000 I 64000
ii6,OOO
\
100S
1 .35-1 .55
Henshaw and Loebenstein (1970)
Rat liver
30->90S
1 .35-1 .45
Perry and Kelley (1968)
L cells (polysomes) (free)
12-60S 25-60S
1 .45 1 .38-1 .44
Blobel (1973)
L cells and Rat liver
After RNAase digestion 4-16S
2
52, 78
Spohr et al . (1970)
HeLa
10-70S
Kwan and Brawerman (1972)
Mouse ascites
12-15S for poly(A) region only
Lee et al . (1971)
Mouse ascites
25->160S
Blobel (1972)
Rabbit reticulocytes
20S
2
52, 78
Lebleu et al . (1971)
Rabbit reticulocytes
Morel at al . (1971)
Duck erythroblasts
15S
2
68, 130
20S
2
Morel et al . (1973)
49, 73
Duck erythroblasts
15S
2 major 6 minor
49, 73 52-64,86-120
Gander et al . (1973)
Duck erythroblasts (free)
12S 20S
7 10
7-65 15-51 all 17 different
Bryan and Hayashi (1973)
Chick embryo brain
3 different size ranges
2 common to all
54, 92 or 48 .4, 78 .5 by discontinuous polyacrylamide gel electrophoresis
4 Normal 4 + 1
56, 68, 78, 125 110
1 .40-1 .48
1 .40-1 .45 1 .40-1 .45
Kumar and Lindberg (1972)
KB Cells (isotonic) (0 .5 M NaCl)
up to 200S
1 .35-1 .47 1 .57-1 .60
Lindberg and Sundquist (1974)
KB Cells (normal) (Adeno viruss infected)
10-100S
1 .38
mRNP Complexes of Kidney Polysomes 163
a 5
4 25% formamide 50% formamide 3
2
1
w 10
20
30 TOP
FRACTION NUMBER d
3 `a
O
Imo . W O
2 V I
FRACTION NUMBER FRACTION NUMBER Figure 7 . Differences in Buoyant Densities of 25% and 50% Formamide-Eluted mRNP EDTA-dissociated polysomes from 16 kidneys were fractionated on oligo(dT)-cellulose, except that T-3 cellulose was used ; fractions were eluted with 25 ml of elution buffer (0 .4 M NaCl) followed by 15 ml of 25% formamide in elution buffer and 15 ml of 50% formamide in elution buffer . (a) Column elution ; (0 0) total radioactivity of 100 µI aliquots from 1 .6 ml fractions . Fractions 23 and 29 were dialyzed separately against RSB for 90 min and fixed in 6% glutaraldehyde, (volumes of fixed dialysates, 2 .3 ml and 2.8 ml, respectively) . Buoyant densities were measured using sample loads of 1 .5 ml . (b) 1 .5 ml of 25% formamide eluate . (c) 1 .5 ml of 50% formamide eluate . (d) 0.5 ml of 25% + 1 .0 ml of 50% formamide eluate mixed . (L-4) density . (0--o) TCA insoluble radioactivity .
Cell 1 64
than the 25% formamide eluate . Another difference between the two mRNP fractions therefore may be that the 50% formamide eluate contains mRNA with longer poly(A) sequences than those of the 25% formamide eluate . An alternative explanation would be that some nonpoly(A)-containing RNA (for example, ribosomal RNA) is present in the 25% formamide eluate . The sizes of the poly(A) sequences have yet to be determined by direct measurement . Experimental Procedures Solutions and Reagents RSB : 0 .01 M Tris, 0 .01 M NaCl, 0.0015 M MgCI2 pH 7.4 at 20°C . HSB .25 : 0 .01 M Tris, 0 .25 M NaCl, 0 .05 M MgCl2 pH 7 .4 at 20°C . NEB .25 : 0 .01 M Tris, 0 .25 M NaCl, 0 .01 M EDTA pH 7 .4 at 20°C. NETS : 0 .01 M Tris, 0 .1 M NaCl, 0 .01 M EDTA, 0.2% SDS . Elution buffer, 0 .01 M Tris or 0 .01 M P043- and 0 .25 M NaCl or 0.4 M NaCl pH 7 .4 at 20°C . TX8 : 2 I Triton X-100, 1 I xylene, 8 g Omnifluor (New England Nuclear Corp ., Boston, Mass .) SUP : 0 .5% SDS, 0 .5 M urea, 0 .01 M P043- pH 7 .0 at 20°C . Formamide (Fisher Scientific Co ., N .J .) was purified according to the procedure of Tibbets, Johansson, and Philipson (1973) except that the ether drying stage was omitted . Preparation of Polysomes Male Charles River mice (50-60 days old) were injected intraperitoneally with either 100 yCi of 3H-orotic acid (orotic-5-3H acid, 11 .1 Ci/mmole, New England Nuclear Corp .) or420tLCi of 3H-adenine (adenine 3H, 675 Ci/mole, New England Nuclear Corp .) . After 1 hr, kidneys were removed and disrupted in 1 ml of RSB per 2 kidneys using a glass Dounce homogenizer (10 strokes of the loose pestle followed by 4 strokes of the tight pestle) . The homogenate was centrifuged at 12,100 x g for 10 min, and the supernatant was layered on discontinuous sucrose gradients of 3 ml 2 M sucrose, 1 ml 1 .5 M sucrose and 1 ml 0 .5 M sucrose in HSB .25 . The gradients were centrifuged at 55,000 rpm for 4 hr in a Spinco 65 rotor at 2-4°C . The supernatant layers were discarded, and the clear polysome pellets were rinsed twice with 2 ml aliquots of RSB .
70% ethanol, dried and counted in 0 .4% Omnifluor in toluene with counting efficiencies greater than 30% . Isopycnic Centrifugation on CsCI Gradients mRNP preparations were fixed in 6% glutaraldehyde (Eastman Kodak Co ., Rochester, N .Y .) neutralized with NaHCO3 just before use . Densities were measured on CsCI gradients containing 0.8% Brij 35 (Fisher Scientific Co.) . Linear gradients were preformed from 2.4 ml of CsCI solution, density 1 .55 g/cm3, and 2 .6 ml of CsCI solution, density 1 .41 g/cm3 . Sample volumes of 1 .0-2 .0 ml were overlaid, and the gradients were centrifuged at 35,000 rpm for 20 hr at 2-4°C in a Spinco SW41 rotor, allowed to decelerate without braking . Twelve-drop fractions were collected from the bottom of the tubes directly onto Whatman 3MM filter discs . Following the first fraction and every sixth fraction thereafter, two drops were collected for density determinations from refractive index measurements . The discs were dried and rinsed in 10% trichloroacetic acid, 5% trichloroacetic acid, and 70% ethanol at 0°C, dried, and counted directly in 0 .4% Omnifluor in toluene. Sucrose Density Gradient Analysis of RNA from mRNP Ethanol was added to the mRNP preparations to a concentration of 70% . The precipitated mRNP was centrifuged at 12,100 x g for 30 min, and the supernatant was discarded . The precipitate was dried under vacuum and redissolved in NETS buffer including unlabeled 28S ribosomal RNA as an internal marker . Aliquots of 2 ml were run on 36 ml 15-30% sucrose (w/w) in NETS gradients at 21,000 rpm for 17 hr in a Spinco SW27 rotor at 23°C. SDS Polyacrylamide Gel Electrophoresis mRNP preparations were dialyzed against SUP and examined on 7.5% acrylamide gels according to Bhorjee and Pederson (1973), except that 6 mm x 10 cm gels were used and acrylamide (Fisher Scientific Co .) was not deionized, but twice recrystallized from chloroform . Bovine serum albumin, chymotrypsin A, and cytochrome c were used as reference standards in parallel gels . Molecular weights were calculated by the method of Weber, Pringle, and Osborn (1972) . Acknowledgment This work was supported by grants from the National Institutes of Health and from the National Science Foundation .
Preparation of mRNP by Affinity Chromotography Polysomes were resuspended in either 10 mM phosphate or 10 mM Tris buffer (pH 7 .4), and 10 mM EDTA . The ribosomal subunitmRNP mixture was centrifuged at 12,100 x g for 10 min to remove undissolved debris . 5 M NaCl was added to a final concentration of either 0 .25 M or 0.4 M NaCl . mRNP was then isolated using oligo(dT)-cellulose based on the method of Lindberg and Sundquist (1974) . A column 7 .5 mm x90 mm was formed in a disposable pipette with 0.8-1 .0 g of oligo(dT)-cellulose (Types T-2 or T-3, Collaborative Research, Inc ., Waltham, Mass .) . The ribosomal subunit-mRNP mixture was loaded on to a column and eluted with elution buffer, followed by 25% formamide in elution buffer, and finally, 50% formamide in elution buffer. The mRNP eluted by the formamide was dialyzed against either 0 .01 M Tris or 0 .01 M P04 3to remove the formamide . All operations were performed at 0-4°C .
Received September 18, 1974 ; revised December 2, 1974
Sucrose Density Gradient Analysis of mRNP mRNP was prepared as described and examined on either 15-30% or 5-45% linear density gradients of sucrose (w/w) in NEB .25 . The A 26p °m was monitored and the approximate S value range was estimated by reference to an unlabeled marker of 60S ribosomal subunits. Fractions with 2% bovine serum albumin coprecipitant were precipitated with equal volumes of 20% trichloroacetic acid . The fractions collected cold on glass fiber discs were rinsed successively with 10% trichloroacetic acid, 5% trichloroacetic acid,
Henshaw, E . C ., and Loebenstein, J . (1970) . Biochim . Biophys . Acta 199, 405 .
References Aviv, H ., and Leder, P . (1972) . Proc. Nat . Acad . Sci . USA 69, 1408 . Bhorjee, J . S ., and Pederson, T. (1973) . Biochemistry 12, 2766 . Blobel, G . (1972) . Biochem . Biophys. Res . Commun . 47, 88 . Blobel, G . (1973) . Proc. Nat. Acad . Sci . USA 70, 924 . Bryan, R . N ., and Hayashi, M . (1973) . Nature New Biol . 244, 271 . Bucher, N . L . R ., and Malt, R . A . (1971) . Regeneration of Liver and Kidney (Boston: Little Brown), p . 179 . Gander, E . S ., Stewart, A . G ., Morel, C. M ., and Scherrer, K . (1973). Eur . J . Biochem . 38, 443 . Henshaw, E . C . (1968) . J . Mol . Biol . 36, 401 .
Hoagland, M . B ., and Askonas, B . A. (1963) . Proc . Nat. Acad . Sci. USA 49, 130 . Kumar, A., and Lindberg, U . (1972) . Proc . Nat. Acad . Sci. USA 69, 681 . Kwan, S- . W ., and Brawerman, G . (1972) . Proc. Nat . Acad . Sci . USA 69, 3247 .
mRNP Complexes of Kidney Polysomes 165
Lebleu, B ., Marbaix, G ., Huez, G., Timmerman, J ., Burny, A ., and Chantrenne, H. (1971) . Eur. J . Biochem . 19, 264 . Lee, S . Y ., Krsmanovic, V ., and Brawerman, G . (1971) . Biochemistry 10, 895 . Lindberg, U ., and Sundquist, B . (1974) . J . Mol . Biol . 86, 451 . McConkey, E . H . (1974) . Proc . Nat . Acad . Sci . USA 71, 1379 . Morel, C ., Kayibanda, B ., and Scherrer, K . (1971) . FEBS Letters 18, 84 . Morel, C ., Gander, E . S ., Herzberg, M ., Dubochet, J ., and Scherrer, K . (1973) . Eur . J . Biochem . 36, 455 . Ouellette, A . J ., Kumar, A ., and Malt, R . A . (1974) . Federation Proceedings 33, 635 . Perry, R . P ., and Kelley, D . E . (1966) . J . Mol . Biol . 16, 255 . Perry, R . P., and Kelley, D . E . (1968) . J . Mol . Biol . 35, 37 . Schochetman, G ., and Perry, R . P . (1972) . J . Mol . Biol . 63, 577 . Spirin, A . S ., and Nemer, M . (1965) . Science 150, 214 . Spohr, G ., Granboulan, N ., Morel, C., and Scherrer, K . (1970) . Eur . J . Biochem . 17, 296 . Tibbetts, C ., Johansson, K ., and Philipson, L . (1973) . J . Virol . 12, 218 . Weber, K ., Pringle, J . R ., and Osborn, M . (1972) . Methods in Enzymology, 26, D . H . W . Hirs and S . N . Timasheff, eds . (New York and London : Academic Press), p. 3 .
