Nucleic Acids Research Volume 6 Number 2 February 1979 eray199NcecAisRsac Voue6NmeI
Ultraviolet light-induced crosslinking of mRNA to proteins Jay R.Greenberg The Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, MA 01545, USA
Received 20 November 1978 ABSTRACT
Irradiation of intact or EDTA-dissociated L-cell polyribosomes with 254 nm UV light at doses of 1-2 x 105 ergs/mm2 extensively crosslinks mRNA to proteins. The crosslinked mRNAprotein complexes can be isolated on the basis of buoyant density in urea-containing Cs2SO4 gradients that dissociate non-covalent complexes. Crosslinking of mRNA can also be assayed by phenolchloroform extraction. mRNA recovered from the crosslinked complexes by digestion with proteinase K has the same electrophoretic mobility in polyacrylamide gels as unirradiated mRNA. Therefore, irradiation does not either crosslink RNA molecules to RNA molecules or break phosphodiester bonds. With these methods it has been found that more than 70% of high molecular weight polydisperse mRNA, but only 25-40% of histone mRNA, can be crosslinked to protein. On the basis of buoyant density the histone mRNA-protein complex had a protein content of 26%, whereas the mean protein content of most non-histone mRNAprotein complexes was 65%. It is concluded that most mRNA in polyribosomes is in close contact with proteins, and that histone mRNA can be crosslinked to many fewer proteins that most other mRNAs. INTRODUCTION The existence of mRNA binding proteins in eukaryotic cells is well known (reviewed by Greenberg (1), Preobrazhensky and Spirin (2). Although some of these proteins have been identified, their significance remains obscure, and there is still considerable difficulty in isolating and working with them. This difficulty arises from the necessity for isolating mRNA-protein complexes (mRNP) under conditions which do not allow either dissociation of native mRNP or fortuitous binding of proteins to RNA. It is usually not possible to be sure that such conditions have been met. Clearly, the study of mRNA-protein interactions would be facilitated by the ability to create specific crossC Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
715
'Nucleic Acids Research links between proteins and mRNA in native mRNP. Crosslinking would make it possible to isolate and work with mRNA-protein complexes under conditions that discourage non-specific binding of proteins to RNA. Furthermore, crosslinking itself could be used as a criterion for identifying specific mRNA-associated proteins. That is, they could be defined as proteins that can readily be crosslinked to mRNA. For lack of a suitable chemical crosslinker we explored the possibility of using photo-induced crosslinking with shortwave UV (254 nm). The ability of shortwave UV radiation to crosslink proteins to nucleic acids is amply documented (reviewed by Smith (3)). In some cases, i.e., those of the lac operator/lac repressor (4), E. coli rRNA/ribosomal proteins (5), E. coli tRNA/ aminoacyl tRNA synthetase (6), and avian retrovirus 35S RNA/pll (7) a high degree of specificity in crosslinking has been demonstrated. In this paper it is shown that most eukaryotic mRNA in polyribosomes, including histone mRNA, can be crosslinked to protein as a result of irradiating isolated polyribosomes with shortwave UV light. Furthermore, the crosslinked mRNA-protein complexes can be readily dissociated from ribosomal subunits, and the mRNA can be recovered and analyzed. However, 4S RNA in polyribosomes was not crosslinked to protein, and histone mRNP behaved differently than other mRNPs. MATERIALS AND METHODS
Materials. Cs2SO4 was obtained from Kawecki-Berylco Industries (New York, New York) and purified by the method of Wilt et al. (8). Ultrapure grade urea was obtained from Schwarz-Mann. Actinomycin D and cytosine arabinoside were purchased from Sigma. Radioactively labeled compounds were obtained from New England Nuclear or Amersham. ACS and Aquasol aqueous counting solutions were obtained from Amersham and New England Nuclear respectively. Protosol gel solubilizer was obtained from New England Nuclear. Proteinase K was obtained from EM labs. Cell culture and labeling. L-cells were grown in suspension cultures and labeled with 3H-uridine in the presence of 0.08 ig/ ml of actinomycin D as described (9). In some experiments cells were preincubated for 30 minutes in medium containing 60 ig/ml of 716
Nucleic Acids Research cytosine arabinoside as well as actinomycin D, then concentrated eight-fold and labeled in the same medium. Labeling with 3Hleucine was carried out as described for 3H-uridine, except that prior to labeling the cells were grown overnight in medium containing 5.25 pg/ml of leucine, which is 1/10 the usual concentration. The medium contained non-dialyzed 4% calf serum and 4% fetal calf serum. Polyribosome isolation. Polyribosomes were isolated as described (9). Briefly, cells were lysed in isotonic buffer containing 0.1% Triton-X-100. Polyribosomes were centrifuged in sucrose gradients containing 0.01 M NaCl, 0.01 M Tris-HCl (pH 7.6), and 0.002 M MgSO4 (RSB), then pelleted by centrifugation through 2 M sucrose in RSB. Irradiation. Pelleted polyribosomes were dissolved in RSB at a concentration of 1-2 A260 units/ml. In some experiments 10% glycerol (v/v) was present. One milliliter samples of polyribosomes were irradiated in polyethylene scintillation vial caps with a 15 watt germicidal bulb (Sylvania G15T8) equipped with an aluminum foil reflector. The radiation intensity was 2000 pW/cm 2 The intensity was measured with an Ultraviolet Products model J-225 meter. The samples were kept on ice during irradiation. The standard dose, used in all experiments reported in this paper, was 105 ergs/mm2, corresponding to about eight minutes of irradiation. Cs2SO4-urea density gradient centrifugation. Irradiated or unirradiated control polyribosomes were precipitated from ethanol. The precipitates were air dried at 37°C, then dissolved in 5 ml of 10 M urea. Water, Cs2SO4 stock solution (1.95 g/cc), 1 M Tris-HCl (pH 7.6), and 0.1 M EDTA were added so as to give a final volume of 10 ml, a Tris-HCl concentration of 0.01 M, and an EDTA concentration of 0.001 M. The final Cs2SO4 concentration was equivalent to a density of 1.35 g/cc in water. The samples were placed in screwcap polycarbonate tubes and centrifuged in a Beckman Ti75 rotor for 65 hours at 58,000 rpm at 250C. The gradients were fractionated by inserting a stainless steel tube to the bottom of the gradient and pumping out the gradient with a peristaltic pump. About 30 equal fractions were collected. The density of every fourth fraction was measured by weighing 0.1 ml. 717
Nucleic Acids Research The density was correlated with the fraction number by fitting a parabolic curve to the data by a least squares procedure with the aid of a computer. The correlation coefficient was greater than 0.995. Samples were recovered from the gradients by pressure ultrafiltering to dryness through a Millipore PTHK 25 mm filter. Residual Cs2SO4 and urea were removed by passing water through the filter. Some 4S RNA passes through the filter in this procedure. The apparatus and filter were rinsed with 0.5% diethylpyrocarbonate and water prior to use. The samples on the filters were recovered by dissolving in 2 ml of 0.5% SDS, 0.1 M NaCl, 0.01 M Tris-HCl (pH 7.6), 0.001 M EDTA (SDS buffer) containing 100 pg/ml of proteinase K. Deproteinization. Samples in SDS buffer were digested for one hour at 370C with 100 vg/ml of proteinase K. The samples were then extracted once with an equal volume of phenol-chloroform mixture (10) by vortexing briefly. The phases were separated by centrifugation for one minute at 10,000 rpm. Polyacrylamide gel electrophoresis. RNA was electrophoresed in 2.7% polyacrylamide gels crosslinked with ethylene diacrylate (9). Electrophoresis was for 2.75 hours at 5 ma/gel. The gels were frozen in hexane/dry ice and sliced into 2 mm segments with an array of razor blades. Scintillation counting. Polyacrylamide gel slices were dissolved in 3% Protosol in toluene containing PPO and POPOP. In double label experiments with 14C marker RNA 3H counts were corrected for 14C spilling into the 3H channel. Other samples were counted in ACS or Aquasol aqueous counting solutions. RESULTS
Evidence for crosslinking of mRNA to proteins. In order to demonstrate crosslinking we isolated polyribosomes from cells which had been labeled with 3H-uridine in the presence of 0.08 ig/ml of actinomycin D. These labeling conditions were chosen so that mRNA would be labeled, but labeling of rRNA would be inhibited (11). The polyribosomes were irradiated at 254 nm and centrifuged in Cs2SO4 density gradients containing 5 M urea (12) in order to resolve crosslinked RNA-protein complexes from RNA and protein. Urea was included to prevent precipitation of RNA 718
Nucleic Acids Research and protein, and also, to enhance the dissociation of noncovalent complexes. These gradients differ from the ones used in our previous work (9,13) in that they are designed to completely dissociate non-covalent mRNA-protein complexes, whereas the gradients previously employed were intended to preserve such complexes. The UV dose was varied, and it was found that a total dose of about 1-2 x 105 ergs/mm2 (8-16 minutes of irradiation at 2000 pW/cm2) gave extensive crosslinking. Representative results are shown in Figure 1. It may be seen that in the absence of irradiation all of the labeled RNA banded in a peak at about 1.65 g/cc, which corresponds to the density of pure RNA. After irradiation, however, there was much radioactivity in a peak at about 1.3-1.35 g/cc. This peak presumably corresponds to crosslinked mRNP. There was also considerable radioactivity at densities intermediate between 1.35 g/cc and 1.65 g/cc which presumably corresponds to less protein-rich complexes. Since after irradiation most of the radioactivity banded at densities lower than the density of pure RNA, and since most of the radioactivity should have been in mRNA, it was tentatively concluded that mRNA was extensively crosslinked to protein. A more quantitative analysis of crosslinking is presented below. Similar results were also obtained by irradiation of EDTA-dissociated polyribosomes (results not shown). In order to demonstrate that the putative crosslinked mRNP contained protein, polyribosomes were isolated from cells labeled with 3H-leucine in the presence of 0.08 pg/ml of actinomycin D, irradiated, and centrifuged in Cs2SO4-urea density gradients. The results are shown in Figure 2. There was a major peak of Hleucine radioactivity at 1.3-1.35 g/cc, which is considered to be the density of mRNP. There was some radioactivity tailing towards higher densities, but none at the density of RNA. There was also a smaller peak at about 1.2 g/cc. This peak corresponds to the density of pure protein. In the absence of irradiation virtually all of the protein label banded at this density (results not shown). Since after irradiation about 2/3 of the leucine label banded at the density of RNP, it is concluded that 2/3 of the label was in protein that could be crosslinked to RNA. The recovery of label from the gradients was 80%. Therefore, even 719
Nucleic Acids Research of labeled RNA 1 (left). and Banding Figureirradiated unirradiated pooly~~~~from
A Ax
20
ribosomes in Cs2SO4-urea from ents. isolatedgradiPolyribosomes were density cells labeled with 3H-uridine (10 pC/ml) for 70 minutes in the presence of 0.08 vpg/ml of actinomycin D. A portion of ~~~the polyribosomes was irradiated, and irradiated and unirradiated samples were centrifuged in Cs2SO4-urea gradients. The gradients were fractionated, and an aliquot of each fraction was counted. Unirradiated polyribosomes. B) Irfx x*A) radiated polyribosomes.
10
F 10 ! \ / \
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1.4 1.6 DENSITY (G/CM3)
I.
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LL' // Figure 2 (right). Banding K of labeled protein from irradiated polyribosomes in oxv a Cs2SO4-urea density gradient. Polyribosomes were isolated from cells labeled 1.8 1.6 1.4 with 3H-leucine (10 pC/ml)
for 70 minutes in the pres-
\
1.2
DENSITY (G/CM3)
ence of 0.08 pg/ml of actinomycin D, irradiated, and centrifuged in a Cs2SO4-urea density gradient. The gradient was fractionated, and an aliquot of each gradient was counted.
if all of the radioactivity unaccounted for were in free protein, it is likely that at least 50% of the labeled protein was cross720
Nucleic Acids Research linked to RNA as a result of irradiation. In other similar experiments the extent of crosslinking of protein to RNA was variable, but generally somewhat lower than the value reported here, e.g. 30%. Under the labeling conditions used incorporation of newly synthesized ribosomal proteins into ribosomes is inhibited (14). However, the metabolism of polyribosomal proteins under these conditions is less well-studied than that of polyribosomal RNA, and it cannot be assumed that all of the label was present in mRNA binding proteins. Therefore, these experiments are a qualitative demonstration of UV-induced crosslinking of proteins to RNA in polyribosomes, rather than a quantitative estimation of the efficiency of crosslinking of proteins to mRNA. Nature of RNA crosslinked to protein. In order to determine whether it is feasible to recover intact mRNA for analysis from UV-crosslinked mRNP, and to gain insight into the kinds of RNA which can be crosslinked to protein, UV-crosslinked mRNP was recovered from Cs2SO4-urea density gradients, deproteinized, and analyzed by means of electrophoresis in polyacrylamide gels. Of particular concern in this experiment was whether histone mRNA can be crosslinked to protein. Previous work from this laboratory done without the use of crosslinking showed that both poly(A) +mRNA and non-histone poly(A) mRNA in polyribosomes are associated with protein (9,13). However, that work did not conclusively establish whether or not histone mRNA is associated with protein. In order to identify histone mRNA cells were labeled with 3H-uridine in the presence of 0.08 pg/ml of actinomycin D either with or without cytosine arabinoside. This drug selectively inhibits the entrance of histone mRNA into polyribosomes (15,16). Therefore, comparison of size classes of mRNA from polyribosomes of control and cytosine arabinoside-treated cells can serve to identify histone mRNA (17). Small polyribosomes (trimers through hexamers) were isolated in sucrose density gradients, UV-irradiated, and centrifuged in Cs2SO4-urea density gradients. The results are shown in Figure 3. The indicated fractions were pooled, desalted by ultrafiltration, treated with proteinase K, phenol-chloroform extracted, and analyzed in 2.7% polyacrylamide gels as shown in Figure 4. The data show that the most dense region of the gradients, corres721
Nucleic Acids Research A z 0
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1. g5~
a. u
B
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~
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% ~~
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1.6 1.4 DENSITY (G/CM3)
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Figure 3. Banding of labeled RNA from irradiated polyribosomes of control and cytosine arabinoside-treated cells in CS2SO4-ureadensity gradients. Small polyribosomes (trimers through hexamers) were isolated from cells labeled for 70 minutes with 3H-uridine in the presence of 0.08 ig/ml of actinomycin D with or without cytosine arabinoside (60 pg/ml). The polyribosomes were irradiated and centrifuged in Cs2SO4-urea gradients, and an aliquot of each fraction was counted. The lettered horizontal bars refer to fractions pooled for further analysis. x-x Control. o-o Cytosine arabinoside.
ponding in density to protein-free RNA, consisted predominately of 4S RNA and histone mRNA. Histone mRNA was identified as the component having an apparent molecular weight of 150,000 which was present in control, but not in cytosine arabinoside-treated cells. There was also some polydisperse radioactivity of higher molecular weight present in this fraction. This was presumably non-histone mRNA not crosslinked to protein. The intermediate density region of the gradients (p = 1.45-1.57 g/cc) contained mostly polydisperse RNA but also some histone mRNA and 4S RNA. The buoyant density of this region is consistent with the presence of protein on the RNA. The low density region (p = 1.25-1.43 g/cc) contained predominantly polydisperse RNA and traces of 4S RNA. There was essentially no histone mRNA. The radioactivity in histone mRNA was measured as the difference 722
eXg4
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Nucleic Acids Research
Figure
4. Polyacrylamide gel electrophoretic analysis of labeled RNA
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from irradiated polyribosomes banded in Cs2SO4-urea gradients. The indicated regions of the gradients shown in Figure 3 were desalted by ultrafiltration. The samples were then treated with proteinase K in the presence of SDS and extracted with phenol-chloroform. The RNA was precipitated with ethanol and electroin 2.7% polyacrylamide gels. phoresed Molecular weight markers of 14C-labeled rRNA were included some of the gels. The gels were in sliced into 2 mm fractions which were dissolved in scintillation fluid and counted. The lettered frames correspond to the lettered gradient regions in Figure 3. The crosshatched areas represent radioactivity considered to be in histone mRNA. The radioactivity in the gels is expressed in units which reflect the relative amount of radioactivity in each region of the gradi~~~ent. The sum of the units for all three gels from each gradient is 100. A) 1.60-1.75 g/cm . B) 1.45-1.58 g/cm3. C) 1.25-1.43 g/cm3. x x Control. o - o Cytosine arabinoside.
FRACTION
between control and cytosine arabinoside-treated samples in appropriate gel fractions (17). On the basis of buoyant density after UV-irradiation it appears that 25% of histone mRNA was crosslinked to protein, whereas >80% of non-histone mRNA was crosslinked to protein. It is likely that this non-histone mRNA consisted of both poly(A)-containing and poly(A)-lacking species (Greenberg, manuscript in preparation). In the experiment described above the protein-free histone mRNA and the putative histone mRNP were found in adjacent fractions of Cs2So4-urea density gradients. It could be argued that these fractions overlapped, and that quantitative separation of histone mRNA from histone mRNP was not obtained. Therefore, the extent of crosslinking of histone mRNA to protein could have been overestimated or underestimated. For this reason another method was sought to estimate the extent of crosslinking. Phenol extraction is a method previously used to quantitate UV-induced crosslinking 723
Nucleic Acids Research of nucleic acids to proteins (6,18). In this method solutions of nucleic acid, crosslinked nucleoprotein, and free protein are extracted with phenol, and the phases are subsequently separated by centrifugation. Denatured free protein and nucleoprotein precipitate at the interphase, whereas protein-free nucleic acid remains in the aqueous phase. We have used this approach to quantitate crosslinking of histone mRNA to protein. Cells were labeled with 3H-uridine in the presence of 0.08 ig/ml of actinomycin D with or without cytosine arabinoside, and total polyribosomes were isolated. A portion of the polyribosomes was UV-irradiated, and irradiated and unirradiated samples were extracted with a mixture of phenol and chloroform (10) in the presence of sodium dodecylsulfate (SDS). The phases were separated, and the organic phase plus the interphase were reextracted with SDS buffer. The resulting aqueous phases were combined, and their RNA was precipitated with ethanol. The organic phase plus the interphase were also mixed with ethanol, and the insoluble material was pelleted by centrifugation. The pellets from the aqueous and organic phases were redissolved in SDS buffer, treated with proteinase K, and reextracted with phenol-chloroform. After phase separation there was little or no visible precipitate or radioactivity at the interphase. The RNA in the aqueous phase was re-precipitated with ethanol and analyzed by means of electrophoresis in polyacrylamide gels. A flow chart of the experiment is shown in Figure 5, and the quantitative aspects of the experiment are summarized in Table 1. The gel electrophoresis results are shown in Figure 6. It may be seen that the labeled RNA from the aqueous phase of the non-TN-treated polyribosomes consisted of three principal components: high molecular weight polydisperse RNA, histone mRNA, and 4S RNA. The aqueous phase contained 85% of the total radioactivity. The RNA from the interphase plus organic phase of the non-TV-treated polyribosomes (15% of the total radioactivity) consisted of high molecular weight polydisperse RNA and 4S RNA. Histone mRNA appeared to be essentially absent. The aqueous phase of the TN-irradiated polyribosomes (40% of the total radioactivity) contained all three principal components, but 4S RNA was predominant. The organic phase plus interphase of the TN-treated polyribosomes 724
Nucleic Acids Research Cells labeled with
H-uridine in the presence of + cytosine arabinoside (60 pg/ml)
0.08pg/ml of actinomycin D
Polyribosomes |
UV (2000
pW/cm
for 8 minutes)
Phenol-chloroform extraction
Organic phase + interphase
Aqueous phase
Reextract with SDS buffer
Combined aqueous phases \
- Aqueous phase
Organic phase +
~~~~~~~~interphase
ethanol precipitation
/ ethanol precipitation
Dissolve precipitate in SDS buffer and
digest with Proteinase K
Phenol-chloroform extraction
Aqueous phase
Organic phase
(discard)
ethanol
precipitation
Analyze RNA in gels
Figure 5. Assay of crosslinking by phenol-chloroform extraction. Polyribosomes were isolated from cellslabeled for 70 minutes with 3H-uridine in the presence of 0.08 ig/ml of actinomycin D with or without 60 jg/ml of cytosine arabinoside. Portions of the polyribosomes were irradiated. All samples were made 0.5% in SDS, 0.1 M in NaCl, 0.005 M in EDTA, and 0.05 M in Tris-HCl (pH 7.6), and extracted with phenol-chloroform as indicated in this figure. Small aliquots of the aqueous phases were counted before and after extraction. The amount of radioactivity in the organic phase plus the interphase was calculated as the difference between the two measurements.
725
Nucleic Acids Research Table 1 Extent of Crosslinking as Assayed by Phenol-Chloroform Extraction Cytosine Arabinoside
Control No UV
Histone mRNA (X of total cpm)
+ UV
No UV
Aqueous phase
Organic phase
Aqueous phase
Organic phase
3.21
0.069
1.05
0.709
High molecular weight polydisperse mRNA (X of total cpm)
64.6
4S RNA (X of total cpm)
18.1
Total
85.9
11.2
2.79 14.1
19.0
18.8 38.9
57.2
3.23 61.1
+ UV
Aqueous phase
Organic phase
Aqueous phase
Organic phase
40a
ob
ob
ob
ob
75a
68.4
11.9
19.1
54.7
X Crosslinking
la,c
16.1 84.5
3.57 15.5
20.6
39.7
X Cross1inking
74a
5.62
3a.c
60.3
aCalculated as (plus UV cpm in organic phase . plus UV cpm in organic phase + plus UV cpm in aqueous phase) x 100.
bAssumed
to be nil.
cCorrected
for X of cpm in No UV organic phase.
(60% of the total radioactivity) contained high molecular weight polydisperse RNA, histone mRNA, and a relatively small amount of 4S RNA. In the case of the UV-treated polyribosomes 40% of the histone mRNA recovered was in the organic phase plus the interphase. This histone mRNA was presumably crosslinked to protein. Similarly, it is estimated that 74-75% of the high molecular weight polydisperse RNA was crosslinked to protein. The 4S RNA did not appear to be crosslinked to protein as a result of irradiation. About 15% of the 4S RNA was found at the interphase after irradiation. However, a virtually identical amount was found at the interphase in the absence of irradiation. Possibly, this was peptidyl tRNA. About 15% of high molecular weight polydisperse mRNA was found at the interphase in the absence of irradiation. Presumably, this mRNA was susceptible to crosslinking. Therefore, in the case of the + UV high molecular weight polydisperse mRNA no attempt was made to correct for the presence of non-crosslinked material at the interphase. DISCUSSION
Characteristics of UV-induced crosslinking. The crosslinking of nucleic acids to proteins by irradiation with shortwave UW light has been extensively investigated. It is known that pyrimidine bases of RNA and DNA can react (19,20), and that up to eleven amino acids can be involved (21,22). The role of purines in 726
Nucleic Acids Research 8.5
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30
40
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40
Figure 6. Polyacrylamide gel electrophoretic analysis of labeled RNA from the aqueous and organic phases of phenol-chloroform extracted polyribosomes. RNA from irradiated or unirradiated polyribosomes of control or cytosine arabinoside-treated cells was fractionated by phenol-chloroform extraction as described in Figure 5. RNA recovered from the aqueous or organic phases was analyzed in 2.7% gels which were sliced into 2 mm fractions and counted. Markers of 14C-labeled rRNA were included in all gels. A) No UV aqueous. B) No UV organic. C) + UV aqueous. D) + UV organic. x--x Control. o-o Cytosine arabinoside.
crosslinking is less well understood than the role of pyrimidines. However, chemical studies have indicated that various alcohols and amines can form photo-adducts with purines (23,24). Since these groups are present in proteins, a role for purines in photoinduced crosslinking is at least a theoretical possibility (K.C. Smith, personal communication). Some studies have shown that UV-induced crosslinking characteristically has a very high degree of specificity. For example, Lin and Riggs (4) showed that the lac repressor protein can be photocrosslinked to DNA that contains the lac operator, but not to other DNA sequences. Moller and Brimacombe (5) showed that 727
Nucleic Acids Research irradiation of E. coli ribosomes results in crosslinking of principally one protein of the large ribosomal subunit and one protein of the small ribosomal subunit to their respective RNAs. Schimmel and coworkers have shown that crosslinks can be formed between E. coli tRNAs and aminoacyl tRNA synthetases, but only if cognate pairs are present. The sites of crosslinking in tRNAs have furthermore been narrowed down to three RNAase T1 oligonucleotides out of a possible fourteen (6,25). More recently, Sen and Todaro (7) have shown that in the case of avian retroviruses only one of the virion proteins, plq, can be photocrosslinked to the virion RNA. This protein appears to be a species- and typespecific RNA binding protein. Apparently, the major requirement for photoinduced crosslinking is close contact between protein and nucleic acid molecules. It may not be absolutely required that the nucleic acid and protein molecules have a high affinity for one another. However, crosslinking of stable complexes should be a first order process which is kinetically favored over crosslinking which results from random collisions. The latter type of crosslinking, if it occurs at all, would obey kinetics that are of second or higher order. The observations discussed above, which suggested that UVinduced crosslinking is both widely applicable and highly specific, encouraged us to attempt UV-induced crosslinking of mRNA to mRNP proteins. The question of specificity in crosslinking is addressed further in a separate paper in which the nature of the proteins crosslinked to mRNA is investigated (Greenberg, manuscript in preparaticn). However, it is noted here that for the most part crosslinking of proteins to mRNA does not seem to involve ribosomal proteins or rRNA, since similar levels of crosslinking could be obtained by irradiating either intact or EDTA-dissociated polyribosomes. Furthermore, 4S RNA did not appear to be crosslinked to protein, and histone mRNA behaved somewhat differently than other mRNAs. It was shown in Figure 6 that mRNA recovered from UV-crosslinked mRNP by treatment with proteinase K had an electrophoretic mobility indistinguishable from that of mRNA from non-crosslinked mRNP. Therefore, UV irradiation did not result in detectable crosslinking between RNA molecules or in detectable cleavage of phosphodiester bonds. However, irradi-
728
Nucleic Acids Research ation does produce effects other than crosslinking, such as pyrimidine dimer formation, and such side reactions may limit the efficiency of crosslinking. The efficiency of UV-induced crosslinking of mRNA to protein appears to be quite high. More than 70% of non-histone mRNA could be crosslinked to protein either by the criterion of buoyant density in Cs2So4-urea gradients or by the criterion of phenol extraction. Presumably, these methods for assaying crosslinking require that at least one protein molecule be crosslinked in order for the RNA to behave as an RNA-protein complex. At present the efficiency of crosslinking of protein to mRNA cannot be evaluated because of the inability to distinguish between labeled mRNA binding proteins and non-mRNA binding proteins present in polyribosomes. However, the efficienck of protein crosslinking may well be lower than that of mRNA crosslinking. If an mRNA molecule is associated with multiple proteins, then the probability of at least one protein molecule being crosslinked is greater than the probability of all protein molecules being crosslinked. An mRNA associated with only a few proteins would have a lower probability of being crosslinked than an mRNA associated with many proteinxs. Kinds of mRNA crosslinked to protein and stoichiometry of crosslinking. In previous work from this laboratory done without crosslinking it was shown that both poly(A)-containing mRNA and non-histone poly(A)-lacking mRNA in polyribosomes were associated with tightly bound protein. That work also showed that if there was any protein associated with histone mRNA, it must have been less stably bound than protein associated with non-histone mRNAs (9,13). In this paper it is shown that both histone and nonhistone mRNAs can be crosslinked to protein. That the nonhistone mRNA includes both poly(A)-containing and poly(A)-lacking species is demonstrated in a separate paper (Greenberg, manuscript in preparation). Histone mRNA behaved somewhat differently than other mRNAs in that the efficiency of crosslinking was lower (2540% as compared with greater than 70%), and also, the buoyant density of the histone mRNP was higher than that of most other mRNP. Both of these observations are consistent with a different stoichiometry of protein binding for histone mRNA. If, using results presented in this paper and our unpublished results, one 729
Nucleic Acids Research assumes buoyant densities of 1.68 g/cc for RNA, 1.22 g/cc for protein, and 1.53 g/cc for histone mRNP in Cs2So4-urea gradients, and one applies the formula of Perry and Kelley (26), then histone mRNP is about 26% protein. If one further assumes a molecular weight of 150,000 for histone mRNA, then histone mRNP contains about 50,000 daltons of protein, or enough protein for one or two typical polypeptides. Similarly, an mRNP particle with a buoyant density of 1.35 g/cc and an RNA content of 700,000 daltons (corresponding to about 18S) would contain about 1.3 x 106 daltons of protein and be about 65% protein by weight. This value agrees rather well with the 60% protein content calculated for EDTA-released formaldehyde-fixed mRNP banded in CsCl gradients (27). Even if these calculations are in error by a factor of two or three, the conclusion remains the same, namely, that UV-crosslinked histone mRNP contains many fewer protein molecules than most non-histone mRNP. As discussed above, an mRNA molecule with a small complement of proteins would have a lower probability of being crosslinked to at least one protein than an mRNA molecule with a large complement of proteins. Perhaps it is for this reason that histone mRNA has a lower efficiency of crosslinking than most other mRNAs. Histone mRNA is a small mRNA with distinctive metabolic properties. In cultured mammalian cells histone mRNA translation is tightly coupled to DNA synthesis (15,16,28). Furthermore, histone mRNA is one of the most rapidly labeling mRNAs (29,30). There are five major histone mRNAs composed of multiple subtypes which are transcribed from non-identical genes (31-34). They are intriguing questions whether the protein or proteins that can be crosslinked to histone mRNA is unique to histone mRNA, and whether the same protein is present on all species of histone mRNA.
ACKNOWLEDGEMENTS The author thanks Thomas J. Goralski for excellent technical assistance. This work was supported by National Cancer Institute Grant P3012708, National Science Foundation Grant PCM77-19394 and the Mimi Aaron Greenberg Fund.
730
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Greenberg, J.R. (1975). J. Cell Biol. 65:269-288. Preobrazhensky, A.A. and Spirin, A.S. (1978). Progr. Nuc. Acid Res. and Molec. Biol. 21:1-37. Smith, K.C. (1976). In Photochemistry and Photobiology-of Nucleic Acids, S.Y. Wang, ed., Academic Press, New York, Vol. 2, pp. 187-218. Lin, S-Y. and Riggs, A. (1974). Proc. Nat. Acad. Sci. USA 71:947-951. Moller, K. and Brimacombe, R. (1975). Molec. Gen. Genet. 141:343-355. Schoemaker, H. and Schimmel, P. (1974). J. Mol. Biol. 84: 503-513. Sen, A. and Todaro, G. (1977). Cell 10;91-99. Wilt, F., Anderson, M. and Ekenberg, E. (1973). Biochemistry 12:959-966. Greenberg, J.R. (1976). Biochem. 15:3516-3523. Perry, R.P., LaTorre, J., Kelley, D.E. and Greenberg, J.R. (1972). Biochim. Biophys. Acta 262:220-226. Perry, R.P. and Kelley, D.E. (1970T. J. Cell Physiol. 76: 127-139. Modak, S.P., Commelin, D., Grosset, L., Imaizumi, M.-T., Monnat, M. and Scherrer, K. (1975). Eur. J. Biochem. 60: 407-421. Greenberg, J.R. (1976). J. Mol. Biol. 108:403-416. Craig, N.C. (1971). J. Mol. Biol. 55:129-134. Borun, T.W., Scharff, M.D. and RobbTlns, E. (1967). Proc. Nat. Acad. Sci. USA 58:1977-1983. Melli, M., Spinelli, G. and Arnold, E. (1977). Cell 12:167174. Perry, R.P. and Kelley, D.E. (1973). J. Mol. Biol. 79:681696. Anderson, E., Nakashima, Y. and Konigsberg, W. (1975). Nucl. Acids Res. 2:361-371. Smith, K.C. and Aplin, R.T. (1966). Biochemistry 5:2125-2130. Smith, K.C. (1970). Biochem. Biophys. Res. Commun. 39:10111016. Smith, K.C. (1969). Biochem. Biophys. Res. Commun. 34:354357. Schott, H.N. and Shetlar, M.D. (1974). Biochem. Biophys. Res. Commun. 59:1112-1116. Salomon, J. and Elad, D. (1973). J. Org. Chem. 38:3420-3421. Salomon, J. and Elad, D. (1974). Photochem. and Photobiol. 19:21-27. Budzik, G.P., Lam, S., Schoemaker, H. and Schimmel, P. (1975). J. Biol. Chem. 250:4433-4439. Perry, R.P. and Kelley, D.E. (1966). J. Mol. Biol. 16:255268. Perry, R.P. and Kelley, D.E. (1968). J. Mol. Biol. 35:37-59. Gallwitz, D. and Mueller, G.C. (1969). J. Biol. Chem. 244: 5947-5952. Schochetman, G. and Perry, R.P. (1972). J. Mol. Biol. 63: 591-596. Adesnik, M. and Darnell, J.E. (1972). J. Mol. Biol. 67:397406.
731
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Cohen, L.H., Newrock, K.M. and Zweidler, A. (1975). Science 190:994-997. Newrock, K.M., Cohen, L.H., Hendricks, M.B., Donnelly, R.J. and Weinberg, E.S. (1978). Cell 14:327-336. Kunkel, N.S. and Weinberg, E.S. (1978). Cell 14:313-326. Schaffner, W., Kunz, G., Daetwyler, H., Telford, H., Smith, H.O. and Birnstiel, M.L. (1978). Cell 14:655-671.
Ultraviolet light-induced crosslinking of mRNA to proteins Jay R.Greenberg The Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, MA 01545, USA
Received 20 November 1978 ABSTRACT
Irradiation of intact or EDTA-dissociated L-cell polyribosomes with 254 nm UV light at doses of 1-2 x 105 ergs/mm2 extensively crosslinks mRNA to proteins. The crosslinked mRNAprotein complexes can be isolated on the basis of buoyant density in urea-containing Cs2SO4 gradients that dissociate non-covalent complexes. Crosslinking of mRNA can also be assayed by phenolchloroform extraction. mRNA recovered from the crosslinked complexes by digestion with proteinase K has the same electrophoretic mobility in polyacrylamide gels as unirradiated mRNA. Therefore, irradiation does not either crosslink RNA molecules to RNA molecules or break phosphodiester bonds. With these methods it has been found that more than 70% of high molecular weight polydisperse mRNA, but only 25-40% of histone mRNA, can be crosslinked to protein. On the basis of buoyant density the histone mRNA-protein complex had a protein content of 26%, whereas the mean protein content of most non-histone mRNAprotein complexes was 65%. It is concluded that most mRNA in polyribosomes is in close contact with proteins, and that histone mRNA can be crosslinked to many fewer proteins that most other mRNAs. INTRODUCTION The existence of mRNA binding proteins in eukaryotic cells is well known (reviewed by Greenberg (1), Preobrazhensky and Spirin (2). Although some of these proteins have been identified, their significance remains obscure, and there is still considerable difficulty in isolating and working with them. This difficulty arises from the necessity for isolating mRNA-protein complexes (mRNP) under conditions which do not allow either dissociation of native mRNP or fortuitous binding of proteins to RNA. It is usually not possible to be sure that such conditions have been met. Clearly, the study of mRNA-protein interactions would be facilitated by the ability to create specific crossC Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
715
'Nucleic Acids Research links between proteins and mRNA in native mRNP. Crosslinking would make it possible to isolate and work with mRNA-protein complexes under conditions that discourage non-specific binding of proteins to RNA. Furthermore, crosslinking itself could be used as a criterion for identifying specific mRNA-associated proteins. That is, they could be defined as proteins that can readily be crosslinked to mRNA. For lack of a suitable chemical crosslinker we explored the possibility of using photo-induced crosslinking with shortwave UV (254 nm). The ability of shortwave UV radiation to crosslink proteins to nucleic acids is amply documented (reviewed by Smith (3)). In some cases, i.e., those of the lac operator/lac repressor (4), E. coli rRNA/ribosomal proteins (5), E. coli tRNA/ aminoacyl tRNA synthetase (6), and avian retrovirus 35S RNA/pll (7) a high degree of specificity in crosslinking has been demonstrated. In this paper it is shown that most eukaryotic mRNA in polyribosomes, including histone mRNA, can be crosslinked to protein as a result of irradiating isolated polyribosomes with shortwave UV light. Furthermore, the crosslinked mRNA-protein complexes can be readily dissociated from ribosomal subunits, and the mRNA can be recovered and analyzed. However, 4S RNA in polyribosomes was not crosslinked to protein, and histone mRNP behaved differently than other mRNPs. MATERIALS AND METHODS
Materials. Cs2SO4 was obtained from Kawecki-Berylco Industries (New York, New York) and purified by the method of Wilt et al. (8). Ultrapure grade urea was obtained from Schwarz-Mann. Actinomycin D and cytosine arabinoside were purchased from Sigma. Radioactively labeled compounds were obtained from New England Nuclear or Amersham. ACS and Aquasol aqueous counting solutions were obtained from Amersham and New England Nuclear respectively. Protosol gel solubilizer was obtained from New England Nuclear. Proteinase K was obtained from EM labs. Cell culture and labeling. L-cells were grown in suspension cultures and labeled with 3H-uridine in the presence of 0.08 ig/ ml of actinomycin D as described (9). In some experiments cells were preincubated for 30 minutes in medium containing 60 ig/ml of 716
Nucleic Acids Research cytosine arabinoside as well as actinomycin D, then concentrated eight-fold and labeled in the same medium. Labeling with 3Hleucine was carried out as described for 3H-uridine, except that prior to labeling the cells were grown overnight in medium containing 5.25 pg/ml of leucine, which is 1/10 the usual concentration. The medium contained non-dialyzed 4% calf serum and 4% fetal calf serum. Polyribosome isolation. Polyribosomes were isolated as described (9). Briefly, cells were lysed in isotonic buffer containing 0.1% Triton-X-100. Polyribosomes were centrifuged in sucrose gradients containing 0.01 M NaCl, 0.01 M Tris-HCl (pH 7.6), and 0.002 M MgSO4 (RSB), then pelleted by centrifugation through 2 M sucrose in RSB. Irradiation. Pelleted polyribosomes were dissolved in RSB at a concentration of 1-2 A260 units/ml. In some experiments 10% glycerol (v/v) was present. One milliliter samples of polyribosomes were irradiated in polyethylene scintillation vial caps with a 15 watt germicidal bulb (Sylvania G15T8) equipped with an aluminum foil reflector. The radiation intensity was 2000 pW/cm 2 The intensity was measured with an Ultraviolet Products model J-225 meter. The samples were kept on ice during irradiation. The standard dose, used in all experiments reported in this paper, was 105 ergs/mm2, corresponding to about eight minutes of irradiation. Cs2SO4-urea density gradient centrifugation. Irradiated or unirradiated control polyribosomes were precipitated from ethanol. The precipitates were air dried at 37°C, then dissolved in 5 ml of 10 M urea. Water, Cs2SO4 stock solution (1.95 g/cc), 1 M Tris-HCl (pH 7.6), and 0.1 M EDTA were added so as to give a final volume of 10 ml, a Tris-HCl concentration of 0.01 M, and an EDTA concentration of 0.001 M. The final Cs2SO4 concentration was equivalent to a density of 1.35 g/cc in water. The samples were placed in screwcap polycarbonate tubes and centrifuged in a Beckman Ti75 rotor for 65 hours at 58,000 rpm at 250C. The gradients were fractionated by inserting a stainless steel tube to the bottom of the gradient and pumping out the gradient with a peristaltic pump. About 30 equal fractions were collected. The density of every fourth fraction was measured by weighing 0.1 ml. 717
Nucleic Acids Research The density was correlated with the fraction number by fitting a parabolic curve to the data by a least squares procedure with the aid of a computer. The correlation coefficient was greater than 0.995. Samples were recovered from the gradients by pressure ultrafiltering to dryness through a Millipore PTHK 25 mm filter. Residual Cs2SO4 and urea were removed by passing water through the filter. Some 4S RNA passes through the filter in this procedure. The apparatus and filter were rinsed with 0.5% diethylpyrocarbonate and water prior to use. The samples on the filters were recovered by dissolving in 2 ml of 0.5% SDS, 0.1 M NaCl, 0.01 M Tris-HCl (pH 7.6), 0.001 M EDTA (SDS buffer) containing 100 pg/ml of proteinase K. Deproteinization. Samples in SDS buffer were digested for one hour at 370C with 100 vg/ml of proteinase K. The samples were then extracted once with an equal volume of phenol-chloroform mixture (10) by vortexing briefly. The phases were separated by centrifugation for one minute at 10,000 rpm. Polyacrylamide gel electrophoresis. RNA was electrophoresed in 2.7% polyacrylamide gels crosslinked with ethylene diacrylate (9). Electrophoresis was for 2.75 hours at 5 ma/gel. The gels were frozen in hexane/dry ice and sliced into 2 mm segments with an array of razor blades. Scintillation counting. Polyacrylamide gel slices were dissolved in 3% Protosol in toluene containing PPO and POPOP. In double label experiments with 14C marker RNA 3H counts were corrected for 14C spilling into the 3H channel. Other samples were counted in ACS or Aquasol aqueous counting solutions. RESULTS
Evidence for crosslinking of mRNA to proteins. In order to demonstrate crosslinking we isolated polyribosomes from cells which had been labeled with 3H-uridine in the presence of 0.08 ig/ml of actinomycin D. These labeling conditions were chosen so that mRNA would be labeled, but labeling of rRNA would be inhibited (11). The polyribosomes were irradiated at 254 nm and centrifuged in Cs2SO4 density gradients containing 5 M urea (12) in order to resolve crosslinked RNA-protein complexes from RNA and protein. Urea was included to prevent precipitation of RNA 718
Nucleic Acids Research and protein, and also, to enhance the dissociation of noncovalent complexes. These gradients differ from the ones used in our previous work (9,13) in that they are designed to completely dissociate non-covalent mRNA-protein complexes, whereas the gradients previously employed were intended to preserve such complexes. The UV dose was varied, and it was found that a total dose of about 1-2 x 105 ergs/mm2 (8-16 minutes of irradiation at 2000 pW/cm2) gave extensive crosslinking. Representative results are shown in Figure 1. It may be seen that in the absence of irradiation all of the labeled RNA banded in a peak at about 1.65 g/cc, which corresponds to the density of pure RNA. After irradiation, however, there was much radioactivity in a peak at about 1.3-1.35 g/cc. This peak presumably corresponds to crosslinked mRNP. There was also considerable radioactivity at densities intermediate between 1.35 g/cc and 1.65 g/cc which presumably corresponds to less protein-rich complexes. Since after irradiation most of the radioactivity banded at densities lower than the density of pure RNA, and since most of the radioactivity should have been in mRNA, it was tentatively concluded that mRNA was extensively crosslinked to protein. A more quantitative analysis of crosslinking is presented below. Similar results were also obtained by irradiation of EDTA-dissociated polyribosomes (results not shown). In order to demonstrate that the putative crosslinked mRNP contained protein, polyribosomes were isolated from cells labeled with 3H-leucine in the presence of 0.08 pg/ml of actinomycin D, irradiated, and centrifuged in Cs2SO4-urea density gradients. The results are shown in Figure 2. There was a major peak of Hleucine radioactivity at 1.3-1.35 g/cc, which is considered to be the density of mRNP. There was some radioactivity tailing towards higher densities, but none at the density of RNA. There was also a smaller peak at about 1.2 g/cc. This peak corresponds to the density of pure protein. In the absence of irradiation virtually all of the protein label banded at this density (results not shown). Since after irradiation about 2/3 of the leucine label banded at the density of RNP, it is concluded that 2/3 of the label was in protein that could be crosslinked to RNA. The recovery of label from the gradients was 80%. Therefore, even 719
Nucleic Acids Research of labeled RNA 1 (left). and Banding Figureirradiated unirradiated pooly~~~~from
A Ax
20
ribosomes in Cs2SO4-urea from ents. isolatedgradiPolyribosomes were density cells labeled with 3H-uridine (10 pC/ml) for 70 minutes in the presence of 0.08 vpg/ml of actinomycin D. A portion of ~~~the polyribosomes was irradiated, and irradiated and unirradiated samples were centrifuged in Cs2SO4-urea gradients. The gradients were fractionated, and an aliquot of each fraction was counted. Unirradiated polyribosomes. B) Irfx x*A) radiated polyribosomes.
10
F 10 ! \ / \
,! z 0> o B
^\
8
1
4 x
1.8
;x./
1.4 1.6 DENSITY (G/CM3)
I.
1.2
z
H~~~~~~~~~~~~~~~~~~ c8
z 0 4
LL' // Figure 2 (right). Banding K of labeled protein from irradiated polyribosomes in oxv a Cs2SO4-urea density gradient. Polyribosomes were isolated from cells labeled 1.8 1.6 1.4 with 3H-leucine (10 pC/ml)
for 70 minutes in the pres-
\
1.2
DENSITY (G/CM3)
ence of 0.08 pg/ml of actinomycin D, irradiated, and centrifuged in a Cs2SO4-urea density gradient. The gradient was fractionated, and an aliquot of each gradient was counted.
if all of the radioactivity unaccounted for were in free protein, it is likely that at least 50% of the labeled protein was cross720
Nucleic Acids Research linked to RNA as a result of irradiation. In other similar experiments the extent of crosslinking of protein to RNA was variable, but generally somewhat lower than the value reported here, e.g. 30%. Under the labeling conditions used incorporation of newly synthesized ribosomal proteins into ribosomes is inhibited (14). However, the metabolism of polyribosomal proteins under these conditions is less well-studied than that of polyribosomal RNA, and it cannot be assumed that all of the label was present in mRNA binding proteins. Therefore, these experiments are a qualitative demonstration of UV-induced crosslinking of proteins to RNA in polyribosomes, rather than a quantitative estimation of the efficiency of crosslinking of proteins to mRNA. Nature of RNA crosslinked to protein. In order to determine whether it is feasible to recover intact mRNA for analysis from UV-crosslinked mRNP, and to gain insight into the kinds of RNA which can be crosslinked to protein, UV-crosslinked mRNP was recovered from Cs2SO4-urea density gradients, deproteinized, and analyzed by means of electrophoresis in polyacrylamide gels. Of particular concern in this experiment was whether histone mRNA can be crosslinked to protein. Previous work from this laboratory done without the use of crosslinking showed that both poly(A) +mRNA and non-histone poly(A) mRNA in polyribosomes are associated with protein (9,13). However, that work did not conclusively establish whether or not histone mRNA is associated with protein. In order to identify histone mRNA cells were labeled with 3H-uridine in the presence of 0.08 pg/ml of actinomycin D either with or without cytosine arabinoside. This drug selectively inhibits the entrance of histone mRNA into polyribosomes (15,16). Therefore, comparison of size classes of mRNA from polyribosomes of control and cytosine arabinoside-treated cells can serve to identify histone mRNA (17). Small polyribosomes (trimers through hexamers) were isolated in sucrose density gradients, UV-irradiated, and centrifuged in Cs2SO4-urea density gradients. The results are shown in Figure 3. The indicated fractions were pooled, desalted by ultrafiltration, treated with proteinase K, phenol-chloroform extracted, and analyzed in 2.7% polyacrylamide gels as shown in Figure 4. The data show that the most dense region of the gradients, corres721
Nucleic Acids Research A z 0
C
1. g5~
a. u
B
~
~
0
% ~~
5
xIO\I
1.8
1.6 1.4 DENSITY (G/CM3)
1.2
Figure 3. Banding of labeled RNA from irradiated polyribosomes of control and cytosine arabinoside-treated cells in CS2SO4-ureadensity gradients. Small polyribosomes (trimers through hexamers) were isolated from cells labeled for 70 minutes with 3H-uridine in the presence of 0.08 ig/ml of actinomycin D with or without cytosine arabinoside (60 pg/ml). The polyribosomes were irradiated and centrifuged in Cs2SO4-urea gradients, and an aliquot of each fraction was counted. The lettered horizontal bars refer to fractions pooled for further analysis. x-x Control. o-o Cytosine arabinoside.
ponding in density to protein-free RNA, consisted predominately of 4S RNA and histone mRNA. Histone mRNA was identified as the component having an apparent molecular weight of 150,000 which was present in control, but not in cytosine arabinoside-treated cells. There was also some polydisperse radioactivity of higher molecular weight present in this fraction. This was presumably non-histone mRNA not crosslinked to protein. The intermediate density region of the gradients (p = 1.45-1.57 g/cc) contained mostly polydisperse RNA but also some histone mRNA and 4S RNA. The buoyant density of this region is consistent with the presence of protein on the RNA. The low density region (p = 1.25-1.43 g/cc) contained predominantly polydisperse RNA and traces of 4S RNA. There was essentially no histone mRNA. The radioactivity in histone mRNA was measured as the difference 722
eXg4
A
3
Nucleic Acids Research
Figure
4. Polyacrylamide gel electrophoretic analysis of labeled RNA
es
28S
4
2
/ . A
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from irradiated polyribosomes banded in Cs2SO4-urea gradients. The indicated regions of the gradients shown in Figure 3 were desalted by ultrafiltration. The samples were then treated with proteinase K in the presence of SDS and extracted with phenol-chloroform. The RNA was precipitated with ethanol and electroin 2.7% polyacrylamide gels. phoresed Molecular weight markers of 14C-labeled rRNA were included some of the gels. The gels were in sliced into 2 mm fractions which were dissolved in scintillation fluid and counted. The lettered frames correspond to the lettered gradient regions in Figure 3. The crosshatched areas represent radioactivity considered to be in histone mRNA. The radioactivity in the gels is expressed in units which reflect the relative amount of radioactivity in each region of the gradi~~~ent. The sum of the units for all three gels from each gradient is 100. A) 1.60-1.75 g/cm . B) 1.45-1.58 g/cm3. C) 1.25-1.43 g/cm3. x x Control. o - o Cytosine arabinoside.
FRACTION
between control and cytosine arabinoside-treated samples in appropriate gel fractions (17). On the basis of buoyant density after UV-irradiation it appears that 25% of histone mRNA was crosslinked to protein, whereas >80% of non-histone mRNA was crosslinked to protein. It is likely that this non-histone mRNA consisted of both poly(A)-containing and poly(A)-lacking species (Greenberg, manuscript in preparation). In the experiment described above the protein-free histone mRNA and the putative histone mRNP were found in adjacent fractions of Cs2So4-urea density gradients. It could be argued that these fractions overlapped, and that quantitative separation of histone mRNA from histone mRNP was not obtained. Therefore, the extent of crosslinking of histone mRNA to protein could have been overestimated or underestimated. For this reason another method was sought to estimate the extent of crosslinking. Phenol extraction is a method previously used to quantitate UV-induced crosslinking 723
Nucleic Acids Research of nucleic acids to proteins (6,18). In this method solutions of nucleic acid, crosslinked nucleoprotein, and free protein are extracted with phenol, and the phases are subsequently separated by centrifugation. Denatured free protein and nucleoprotein precipitate at the interphase, whereas protein-free nucleic acid remains in the aqueous phase. We have used this approach to quantitate crosslinking of histone mRNA to protein. Cells were labeled with 3H-uridine in the presence of 0.08 ig/ml of actinomycin D with or without cytosine arabinoside, and total polyribosomes were isolated. A portion of the polyribosomes was UV-irradiated, and irradiated and unirradiated samples were extracted with a mixture of phenol and chloroform (10) in the presence of sodium dodecylsulfate (SDS). The phases were separated, and the organic phase plus the interphase were reextracted with SDS buffer. The resulting aqueous phases were combined, and their RNA was precipitated with ethanol. The organic phase plus the interphase were also mixed with ethanol, and the insoluble material was pelleted by centrifugation. The pellets from the aqueous and organic phases were redissolved in SDS buffer, treated with proteinase K, and reextracted with phenol-chloroform. After phase separation there was little or no visible precipitate or radioactivity at the interphase. The RNA in the aqueous phase was re-precipitated with ethanol and analyzed by means of electrophoresis in polyacrylamide gels. A flow chart of the experiment is shown in Figure 5, and the quantitative aspects of the experiment are summarized in Table 1. The gel electrophoresis results are shown in Figure 6. It may be seen that the labeled RNA from the aqueous phase of the non-TN-treated polyribosomes consisted of three principal components: high molecular weight polydisperse RNA, histone mRNA, and 4S RNA. The aqueous phase contained 85% of the total radioactivity. The RNA from the interphase plus organic phase of the non-TV-treated polyribosomes (15% of the total radioactivity) consisted of high molecular weight polydisperse RNA and 4S RNA. Histone mRNA appeared to be essentially absent. The aqueous phase of the TN-irradiated polyribosomes (40% of the total radioactivity) contained all three principal components, but 4S RNA was predominant. The organic phase plus interphase of the TN-treated polyribosomes 724
Nucleic Acids Research Cells labeled with
H-uridine in the presence of + cytosine arabinoside (60 pg/ml)
0.08pg/ml of actinomycin D
Polyribosomes |
UV (2000
pW/cm
for 8 minutes)
Phenol-chloroform extraction
Organic phase + interphase
Aqueous phase
Reextract with SDS buffer
Combined aqueous phases \
- Aqueous phase
Organic phase +
~~~~~~~~interphase
ethanol precipitation
/ ethanol precipitation
Dissolve precipitate in SDS buffer and
digest with Proteinase K
Phenol-chloroform extraction
Aqueous phase
Organic phase
(discard)
ethanol
precipitation
Analyze RNA in gels
Figure 5. Assay of crosslinking by phenol-chloroform extraction. Polyribosomes were isolated from cellslabeled for 70 minutes with 3H-uridine in the presence of 0.08 ig/ml of actinomycin D with or without 60 jg/ml of cytosine arabinoside. Portions of the polyribosomes were irradiated. All samples were made 0.5% in SDS, 0.1 M in NaCl, 0.005 M in EDTA, and 0.05 M in Tris-HCl (pH 7.6), and extracted with phenol-chloroform as indicated in this figure. Small aliquots of the aqueous phases were counted before and after extraction. The amount of radioactivity in the organic phase plus the interphase was calculated as the difference between the two measurements.
725
Nucleic Acids Research Table 1 Extent of Crosslinking as Assayed by Phenol-Chloroform Extraction Cytosine Arabinoside
Control No UV
Histone mRNA (X of total cpm)
+ UV
No UV
Aqueous phase
Organic phase
Aqueous phase
Organic phase
3.21
0.069
1.05
0.709
High molecular weight polydisperse mRNA (X of total cpm)
64.6
4S RNA (X of total cpm)
18.1
Total
85.9
11.2
2.79 14.1
19.0
18.8 38.9
57.2
3.23 61.1
+ UV
Aqueous phase
Organic phase
Aqueous phase
Organic phase
40a
ob
ob
ob
ob
75a
68.4
11.9
19.1
54.7
X Crosslinking
la,c
16.1 84.5
3.57 15.5
20.6
39.7
X Cross1inking
74a
5.62
3a.c
60.3
aCalculated as (plus UV cpm in organic phase . plus UV cpm in organic phase + plus UV cpm in aqueous phase) x 100.
bAssumed
to be nil.
cCorrected
for X of cpm in No UV organic phase.
(60% of the total radioactivity) contained high molecular weight polydisperse RNA, histone mRNA, and a relatively small amount of 4S RNA. In the case of the UV-treated polyribosomes 40% of the histone mRNA recovered was in the organic phase plus the interphase. This histone mRNA was presumably crosslinked to protein. Similarly, it is estimated that 74-75% of the high molecular weight polydisperse RNA was crosslinked to protein. The 4S RNA did not appear to be crosslinked to protein as a result of irradiation. About 15% of the 4S RNA was found at the interphase after irradiation. However, a virtually identical amount was found at the interphase in the absence of irradiation. Possibly, this was peptidyl tRNA. About 15% of high molecular weight polydisperse mRNA was found at the interphase in the absence of irradiation. Presumably, this mRNA was susceptible to crosslinking. Therefore, in the case of the + UV high molecular weight polydisperse mRNA no attempt was made to correct for the presence of non-crosslinked material at the interphase. DISCUSSION
Characteristics of UV-induced crosslinking. The crosslinking of nucleic acids to proteins by irradiation with shortwave UW light has been extensively investigated. It is known that pyrimidine bases of RNA and DNA can react (19,20), and that up to eleven amino acids can be involved (21,22). The role of purines in 726
Nucleic Acids Research 8.5
A
IA[B
.1 J {.A
6.8
28S
8S
J
~~~~~~~~0.6\A'c\/T
5.1
0.8
3A
8~~~~~
28DS2 188 4
42 4
X
Ti
1.2
0.812 / 2 0.4 10
20 FRACTION
30
40
10
20 FRACTION
30
40
Figure 6. Polyacrylamide gel electrophoretic analysis of labeled RNA from the aqueous and organic phases of phenol-chloroform extracted polyribosomes. RNA from irradiated or unirradiated polyribosomes of control or cytosine arabinoside-treated cells was fractionated by phenol-chloroform extraction as described in Figure 5. RNA recovered from the aqueous or organic phases was analyzed in 2.7% gels which were sliced into 2 mm fractions and counted. Markers of 14C-labeled rRNA were included in all gels. A) No UV aqueous. B) No UV organic. C) + UV aqueous. D) + UV organic. x--x Control. o-o Cytosine arabinoside.
crosslinking is less well understood than the role of pyrimidines. However, chemical studies have indicated that various alcohols and amines can form photo-adducts with purines (23,24). Since these groups are present in proteins, a role for purines in photoinduced crosslinking is at least a theoretical possibility (K.C. Smith, personal communication). Some studies have shown that UV-induced crosslinking characteristically has a very high degree of specificity. For example, Lin and Riggs (4) showed that the lac repressor protein can be photocrosslinked to DNA that contains the lac operator, but not to other DNA sequences. Moller and Brimacombe (5) showed that 727
Nucleic Acids Research irradiation of E. coli ribosomes results in crosslinking of principally one protein of the large ribosomal subunit and one protein of the small ribosomal subunit to their respective RNAs. Schimmel and coworkers have shown that crosslinks can be formed between E. coli tRNAs and aminoacyl tRNA synthetases, but only if cognate pairs are present. The sites of crosslinking in tRNAs have furthermore been narrowed down to three RNAase T1 oligonucleotides out of a possible fourteen (6,25). More recently, Sen and Todaro (7) have shown that in the case of avian retroviruses only one of the virion proteins, plq, can be photocrosslinked to the virion RNA. This protein appears to be a species- and typespecific RNA binding protein. Apparently, the major requirement for photoinduced crosslinking is close contact between protein and nucleic acid molecules. It may not be absolutely required that the nucleic acid and protein molecules have a high affinity for one another. However, crosslinking of stable complexes should be a first order process which is kinetically favored over crosslinking which results from random collisions. The latter type of crosslinking, if it occurs at all, would obey kinetics that are of second or higher order. The observations discussed above, which suggested that UVinduced crosslinking is both widely applicable and highly specific, encouraged us to attempt UV-induced crosslinking of mRNA to mRNP proteins. The question of specificity in crosslinking is addressed further in a separate paper in which the nature of the proteins crosslinked to mRNA is investigated (Greenberg, manuscript in preparaticn). However, it is noted here that for the most part crosslinking of proteins to mRNA does not seem to involve ribosomal proteins or rRNA, since similar levels of crosslinking could be obtained by irradiating either intact or EDTA-dissociated polyribosomes. Furthermore, 4S RNA did not appear to be crosslinked to protein, and histone mRNA behaved somewhat differently than other mRNAs. It was shown in Figure 6 that mRNA recovered from UV-crosslinked mRNP by treatment with proteinase K had an electrophoretic mobility indistinguishable from that of mRNA from non-crosslinked mRNP. Therefore, UV irradiation did not result in detectable crosslinking between RNA molecules or in detectable cleavage of phosphodiester bonds. However, irradi-
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Nucleic Acids Research ation does produce effects other than crosslinking, such as pyrimidine dimer formation, and such side reactions may limit the efficiency of crosslinking. The efficiency of UV-induced crosslinking of mRNA to protein appears to be quite high. More than 70% of non-histone mRNA could be crosslinked to protein either by the criterion of buoyant density in Cs2So4-urea gradients or by the criterion of phenol extraction. Presumably, these methods for assaying crosslinking require that at least one protein molecule be crosslinked in order for the RNA to behave as an RNA-protein complex. At present the efficiency of crosslinking of protein to mRNA cannot be evaluated because of the inability to distinguish between labeled mRNA binding proteins and non-mRNA binding proteins present in polyribosomes. However, the efficienck of protein crosslinking may well be lower than that of mRNA crosslinking. If an mRNA molecule is associated with multiple proteins, then the probability of at least one protein molecule being crosslinked is greater than the probability of all protein molecules being crosslinked. An mRNA associated with only a few proteins would have a lower probability of being crosslinked than an mRNA associated with many proteinxs. Kinds of mRNA crosslinked to protein and stoichiometry of crosslinking. In previous work from this laboratory done without crosslinking it was shown that both poly(A)-containing mRNA and non-histone poly(A)-lacking mRNA in polyribosomes were associated with tightly bound protein. That work also showed that if there was any protein associated with histone mRNA, it must have been less stably bound than protein associated with non-histone mRNAs (9,13). In this paper it is shown that both histone and nonhistone mRNAs can be crosslinked to protein. That the nonhistone mRNA includes both poly(A)-containing and poly(A)-lacking species is demonstrated in a separate paper (Greenberg, manuscript in preparation). Histone mRNA behaved somewhat differently than other mRNAs in that the efficiency of crosslinking was lower (2540% as compared with greater than 70%), and also, the buoyant density of the histone mRNP was higher than that of most other mRNP. Both of these observations are consistent with a different stoichiometry of protein binding for histone mRNA. If, using results presented in this paper and our unpublished results, one 729
Nucleic Acids Research assumes buoyant densities of 1.68 g/cc for RNA, 1.22 g/cc for protein, and 1.53 g/cc for histone mRNP in Cs2So4-urea gradients, and one applies the formula of Perry and Kelley (26), then histone mRNP is about 26% protein. If one further assumes a molecular weight of 150,000 for histone mRNA, then histone mRNP contains about 50,000 daltons of protein, or enough protein for one or two typical polypeptides. Similarly, an mRNP particle with a buoyant density of 1.35 g/cc and an RNA content of 700,000 daltons (corresponding to about 18S) would contain about 1.3 x 106 daltons of protein and be about 65% protein by weight. This value agrees rather well with the 60% protein content calculated for EDTA-released formaldehyde-fixed mRNP banded in CsCl gradients (27). Even if these calculations are in error by a factor of two or three, the conclusion remains the same, namely, that UV-crosslinked histone mRNP contains many fewer protein molecules than most non-histone mRNP. As discussed above, an mRNA molecule with a small complement of proteins would have a lower probability of being crosslinked to at least one protein than an mRNA molecule with a large complement of proteins. Perhaps it is for this reason that histone mRNA has a lower efficiency of crosslinking than most other mRNAs. Histone mRNA is a small mRNA with distinctive metabolic properties. In cultured mammalian cells histone mRNA translation is tightly coupled to DNA synthesis (15,16,28). Furthermore, histone mRNA is one of the most rapidly labeling mRNAs (29,30). There are five major histone mRNAs composed of multiple subtypes which are transcribed from non-identical genes (31-34). They are intriguing questions whether the protein or proteins that can be crosslinked to histone mRNA is unique to histone mRNA, and whether the same protein is present on all species of histone mRNA.
ACKNOWLEDGEMENTS The author thanks Thomas J. Goralski for excellent technical assistance. This work was supported by National Cancer Institute Grant P3012708, National Science Foundation Grant PCM77-19394 and the Mimi Aaron Greenberg Fund.
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