- Email: [email protected]
[5] In vitro translation of procollagen messenger RNAs
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[5] I n V i t r o T r a n s l a t i o n of P r o c o l l a g e n M e s s e n g e r R N A s By SHERRILL L. ADAMS In vitro translation is a simple, convenient assay for the identification and quantitation of messenger RNAs (mRNAs). It provides a useful tool in the analysis of collagen gene expression, particularly when used in conjunction with other techniques such as hybridization analysis. Many investigators have used in vitro translation to demonstrate differences in the amounts of (translatable) mRNAs among various cell types. In most cases, mRNA translatability correlates well with the steady-state mRNA level and with the rate of protein synthesis, suggesting that gene expression is controlled predominantly by controlling mRNA transcription and degradation. A well-characterized example of this type of regulation is the decreased type ! collagen synthesis in chicken embryo fibroblasts transformed by Rous sarcoma virus (reviewed by Adams et all). Several examples have been described, however, in which the amount of translatable collagen m R N A does not correlate either with synthesis of collagen protein2-5 or with the steady-state mRNA level, 5-9 suggesting that posttranscriptional mechanisms may also be important in the control of collagen gene expression. The use of in vitro translation as an analytical tool has become greatly simplified in recent years, due to the commercial availability of efficient in vitro translation systems. Lysate preparations from wheat germ,l° rabbit reticulocytes,it and Chinese hamster lung cells, ~2for use in translation of S. L. Adams, M. Pacifici, R. J. Focht, E. S. Allebach, and D. Boettiger, in "Collagen: Biology, Chemistry and Pathology" (R. Fleischmajer, B. R. Olsen, and K. Ktihn, eds.), pp. 202-213. New York Academy of Sciences, New York, 1985. 2 p. Tolstoshev, R. Haber, B. Trapnell, and R. G. Crystal, J. Biol. Chem. 256, 9672 (1981). 3 p. Tolstoshev, R. A. Berg, S. I. Rennard, K. H. Bradley, B. C. Trapnell, and R. G. Crystal, J. Biol. Chem. 256, 3135 (1981). 4 S. L. Adams, D. Boettiger, R. J. Focht, H. Holtzer, and M. Pacifici, Cell 30, 373 (1982). 5 E. S. Allebach, D. Boettiger, M. Pacifici, and S. L. Adams, Mol. Cell. Biol. 5, 1002 (1985). 6 R. J. Focht and S. L. Adams, Mol. Cell. Biol. 4, 1843 (1984). 7 S. A. Saxe, L. N. Lukens, and P. J. Pawlowski, J. Biol. Chem. 260, 3812 (1985). 8 M. H. Finer, L. C. Gerstenfeld, D. Young, P. Doty, and H. Boedtker, Mol. Cell. Biol. 5, 1415 (1985). 9 L. B. Rowe and R. I. Schwarz, Mol. Cell. Biol. 3, 241 (1983). 10 S. L. Adams, M. E. Sobel, B. H. Howard, K. Olden, K. M. Yamada, I, Pastan, and B. de Crombrugghe, Proc. Natl. A tad. Sci. U.S.A. 74, 3399 (1977). 1l j. M. Monson and H. M. Goodman, Biochemistry 17, 5122 (1978). 12 M. A. Haralson, this series, Vol. 82, p. 225.
METHODS IN ENZYMOLOGY.VOL. 144
Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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collagen mRNAs, have been described in detail. Since most investigators are no longer likely to prepare their own extracts for in vitro translation, we felt that a description of our small-scale adaptation of the commercially available systems might be useful to others. Described below are the procedures used in our laboratory for analysis of types I and II collagen mRNAs by translation of both purified (deproteinized) total cellular RNAs and polysome-associated RNAs. We have particularly emphasized the precautions one must take in performing these assays and in interpreting the data generated by them. The use of in vitro translation experiments in examining the roles of transcriptional and posttranscriptional events in the control of collagen gene expression will be discussed. In Vitro Translation of Collagen mRNAs General N u c l e a s e Precautions
Since the collagen mRNAs are quite large in size, it is extremely important to minimize RNA degradation due to the presence of nucleases, and to assay RNA preparations qualitatively for degradation. We have found that autoclaving is usually sufficient for disposable items such as microcentrifuge tubes and pipet tips. Glassware should be baked at 200° for at least 2 hr. Solutions may be sterilized either by autoclaving (preferably after addition of diethyl pyrocarbonate to 0.1%) or by filtering through a 0.2-/zm nitrocellulose filter in a sterile disposable filter unit. Inactivation of nucleases during cell lysis for isolation of RNAs and polysomes presents special problems and will be dealt with in the appropriate sections below. R N A Isolation
Lysis of cells for RNA isolation releases nucleases which are normally sequestered by compartmentation within the intact cell. This presents a potentially serious problem for the isolation of intact high-molecularweight RNAs such as the collagen mRNAs. This problem may be resolved by lysing the cells in the presence of reagents which will immediately inactivate the nucleases (for example, chaotropic agents such as guanidine hydrochloride). Several procedures have been described in detail for the successful isolation of translatable collagen mRNAs. 10-15 ~3 H. Boedtker, A. M. Frischauf, and H. Lehrach, Biochemistry 15, 4765 (1976), 14 D. W. Rowe, R. C. Moen, J, M. Davidson, P. H. Byers, P. Bornstein, and R. D. Palmiter, Biochemistry 17, 1581 (1978). ~5j. M. Monson, this series, Vol. 82, p. 218.
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To ensure that high-molecular-weight RNAs remain intact, we routinely analyze all RNA preparations by denaturation, followed by agarose gel electrophoresis and ethidium bromide staining; several denaturing agents may be used for this purpose? 6-~8 The ethidium bromide-stained large (28 S in mammalian species, 27 S in avian species) and small (18 S) ribosomal RNAs (rRNAs) should be present in a 2 : 1 ratio (since the 2728 S rRNA is approximately double the 18 S rRNA in size). The 45 S rRNA precursor may often be observed as well. RNA preparations which do not meet these criteria should not be used, since, if the preparation has suffered degradation of 27-28 S rRNA, the larger collagen mRNAs will also be degraded. In Vitro Translation Reactions
Two types of in vitro translation system are commercially available, lysates from rabbit reticulocytes and from wheat germ. Both have been treated with calcium-dependent micrococcal nuclease, to decrease the amount of endogenous mRNA~9; the nuclease has then been inactivated by chelation of the calcium. In addition, both are provided with a reaction mix which contains most of the reagents necessary for protein synthesis; one only needs to add the RNA of interest, a radioactive amino acid, and the appropriate amounts of magnesium acetate (MgAc2) and potassium acetate (KAc) to optimize translation of that RNA preparation. Translation of large mRNAs (such as the collagen mRNAs) is generally more efficient with the rabbit reticulocyte lysate (although wheat germ lysates have been used successfullyJ°,~2,:°). We have used the rabbit reticulocyte lysate in vitro translation systems from Bethesda Research Laboratories (BRL), New England Nuclear Corporation, Amersham Corporation, and Promega Biotec; for the last several years, all lots we have tested from these four suppliers have been quite reproducible, and quite efficient in translation of RNAs encoding high-molecular-weight proteins. Described below is the simple, small-scale procedure we use with the system from BRL; with only minor modifications, this protocol can be adapted to all the systems mentioned above. For most experiments in which the added mRNA is the major variable, a cocktail may be made which contains all the necessary ingredients ~6G. K. McMaster and G. G. Carmichael, Proc. Natl. Acad. Sci. U.S.A. U , 4835 (1977). 17 H. Lehrach, D. Diamond, J. M. Wozney, and H. Boedtker, Biochemistry 16, 4743 (1977). ~8j. M. Bailey and N. Davidson, Anal. Biochem. 70~ 75 (1976). ~9H. R. B. Pelham and R. J. Jackson, Fur. J. Biochem. 67, 247 (1976). 20 E. Vuorio, L. Sandell, D. Kravis, V. C. Sheffield, T. Vuorio, A. Dorfman, and W. B. Upholt, Nucleic Acids Res. I0, 1175 (1982).
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except the mRNA. Assembly of the cocktail and all subsequent steps prior to incubation should be performed at 4° . For each reaction, the cocktail consists of 3.75 /xl of nuclease-treated lysate, 1 /xl of 10x reaction mix (with methionine omitted), 1.25/xl of [35S]methionine (specific activity 1000-1500 Ci/mmol), the predetermined amounts of MgAc2 and KAc, and sterile water to bring the final volume to 10 /xl. [3H]Proline (specific activity 100-130 Ci/mmol) may also be used as the radioactive amino acid by drying 25/xCi under vacuum prior to addition of the remaining reagents; in this case the 10x reaction mix with proline omitted should be used. The total amount of each ingredient in the cocktail consists of the volumes specified above multiplied by a factor which is one more than the number of reactions in the experiment. The precise amounts of KAc and MgAc2 which must be added to the translation reactions should be determined by titration, using the sterile solutions provided with the system. In the reactions described above, the lysate contributes 26 mM KC1 and 1.3 mM MgCI2, and the reaction mix contributes 40 mM KCI. Translation will proceed at these salt concentrations, but probably not at an optimal rate; for translation of deproteinized RNA in our current lysate preparation, we add KAc to make the final K* concentration 105 mM and MgAc2 to make the final Mg 2+ concentration 2.3 mM. Once the optimum amounts have been determined, the additional MgAc: and KAc may be added to the cocktail. One to two micrograms of total cellular RNA (or a proportionate amount of polyadenylated RNA) is put into a sterile microfuge tube in a volume of 2/xl. The cocktail containing the lysate is then added to the RNA, and the reactions are incubated for 90 min at 30°. Protein synthesis is somewhat more efficient at 37°; however, at this temperature some proteolysis takes place, resulting in a lower net yield of radioactive protein (unpublished observations). Unless the in vitro translation products are to undergo subsequent enzymatic treatments, reactions are then terminated by addition of electrophoresis sample buffer. The addition of at least 4 volumes of sample buffer containing 2.5% sodium dodecyl sulfate (SDS) is necessary to completely solubilize the protein in the reticulocyte lysate; use of smaller volumes may result in streaking of radioactivity or aggregation of material in the stacking gel. The samples should be boiled after addition of sample buffer, since freezing of unboiled samples sometimes results in aggregation of radioactive protein. After addition of sample buffer, a 2-/xl aliquot is removed to quantitate incorporation of the radioactive amino acid into acid-insoluble material. 19 The in vitro translation products may be analyzed by any electrophoretic procedure used for analysis of in vivo synthesized collagens. We
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routinely analyze one-half of each reaction (about 50,000 cpm of acidprecipitable [35S]methionine-labeled protein) by electrophoresis on an 8% SDS polyacrylamide gel with a 4% stacking gel. 21 The gels are then enhanced by fluorography22,23 and X-ray film is exposed at - 8 0 °. [35S]Methionine-labeled preprocollagens can usually be visualized easily with an exposure of 15-24 hr, while [3H]proline-labeled preprocollagens require an exposure of approximately 7 days. Identification o f Preprocollagens One of the simplest methods for the identification of collagenous proteins is digestion with bacterial collagenase; detailed collagenase digestion protocols have been described previously both for in oivo 24 and in vitro llJ4 synthesized proteins. At the end of the in vitro translation reaction (prior to addition of sample buffer and removal of aliquots for TCA precipitation) each I0 /.d reaction to be digested receives 1 /zl of I% aprotinin (Sigma; diluted in water from the stock, for a final concentration of 0.1%), 1/xl of 62.5 mM CaCI2 (for a final concentration of 5 mM), and 1 tzl of collagenase (Advance Biofactures Form III, 2080 units/ml, for a final concentration of 2.08 units per reaction). Incubation is for 45 min at 37°. The reaction is then terminated as described above, and an aliquot is removed for TCA precipitation. The final concentration of collagenase in these reactions is 160 units/ ml, or 2.94/zg/ml. This concentration is somewhat lower than that used to digest in vivo synthesized collagens with comparable enzyme preparations; for example, Peterkofskyz4 used 6.95 ~g/ml of Advanced Biofactures collagenase for 90 min at 37°. Even with the low enzyme concentration and short digestion times we use, in the presence of a protease inhibitor, a small amount of degradation of noncollagen proteins sometimes occurs. Therefore a simple determination of the number of acidprecipitable counts before and after collagenase digestion may lead to an overestimate of the amount of preprocollagen synthesized in vitro; electrophoretic analysis is probably necessary to accurately determine the fraction of in vitro protein synthesis devoted to preprocollagens. Lanes 1 and 6 in Fig. 1 show typical in vitro protein synthesis reactions directed by total cellular RNA from tendon fibroblasts and vertebral chondroblasts, respectively; lane 3 shows the products of tendon RNA which have been digested with collagenase. Type I collagen mRNAs in the 25 U. K. Laemmli, Nature (London) 277, 680 (1970). 22 W. M. Bonner and R. A. Laskey, Eur. J. Biochern. 46, 83 (1974). 23 R. A. Laskey and A. D. Mills, Eur. J. Biochem. 56, 335 (1975). 24 B. Peterkofsky, this series, Vol. 82, p. 453.
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In Vitro TRANSLATIONOF COLLAGENmRNAs
1 2 3 4 5
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8
89
9
CP c~1 (II}
51(I) ~2(I)
a2(I)
ii!i
FIG. 1. In vitro translation reactions were performed as described in the text, with the following additions: lane 1, total cellular RNA from tendon fibroblasts; lane 2, tendon RNA plus ATA; lane 3, tendon RNA plus collagenase; lane 4, cytoplasm from skin fibroblasts; lane 5, fibroblast cytoplasm plus ATA; lane 6, total cellular RNA from vertebral chondroblasts; lane 7, chondroblast RNA plus ATA; lane 8, chondroblast cytoplasm; lane 9, chondroblast cytoplasm plus ATA. cd(1), a2(I), and al(II), the subunit of types I and II preprocollagens; CP, cartilage proteoglycan core protein.
tendon fibroblast R N A preparation are translated quite efficiently, while type II collagen mRNA is translated very inefficiently. The poor translation of type II collagen mRNA is not due to RNA degradation, since the mRNA encoding the core protein of the cartilage proteoglycan, which has a molecular weight of 340,000, 4'2° is translated quite efficiently. This attests both to the quality of the RNA preparation and to the efficiency of
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the in vitro translation reaction. The poor in vitro translational efficiency of type II collagen mRNA will be discussed further below. Another simple and commonly used method of identifying collagenous proteins among in vitro translation products involves the use of either [3H]- or [~4C]proline as the radioactive amino acid. Since the proline content of collagens is very high, these proteins are preferentially labeled. In fact, they may appear to be the only proteins synthesized in an in vitro translation reaction directed by RNA from tissues such as embryonic chicken tendon or calvaria (see Monsonl5 for an example), making identification quite straightforward. There are certain circumstances, however, in which proline labeling of preprocollagens may not be desirable. For example, in vitro translation of type II collagen mRNA is so inefficient (Fig. l, lane 6) that its [3H]proline-labeled protein product is barely detectable, even after a 7 day exposure (data not shown). Furthermore, if one wishes to estimate the fraction of in vitro translation products represented by preprocollagen, or to examine the translation of noncollagen mRNAs as well, it may be preferable to use an amino acid whose distribution is more uniform among cellular proteins. Other methods of identifying collagens among in vitro translation products include pepsin digestion, ~5 immunoprecipitation, IL14 column chromatography, tryptic peptide mapping, and amino acid sequencing. 25 All these procedures can be applied to the protocol described here. However, the needs of many investigators can probably be met by the simple procedures described above. Signal Peptide Processing by Microsomal Membranes
Since the collagens are secreted proteins which are synthesized on ribosomes associated with the endoplasmic reticulum, the amino-terminus of each collagen subunit consists of a hydrophobic signal or leader peptide. 26 Analysis of the structure and function of these signal peptides previously required isolation of pancreatic microsomal membranes for addition to the in vitro translation reactions. These membrane preparations are now available commercially, both from Amersham and New England Nuclear Corporations. Signal peptide cleavage by microsomal membranes provides a useful tool, particularly in conjunction with the translational runoff experiments described below, for analyzing subcellular compartmentation of collagen mRNAs. 25 p. N. Graves, B. R. Olsen, P. D. Fietzek, D. J. Prockop, and J. M. Monson, Eur. J. Biochem. 118, 363 (1981). 26 R. D. Palmiter, J. M. Davidson, J. Gagnon, D. W. Rowe, and P. Bornstein, J. Biol. Chem. 2~1, 1433 (1979).
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The reactions for analysis of signal peptide cleavage are nearly identical to the in vitro translation reactions described above. The volume of added RNA is reduced from 2 to 1 /A; the 8 /xl cocktail and 1 /.d of microsomal membranes are added to the appropriate reactions. The Amersham microsomal membranes are supplied in a solution containing 184 mM KAc and 1.1 mM MgAc2; thus the amounts of KAc and MgAc2 added to the cocktail may require slight adjustment for optimal synthesis. Incubation, termination, and analysis of the reactions proceed exactly as described above. Addition of the microsomal membranes to an in vitro translation reaction results in synthesis of collagens from which the signal peptides have been cleaved. Thus these products are usually slightly smaller than the normal primary translation products, and in some cases may closely resemble the sizes of the in uivo synthesized proteins. For an example, compare the unprocessed in vitro synthesized type I preprocoilagen (Fig. 1, lane 1) with the processed type I procollagen (Fig. 1, lanes 4 and 5). A precise determination of the size of the type I preprocollagens, and comparison with the procollagens synthesized in vivo, has been described by Sandell and Veis. 27 RNA Quantitation by in Vitro Translation In vitro translation has been used by many investigators to compare the amounts of collagen mRNAs among several RNA preparatiofls. 2'3'6'1°'14'28 This type of assay provides a useful adjunct to protein biosynthesis and RNA hybridization studies in determining the control mechanisms for collagen gene expression. However, such experiments should be designed very carefully and data interpreted with caution, since there are a number of potential artifacts which may be encountered. When using in vitro translation for RNA quantitation, it is important to determine that the translatability of the mRNA of interest is not altered by changes in RNA concentration. The effects of competition on mRNA translatability have been studied extensively (reviewed by Moldave29). These studies have shown that, at high RNA concentrations, translation of mRNAs which display low translation initiation efficiency (relative to other mRNAs in the population) will be inhibited. Thus when one wants to compare the amount of translatable collagen mRNA among several cell types, as is often the case, one should use subsaturating amounts of RNA 27 L. Sandell and A. Veis, Biochem. Biophys. Res. Commun. 92, 554 (1980). 2s S. Sandmeyer and P. Bornstein, J. Biol. Chem. 254, 4950 (1979). 29 K. Moldave, Annu. Rev. Biochem. 54, 1109 (1985).
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(or show that the relative translatability of the mRNA of interest does not change with R N A concentration). Thus we routinely analyze 1-2 ~g of total cellular RNA per 10/zl reaction, which is a saturating concentration, i.e., further increase in the amount of RNA does not lead to a further increase in incorporation of radioactive amino acids. However, for quantitative comparisons, we also analyze at least two subsaturating concentrations, for example 0.3 and 0.6/xg per 10/xl reaction. 6 A quantitative comparison of two different mRNAs within the same RNA population [for example, al(I) and ot2(I) collagen] by in vitro translation is more difficult than comparing the abundance of a single mRNA among several RNA populations, and in most cases probably should not be attempted. Since mRNAs have different intrinsic translational efficiencies, 29 the fact that one translation product appears to be more abundant than another may not be an indication of the relative amounts of the RNAs, but instead may be an indication of relative translational efficiencies. Furthermore, the mRNAs may be affected differently by extrinsic factors which alter translational efficiencies. For example, translation of the a2(I) collagen mRNA is much more sensitive to inhibition than the al(I) collagen mRNA, both in vivo 3° and in vitro (unpublished observations). In the presence of an inhibitory agent, the ratio of al(I) to a2(I) preprocollagen synthesized in vitro will be altered and will not represent the true ratio of the mRNAs. Thus for quantitative studies of this kind hybridization analysis is preferable if the appropriate probes are available. Even when the in vitro translation assay is used properly to compare the abundance of a single mRNA species among several RNA preparations, in vitro translation data, evaluated in the absence of corroborating protein biosynthesis and RNA hybridization data, can be misleading. There are several examples of disagreement between steady-state collagen mRNA levels, assayed by both in vitro translation and hybridization, and collagen protein biosynthesis, 2-6 implying the existence of cellular factors which alter collagen mRNA translatability. There are also examples of discordance between the abundance of collagen mRNAs and their translatability, indicating that intrinsic features of the RNA populations may alter the translatability of the type I collagen mRNAs. 5-9 A resolution of the contradictions among protein biosynthesis, in vitro translation, and hybridization data is emerging in the study of type I collagen mRNAs in chondroblasts. Hybridization data indicate that the type I collagen mRNAs in these cells are quite abundant, although very little type I collagen protein is detected either in vivo or in vitro 4-8 (also see 30 p. j. Pawlowski, Biochemistry 21, 34 (1982).
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Ill Vitro TRANSLATIONOF COLLAGEN mRNAs
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Fig 1, lane 6). Recent experiments in our laboratory indicate that the o~2(I) collagen mRNAs in chondroblasts display an altered electrophoretic mobility compared with the same mRNAs in other types of cells6; this altered mobility appears to be due to alternative processing at the 5' end of the mRNA (I. Weiss and S. L. Adams, unpublished observations). This mRNA initiates translation poorly in vivo, as indicated by its localization on small polysomes in the chondroblast cytoplasm (V. Bennett and S. Adams, unpublished observations). These results suggest that the lack of type I collagen synthesis in chondroblasts is dictated largely by posttranscriptional control mechanisms such as alternative RNA processing and translational discrimination. Thus in vitro translation data in conjunction with hybridization data can be used to determine the relative contributions of transcriptional and posttranscriptional control mechanisms in collagen gene expression. I n V i t r o T r a n s l a t i o n of P o l y s o m a l m R N A s
Cytoplasm Isolation Subcellular localization of actively translated collagen mRNAs requires the isolation of cytoplasmic or polysomal fractions with the collagen mRNAs intact. This is more difficult than the isolation of purified (deproteinized) RNAs described above, since the procedures for isolation of deproteinized RNA, which provide optimal nuclease inhibition, destroy the protein-RNA interactions which must be maintained for polysome isolation. The nature and severity of the nuclease problem vary from one cell type to another, and trial and error may be necessary to find a suitable inhibitor. Some potentially useful nuclease inhibitors are hepatin (Sigma), vanadium ribonucleoside complex (BRL), placental ribonuclease inhibitor (BRL, Promega Biotec), and rat liver supernatant. 31 The procedure described below utilizes only heparin, since the chicken embryo cells we have worked with appear to contain a nuclease activity which is refractory to other inhibitors. Many procedures for isolation of cytoplasms and polysomes describe cell lysis in the absence of detergents, or in the presence of a nonionic detergent, such as Triton X-100 or NP-40. These procedures are not adequate for analysis of mRNAs encoding secreted proteins such as collagens (or other extracellular matrix proteins such as fibronectin and proteoglycan core protein). The combination of a nonionic detergent and a 31 G. Blobel and V. R. Potter, Proc. Natl. Acad. Sci. U.S.A. 55, 1238 (1966).
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weakly ionic detergent such as sodium deoxycholate is necessary to obtain complete release ofpolysomes. Lysis in the absence of deoxycholate (with or without a nonionic detergent) results in loss of at least 90% of the collagen-synthesizing polysomes (unpublished observations). Substrate-attached cells are scraped into the medium and transferred to a 50-ml sterile polypropylene tube on ice; cells grown in suspension can be collected directly into a sterile tube. The cells are pelleted by centrifugation, the medium is removed, and the cell pellet is rinsed twice with a balanced salt solution. The cells are then lysed by addition of 20 mM TrisC1, pH 7.5, 10 mM NaC1, 3 mM MgCI2, 1 mM DTT, and 0.25 M sucrose, containing 0.2% Triton X-100, 0.05% sodium deoxycholate, and 200/zg/ ml heparin. We use 1 ml of lysis solution per 15-30 x 106 cells, which provides complete lysis while maintaining a high concentration of polysomes. The lysate is vortexed very lightly, transferred to a sterile microfuge tube, and centrifuged for 10 min at 12,000 g to pellet nuclei and mitochondria. The supernatant is then stored in small aliquots in sterile freezing vials at -146 °, where it is stable for at least 2 years.
Translation of Polysome Bound Collagen mRNAs The functional association of collagen mRNAs with polysomes may be conveniently assayed without actual isolation of polysomes. Total membrane-free cytoplasm can be analyzed in an in vitro translation reaction identical to those described above, replacing the purified RNA preparation with the cytoplasm (Fig. 1, lanes 4, 5, 8, and 9). In our experience the Mgz+ and K ÷ optima for these translational runoff reactions are somewhat different from those for purified RNAs, hence should be checked for each preparation. To ensure that the synthesis of proteins in these reactions is directed by mRNAs already associated with ribosomes (in the added cytoplasm), control reactions should be performed in the presence of an inhibitor of translation initiation such as aurintricarboxylic acid (ATA; Sigma). The addition of 75 ~M ATA in our system provides complete inhibition of translation of added purified mRNAs (Fig. I, lanes 2 and 7), but minimal inhibition of translation of polysome-bound mRNAs in a cytoplasmic extract (Fig. 1, lanes 5 and 9). Thus any translation observed in the presence of 75/~M ATA represents runoff translation of mRNAs which were already functionally associated with polysomes at the time the cells were lysed for cytoplasm preparation. This is confirmed by the fact that signal peptide cleavage has already taken place in these translational runoff experiments (Fig. 1; compare the translational runoffs in lanes 4 and 5
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with the preprocollagens in lane 1). Since cleavage presumably cannot take place in the rabbit reticulocyte lysate, because of the absence of the endoplasmic reticulum, cleavage must have taken place prior to cell lysis. Analysis of Translational Runoff Assays
There are two types of experiments for which translational runoff assays are particularly useful: cytoplasmic localization of mRNAs, and identification of translational control mechanisms. Their use in cytoplasmic localization of mRNAs is shown in Fig. I, lanes 4 and 5. In the cytoplasm of skin fibroblasts, virtually all the translatable type I collagen mRNAs are found on polysomes associated with the endoplasmic reticulure. This is indicated by the fact that all the procollagen synthesized in the translational runoff reactions has had the signal peptide removed. The use of translational runoff assays in identifying translational control mechanisms can be seen most clearly when comparing the in vitro translation products of deproteinized chondroblast RNAs with the runoff translation products of chondroblast polysomal RNAs. Type II collagen synthesis is very efficient in oioo4 and in the translational runoff (Fig. 1, lanes 8 and 9), comprising 10-20% of the protein synthesis. In contrast, type II collagen comprises less than 1% of in vitro synthesized protein (Fig. 1, lane 6). These results imply that there are cytoplasmic factors associated either with the type II collagen mRNA or with the chondroblast ribosomes which are required for collagen mRNA translation. Acknowledgments Experiments performed in this laboratory were supported by NIH Grant GM 28840 and March of Dimes Grant 1-892. I gratefully acknowledge the contributions of everyone in the laboratory, with particular thanks to Vickie Bennett for providing the data included herein and for critical reading of the manuscript. I would also like to thank Mary Lou Pardue and Richard Morimoto for sending us their translational runoff protocols.
MAJOR COMPONENTS OF THE EXTRACELLULAR MATRIX
[5]
[5] I n V i t r o T r a n s l a t i o n of P r o c o l l a g e n M e s s e n g e r R N A s By SHERRILL L. ADAMS In vitro translation is a simple, convenient assay for the identification and quantitation of messenger RNAs (mRNAs). It provides a useful tool in the analysis of collagen gene expression, particularly when used in conjunction with other techniques such as hybridization analysis. Many investigators have used in vitro translation to demonstrate differences in the amounts of (translatable) mRNAs among various cell types. In most cases, mRNA translatability correlates well with the steady-state mRNA level and with the rate of protein synthesis, suggesting that gene expression is controlled predominantly by controlling mRNA transcription and degradation. A well-characterized example of this type of regulation is the decreased type ! collagen synthesis in chicken embryo fibroblasts transformed by Rous sarcoma virus (reviewed by Adams et all). Several examples have been described, however, in which the amount of translatable collagen m R N A does not correlate either with synthesis of collagen protein2-5 or with the steady-state mRNA level, 5-9 suggesting that posttranscriptional mechanisms may also be important in the control of collagen gene expression. The use of in vitro translation as an analytical tool has become greatly simplified in recent years, due to the commercial availability of efficient in vitro translation systems. Lysate preparations from wheat germ,l° rabbit reticulocytes,it and Chinese hamster lung cells, ~2for use in translation of S. L. Adams, M. Pacifici, R. J. Focht, E. S. Allebach, and D. Boettiger, in "Collagen: Biology, Chemistry and Pathology" (R. Fleischmajer, B. R. Olsen, and K. Ktihn, eds.), pp. 202-213. New York Academy of Sciences, New York, 1985. 2 p. Tolstoshev, R. Haber, B. Trapnell, and R. G. Crystal, J. Biol. Chem. 256, 9672 (1981). 3 p. Tolstoshev, R. A. Berg, S. I. Rennard, K. H. Bradley, B. C. Trapnell, and R. G. Crystal, J. Biol. Chem. 256, 3135 (1981). 4 S. L. Adams, D. Boettiger, R. J. Focht, H. Holtzer, and M. Pacifici, Cell 30, 373 (1982). 5 E. S. Allebach, D. Boettiger, M. Pacifici, and S. L. Adams, Mol. Cell. Biol. 5, 1002 (1985). 6 R. J. Focht and S. L. Adams, Mol. Cell. Biol. 4, 1843 (1984). 7 S. A. Saxe, L. N. Lukens, and P. J. Pawlowski, J. Biol. Chem. 260, 3812 (1985). 8 M. H. Finer, L. C. Gerstenfeld, D. Young, P. Doty, and H. Boedtker, Mol. Cell. Biol. 5, 1415 (1985). 9 L. B. Rowe and R. I. Schwarz, Mol. Cell. Biol. 3, 241 (1983). 10 S. L. Adams, M. E. Sobel, B. H. Howard, K. Olden, K. M. Yamada, I, Pastan, and B. de Crombrugghe, Proc. Natl. A tad. Sci. U.S.A. 74, 3399 (1977). 1l j. M. Monson and H. M. Goodman, Biochemistry 17, 5122 (1978). 12 M. A. Haralson, this series, Vol. 82, p. 225.
METHODS IN ENZYMOLOGY.VOL. 144
Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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collagen mRNAs, have been described in detail. Since most investigators are no longer likely to prepare their own extracts for in vitro translation, we felt that a description of our small-scale adaptation of the commercially available systems might be useful to others. Described below are the procedures used in our laboratory for analysis of types I and II collagen mRNAs by translation of both purified (deproteinized) total cellular RNAs and polysome-associated RNAs. We have particularly emphasized the precautions one must take in performing these assays and in interpreting the data generated by them. The use of in vitro translation experiments in examining the roles of transcriptional and posttranscriptional events in the control of collagen gene expression will be discussed. In Vitro Translation of Collagen mRNAs General N u c l e a s e Precautions
Since the collagen mRNAs are quite large in size, it is extremely important to minimize RNA degradation due to the presence of nucleases, and to assay RNA preparations qualitatively for degradation. We have found that autoclaving is usually sufficient for disposable items such as microcentrifuge tubes and pipet tips. Glassware should be baked at 200° for at least 2 hr. Solutions may be sterilized either by autoclaving (preferably after addition of diethyl pyrocarbonate to 0.1%) or by filtering through a 0.2-/zm nitrocellulose filter in a sterile disposable filter unit. Inactivation of nucleases during cell lysis for isolation of RNAs and polysomes presents special problems and will be dealt with in the appropriate sections below. R N A Isolation
Lysis of cells for RNA isolation releases nucleases which are normally sequestered by compartmentation within the intact cell. This presents a potentially serious problem for the isolation of intact high-molecularweight RNAs such as the collagen mRNAs. This problem may be resolved by lysing the cells in the presence of reagents which will immediately inactivate the nucleases (for example, chaotropic agents such as guanidine hydrochloride). Several procedures have been described in detail for the successful isolation of translatable collagen mRNAs. 10-15 ~3 H. Boedtker, A. M. Frischauf, and H. Lehrach, Biochemistry 15, 4765 (1976), 14 D. W. Rowe, R. C. Moen, J, M. Davidson, P. H. Byers, P. Bornstein, and R. D. Palmiter, Biochemistry 17, 1581 (1978). ~5j. M. Monson, this series, Vol. 82, p. 218.
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To ensure that high-molecular-weight RNAs remain intact, we routinely analyze all RNA preparations by denaturation, followed by agarose gel electrophoresis and ethidium bromide staining; several denaturing agents may be used for this purpose? 6-~8 The ethidium bromide-stained large (28 S in mammalian species, 27 S in avian species) and small (18 S) ribosomal RNAs (rRNAs) should be present in a 2 : 1 ratio (since the 2728 S rRNA is approximately double the 18 S rRNA in size). The 45 S rRNA precursor may often be observed as well. RNA preparations which do not meet these criteria should not be used, since, if the preparation has suffered degradation of 27-28 S rRNA, the larger collagen mRNAs will also be degraded. In Vitro Translation Reactions
Two types of in vitro translation system are commercially available, lysates from rabbit reticulocytes and from wheat germ. Both have been treated with calcium-dependent micrococcal nuclease, to decrease the amount of endogenous mRNA~9; the nuclease has then been inactivated by chelation of the calcium. In addition, both are provided with a reaction mix which contains most of the reagents necessary for protein synthesis; one only needs to add the RNA of interest, a radioactive amino acid, and the appropriate amounts of magnesium acetate (MgAc2) and potassium acetate (KAc) to optimize translation of that RNA preparation. Translation of large mRNAs (such as the collagen mRNAs) is generally more efficient with the rabbit reticulocyte lysate (although wheat germ lysates have been used successfullyJ°,~2,:°). We have used the rabbit reticulocyte lysate in vitro translation systems from Bethesda Research Laboratories (BRL), New England Nuclear Corporation, Amersham Corporation, and Promega Biotec; for the last several years, all lots we have tested from these four suppliers have been quite reproducible, and quite efficient in translation of RNAs encoding high-molecular-weight proteins. Described below is the simple, small-scale procedure we use with the system from BRL; with only minor modifications, this protocol can be adapted to all the systems mentioned above. For most experiments in which the added mRNA is the major variable, a cocktail may be made which contains all the necessary ingredients ~6G. K. McMaster and G. G. Carmichael, Proc. Natl. Acad. Sci. U.S.A. U , 4835 (1977). 17 H. Lehrach, D. Diamond, J. M. Wozney, and H. Boedtker, Biochemistry 16, 4743 (1977). ~8j. M. Bailey and N. Davidson, Anal. Biochem. 70~ 75 (1976). ~9H. R. B. Pelham and R. J. Jackson, Fur. J. Biochem. 67, 247 (1976). 20 E. Vuorio, L. Sandell, D. Kravis, V. C. Sheffield, T. Vuorio, A. Dorfman, and W. B. Upholt, Nucleic Acids Res. I0, 1175 (1982).
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except the mRNA. Assembly of the cocktail and all subsequent steps prior to incubation should be performed at 4° . For each reaction, the cocktail consists of 3.75 /xl of nuclease-treated lysate, 1 /xl of 10x reaction mix (with methionine omitted), 1.25/xl of [35S]methionine (specific activity 1000-1500 Ci/mmol), the predetermined amounts of MgAc2 and KAc, and sterile water to bring the final volume to 10 /xl. [3H]Proline (specific activity 100-130 Ci/mmol) may also be used as the radioactive amino acid by drying 25/xCi under vacuum prior to addition of the remaining reagents; in this case the 10x reaction mix with proline omitted should be used. The total amount of each ingredient in the cocktail consists of the volumes specified above multiplied by a factor which is one more than the number of reactions in the experiment. The precise amounts of KAc and MgAc2 which must be added to the translation reactions should be determined by titration, using the sterile solutions provided with the system. In the reactions described above, the lysate contributes 26 mM KC1 and 1.3 mM MgCI2, and the reaction mix contributes 40 mM KCI. Translation will proceed at these salt concentrations, but probably not at an optimal rate; for translation of deproteinized RNA in our current lysate preparation, we add KAc to make the final K* concentration 105 mM and MgAc2 to make the final Mg 2+ concentration 2.3 mM. Once the optimum amounts have been determined, the additional MgAc: and KAc may be added to the cocktail. One to two micrograms of total cellular RNA (or a proportionate amount of polyadenylated RNA) is put into a sterile microfuge tube in a volume of 2/xl. The cocktail containing the lysate is then added to the RNA, and the reactions are incubated for 90 min at 30°. Protein synthesis is somewhat more efficient at 37°; however, at this temperature some proteolysis takes place, resulting in a lower net yield of radioactive protein (unpublished observations). Unless the in vitro translation products are to undergo subsequent enzymatic treatments, reactions are then terminated by addition of electrophoresis sample buffer. The addition of at least 4 volumes of sample buffer containing 2.5% sodium dodecyl sulfate (SDS) is necessary to completely solubilize the protein in the reticulocyte lysate; use of smaller volumes may result in streaking of radioactivity or aggregation of material in the stacking gel. The samples should be boiled after addition of sample buffer, since freezing of unboiled samples sometimes results in aggregation of radioactive protein. After addition of sample buffer, a 2-/xl aliquot is removed to quantitate incorporation of the radioactive amino acid into acid-insoluble material. 19 The in vitro translation products may be analyzed by any electrophoretic procedure used for analysis of in vivo synthesized collagens. We
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routinely analyze one-half of each reaction (about 50,000 cpm of acidprecipitable [35S]methionine-labeled protein) by electrophoresis on an 8% SDS polyacrylamide gel with a 4% stacking gel. 21 The gels are then enhanced by fluorography22,23 and X-ray film is exposed at - 8 0 °. [35S]Methionine-labeled preprocollagens can usually be visualized easily with an exposure of 15-24 hr, while [3H]proline-labeled preprocollagens require an exposure of approximately 7 days. Identification o f Preprocollagens One of the simplest methods for the identification of collagenous proteins is digestion with bacterial collagenase; detailed collagenase digestion protocols have been described previously both for in oivo 24 and in vitro llJ4 synthesized proteins. At the end of the in vitro translation reaction (prior to addition of sample buffer and removal of aliquots for TCA precipitation) each I0 /.d reaction to be digested receives 1 /zl of I% aprotinin (Sigma; diluted in water from the stock, for a final concentration of 0.1%), 1/xl of 62.5 mM CaCI2 (for a final concentration of 5 mM), and 1 tzl of collagenase (Advance Biofactures Form III, 2080 units/ml, for a final concentration of 2.08 units per reaction). Incubation is for 45 min at 37°. The reaction is then terminated as described above, and an aliquot is removed for TCA precipitation. The final concentration of collagenase in these reactions is 160 units/ ml, or 2.94/zg/ml. This concentration is somewhat lower than that used to digest in vivo synthesized collagens with comparable enzyme preparations; for example, Peterkofskyz4 used 6.95 ~g/ml of Advanced Biofactures collagenase for 90 min at 37°. Even with the low enzyme concentration and short digestion times we use, in the presence of a protease inhibitor, a small amount of degradation of noncollagen proteins sometimes occurs. Therefore a simple determination of the number of acidprecipitable counts before and after collagenase digestion may lead to an overestimate of the amount of preprocollagen synthesized in vitro; electrophoretic analysis is probably necessary to accurately determine the fraction of in vitro protein synthesis devoted to preprocollagens. Lanes 1 and 6 in Fig. 1 show typical in vitro protein synthesis reactions directed by total cellular RNA from tendon fibroblasts and vertebral chondroblasts, respectively; lane 3 shows the products of tendon RNA which have been digested with collagenase. Type I collagen mRNAs in the 25 U. K. Laemmli, Nature (London) 277, 680 (1970). 22 W. M. Bonner and R. A. Laskey, Eur. J. Biochern. 46, 83 (1974). 23 R. A. Laskey and A. D. Mills, Eur. J. Biochem. 56, 335 (1975). 24 B. Peterkofsky, this series, Vol. 82, p. 453.
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In Vitro TRANSLATIONOF COLLAGENmRNAs
1 2 3 4 5
67
8
89
9
CP c~1 (II}
51(I) ~2(I)
a2(I)
ii!i
FIG. 1. In vitro translation reactions were performed as described in the text, with the following additions: lane 1, total cellular RNA from tendon fibroblasts; lane 2, tendon RNA plus ATA; lane 3, tendon RNA plus collagenase; lane 4, cytoplasm from skin fibroblasts; lane 5, fibroblast cytoplasm plus ATA; lane 6, total cellular RNA from vertebral chondroblasts; lane 7, chondroblast RNA plus ATA; lane 8, chondroblast cytoplasm; lane 9, chondroblast cytoplasm plus ATA. cd(1), a2(I), and al(II), the subunit of types I and II preprocollagens; CP, cartilage proteoglycan core protein.
tendon fibroblast R N A preparation are translated quite efficiently, while type II collagen mRNA is translated very inefficiently. The poor translation of type II collagen mRNA is not due to RNA degradation, since the mRNA encoding the core protein of the cartilage proteoglycan, which has a molecular weight of 340,000, 4'2° is translated quite efficiently. This attests both to the quality of the RNA preparation and to the efficiency of
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the in vitro translation reaction. The poor in vitro translational efficiency of type II collagen mRNA will be discussed further below. Another simple and commonly used method of identifying collagenous proteins among in vitro translation products involves the use of either [3H]- or [~4C]proline as the radioactive amino acid. Since the proline content of collagens is very high, these proteins are preferentially labeled. In fact, they may appear to be the only proteins synthesized in an in vitro translation reaction directed by RNA from tissues such as embryonic chicken tendon or calvaria (see Monsonl5 for an example), making identification quite straightforward. There are certain circumstances, however, in which proline labeling of preprocollagens may not be desirable. For example, in vitro translation of type II collagen mRNA is so inefficient (Fig. l, lane 6) that its [3H]proline-labeled protein product is barely detectable, even after a 7 day exposure (data not shown). Furthermore, if one wishes to estimate the fraction of in vitro translation products represented by preprocollagen, or to examine the translation of noncollagen mRNAs as well, it may be preferable to use an amino acid whose distribution is more uniform among cellular proteins. Other methods of identifying collagens among in vitro translation products include pepsin digestion, ~5 immunoprecipitation, IL14 column chromatography, tryptic peptide mapping, and amino acid sequencing. 25 All these procedures can be applied to the protocol described here. However, the needs of many investigators can probably be met by the simple procedures described above. Signal Peptide Processing by Microsomal Membranes
Since the collagens are secreted proteins which are synthesized on ribosomes associated with the endoplasmic reticulum, the amino-terminus of each collagen subunit consists of a hydrophobic signal or leader peptide. 26 Analysis of the structure and function of these signal peptides previously required isolation of pancreatic microsomal membranes for addition to the in vitro translation reactions. These membrane preparations are now available commercially, both from Amersham and New England Nuclear Corporations. Signal peptide cleavage by microsomal membranes provides a useful tool, particularly in conjunction with the translational runoff experiments described below, for analyzing subcellular compartmentation of collagen mRNAs. 25 p. N. Graves, B. R. Olsen, P. D. Fietzek, D. J. Prockop, and J. M. Monson, Eur. J. Biochem. 118, 363 (1981). 26 R. D. Palmiter, J. M. Davidson, J. Gagnon, D. W. Rowe, and P. Bornstein, J. Biol. Chem. 2~1, 1433 (1979).
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The reactions for analysis of signal peptide cleavage are nearly identical to the in vitro translation reactions described above. The volume of added RNA is reduced from 2 to 1 /A; the 8 /xl cocktail and 1 /.d of microsomal membranes are added to the appropriate reactions. The Amersham microsomal membranes are supplied in a solution containing 184 mM KAc and 1.1 mM MgAc2; thus the amounts of KAc and MgAc2 added to the cocktail may require slight adjustment for optimal synthesis. Incubation, termination, and analysis of the reactions proceed exactly as described above. Addition of the microsomal membranes to an in vitro translation reaction results in synthesis of collagens from which the signal peptides have been cleaved. Thus these products are usually slightly smaller than the normal primary translation products, and in some cases may closely resemble the sizes of the in uivo synthesized proteins. For an example, compare the unprocessed in vitro synthesized type I preprocoilagen (Fig. 1, lane 1) with the processed type I procollagen (Fig. 1, lanes 4 and 5). A precise determination of the size of the type I preprocollagens, and comparison with the procollagens synthesized in vivo, has been described by Sandell and Veis. 27 RNA Quantitation by in Vitro Translation In vitro translation has been used by many investigators to compare the amounts of collagen mRNAs among several RNA preparatiofls. 2'3'6'1°'14'28 This type of assay provides a useful adjunct to protein biosynthesis and RNA hybridization studies in determining the control mechanisms for collagen gene expression. However, such experiments should be designed very carefully and data interpreted with caution, since there are a number of potential artifacts which may be encountered. When using in vitro translation for RNA quantitation, it is important to determine that the translatability of the mRNA of interest is not altered by changes in RNA concentration. The effects of competition on mRNA translatability have been studied extensively (reviewed by Moldave29). These studies have shown that, at high RNA concentrations, translation of mRNAs which display low translation initiation efficiency (relative to other mRNAs in the population) will be inhibited. Thus when one wants to compare the amount of translatable collagen mRNA among several cell types, as is often the case, one should use subsaturating amounts of RNA 27 L. Sandell and A. Veis, Biochem. Biophys. Res. Commun. 92, 554 (1980). 2s S. Sandmeyer and P. Bornstein, J. Biol. Chem. 254, 4950 (1979). 29 K. Moldave, Annu. Rev. Biochem. 54, 1109 (1985).
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(or show that the relative translatability of the mRNA of interest does not change with R N A concentration). Thus we routinely analyze 1-2 ~g of total cellular RNA per 10/zl reaction, which is a saturating concentration, i.e., further increase in the amount of RNA does not lead to a further increase in incorporation of radioactive amino acids. However, for quantitative comparisons, we also analyze at least two subsaturating concentrations, for example 0.3 and 0.6/xg per 10/xl reaction. 6 A quantitative comparison of two different mRNAs within the same RNA population [for example, al(I) and ot2(I) collagen] by in vitro translation is more difficult than comparing the abundance of a single mRNA among several RNA populations, and in most cases probably should not be attempted. Since mRNAs have different intrinsic translational efficiencies, 29 the fact that one translation product appears to be more abundant than another may not be an indication of the relative amounts of the RNAs, but instead may be an indication of relative translational efficiencies. Furthermore, the mRNAs may be affected differently by extrinsic factors which alter translational efficiencies. For example, translation of the a2(I) collagen mRNA is much more sensitive to inhibition than the al(I) collagen mRNA, both in vivo 3° and in vitro (unpublished observations). In the presence of an inhibitory agent, the ratio of al(I) to a2(I) preprocollagen synthesized in vitro will be altered and will not represent the true ratio of the mRNAs. Thus for quantitative studies of this kind hybridization analysis is preferable if the appropriate probes are available. Even when the in vitro translation assay is used properly to compare the abundance of a single mRNA species among several RNA preparations, in vitro translation data, evaluated in the absence of corroborating protein biosynthesis and RNA hybridization data, can be misleading. There are several examples of disagreement between steady-state collagen mRNA levels, assayed by both in vitro translation and hybridization, and collagen protein biosynthesis, 2-6 implying the existence of cellular factors which alter collagen mRNA translatability. There are also examples of discordance between the abundance of collagen mRNAs and their translatability, indicating that intrinsic features of the RNA populations may alter the translatability of the type I collagen mRNAs. 5-9 A resolution of the contradictions among protein biosynthesis, in vitro translation, and hybridization data is emerging in the study of type I collagen mRNAs in chondroblasts. Hybridization data indicate that the type I collagen mRNAs in these cells are quite abundant, although very little type I collagen protein is detected either in vivo or in vitro 4-8 (also see 30 p. j. Pawlowski, Biochemistry 21, 34 (1982).
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93
Fig 1, lane 6). Recent experiments in our laboratory indicate that the o~2(I) collagen mRNAs in chondroblasts display an altered electrophoretic mobility compared with the same mRNAs in other types of cells6; this altered mobility appears to be due to alternative processing at the 5' end of the mRNA (I. Weiss and S. L. Adams, unpublished observations). This mRNA initiates translation poorly in vivo, as indicated by its localization on small polysomes in the chondroblast cytoplasm (V. Bennett and S. Adams, unpublished observations). These results suggest that the lack of type I collagen synthesis in chondroblasts is dictated largely by posttranscriptional control mechanisms such as alternative RNA processing and translational discrimination. Thus in vitro translation data in conjunction with hybridization data can be used to determine the relative contributions of transcriptional and posttranscriptional control mechanisms in collagen gene expression. I n V i t r o T r a n s l a t i o n of P o l y s o m a l m R N A s
Cytoplasm Isolation Subcellular localization of actively translated collagen mRNAs requires the isolation of cytoplasmic or polysomal fractions with the collagen mRNAs intact. This is more difficult than the isolation of purified (deproteinized) RNAs described above, since the procedures for isolation of deproteinized RNA, which provide optimal nuclease inhibition, destroy the protein-RNA interactions which must be maintained for polysome isolation. The nature and severity of the nuclease problem vary from one cell type to another, and trial and error may be necessary to find a suitable inhibitor. Some potentially useful nuclease inhibitors are hepatin (Sigma), vanadium ribonucleoside complex (BRL), placental ribonuclease inhibitor (BRL, Promega Biotec), and rat liver supernatant. 31 The procedure described below utilizes only heparin, since the chicken embryo cells we have worked with appear to contain a nuclease activity which is refractory to other inhibitors. Many procedures for isolation of cytoplasms and polysomes describe cell lysis in the absence of detergents, or in the presence of a nonionic detergent, such as Triton X-100 or NP-40. These procedures are not adequate for analysis of mRNAs encoding secreted proteins such as collagens (or other extracellular matrix proteins such as fibronectin and proteoglycan core protein). The combination of a nonionic detergent and a 31 G. Blobel and V. R. Potter, Proc. Natl. Acad. Sci. U.S.A. 55, 1238 (1966).
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weakly ionic detergent such as sodium deoxycholate is necessary to obtain complete release ofpolysomes. Lysis in the absence of deoxycholate (with or without a nonionic detergent) results in loss of at least 90% of the collagen-synthesizing polysomes (unpublished observations). Substrate-attached cells are scraped into the medium and transferred to a 50-ml sterile polypropylene tube on ice; cells grown in suspension can be collected directly into a sterile tube. The cells are pelleted by centrifugation, the medium is removed, and the cell pellet is rinsed twice with a balanced salt solution. The cells are then lysed by addition of 20 mM TrisC1, pH 7.5, 10 mM NaC1, 3 mM MgCI2, 1 mM DTT, and 0.25 M sucrose, containing 0.2% Triton X-100, 0.05% sodium deoxycholate, and 200/zg/ ml heparin. We use 1 ml of lysis solution per 15-30 x 106 cells, which provides complete lysis while maintaining a high concentration of polysomes. The lysate is vortexed very lightly, transferred to a sterile microfuge tube, and centrifuged for 10 min at 12,000 g to pellet nuclei and mitochondria. The supernatant is then stored in small aliquots in sterile freezing vials at -146 °, where it is stable for at least 2 years.
Translation of Polysome Bound Collagen mRNAs The functional association of collagen mRNAs with polysomes may be conveniently assayed without actual isolation of polysomes. Total membrane-free cytoplasm can be analyzed in an in vitro translation reaction identical to those described above, replacing the purified RNA preparation with the cytoplasm (Fig. 1, lanes 4, 5, 8, and 9). In our experience the Mgz+ and K ÷ optima for these translational runoff reactions are somewhat different from those for purified RNAs, hence should be checked for each preparation. To ensure that the synthesis of proteins in these reactions is directed by mRNAs already associated with ribosomes (in the added cytoplasm), control reactions should be performed in the presence of an inhibitor of translation initiation such as aurintricarboxylic acid (ATA; Sigma). The addition of 75 ~M ATA in our system provides complete inhibition of translation of added purified mRNAs (Fig. I, lanes 2 and 7), but minimal inhibition of translation of polysome-bound mRNAs in a cytoplasmic extract (Fig. 1, lanes 5 and 9). Thus any translation observed in the presence of 75/~M ATA represents runoff translation of mRNAs which were already functionally associated with polysomes at the time the cells were lysed for cytoplasm preparation. This is confirmed by the fact that signal peptide cleavage has already taken place in these translational runoff experiments (Fig. 1; compare the translational runoffs in lanes 4 and 5
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with the preprocollagens in lane 1). Since cleavage presumably cannot take place in the rabbit reticulocyte lysate, because of the absence of the endoplasmic reticulum, cleavage must have taken place prior to cell lysis. Analysis of Translational Runoff Assays
There are two types of experiments for which translational runoff assays are particularly useful: cytoplasmic localization of mRNAs, and identification of translational control mechanisms. Their use in cytoplasmic localization of mRNAs is shown in Fig. I, lanes 4 and 5. In the cytoplasm of skin fibroblasts, virtually all the translatable type I collagen mRNAs are found on polysomes associated with the endoplasmic reticulure. This is indicated by the fact that all the procollagen synthesized in the translational runoff reactions has had the signal peptide removed. The use of translational runoff assays in identifying translational control mechanisms can be seen most clearly when comparing the in vitro translation products of deproteinized chondroblast RNAs with the runoff translation products of chondroblast polysomal RNAs. Type II collagen synthesis is very efficient in oioo4 and in the translational runoff (Fig. 1, lanes 8 and 9), comprising 10-20% of the protein synthesis. In contrast, type II collagen comprises less than 1% of in vitro synthesized protein (Fig. 1, lane 6). These results imply that there are cytoplasmic factors associated either with the type II collagen mRNA or with the chondroblast ribosomes which are required for collagen mRNA translation. Acknowledgments Experiments performed in this laboratory were supported by NIH Grant GM 28840 and March of Dimes Grant 1-892. I gratefully acknowledge the contributions of everyone in the laboratory, with particular thanks to Vickie Bennett for providing the data included herein and for critical reading of the manuscript. I would also like to thank Mary Lou Pardue and Richard Morimoto for sending us their translational runoff protocols.