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Substructure of Nuclear Ribonucleoprotein Complexes T. MARTIN, P. BILLINGS, J. PULLMAN, B. STEVENS, AND A. KINNIBURGH* Department of Biology, University of Chicago, Chicago, Illinois 60637
primarily of two to four distinct species, all having molecular weights in the range 35,000-40,000 (Martin et al. 1974; Billings and Martin 1977; see also LeStourgeon et al., this volume). These polypeptides appear to have been conserved in size and amino acid composition during the evolution ofeukaryotes (Martin et al. 1974). Most of our studies, including all of the experiments described in this paper, have employed the Taper hepatoma of mouse in the ascites form as a matter of convenience. The rapidity and extent of binding of newly synthesized RNA into a 30S subcomplex form suggest that the specific proteins associate with and fold the RNA chain while still in nascent form. The bulk of the bound RNA turns over with the half-life characteristic of hnRNA (Martin and McCarthy 1972), but the nuclear 30S RNP structures also contain the majority (and probably all) of sequences represented in cytoplasmic mRNA (Kinniburgh and Martin 1976a). We therefore infer that the simple set of polypeptides present in the 30S complex act as generalized "folding proteins" for most nascent RNA and aid in the formation of the native template for processing. I f the polypeptide components are to fold RNA effectively into distinctive unit structures, we may expect them to have definable protein-protein interactions. Furthermore, if the RNA-protein complex formed is to act as a substrate for specific processing events, then some hierarchy of binding affinity of specific types of nuclear RNA sequence for the prorein complex would seem likely. Certain regions of the large mRNA molecules should be preferentially bound or exposed by the protein complex if it is to participate in regulating the specificity of processing events. The presence of nuclear poly(A) in the form of a 15S subcomplex, very different in size and in polypeptide composition to the 30S particle (Quinlan et ai. 1974, 1977), yields an obvious example of the in vivo specificity of nuclear RNA-protein interactions. More relevant to hnRNA processing, however, would be the definition of interactions of nuclear proteins with specific internal regions of hnRNA such as oligo(U), oligo(A), and double-stranded (ds) RNA sequences. We have begun to approach this problem with reference to the 30S RNP (Kinniburgh et al. 1976), and the following experiments represent further attempts to understand the actual and potential protein-protein and RNA-protein interactions in this subunit of the hnRNP complex.
Nascent heterogeneous nuclear RNA (hnRNA) synthesized in regions of active chromatin rapidly becomes associated with protein; the presence of protein on the growing RNA chains has been visualized in spread chromatin by electron microscopy (EM) (see, e.g., Miller and Bakken 1972; Malcolm and Sommerville 1974; Laird et al. 1976; McKnight et al., this volume). It is reasonable to presume that this protein folds the lengthening RNA chains, facilitates their removal from the template, and later participates in the processing and maturation events which lead to the turnover of nucleus-restricted sequences while mature mRNA molecules are transported to the cytoplasm. There have been very extensive studies on the pathway of nuclear RNA molecules from synthesis to the appearance of mRNA on cytoplasmic polyribosomes. It is fair to say that, at present, the wealth of information produced promises rather than achieves a satisfying description of this complex process. These studies have centered on the characteristics of purified RNA molecules. Far fewer attempts have been made to examine the native forms of nuclear RNA molecules undergoing processing, i.e., as ribonucleoprotein (RNP) complexes, which are presumably the true substrates for the processing enzymes.
Questions of Structure and Function of hnRNP We have been concerned for a number of years with extending and refining the observations of Georgiev and his colleagues that rapidly labeled hnRNA could be extracted from nuclei of mammalian cells in the form of RNP complexes, thus enabling a biochemical analysis of the structures (Samarina et al. 1966). The view that has emerged from these studies is that the bulk of newly synthesized RNA in the eukaryotic nucleus consists of large molecules folded by protein to form a chain of linked RNP substructures which, when cleaved by endogenous or exogenous nuclease, yield 30S particles (Martin and McCarthy 1972; Kinniburgh et al. 1976). Such a model was originally proposed by Samarina et al. (1968). The protein responsible for maintaining the 30S RNP substructure of hnRNA has a very simple polypeptide composition consisting * Present address: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706.
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MARTIN ET AL. Morphology of Nuclear 30S and 15S RNP
Nascent RNP fibers observed by electron microscopy of spread chromatin have shown varying proportions of beaded and folded regions, presumably dependent upon the conditions employed (Miller and Bakken 1972; Malcolm and Sommerville 1974; Laird et al. 1976; McKnight et al., this volume). The micrographs imply a high degree of particulate character in the fibers, although admittedly it has not been as apparent in this case as in electron microscope studies of polyribosomes or, more recently, of the nucleosome form of chromatin. In regions not confused by secondary folding, the fibers have generally been assigned a diameter of approximately 200/k. We previously reported a diameter of 150-250 ]k for negatively stained 30S RNP particles from mouse ascites cells (Martin et al. 1974), and recently we have begun a detailed electron microscope study of nuclear RNP complexes that can be sufficiently purified to justify such analysis. One fact emerges immediately from attempts to observe unfixed 30S particles and that is the far greater lability of these structures when compared with ribosomes or ribosomal subunits. In the absence of fixative, 30S particles appear as amorphous or "unfolded" structures (results not shown). When fixed with glutaraldehyde and negatively stained with uranyl acetate, the particulate character of 30S RNP preparations is readily apparent (Fig. 1A,B). Under these conditions the majority of 30S RNP appear as regular compact structures of varying morphology but relatively consistent size, 235 • 195/k average dimensions. As expected, the morphology of the 15S poly(A)-containing RNP subcomplex is distinctly different. After glutaraldehyde fixation and negative staining, these particles appear as ellipsoids with dimensions of 165 • 120/k (Fig. 1C). The 15S RNP are more difficult to purify than 30S complexes, and the preparation also shows the presence of elongated and crescent structures as well as 100-/k rings and "stacked disks" from the adjacent 17S peak (see Quinlan et al. 1974). Shadowed 30S particles (prepared by freeze-drying) appear somewhat larger; our results for rotary shadowing with platinum give values of 250 • 195 /~, whereas those for unidirectional shadowing are 280 • 250 • 215 ,k (results not shown). The latter estimates indicate that the 30S RNP are slightly flattened structures, though this may arise from distortion of a relatively flexible particle in binding to the EM grid film. The generally larger size values obtained by shadowing compared with negative staining techniques are consistent with results from other systems, notably for ribosomes (Huxley and Zubay 1960; Vasiliev 1971). We have begun to catalog the forms apparent in the negatively stained preparations of 30S RNP. Among the various shapes observed, the most clearly defined were round, ellipseidal, rectangular, and triangular (Fig. 2a-e). The similarity among the mere-
bers of each category, both in negatively stained and in shadowed preparations, suggests a limited range of potential morphological forms. The particles which do not fit into these categories appear as intermediate forms, e.g., rectangles with rounded corners. It is unlikely that all the profiles represent different projections of a single three-dimensional configuration; the shadows cast by particles of ditferent morphologies cannot be rationalized on the basis of a single model, but rather suggest a limited diversity of shapes. If, in fact, these structures are subsections of the nascent RNA fibers, a variety of configurations would be consistent with the somewhat irregular folding apparent in electron micrographs of spread chromatin (see, e.g., Laird et al. 1976). Interactions between the polypeptides of the complexes and RNA sequences having distinctive properties could yield a set of conformational isomers. It is not surprising that RNP complexes of such a dynamic metabolic character are less rigid and consistent structures than ribosomes, for example. In attempting to understand the internal structure of these nuclear particles, it may be of value to carry out a more detailed analysis of the unfolded forms of 30S RNP apparent in negatively stained particles (Fig. 2f). We have not yet completed such a study, which may be expected to contribute to a decision between the models of 30S RNP either as consisting of RNA coiled on a protein core or as the result of a regular folding of an RNP fibril. These latter alternatives arise from the investigation of protein-protein and protein-RNA interactions such as described in the following sections. Protein-Protein Interactions and the Integrity of 30S RNP
Models have been constructed in which the native hnRNP structure has been interpreted as either particulate or as folded fibril. In the first model (Samarina et al. 1968; Lukanidin et al. 1972b), nascent hnRNA is thought to complex with surface binding sites on a number of stable globular protein complexes Cinformofers"). Cleavage between particles would yield individual informofers, each carrying a segment of RNA, and would thus explain the nature and origin of 30S RNP (Fig. 3a). An alternative model implies that smaller protein complexes bind to the nascent RNA, forming an RNP fibril which is subsequently periodically folded into a more compact structure (Stevenin and Jacob 1974) and possibly maintained in this conformation by protein-protein interactions of varying degrees of stability (Fig. 3b). These various alternatives for the organization of nascent RNP fibers have been discussed by Malcolm and Sommerville (1974) in the context of amphibian lampbrush-loop fine structure. In considering the interactions that maintain the integrity of the 30S RNP subcomplex, several observations tend to diminish the importance of the RNA
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Figure 1. Electron micrographs of fields of 30S and 15S RNP particles from mouse ascites cells (Quinlan et al. 1974). Specimens were fixed with 0.5% glutaraldehyde and negatively stained with 1% uranyl acetate. (.4)Crude 30S preparation; (B) purified 30S preparation; (C) 15S preparation. Lamellar structures and disks with central holes (arrows) are contaminants from the 17S peak. Scale bars represent 500/k. 901
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MARTIN ET AL.
Figure 2. Gallery showing the different projections of negatively stained 30S particles that were observed: (a) Triangular forms; (b) rectangular and square forms; (c) circular forms; (d) small circular forms (found mainly in material from the light shoulder of the 30S peak); (e) ellipsoidal forms; (f) disintegrating forms. component. First, a large part of the R N A is relatively sensitive to RNase (see below). This suggests, at least operationally, a surface location of the nucleic acid. At moderate salt concentrations, 30S R N P proteins aggregate on treatment with nuclease, implying a considerable tendency for protein-protein interactions. Second, the 30S R N P contains approxi-
RNase Sensitive Sites
.
(o) Protein Core
(b) Folded Fibril
Figure 3. Two distinct models for the structure of the 30S RNP subcomplex of hnRNP: (a)RNA coiled on the surface of a protein core (the informofer of Samarina et al. [1968]); (b) condensed or folded region of a continuous RNP fibril. These alternativeforms have also been discussed by Malcolm and Sommerville (1974).
mately 1000 nucleotides of RNA (Samarina et al. 1968; Martin et al. 1974); however, whether this is relatively intact or internally nicked to 50-100-nucleotide segments does not affect the integrity of 30S RNP at physiological salt concentrations (Kinniburgh and Martin 1976a). Thus, given the lack of a requirement for RNA continuity within the particle, our attention is focused on the protein component. The concept of a reutilizable protein core complex (the informofer of Samarina et al. [1968]) is an attractive one and resembles in many ways the more recently evolved model for the nucleosome histone core in chromatin structure (see, e.g., Kornberg 1974; other papers, this volume). Major support for this model of the formation of hnRNP, however, comes from the observation that chemically radioiodinated 30S RNP protein cores, stripped of RNA and maintained in solution by high concentrations of salt, continue to sediment in approximately the same position in sucrose gradients as native 30S RNP (Lukanidin et al. 1972b). The stability of these cores suggests that the protein complexes could be reutilized intact in the nucleus.
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SUBSTRUCTURE OF NUCLEAR RNP COMPLEXES In our first experiments on the salt stability of 30S RNP, we employed the lactoperoxidase-catalyzed radioiodination technique of Marchalonis (1969) to monitor the fate of the protein moiety. The 30S RNP were iodinated either directly after extraction from the nuclei (crude 30S RNP) or after resuspending pelleted RNP recovered from the 30S region of a first gradient (purified 30S RNP). RNP particles were then centrifuged on a sucrose gradient as usual to separate them from enzyme and free iodine. Portions of such 30S RNP were adjusted to a final salt concentration varying from 0.1 M to 2 M NaC1 before analysis on sucrose gradients containing the same salt concentration. We found that iodinated 30S RNP-protein was recovered from such gradients in approximately the 30S region as reported earlier by Lukanidin et al. (1972b), indicating that under these conditions the protein moiety of 30S RNP remained intact even in salt concentrations as high as 2 M NaC1. (An iodinated 30S RNP gradient profile in which the sucrose gradient contained 0.5 M NaC1 is shown in the top panel of Fig. 4.) Parallel experiments with uridine- or adenosine-labeled 30S RNP demonstrated that RNA was progressively lost from the 30S region as the salt concentration was raised above 0.2 M NaC1. The RNA displaced by high salt was recovered in a very slowly sedimenting peak at the top of the gradient (see top panel in Fig. 5). Several observations, however, were somewhat inconsistent with this salt-resistant protein core model. First, the degree of stability as indicated by radioactivity in iodinated crude 30S RNP was invariably considerably less than that of iodinated purified particles. This was originally interpreted as reflecting a variable recovery of some minor, high-molecular-weight, cosedimenting or loosely bound proteins, perhaps more accessible to the enzyme, but which are largely removed in pelleting and repurifying 30S RNP (Martin et al. 1974). However, SDS-polyacrylamide gel analysis of the unstable protein displaced from the 30S region on high-salt sucrose gradients indicated that it was representative of the total protein composition of such 30S RNP and not enriched in high-molecular-weight species. Furthermore, the specific activity of the dissociated protein was not notably higher than that of the corresponding protein remaining with stable particles. Second, attempts to isolate RNA-free protein cores labeled in vivo with [3H]leucine clearly indicated that the bulk of the protein was easily dissociated in 0.5 M NaC1. These results suggested that iodination of the protein somehow rendered the protein complex stable to high salt. We have now monitored the distribution of 30S RNP polypeptides across high-salt gradients by SDSacrylamide gel electrophoresis for beth iodinated and noniodinated preparations (Figs. 4 and 5). The results verify the conclusions of the [SH]leucinelabeling experiments, namely, that noniodinated RNPs do not contain a salt-stable core, but rather
903
that, in high salt, the 35,000-40,000-dalton proteins are released to the top of the gradient coordinately with [SH]uridine-labeled RNA (Fig. 5). In contrast, 30S RNP iodinated by the lactoperoxidase procedure does contain a salt-stable protein complex. However, most of the iodinated protein fails to enter the separation gel; instead it is trapped
Figure 4. Iodinated 30S RNP centrifuged on sucrose gradients containing 0.5 M NaCI. Pelleted 30S RNPs were resuspended in 1 ml of STM, pH 8 (Martin and McCarthy 1972), and iodinated on ice with carrier-free [125I]NaIby the lactoperoxidase-H202 procedure of Marchalonis (1969). The iodination reaction was terminated after 1 hr with 2-mercaptoethanol, and the suspension was adjusted to 0.5 M NaC1 and recentrifuged on 15-30% sucrose gradients containing 0.5 M NaCl (SW 27 rotor, 25,000 rpm for 15 hr). The gradient was collected in 36 fractions. (Toppanel) Acid-insoluble radioactivity of 20-/~1 aliquots of each fraction is shown (~-----~)superimposed on the A2,4absorption profile. Remaining fractions were pooled in 3-ml portions and TCA was added to 7%. The precipitates were collected by centrifugation, washed with ethanol, and solubilized by boiling for 10 rain in buffered 2% SDS containing 8 M urea and 0.1 M dithiothreitel. Equal portions of each sample across the gradient were analyzed on 10% pelyacrylamide SDS gels (shown in the lower panel beneath the equivalent pooled fractions). The 5% polyacrylamide stacker gel at the top is retained to demonstrate the crosslinked protein not entering the separation gel. Molecularweight marker proteins in the first and last slots are (top to bottom) ~-galactosidase (130,000); phosphorylase a (92,500); serum albumin (68,000); ovalbumin (43,000); glyceraldehyde-3-phosphate dehydrogenase (36,000); and globin (15,500). Lactoperoxidase appears in pooled fraction 2 as a polypeptide doublet of molecular weight ~78,000.
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MARTIN ET AL.
Figure 5. RNA-labeled 30S RNP centrifuged on sucrose gradients containing 0.5 M NaC1. Mouse ascites cells were pulse-labeled for 30 rain in vitro with 15 pCi/ml [3H]uridine (Quinlan et al. 1974). Nuclear extracts were made and centrifuged on sucrose gradients in STM, pH 8 (Martin et al. 1974). The 30S RNPs recovered were pelleted overnight and resuspended in STM, pH 8. After the final salt concentration was adjusted to 0.5 MNaC1, the particle suspension was recentrifuged on sucrose gradients containing 0.5 M NaC1 as in Fig. 4. (~----$) One-milliliter fractions were recovered and 0.1-ml aliquots were assayed for acid precipitable radioactivity; ( ) UV absorption at 254 nm. The distribution of protein across the gradient is shown in the lower panel. Remaining fractions were pooled (1-3, 4-6, etc.), precipitated on ice with TCA, and dissolved in 2% SDS buffer. Equal volumes were assayed on SDS-polyacrylamide gels with molecular-weight markers in the first and last slots as described in Fig. 4.
in the stacking gel or at the interface of the stacking and separation gels (Fig. 4). Dimer and higher-order aggregates of the 35,000-40,000-dalton polypeptides are visible on the gel. By autoradiography or protein stain, it can be estimated that more than 90% of the protein, which is stable to high salt and is recovered in the 30S region, cannot be dissociated to complexes small enough to migrate into a 10% polyacrylamide gel even after prolonged boiling in 2% SDS, 0.1 M dithiothreitol, and 8 M urea. The relatively small amount of protein that is dissociated from iodinated 30S RNP on high-salt sucrose gradients fails to show this extensive aggregation or cross-linking (Fig. 4). Although the exact nature of the protein-protein
cross-linking induced by the iodination conditions is still under investigation, its mediation by free radicals appears likely since they have been implicated in peroxidase-H202 reactions including iodination (Gross and Sizer 1959; Yamazaki et al. 1960; Yip and Hadley 1967). The demonstration that diphenyl dimers may be formed under similar reaction conditions (Gross and Sizer 1959) suggests that if the steric arrangement of tyrosine residues in the RNP-protein complexes allows sufficiently close approach, stable dityrosyl bridges may be formed. Dityrosine bridges may also be produced by UV-irradiation (Lehrer and Fasman 1967), and such cross-linking has been used to advantage in probing histone-histone binding sites in nucleosomes (Martinson and McCarthy 1976). Finally, we have examined the fate of the pulselabeled RNA of 30S RNP in which the proteins have been cross-linked by iodination to form a salt-stable complex. After determining that the iodination procedure did not affect the RNA remaining bound to cross-linked particles on gradients containing low salt, we monitored the distribution of labeled RNA on high-salt sucrose gradients. As found with uncross-linked 30S RNP on identical gradients, most of the RNA label is dissociated from the 30S region where the cross-linked protein cores were recovered (data not shown). These data suggest that most if not all RNA binding sites are exposed on the surface of the protein core and may be easily disrupted by moderate changes in the ionic strength without the necessity of completely unfolding the particle for release. Whether a surface location of RNA binding sites reflects an in vivo folding of RNP fibrils after protein is bound, bringing protein-protein interaction sites into apposition, or whether preformed protein complexes bind RNA, perhaps with additional protein associated with the interparticle RNasesensitive regions, remains to be determined. Although the salt stability of the iodinated protein core (informofer) appears to be an artifact of the in vitro labeling, the ability to zero-length cross-link a large proportion of the 20 or so component 35,00040,000-dalton polypeptides of the 30S complex in this manner indicates a very close approach of these polypeptides in the native structure. The intimate protein-protein interactions responsible undoubtedly contribute substantially to the conformation and integrity of the RNP complex.
Specificity of the Interaction of Nucleic Acid Sequence Classes with RNP-Proteins As mentioned earlier, there is evidence that a large part of the RNA of 30S RNP complexes is on the surface of the particles, at least in an operational sense. In addition, there appear to be unoccupied sites for RNA binding on the RNP as isolated from sucrose gradients. The binding of added purified RNA has previously been shown to exhibit a limited degree of specificity (Samarina et al. 1967a,b;
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SUBSTRUCTURE OF NUCLEAR RNP COMPLEXES Martin et al. 1974). The capacity to bind RNA is increased greatly i f the salt concentration is raised to a point where it appears t h a t the complex dissociates (see preceding section), the RNA added, then the salt concentration lowered. In the case of direct addition and in the latter "reconstitution" experiments, the bound RNA sediments in 30S RNP form. Although the 30S RNPs have been shown to have low Afl~rdty for mature ribosomal RNA and tRNA, they bind pulse-labeled hnRNA, cytoplasmic poly(A)+ mRNA, and histone mRNA quite avidly (Martin et al. 1974). We have recently attempted to extend the analysis of RNA sequence affinities to 30S RNP proteins in order to understand more precisely the RNA-protein interaction and to assess which regions of the large hnRNA molecules would be expected to be tightly bound and which would be exposed from the surface or between the 30S subcomplexes. Since we have shown that the poly(A) segment of nuclear RNA is bound in vivo in a 15S subcomplex form having a completely different polypeptide composition to the 30S RNP (Quinlan et al. 1974), we have sought in vitro evidence of this simple difference in binding specificity. Experiments in which the binding of poly(A) to 30S RNP is compared with that of other RNA species has revealed a much lower affinity than that of poly(U), hnRNA, or mRNA (results not shown). More dramatic evidence of the selectivity of binding is obtained when [3H]adenosinelabeled RNA is extracted and purified from 15S and 30S RNP and then each is incubated in tracer amounts with fresh nuclei under normal RNP extraction conditions. It is found that the poly(A) of 15S RNP is bound once more into a 15S form, while the 30S RNP-RNA sediments at 30S (Fig. 6). Thus a high degree of selectivity of this simple kind is apparent under these competitive conditions. It is not known at present whether the relatively low affinity of 30S RNP-proteins for homopolymer(A) will affect the orientation of hnRNA-oligo(A) sequences in the complexes. In this regard, however, it is worth noting that approximately 45% of the nuclear oligo(A) of the mouse ascites cells is found in 30S RNP form, a somewhat lower fraction than the value (70%) for extractable hnRNA sequences in general (Kinniburgh et al. 1976). In considering other possible sequences in hnRNA which m a y be expected to have potential significance in nuclear functions, double-stranded (or at least complementary) RNA sequences emerge as strong candidates. Double-stranded RNA (dsRNA) regions have been shown to be sites of ribosomal and messenger RNA processing in prokaryotes (Dunn and Studier 1973; Robertson et al. 1977), and a similar role has been suggested for the cleavage of mRNA from eukaryotic hnRNA (Darnell 1976; Ryskov et al. 1976). We have therefore analyzed the distribution ofdsRNA regions in sucrose gradients of the nuclear extracts containing 30S RNP complexes. In these experiments dsRNA is determined by resistance to
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Substructure of Nuclear Ribonucleoprotein Complexes T. MARTIN, P. BILLINGS, J. PULLMAN, B. STEVENS, AND A. KINNIBURGH* Department of Biology, University of Chicago, Chicago, Illinois 60637
primarily of two to four distinct species, all having molecular weights in the range 35,000-40,000 (Martin et al. 1974; Billings and Martin 1977; see also LeStourgeon et al., this volume). These polypeptides appear to have been conserved in size and amino acid composition during the evolution ofeukaryotes (Martin et al. 1974). Most of our studies, including all of the experiments described in this paper, have employed the Taper hepatoma of mouse in the ascites form as a matter of convenience. The rapidity and extent of binding of newly synthesized RNA into a 30S subcomplex form suggest that the specific proteins associate with and fold the RNA chain while still in nascent form. The bulk of the bound RNA turns over with the half-life characteristic of hnRNA (Martin and McCarthy 1972), but the nuclear 30S RNP structures also contain the majority (and probably all) of sequences represented in cytoplasmic mRNA (Kinniburgh and Martin 1976a). We therefore infer that the simple set of polypeptides present in the 30S complex act as generalized "folding proteins" for most nascent RNA and aid in the formation of the native template for processing. I f the polypeptide components are to fold RNA effectively into distinctive unit structures, we may expect them to have definable protein-protein interactions. Furthermore, if the RNA-protein complex formed is to act as a substrate for specific processing events, then some hierarchy of binding affinity of specific types of nuclear RNA sequence for the prorein complex would seem likely. Certain regions of the large mRNA molecules should be preferentially bound or exposed by the protein complex if it is to participate in regulating the specificity of processing events. The presence of nuclear poly(A) in the form of a 15S subcomplex, very different in size and in polypeptide composition to the 30S particle (Quinlan et ai. 1974, 1977), yields an obvious example of the in vivo specificity of nuclear RNA-protein interactions. More relevant to hnRNA processing, however, would be the definition of interactions of nuclear proteins with specific internal regions of hnRNA such as oligo(U), oligo(A), and double-stranded (ds) RNA sequences. We have begun to approach this problem with reference to the 30S RNP (Kinniburgh et al. 1976), and the following experiments represent further attempts to understand the actual and potential protein-protein and RNA-protein interactions in this subunit of the hnRNP complex.
Nascent heterogeneous nuclear RNA (hnRNA) synthesized in regions of active chromatin rapidly becomes associated with protein; the presence of protein on the growing RNA chains has been visualized in spread chromatin by electron microscopy (EM) (see, e.g., Miller and Bakken 1972; Malcolm and Sommerville 1974; Laird et al. 1976; McKnight et al., this volume). It is reasonable to presume that this protein folds the lengthening RNA chains, facilitates their removal from the template, and later participates in the processing and maturation events which lead to the turnover of nucleus-restricted sequences while mature mRNA molecules are transported to the cytoplasm. There have been very extensive studies on the pathway of nuclear RNA molecules from synthesis to the appearance of mRNA on cytoplasmic polyribosomes. It is fair to say that, at present, the wealth of information produced promises rather than achieves a satisfying description of this complex process. These studies have centered on the characteristics of purified RNA molecules. Far fewer attempts have been made to examine the native forms of nuclear RNA molecules undergoing processing, i.e., as ribonucleoprotein (RNP) complexes, which are presumably the true substrates for the processing enzymes.
Questions of Structure and Function of hnRNP We have been concerned for a number of years with extending and refining the observations of Georgiev and his colleagues that rapidly labeled hnRNA could be extracted from nuclei of mammalian cells in the form of RNP complexes, thus enabling a biochemical analysis of the structures (Samarina et al. 1966). The view that has emerged from these studies is that the bulk of newly synthesized RNA in the eukaryotic nucleus consists of large molecules folded by protein to form a chain of linked RNP substructures which, when cleaved by endogenous or exogenous nuclease, yield 30S particles (Martin and McCarthy 1972; Kinniburgh et al. 1976). Such a model was originally proposed by Samarina et al. (1968). The protein responsible for maintaining the 30S RNP substructure of hnRNA has a very simple polypeptide composition consisting * Present address: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706.
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MARTIN ET AL. Morphology of Nuclear 30S and 15S RNP
Nascent RNP fibers observed by electron microscopy of spread chromatin have shown varying proportions of beaded and folded regions, presumably dependent upon the conditions employed (Miller and Bakken 1972; Malcolm and Sommerville 1974; Laird et al. 1976; McKnight et al., this volume). The micrographs imply a high degree of particulate character in the fibers, although admittedly it has not been as apparent in this case as in electron microscope studies of polyribosomes or, more recently, of the nucleosome form of chromatin. In regions not confused by secondary folding, the fibers have generally been assigned a diameter of approximately 200/k. We previously reported a diameter of 150-250 ]k for negatively stained 30S RNP particles from mouse ascites cells (Martin et al. 1974), and recently we have begun a detailed electron microscope study of nuclear RNP complexes that can be sufficiently purified to justify such analysis. One fact emerges immediately from attempts to observe unfixed 30S particles and that is the far greater lability of these structures when compared with ribosomes or ribosomal subunits. In the absence of fixative, 30S particles appear as amorphous or "unfolded" structures (results not shown). When fixed with glutaraldehyde and negatively stained with uranyl acetate, the particulate character of 30S RNP preparations is readily apparent (Fig. 1A,B). Under these conditions the majority of 30S RNP appear as regular compact structures of varying morphology but relatively consistent size, 235 • 195/k average dimensions. As expected, the morphology of the 15S poly(A)-containing RNP subcomplex is distinctly different. After glutaraldehyde fixation and negative staining, these particles appear as ellipsoids with dimensions of 165 • 120/k (Fig. 1C). The 15S RNP are more difficult to purify than 30S complexes, and the preparation also shows the presence of elongated and crescent structures as well as 100-/k rings and "stacked disks" from the adjacent 17S peak (see Quinlan et al. 1974). Shadowed 30S particles (prepared by freeze-drying) appear somewhat larger; our results for rotary shadowing with platinum give values of 250 • 195 /~, whereas those for unidirectional shadowing are 280 • 250 • 215 ,k (results not shown). The latter estimates indicate that the 30S RNP are slightly flattened structures, though this may arise from distortion of a relatively flexible particle in binding to the EM grid film. The generally larger size values obtained by shadowing compared with negative staining techniques are consistent with results from other systems, notably for ribosomes (Huxley and Zubay 1960; Vasiliev 1971). We have begun to catalog the forms apparent in the negatively stained preparations of 30S RNP. Among the various shapes observed, the most clearly defined were round, ellipseidal, rectangular, and triangular (Fig. 2a-e). The similarity among the mere-
bers of each category, both in negatively stained and in shadowed preparations, suggests a limited range of potential morphological forms. The particles which do not fit into these categories appear as intermediate forms, e.g., rectangles with rounded corners. It is unlikely that all the profiles represent different projections of a single three-dimensional configuration; the shadows cast by particles of ditferent morphologies cannot be rationalized on the basis of a single model, but rather suggest a limited diversity of shapes. If, in fact, these structures are subsections of the nascent RNA fibers, a variety of configurations would be consistent with the somewhat irregular folding apparent in electron micrographs of spread chromatin (see, e.g., Laird et al. 1976). Interactions between the polypeptides of the complexes and RNA sequences having distinctive properties could yield a set of conformational isomers. It is not surprising that RNP complexes of such a dynamic metabolic character are less rigid and consistent structures than ribosomes, for example. In attempting to understand the internal structure of these nuclear particles, it may be of value to carry out a more detailed analysis of the unfolded forms of 30S RNP apparent in negatively stained particles (Fig. 2f). We have not yet completed such a study, which may be expected to contribute to a decision between the models of 30S RNP either as consisting of RNA coiled on a protein core or as the result of a regular folding of an RNP fibril. These latter alternatives arise from the investigation of protein-protein and protein-RNA interactions such as described in the following sections. Protein-Protein Interactions and the Integrity of 30S RNP
Models have been constructed in which the native hnRNP structure has been interpreted as either particulate or as folded fibril. In the first model (Samarina et al. 1968; Lukanidin et al. 1972b), nascent hnRNA is thought to complex with surface binding sites on a number of stable globular protein complexes Cinformofers"). Cleavage between particles would yield individual informofers, each carrying a segment of RNA, and would thus explain the nature and origin of 30S RNP (Fig. 3a). An alternative model implies that smaller protein complexes bind to the nascent RNA, forming an RNP fibril which is subsequently periodically folded into a more compact structure (Stevenin and Jacob 1974) and possibly maintained in this conformation by protein-protein interactions of varying degrees of stability (Fig. 3b). These various alternatives for the organization of nascent RNP fibers have been discussed by Malcolm and Sommerville (1974) in the context of amphibian lampbrush-loop fine structure. In considering the interactions that maintain the integrity of the 30S RNP subcomplex, several observations tend to diminish the importance of the RNA
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Figure 1. Electron micrographs of fields of 30S and 15S RNP particles from mouse ascites cells (Quinlan et al. 1974). Specimens were fixed with 0.5% glutaraldehyde and negatively stained with 1% uranyl acetate. (.4)Crude 30S preparation; (B) purified 30S preparation; (C) 15S preparation. Lamellar structures and disks with central holes (arrows) are contaminants from the 17S peak. Scale bars represent 500/k. 901
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Figure 2. Gallery showing the different projections of negatively stained 30S particles that were observed: (a) Triangular forms; (b) rectangular and square forms; (c) circular forms; (d) small circular forms (found mainly in material from the light shoulder of the 30S peak); (e) ellipsoidal forms; (f) disintegrating forms. component. First, a large part of the R N A is relatively sensitive to RNase (see below). This suggests, at least operationally, a surface location of the nucleic acid. At moderate salt concentrations, 30S R N P proteins aggregate on treatment with nuclease, implying a considerable tendency for protein-protein interactions. Second, the 30S R N P contains approxi-
RNase Sensitive Sites
.
(o) Protein Core
(b) Folded Fibril
Figure 3. Two distinct models for the structure of the 30S RNP subcomplex of hnRNP: (a)RNA coiled on the surface of a protein core (the informofer of Samarina et al. [1968]); (b) condensed or folded region of a continuous RNP fibril. These alternativeforms have also been discussed by Malcolm and Sommerville (1974).
mately 1000 nucleotides of RNA (Samarina et al. 1968; Martin et al. 1974); however, whether this is relatively intact or internally nicked to 50-100-nucleotide segments does not affect the integrity of 30S RNP at physiological salt concentrations (Kinniburgh and Martin 1976a). Thus, given the lack of a requirement for RNA continuity within the particle, our attention is focused on the protein component. The concept of a reutilizable protein core complex (the informofer of Samarina et al. [1968]) is an attractive one and resembles in many ways the more recently evolved model for the nucleosome histone core in chromatin structure (see, e.g., Kornberg 1974; other papers, this volume). Major support for this model of the formation of hnRNP, however, comes from the observation that chemically radioiodinated 30S RNP protein cores, stripped of RNA and maintained in solution by high concentrations of salt, continue to sediment in approximately the same position in sucrose gradients as native 30S RNP (Lukanidin et al. 1972b). The stability of these cores suggests that the protein complexes could be reutilized intact in the nucleus.
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SUBSTRUCTURE OF NUCLEAR RNP COMPLEXES In our first experiments on the salt stability of 30S RNP, we employed the lactoperoxidase-catalyzed radioiodination technique of Marchalonis (1969) to monitor the fate of the protein moiety. The 30S RNP were iodinated either directly after extraction from the nuclei (crude 30S RNP) or after resuspending pelleted RNP recovered from the 30S region of a first gradient (purified 30S RNP). RNP particles were then centrifuged on a sucrose gradient as usual to separate them from enzyme and free iodine. Portions of such 30S RNP were adjusted to a final salt concentration varying from 0.1 M to 2 M NaC1 before analysis on sucrose gradients containing the same salt concentration. We found that iodinated 30S RNP-protein was recovered from such gradients in approximately the 30S region as reported earlier by Lukanidin et al. (1972b), indicating that under these conditions the protein moiety of 30S RNP remained intact even in salt concentrations as high as 2 M NaC1. (An iodinated 30S RNP gradient profile in which the sucrose gradient contained 0.5 M NaC1 is shown in the top panel of Fig. 4.) Parallel experiments with uridine- or adenosine-labeled 30S RNP demonstrated that RNA was progressively lost from the 30S region as the salt concentration was raised above 0.2 M NaC1. The RNA displaced by high salt was recovered in a very slowly sedimenting peak at the top of the gradient (see top panel in Fig. 5). Several observations, however, were somewhat inconsistent with this salt-resistant protein core model. First, the degree of stability as indicated by radioactivity in iodinated crude 30S RNP was invariably considerably less than that of iodinated purified particles. This was originally interpreted as reflecting a variable recovery of some minor, high-molecular-weight, cosedimenting or loosely bound proteins, perhaps more accessible to the enzyme, but which are largely removed in pelleting and repurifying 30S RNP (Martin et al. 1974). However, SDS-polyacrylamide gel analysis of the unstable protein displaced from the 30S region on high-salt sucrose gradients indicated that it was representative of the total protein composition of such 30S RNP and not enriched in high-molecular-weight species. Furthermore, the specific activity of the dissociated protein was not notably higher than that of the corresponding protein remaining with stable particles. Second, attempts to isolate RNA-free protein cores labeled in vivo with [3H]leucine clearly indicated that the bulk of the protein was easily dissociated in 0.5 M NaC1. These results suggested that iodination of the protein somehow rendered the protein complex stable to high salt. We have now monitored the distribution of 30S RNP polypeptides across high-salt gradients by SDSacrylamide gel electrophoresis for beth iodinated and noniodinated preparations (Figs. 4 and 5). The results verify the conclusions of the [SH]leucinelabeling experiments, namely, that noniodinated RNPs do not contain a salt-stable core, but rather
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that, in high salt, the 35,000-40,000-dalton proteins are released to the top of the gradient coordinately with [SH]uridine-labeled RNA (Fig. 5). In contrast, 30S RNP iodinated by the lactoperoxidase procedure does contain a salt-stable protein complex. However, most of the iodinated protein fails to enter the separation gel; instead it is trapped
Figure 4. Iodinated 30S RNP centrifuged on sucrose gradients containing 0.5 M NaCI. Pelleted 30S RNPs were resuspended in 1 ml of STM, pH 8 (Martin and McCarthy 1972), and iodinated on ice with carrier-free [125I]NaIby the lactoperoxidase-H202 procedure of Marchalonis (1969). The iodination reaction was terminated after 1 hr with 2-mercaptoethanol, and the suspension was adjusted to 0.5 M NaC1 and recentrifuged on 15-30% sucrose gradients containing 0.5 M NaCl (SW 27 rotor, 25,000 rpm for 15 hr). The gradient was collected in 36 fractions. (Toppanel) Acid-insoluble radioactivity of 20-/~1 aliquots of each fraction is shown (~-----~)superimposed on the A2,4absorption profile. Remaining fractions were pooled in 3-ml portions and TCA was added to 7%. The precipitates were collected by centrifugation, washed with ethanol, and solubilized by boiling for 10 rain in buffered 2% SDS containing 8 M urea and 0.1 M dithiothreitel. Equal portions of each sample across the gradient were analyzed on 10% pelyacrylamide SDS gels (shown in the lower panel beneath the equivalent pooled fractions). The 5% polyacrylamide stacker gel at the top is retained to demonstrate the crosslinked protein not entering the separation gel. Molecularweight marker proteins in the first and last slots are (top to bottom) ~-galactosidase (130,000); phosphorylase a (92,500); serum albumin (68,000); ovalbumin (43,000); glyceraldehyde-3-phosphate dehydrogenase (36,000); and globin (15,500). Lactoperoxidase appears in pooled fraction 2 as a polypeptide doublet of molecular weight ~78,000.
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Figure 5. RNA-labeled 30S RNP centrifuged on sucrose gradients containing 0.5 M NaC1. Mouse ascites cells were pulse-labeled for 30 rain in vitro with 15 pCi/ml [3H]uridine (Quinlan et al. 1974). Nuclear extracts were made and centrifuged on sucrose gradients in STM, pH 8 (Martin et al. 1974). The 30S RNPs recovered were pelleted overnight and resuspended in STM, pH 8. After the final salt concentration was adjusted to 0.5 MNaC1, the particle suspension was recentrifuged on sucrose gradients containing 0.5 M NaC1 as in Fig. 4. (~----$) One-milliliter fractions were recovered and 0.1-ml aliquots were assayed for acid precipitable radioactivity; ( ) UV absorption at 254 nm. The distribution of protein across the gradient is shown in the lower panel. Remaining fractions were pooled (1-3, 4-6, etc.), precipitated on ice with TCA, and dissolved in 2% SDS buffer. Equal volumes were assayed on SDS-polyacrylamide gels with molecular-weight markers in the first and last slots as described in Fig. 4.
in the stacking gel or at the interface of the stacking and separation gels (Fig. 4). Dimer and higher-order aggregates of the 35,000-40,000-dalton polypeptides are visible on the gel. By autoradiography or protein stain, it can be estimated that more than 90% of the protein, which is stable to high salt and is recovered in the 30S region, cannot be dissociated to complexes small enough to migrate into a 10% polyacrylamide gel even after prolonged boiling in 2% SDS, 0.1 M dithiothreitol, and 8 M urea. The relatively small amount of protein that is dissociated from iodinated 30S RNP on high-salt sucrose gradients fails to show this extensive aggregation or cross-linking (Fig. 4). Although the exact nature of the protein-protein
cross-linking induced by the iodination conditions is still under investigation, its mediation by free radicals appears likely since they have been implicated in peroxidase-H202 reactions including iodination (Gross and Sizer 1959; Yamazaki et al. 1960; Yip and Hadley 1967). The demonstration that diphenyl dimers may be formed under similar reaction conditions (Gross and Sizer 1959) suggests that if the steric arrangement of tyrosine residues in the RNP-protein complexes allows sufficiently close approach, stable dityrosyl bridges may be formed. Dityrosine bridges may also be produced by UV-irradiation (Lehrer and Fasman 1967), and such cross-linking has been used to advantage in probing histone-histone binding sites in nucleosomes (Martinson and McCarthy 1976). Finally, we have examined the fate of the pulselabeled RNA of 30S RNP in which the proteins have been cross-linked by iodination to form a salt-stable complex. After determining that the iodination procedure did not affect the RNA remaining bound to cross-linked particles on gradients containing low salt, we monitored the distribution of labeled RNA on high-salt sucrose gradients. As found with uncross-linked 30S RNP on identical gradients, most of the RNA label is dissociated from the 30S region where the cross-linked protein cores were recovered (data not shown). These data suggest that most if not all RNA binding sites are exposed on the surface of the protein core and may be easily disrupted by moderate changes in the ionic strength without the necessity of completely unfolding the particle for release. Whether a surface location of RNA binding sites reflects an in vivo folding of RNP fibrils after protein is bound, bringing protein-protein interaction sites into apposition, or whether preformed protein complexes bind RNA, perhaps with additional protein associated with the interparticle RNasesensitive regions, remains to be determined. Although the salt stability of the iodinated protein core (informofer) appears to be an artifact of the in vitro labeling, the ability to zero-length cross-link a large proportion of the 20 or so component 35,00040,000-dalton polypeptides of the 30S complex in this manner indicates a very close approach of these polypeptides in the native structure. The intimate protein-protein interactions responsible undoubtedly contribute substantially to the conformation and integrity of the RNP complex.
Specificity of the Interaction of Nucleic Acid Sequence Classes with RNP-Proteins As mentioned earlier, there is evidence that a large part of the RNA of 30S RNP complexes is on the surface of the particles, at least in an operational sense. In addition, there appear to be unoccupied sites for RNA binding on the RNP as isolated from sucrose gradients. The binding of added purified RNA has previously been shown to exhibit a limited degree of specificity (Samarina et al. 1967a,b;
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SUBSTRUCTURE OF NUCLEAR RNP COMPLEXES Martin et al. 1974). The capacity to bind RNA is increased greatly i f the salt concentration is raised to a point where it appears t h a t the complex dissociates (see preceding section), the RNA added, then the salt concentration lowered. In the case of direct addition and in the latter "reconstitution" experiments, the bound RNA sediments in 30S RNP form. Although the 30S RNPs have been shown to have low Afl~rdty for mature ribosomal RNA and tRNA, they bind pulse-labeled hnRNA, cytoplasmic poly(A)+ mRNA, and histone mRNA quite avidly (Martin et al. 1974). We have recently attempted to extend the analysis of RNA sequence affinities to 30S RNP proteins in order to understand more precisely the RNA-protein interaction and to assess which regions of the large hnRNA molecules would be expected to be tightly bound and which would be exposed from the surface or between the 30S subcomplexes. Since we have shown that the poly(A) segment of nuclear RNA is bound in vivo in a 15S subcomplex form having a completely different polypeptide composition to the 30S RNP (Quinlan et al. 1974), we have sought in vitro evidence of this simple difference in binding specificity. Experiments in which the binding of poly(A) to 30S RNP is compared with that of other RNA species has revealed a much lower affinity than that of poly(U), hnRNA, or mRNA (results not shown). More dramatic evidence of the selectivity of binding is obtained when [3H]adenosinelabeled RNA is extracted and purified from 15S and 30S RNP and then each is incubated in tracer amounts with fresh nuclei under normal RNP extraction conditions. It is found that the poly(A) of 15S RNP is bound once more into a 15S form, while the 30S RNP-RNA sediments at 30S (Fig. 6). Thus a high degree of selectivity of this simple kind is apparent under these competitive conditions. It is not known at present whether the relatively low affinity of 30S RNP-proteins for homopolymer(A) will affect the orientation of hnRNA-oligo(A) sequences in the complexes. In this regard, however, it is worth noting that approximately 45% of the nuclear oligo(A) of the mouse ascites cells is found in 30S RNP form, a somewhat lower fraction than the value (70%) for extractable hnRNA sequences in general (Kinniburgh et al. 1976). In considering other possible sequences in hnRNA which m a y be expected to have potential significance in nuclear functions, double-stranded (or at least complementary) RNA sequences emerge as strong candidates. Double-stranded RNA (dsRNA) regions have been shown to be sites of ribosomal and messenger RNA processing in prokaryotes (Dunn and Studier 1973; Robertson et al. 1977), and a similar role has been suggested for the cleavage of mRNA from eukaryotic hnRNA (Darnell 1976; Ryskov et al. 1976). We have therefore analyzed the distribution ofdsRNA regions in sucrose gradients of the nuclear extracts containing 30S RNP complexes. In these experiments dsRNA is determined by resistance to
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