Molec. Biol. Rep. Vol. 5, 1-2: 37-42, 1979

I-InRNP CORE PROTEINS: SYNTHESIS, TURNOVER AND INTRACELLULAR DISTRIBUTION Terence MARTIN, Richard JONES & Peter BILLINGS

Department of Biology, University of Chicago, Chicago, Illinois 60637, U.S.A.

Abstract Our present data indicate that the Mr 34-40,000 polypeptides which are involved in the binding of a large fraction of hnRNA sequences, including mRNA, are for the most part metabolically stable species in mouse ascites tumor cells. An exception to this generalization is the smallest of 30S RNP core polypeptides, the Mr 34,000 protein, which has a relatively high turnover rate. The relationship of the various synthesis and degradation rates to the physiological state of mammalian cells remains to be determined, as does the pathway of assembly and disassembly of RNP substructures during re-utilization of the proteins and during their turnover. Immunofluorescent studies, which have confirmed the expected nucleoplasmic or euchromatic localization of the RNP core proteins, have also indicated that these species are stable during mitosis, at which time they are dispersed through the cell away from the condensed chromosomes. The proteins appear to relocate in the nucleus as soon as the nuclear envelope is reformed.

Introduction Electron microscopy of spread chromatin of eukaryotes has indicated that most nascent RNA molecules, including ribosomal RNA precursor and heterogeneous nuclear RNA, are already complexed with protein (1,3). Biochemical evidence also suggests the rapid association of hnRNA with protein to form ribonucleoprotein complexes (4, 5, and for review 6). Workers in a number of laboratories have been led to the conclusion that a large proportion of hnRNA is bound by a relatively simple set of polypeptides to form a chain of linked RNP substructures, which when cleaved by endogenous, or low levels of exogenous nuclease, yields 30-40S particles (1, 7, 8). A considerable fraction of the pulse-labeled RNA is recovered in the 30S RNP complexes, which when

purified contain 2-4 polypeptide species in the range Mr 34,000-40,000. These polypeptides appear to have been conserved in size and amino acid composition during the evolution of higher eukaryotes (7). A variety of evidence suggests that interactions between the multiple copies of the Mr 34,000-40,000 polypeptides present in the 30S complex are largely responsible for defining and maintaining the integrity of this hnRNP substructure. We will refer to these proteins as the hnRNP core proteins, even though the precise organization of RNA and protein in these structures, admittedly, remains to be resolved. We also accept the likelihood that other proteins interact with certain regions of hnRNA. In view of these considerations, and the determination of the cellular and cell-free affinities of the core proteins for distinct types of nucleic acid sequence; we have proposed a simple model (9) depicting the possible organization of hnRNA sequence in relation to the 30S substructures (Figure 1). It is clear from this representation that we envisage certain distinctive RNA sequence types, including oligo(A) and potentially double-stranded regions, to be exposed from the 30S cores, and therefore available for binding of other specific proteins, possibly processing enzymes. Since the bulk of hnRNA and mRNA sequences have high affinity for the proteins of the 30S RNP subcomplex, the specificity of these polypeptides for RNA can be more readily defined as resulting from specific exclusion rather than selection of sequence classes.

Synthesis and Turnover of 30S RNP Proteins The RNA which can be extracted from isolated nuclei in the form of 30S subcomplexes has previously been shown to be comprised in large part of sequences characteristic of the bulk of hnRNA which are rapidly turning over without reaching the cytoplasm (5). Relatively little is known however, about the fate 37

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Figure t. Simple hypothetical model for the structure of imRNP fibers, indicating the 30S RNP substructure and the presumed relationship of identifiable RNA sequences to the 30S particles (9). Oligo(A) and double-stranded regions are shown, as is the poly(A) of hnRNA which is bound in a 15S subunit containing a group of proteins completely distinct from the simple set of Mr 34-40,000 polypeptides which, as a multimer, lorm the 30S complex. The model is an extension of previous proposals (1,2, 10).

o f 30S RNP core polypeptides during the processing events which result in the intranuclear turnover o f much o f the h n R N A while only a small subfraction o f the sequences is conserved for transport to the cytoplasm. The problem o f the mechanism o f formation o f hnRNP is closely tied to questions o f the reutilization and turnover o f the individual proteins, either as large protein complexes or as free subunits. Reutilization may involve complex cycles o f protein disassembly, equilibration with pools, proteins modification and reassembly into complexes with new RNA. As a very preliminary approach to the question o f the formation o f hnRNP complexes, we wished to estimate the rates o f synthesis and turnover o f the simple set o f readily identifiable polypeptides in the molecular weight range 34-40,000 comprising the 30S RNP protein core. During a very short labeling period before degradation becomes significant, the protein specific activity is directly related to the half-life; however, for quantitative determinations o f the half-lives by this method, detailed knowledge o f the labeled precursor pool is required. Relative data can, however, be obtained. We, therefore, compared the specific activities o f 30S RNP proteins, histones, nucleoplasmic and cytosol proteins prepared from cells incubated in culture with [S H] -leucine for various times. These results, displayed in Figure 2, are not corrected for varying leucine content. Total histone, for example, has more than twice the leucine content o f mouse ascites RNP protein (7.3 vs 3.3 mole %),

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Figure 2. A comparison of the initial kinetics of synthesis of 30S RNP core proteins, histones and nuclear and cytoplasmic soluble proteins. Mouse Taper aseites cells were removed from the host and cultured at 37~ in MEM. After 15 min equilibration, [3HI-leucine was added and at various times equal volumes of the suspension were removed and rapidly chilled. Nuclear extracts were prepared and 30S RNP subeomplexes were purified by centrifugation through sucrose gradients (5, 7). The 3-5S region of the gradient was taken as a crude nucleoplasmic fraction and the supernatant fraction from the nuclear isolation was centrifuged for 4 hr at 100,000 x g to recover post-ribosomal supernatant proteins (crude cytoplasmic fraction). Histones were precipitated with acetone from 0.4 N H~ SO4 extracts made on the residual nuclei following RNP extraction. Protein concentrations were estimated by the Bio-Rad protein dye-binding assay; the historic concentration was based on an extinction coefficient of 3.50 at 230 nm after dissolving in 4 M urea. The specific activities were calculated using hot TCA insoluble radioactivities (10% TCA in 100 mM leueine; 90~ 15 min). Soluble proteins of the cytoplasm, i - - , ; soluble nucleoplasmic proteins, 9 ...... 9 histones, o....o; 30S RNP, O--Q.

and this could account for the apparently higher synthesis rate o f the crude histone fraction 9 However, the results do suggest that the core proteins o f the 30S RNP are relatively stable and are very likely reutilized extensively in the nucleus. A dual isotope labeling procedure was used to obtain more information on the relative half-lives o f the major 30S RNP proteins. Ascites cells were

labeled in the mice by injection of [3 H] -leucine and after 23 hr the cells were removed from the host and incubated in culture with [14C]-leucine for 90 min. The short-term label should then reflect the initial rate o f synthesis, while the long-term label is an index of decay of radioactivity in previously synthesized protein. Nuclear extracts were prepared from the cells and the proteins of the 30S particles were resolved on SDS-polyacrylamide gels. The use of the same precursor labeled with 3H or 14 C eliminates the necessity o f correction for variable leucine content in the analyzed proteins. The higher shortterm/long-term incorporation ratios (14C/3H) are indicative of proteins synthesized rapidly but with short half-lives, while low ratios characterize more stable species (11). Of the major 30S RNP proteins, only the M r 34,000 protein showed a conspicuous difference in the relative incorporation of the two leucine isotopes (Figure 3). From the considerably greater incorporation of the short-term label it may be interpreted that this protein has a distinctly faster turnover rate than the other 30S RNP proteins. The apparent order o f turnover of the various 30S proteins was: Mr 34,000 ~, 37,000 ~ 38,000 > 35,000. The nucleosome core histones as expected showed greater stability; however, histone HI had a lower stability, in the range o f the M r 34,000 protein o f the 30S complex. Turnover o f histone H1 has been reported to occur during the Gl phase before DNA synthesis (13). The apparent turnover rates o f the Mr 34,000 RNP protein and H1 were slightly greater than the weight average o f cytoplasmic post-ribosomal supernatant proteins in these cells; however, there was wide variation amongst the individual soluble proteins and some possessed exceedingly high turnover rates. Several methodological considerations prevent a conversion of these isotope ratios into absolute half-lives. However, in that the proteins are labeled under the identical conditions, the comparative values are probably valid. If the protein core is conserved as a functional unit remaining intact and being reutilized as such, it would be expected that the individual protein components would be co-ordinately synthesized and degraded as a unit. This should be reflected in uniform turnover times o f the polypeptides. On the other hand, if the half-lives differ, it suggests that each protein is independently made, and that at least some o f the proteins are exchanged or replaced in the particle and turnover at their own characteristic rates. Alternatively, one may postulate that the

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Figure 3. Relative turnover rates of the major 30S RNP proteins. Mice bearing ascites tumors were injected IP with [s H] qeucine and after 23 hr the ascites cells were removed from the peritoneum and cultured for 90 min in MEM containing [14Clqeueine. Nuclear extracts were centrifuged through sucrose gradients to purify the 30S hnRNP subcomplexes (5, 7). Histories were extracted from residual nuclei with 0.4N H2 SO,. The nuclear supernatant fraction after homogenization of the cells was used as a source of monoribosomes, which w e r e purified on sucrose gradients, and of crude cytosolic proteins (4 hr, 100,000 x g supernatant proteins). The proteins were resolved by electrophoresis in 10% polyacrylamide SDS gels, and individual 30S RNP core proteins and histones were excised and combusted in a Packard Model 306 Sample Oxidizer to physically separate the short-term t 4 C and long-term s H isotopes. The x4C/SH ratio for monoribosomes was determined by oxidation of the entire low MW portion of that gel, and for cytosolic proteins, the range was assessed by slicing the gel and including only those slices having greater than 1000 cpm in either isotope. The average 14C/SH ratio is indicated in each case by a vertical line. The number of determinations is given in parenthesis. Note that the number of Mr 34-40,000 RNP polypeptides resolved and their apparent MWs are critically dependent on the electrophoresis conditions (ef. 12). 30S RNP population is non-uniform, being comprised o f particles o f varying protein stoichiometries, with subpopulations turning over at different rates. A further possibility is that the individual polypeptides, which are similar in amino acid composition and some other properties, are related to each other as precursors and products (perhaps involving modifications). Tile preliminary results presented here indicate that the synthesis and degradation rates of the major 30S RNP-proteins are not uniform. Therefore, unless the proteins showing the lower labeling exist in a relatively large pool in the nucleus, or discrete subpopulations of 30S RNP exist, it would seem unlikely that once assembled, the RNP protein core is reutilized intact. Possibly the more rapidly turning over 39

protein of Mr 34,000 exchanges with a more basic unit comprised of the more stable species. In view of these considerations, it is interesting that, of the major proteins, the most stable protein (Mr 35,000) shows the least variance in the stoichiometry of the particles from mouse ascites cells, while the most rapidly turning over protein of Mr 34,000 shows a considerable fluctuation in its relative abundance (unpublished observations). The variable appearance of the Mr 34,000 protein is possibly a consequence of its more rapid turnover rate and may reflect the involvement of this protein in potential regulatory functior.s in the hnRNA metabolism of the cell. It should be noted that LeStourgeon et al. (8) have reported a correlation between the abundance of this protein and the mitotic capacity of the cells. More detailed studies are required to determine the relationship of the individual proteins to each other and to the putative precursor pool for re-utilization. The availability of specific antibodies for RNP proteins would greatly facilitate such investigations.

Immunochemical Studies on Ribonucleoprotein Complexes

1. Production and Characterization of Antibodies Against RNP Proteins Recently, we have developed specific immunological probes to complement our biochemical and molecular studies on hnRNP complexes. Mouse tmRNP proteins were found to be poor immunogens in rabbits, apparently reflecting the high degree of conservation of RNP proteins throughout evolution (cf. 14). Antibodies against proteins which bind hnRNA were however produced by immunizing White Leghorn chickens with either whole purified 30S RNP complexes from mouse Taper hepatoma ceils, or with just the four Mr 34-40,000 RNP-core proteins obtained by elution from SDS-polyacrylamide gels. Figure 4 shows a typical Ouchterlony double diffusion analysis demonstrating that the immune sera reacted strongly with 30S RNP particles, while sera obtained prior to immunization produced no precipitin lines. In other assays, it was established that antibodies against the whole 30S RNP complex could precipitate RNP polypeptides derived from treating 30S RNP with SDS. Furthermore, antibodies against just the SDS-derived RNP-core polypeptides were found to precipitate intact 30S RNP particles which never had been exposed to detergent. 0

Figure 4. Ouchterlony analysisof reactions between 30S RNP complexes and anti-RNP antibodies. Center wells contained 10 ttg of 30S RNP particles isolated from mouse Taper hepatoma cells (7). Outer wells contained serial dilutions of either pre-immune serum (left set) or anti-serum (right set) from a chicken immunized with whole purified 30S RNP complexes. Serum dilutions (clockwise) were 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32. Diffusion was for 48 hr at 0-4~ through 1% agarose containing 1.5 M NaC1 in Trisglycine-barbital buffer, pH 8.6 (15). Double diffusion assays of sera from chicken immunized with just the major 30S RNP polypeptides resulted in Ouchterlony patterns similar to those shown above. For routine tests, RNase-free gamma globulin was isolated from each preparation of immune and pre-immune serum by successive precipitations with sodium sulfate (18%, 14%, 12.5%). Purity of the preparations was confirmed by crossed immunoelectrophoresis. The purified anti-RNP gamma globulins effectively precipitated 30S RNP complexes in Ouchterlony assays and in immunotitration experiments. Tests of antibody specificity involving the binding of antibodies to specific bands in SDSpolyacrylamide gels demonstrated that all of four preparations of anti-RNP antibodies had a strong selective affinity for the major 30S RNP polypeptides in the 34-40,000 dalton region of the gels (unpublished results). The antibodies did not bind ribosomal proteins. With the establishment of antibody specificity, we next undertook indirect immunofluorescence studies to determine the intracellular distribution of the 30S RNP antigens in Taper hepatoma cells.

2. Immunofluorescent Localization of 30S RNP Antigens in Taper Hepatoma Cells Intact hepatoma cells were separated from ascites fluid and rinsed thoroughly with Earle's balanced salt solution. The cells were then suspended in Eagle's minimal essential medium with Earle's salts, allowed to attach to poly-L-lysine-coated coverslips, fixed, and treated with chicken anti-RNP gamma globulin. Binding of the primary antibodies was detected by fluorescence microscopy after treatment

of the cell layer with fluorescein isothiocyanateconjugated rabbit anti-chicken IgG. The results showed that the nuclei of cells treated with immune gamma globulins were brightly fluorescent (Figure 5). Moreover, nucleolar areas were free of fluorescence. A similar distribution of fluorescence was also found in other types of eukaryotic cells examined by the indirect immunofluorescence assay. Cells treated with gamma globulins isolated from the animals prior to immunization did not fluoresce. Similarly, there was no fluorescence detectable in cells where treatment with the primary antibody was deleted. As another control, replicate coverslip preparations of Taper hepatoma cells were prepared for indirect irnmunofluorescence microscopy using selected human autoimmune sera known to contain antinucleolar antibodies. Under these conditions, nucleoli were found to be brightly fluorescent. Thus, the absence of nucleolar fluorescence in cells treated without immune gamma globulins represents an absence of 30S RNP antigens at these sites, rather than problems related to lack of nucleolar fixation or antibody penetration.

Figure 5. Immunofluorescent localization of 30S RNP antigens in interphase hepatoma cells. Mouse Taper hepatoma cells were plated on poly-Lqysine-coated eoverslips and fixed for 20 min at 0-4~ with 4% forrnalin-85.5% EtOH (FA). The cell sheet was then treated sequentially with the following reagents: 50% FA-50% acetone (0-4~ 20 min), 100% acetone (0-4~ 30 rain), three changes of 10 mM phosphate buffer-140 mM NaC1, pH 7.2 (PBS wash), 3% Tween-80 (23~ 1 min), PBS wash, normal rabbit serum (1:32 dilution, 37~ 45 min), PBS wash, chicken anti-RNP gamma globulin (20 tag, 37~ 1 hr in humidified chamber), PBS wash, and fluorescein isothiocyanate-conjugated rabbit IgG anti-chicken IgG specific for heavy and light chains (1:32 dilution, 37~ 1 hr in humidified chamber). The cells were then rinsed extensively in PBS, mounted in elvanol, and photographed under phase-contrast microscopy (upper panels) or epifluorescent illumination (lower panels). 1600 X.

While nucleus-restricted fluorescence was characteristic of cells in interphase (Figure 5), cells in rectaphase had immunofluorescent sites distributed throughout the cell, and chromosomes were free of fluorescence (Figure 6). Thus, these results establish that the distribution of RNP antigens changes dramatically during mitosis. A more complete study following the sequential alterations at different stages of the mitotic cycle has been undertaken, and will be reported elsewhere. Our preliminary results suggest that dispersal of the 30S RNP antigens accompanies breakdown of the nuclear membrane, and that nucleus-restricted fluorescence returns as the nuclear envelope is reassembled.

Figure 6. lmmunofluorescent localization of 30S RNP antigens in metaphase hepatoma cells. Mouse Taper hepatoma cells were prepared for indirect immunofluorescence microscopy as in Figure 5. Upper panels: phase-contrast micrographs of cells in metaphase. Lower panels: same tells photographed under epi-fluorescent illumination. 2100 X.

Acknowledgements The research described was supported by USHPS Research Grant CA-12550 and The University of Chicago Cancer Research Center Grant CA-19265. R.J. was a postdoctoral fellow of USPHS Training Grant GM-07543.

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References 1. Miller, O.L., Jr., and Bakken, A.H., Karolinska Syrup. Res. Methods Reprod. Endocrinol. 5, 155 (1972). 2. Malcolm, D.B., ~ 7d Sommerville, J., Chromosoma 48, 137 (1974). 3. Laird, C.D., Wilkinson, L.E., Foe, V.E., ~nd Chooi Y. Chromosoma 58, 169 (1976). 4. Samarina, O.P., Lukanidin, E.M., Molnar, J. and Georgiev, G.P.J. Mol. Biol. 33,241 (1968). 5. Martin, T.E. and McCarthy, B.J., Biochlm. Biophys. Acta 277,354 (1972). 6. Van Vertrooij, W.J., and Jan~sen, D.B., Mol. Biol. Rep. 4, 3 (1978). 7. Martin, T., Billings, P., Levey, A., Ozarslan, S., Quinlan, T., Swift, H., and Urbas, L., Cold Spring Harbor Syrup. Quant. Biol. 38,921 (1973). 8. LeStourgeon, W.M., Beyer, A.L., Christensen, M.E., Walker, B.W., Poupore, S.M., and Daniels, L.P., Cold Spring Harbor Syrup. Quant. Biol. 42, 885 (1977). 9. Martin, T., Billings, P., Pullman, J., Stevens, B., and Kinniburgh, A., Cold Spring Harbor Syrup. Quant. Biol. 42, 899 (1977). 10. Kinniburgh, A.J., Billings, P.B., Quinlan, T.J., and Martin, T.E.: in W.E. Cohn and E. Volkin (eds.) Progress in Nucleic Acid Research and Molecular Biology, Academic Press, New York, 1976, Vol. 19, pp. 335-351. 11. Arias, I.M., Doyle, D., and Schimke, R.T., J. Biol. Chem. 244, 3303 (1969). 12. Billings, P.B., and Martin, T.E., Methods Ceil Biol. 17,349 (1978). 13. Gurley, L.R., Enger, M.D., and Waiters, R.A., Biochemistry 12, 237 (1973). 14. Lukanidin, E.M., Olsnes, S., and Pihl, A., Nature New Biol. 240, 90 (1972). 15. Weeke, B., Scand. J. Immunol. 2, Suppl. 1, 15 (1973).

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