Ultrastructural and biochemical studies on ribonucleoprotein particles from isolated nucleoli of thioacetamide-treated rat liver

The morphology of isolated nucleolar ribonucleoprotein particles from thioacetamide-treated rat livers was found to be very similar to those in situ. The sedimentation profiles of these nucleolar ribonucleoprotein particles in sucrose density gradients showed the presence of three components. The particles in these peaks were electron opaque spherical particles that were quite homogeneous in size (200–250 Å). The ultrastructure of these RNP particles from thioacetamide-treated livers is similar to that of both ribosomes and intranucleolar RNP particles inasmuch as at high magnifications a convoluted, linear strand of RNA was observed to be present in each of the particles.     

Ribonucleoproteins containing mRNA in the cytoplasm of mouse plasmacytoma cells]

The rapidly labelled postribosomal ribonucleoprotein (RNP) found in the cytoplasm of mouse plasmacytoma cells were investigated. It has been shown that 45S and 80S particles contain relatively high molecular weight (approximately 12-17S) pulse-labelled RNA similar to the polyribosomal mRNA. No other postribosomal RNP was found which would contain an RNA with similar sedimentation characteristics. In CsC1 density gradients, the postribosomal RNP gives two peaks. One of them, the rapidly labelled component (rho 1.52 g/cm3) is found only in 45S RNP. The other rapidly labelled component (rho 1.36-1.41 g/cm3) is revealed in all investigated regions of sucrose gradients. The latter contains relatively low molecular weight RNA (approximately7-9S). These RNP are supposed to be informosome-like particles. The components with a buoyant density of 1.52 g/cm3 may represent an mRNP-45S subparticles complex. The rapidly labelled mRNA of 80S particles is released after EDTA treatment in the form of mRNP with a buoyant density of 1.45-1.47 g/cm3.     

Ribosomal precursor particles of Bacillus megaterium.

Pulse-labeled cells of Bacillus megaterium were converted to protoplasts, and lysates of the protoplasts were analyzed by sucrose gradient sedimentation. Precursor ribonucleoprotein (RNP) particles then appeared predominantly as 50S and 30S precursor ribosomal subunits. Polyacrylamide gel electrophoresis of the ribosomal ribonucleic acid from the 50S and 30S RNP particles confirmed their precursor nature since they were shown to contain precursor 23S and 16S ribosomal ribonucleic acid, respectively. Treatment of protoplast lysates with 0.5% deoxycholate prior to sedimentation analysis resulted in a markedly different radioactivity profile. The 50S RNP particles were no longer present, but 43S particles were observed in addition to increased amounts of pulse-labeled material sedimenting at 30S and slower. Extracts from cells broken in a French press showed a profile from sucrose gradient sedimentation similar to that of the deoxycholate-treated protoplast lysate. These data suggest that the nature of the precursor ribosomal particles appears to be a function of the method of cell disruption or detergent treatment of the cell extract preparation. The observed 50S and 30S RNP particles may be the major precursor ribosomal subunits in vivo; the slower-sedimenting species could result from some form of breakdown or change in the configuration of the 50S and 30S precursors.     

Poly a blocks in the nuclear ribonucleoprotein complexes,containing pre-mRNA

Poly A was found in nuclear particles containing pre-mRNA. It was shown that during the isolation of 30S particles from rat liver or Ehrlich ascites carcinoma nuclei, all poly A is detached from the particles containing pre-mRNA and is found in the form of RNP with a sedimentation coefficient of about 14S. When RNP polyparticles are isolated in the presence of RNase inhibitor poly A is distributed among the particles of higher molecular weights.Since the sedimentation properties and buoyant density of the poly A-containing particles are different from the 30S particles it was suggested that the poly A fragments are bound not with informofers, but with another kind of protein.     

Cross-linked informofers.

The proteins of 30S RNP particles containing pre-mRNA (hnRNA) were cross-linked with bifunctional reagents (dimethyl-suberimidate and dimethyl-3,3'-dithiobispropionimidate). Further treatment with 1 or 2 M NaCl dissociates all RNA from protein. However, a significant part of protein particles--informofers being cross-linked survived high salt treatment. Their sedimentation coefficients were close to those of original particles. No RNA could be detected in the informofers even after labeling the cells with a precursor for a long period of time. Sodium dodecylsulfate or urea dissociated cross-linked informofers into oligomeric polypeptides. They could be dissociated by beta-mercaptoethanol treatment if a reversible cross-linked reagent had been used. The resulting polypeptides were represented by informatin. RNP particles (30S RNP or poly-particles) were reconstituted upon mixing of cross-linked informofers with pre-mRNA and removal of 2 M NaCl.     

Multiple states of U3 RNA in Novikoff hepatoma nucleoli

U3 RNA, a capped small nuclear RNA found thus far only in the nucleolus, has been implicated in the processing and/or transport of preribosomal RNA [Busch, H., Reddy, R., Rothblum, L., & Choi, Y. C. (1982) Annu. Rev. Biochem. 51, 617-654]. Tris(hydroxymethyl)aminomethane (Tris) (10 mM, pH 7.0) extracts of Novikoff hepatoma nucleoli, which contained about 80% of total nucleolar U3 RNA, were analyzed by sucrose density gradient centrifugation. Approximately 65% of the U3 RNA was bound to greater than 60S preribosomal ribonucleoprotein (RNP) particles, and about 15% sedimented at less than 20 S. The association between the 65% of U3 RNA that was bound to the preribosomal RNP particles was stable up to 55 degrees C. About 10% of U3 RNA was base paired to preribosomal RNA after deproteinization at 22 degrees C. The base-paired fraction of U3 RNA was released from the preribosomal RNA by heating to 45 degrees C or treating with 4 M urea. These results show that of the total nucleolar U3 RNP, (a) about 55% is bound to preribosomal RNP particles primarily by protein interactions, (b) about 10% is base paired to preribosomal RNA, (c) approximately 15% sedimented slowly and consisted presumably of free U3 RNP particles, and (d) the remaining 20% of U3 RNP was not extractable using 10 mM Tris buffer. On the basis of the different association states of U3 RNP particles, a model is proposed for the binding and dissociation events which take place between U3 RNP and preribosomal RNP particles.     

Globin messenger sequences in nuclear ribonucleoprotein particles of avian erythroblasts

Nuclear ribonucleoprotein particles were isolated from chick erythroblast nuclei. The particles were found to sediment as heterogeneous material. The major fraction of the rapidly synthesized RNP sedimented at 30 S, whereas the nuclei were found to contain a major, apparently more stable, RNP component sedimenting at about 40 S. The RNA isolated from the RNP particles was assayed for globin messenger activity in a wheat germ cell-free system. RNP sedimenting at relatively low S values (approx. 15 S) as well as RNP-particles of larger size code for globin. In addition to globin, the RNA of the particles codes also for other, not yet identified, proteins.     

Inhibition of cell-free protein synthesis by low-molecular-weight RNAs from free cytoplasmic ribonucleoprotein particles

Free cytoplasmic messenger ribonucleoprotein (mRNP) particles from rat liver were treated with EDTA and separated into two populations of RNP particles with sedimentation maxima of 20 S and 35 S respectively. The 20-S and 35-S RNP particles, treated with 0.5 M KCl, have protein-to-RNA ratios of 0.31:1 and 5.7:1 respectively. Whereas 20-S and 35-S RNP particles exhibit a similar protein complement of seven major polypeptides, the low-molecular-weight RNA components of the two particle populations are different. A characteristic set of distinct low-molecular-weight RNAs is found for 20-S and 35-S RNP particles. When the individual low-molecular-weight RNAs of 20-S and 35-S RNP particles isolated from preparative polyacrylamide gels were assayed for their capability to inhibit protein synthesis in vitro, several potent translational inhibitory RNAs were detected. In particular, the low-molecular-weight RNAs of 147, 203 and 263 nucleotides in length associated with the 35-S RNP particles turned out to be strong inhibitors of protein synthesis.     

Messenger ribonucleoprotein particles in unfertilized sea urchin eggs

The properties of poly(A)-containing messenger ribonucleoprotein particles (mRNPs) from unfertilized sea urchin eggs isolated under various ionic conditions were studied. Poly(A)-containing RNPs of eggs sediment with a modal value of 60–65 S under all conditions used. However, buoyant densities vary strikingly with conditions of particle preparation. Deproteinized poly(A)-containing mRNA has an average molecular weight of about 1 × 10⁶. RNPs prepared in 0.35 M Na⁺ in the absence of Mg²⁺ contain an average of 0.25 × 10⁶ daltons of protein, while particles prepared in 0.05 M Na⁺ in the absence of Mg²⁺ contain 0.35 to 11 × 10⁶ daltons of protein per RNA molecule. Particles prepared in 0.35 M Na⁺ plus 5 mM Mg²⁺ contain 1.4 × 10⁶ daltons of protein suggesting that Mg²⁺ may be necessary for maintenance of RNP intergrity if high Na⁺ concentrations are used to prevent nonspecific RNA-protein interactions. Particles prepared in 0.35 M K⁺ contain 0.9 × 10⁶ daltons of protein in both Mg²⁺ and EDTA. Mg²⁺ does not cause significant aggregation of particles, since the size of RNA extracted from RNPs is proportional to RNP sedimentation rate. Monovalent cation concentrations normally used in analysis of RNPs by sedimentation cause deproteinized poly(A)-containing RNA to sediment with abnormally high sedimentation coefficients, indicating that high sedimentation rates alone do not indicate that RNA is contained in an RNP.     

A cytoplasmic ribonucleoprotein complex containing a small RNA inhibitor of protein synthesis

Ribonucleoprotein complexes (RNP) sedimenting between 10 and 15 S were isolated from the postpolysomal cytoplasmic fraction of embryonic chicken muscle. These RNP complexes lack mRNA but contain RNA with a sedimentation coefficient of 4.4 S. The 4.4 S RNA did not arise as a product of degradation during the course of the isolation procedure nor did it contain oligo(U)- or poly(A)-rich regions. Furthermore, the 4.4 S RNA-containing RNP complex was easily separable from free mRNPs and, therefore, is not considered as part of the free mRNP complexes. Both the 4.4 S RNA and 10 to 15 S RNP were able to inhibit translation of either "capped" or "uncapped" mRNA in a heterologous cell-free system. This inhibitory effect may result from interference of 4.4 S RNA with an early event in mRNA translation. A large number of polypeptides of Mr = 14,000 to 220,000 were present in the 10 to 15 S RNP. Among these, the most prominent polypeptides were of Mr = 36,000; 48,000; 52,000; 58,000; 65,000; 78,000; 84,000; 96,000; 105,000; 165,000; and 220,000. With the exception of the Mr = 36,000 polypeptide, these major components were also found in the nonpolysomal cytoplasmic mRNA protein complexes (free mRNP).     

Messenger ribonucleoprotein complexes of cryptobiotic embryos of Artemia salina.

Poly(A)-containing ribonucleoprotein (poly(A)+-RNP) particles in the post-mitochondrial supernatant of cryptobiotic embryos of Artemia salina were characterized by hybridization to [3H]-poly(U). By sucrose isopycnic centrifugation, approximately 2/3 of poly(A)+-RNPs was found to band at 1.27-1.30 (g/cm3) and the rest 1+/3 at 1.20-1.23 (g/cm3) and below 1.20 (g/cm3). The 1.27-1.30 RNPs could be separated into two density classes, 1.27-1.28 and 1.30 (g/cm3) respectively. The latter RNP class was apparently complexed with ribosomal components because they were completely converted to the former RNP class (free RNPs) by 25 mM EDTA treatment. Further, the 1.30 (g/cm3) RNPs were resolved into several RNP species having sedimentation coefficients above 50 S. which were transformed mostly to 20-30 S rnps in the presence of 25 mM EDTA. The free 20-30 S RNPs contained 8-14 S poly(A)+-RNAs, having the highest template activity in a wheat embryo cell-free system, whereas the 1.20-1.23 poly(A)+-RNPs consisted of 10 S and 16 S RNPs, both of which contained 4 S poly(A)-containing sequences without any template activity.     

Preribosomal ribonucleoprotein particles are a major component of a nucleolar matrix fraction

Biochemical and morphological studies were performed on Novikoff hepatoma ascites cell nucleolar matrix fractions prepared by deoxyribonuclease I digestion and high-molarity salt extractions essentially according to a published method [Berezney, R., & Buchholz, L. A. (1981) Exp. Cell Res. 20, 4995-5002]. The nucleolar matrix fraction was enriched in polypeptides of molecular mass of 28, 37.5, 40, 70, 72, 110 (protein C23), and 160 kDa, compared to the nuclear fraction in which polypeptides of molecular mass of 31, 33.5, 43.5, 46, 50, 56, and 59 kDa were predominant. About one-fourth of the protein, half of the RNA, and less than 4% of the DNA originally present in the nucleoli remained in the matrix fraction. Addition of single agents such as ethylenediaminetetraacetic acid, ribonuclease A, or mercaptoethanol during preparation had no significant effect on the polypeptide composition of the nucleolar matrix fraction. However, the combination of mercaptoethanol and ribonuclease A caused most of the RNA and protein to be removed, including protein C23 and the 160-kDa polypeptide, with polypeptides in the range of Mr 30 000-50 000 remaining. Electron microscopy of nucleolar matrix fractions revealed the presence of particles similar in size to the granular elements of nucleoli. However, when ribonuclease A and mercaptoethanol were included in the procedure, only amorphous material remained. Many proteins of nucleolar preribosomal RNP particles were also associated with the nucleolar matrix fraction. RNA from the nucleolar matrix fraction was enriched in sequences from 18S and 28S ribosomal RNA. These results indicate that preribosomal RNP particles are major constituents of a nucleolar matrix fraction prepared by the deoxyribonuclease I-high-molarity salt method.     

Polysomal and cytoplasmic mRNP particles containing 7S(L) RNA

Nuclear RNP complexes, cytoplasmic mRNP particles and free and membrane-bound polysomes were prepared from rat liver and their low-molecular-mass RNA components were analyzed on polyacrylamide/formamide gels. The separated small RNAs transferred to diazophenylthioether paper were hybridized to the nick-translated recombinant plasmid pA6 containing cDNA sequences for the low-Mr RNA called 7S(L) RNA. Nuclear RNP particles and free and membrane-bound polysomes were found to contain 7S(L) RNA. In the cytoplasm 7S(L) RNA could be identified as the major small RNA in 20-S cmRNP particles.     

Cytogenetic analysis of oocytes and early preimplantation embryos from XO mice.

The cytoplasm of early sea urchin embryos contains nonribosomal, high molecular weight RNA both associated with ribosomes in polysomes and free of ribosomes in particles termed free RNP. In a 1-hr labeling period, 50% of the newly synthesized RNA enters the pool of ribosome-free RNP particles during the cleavage stages, and this percentage decreases until less than 20% of the new RNA in the mesenchyme blastula stage is found in the free RNP. mRNA from both polysomes and free RNP contain poly(A)(+) and poly(A)(?) species. During the cleavage stages only 8–10% of the RNA from each fraction is polyadenylated; however, in the blastula, 40–50% of the nonhistone polysomal RNA is polyadenylated while only 22–30% of the free RNP RNA is polyadenylated. At any developmental stage, the poly(A)(+)RNA from the free RNA and polysomes have identical sedimentation profiles; this is also the case for the poly(A)(?)RNA except for the absence of the 9 S histone mRNA from the free RNP. Changes in poly(A)(+)RNA content and sedimentation profiles during development occur simultaneously in the free RNP and the polysomes. Kinetic studies of these two RNP populations as well as nuclear RNP show that the bulk of the free RNP are not unusually stable cytoplasmic components. The free RNP decay with a half-life of about 40 min while nuclear RNA and polysomal RNA display half-lives of about 12 and 65 min, respectively. Further, the rate of synthesis of the free RNP is not consistent with their being the only precursors for polysomes. Our estimates of the rates of synthesis for nuclear RNA, polysomes, and free RNP are, respectively, 1.1 × 10^?15, 2.2 × 10^?16, and 5.0 × 15^?17 g/min/nucleus. The data on free RNP is discussed in terms of translational regulation of protein synthesis in the developing sea urchin.     

Nuclear ribonucleoprotein particles in the cellular slime mold Dictyostelium discoideum

The nuclear ribonucleoprotein (RNP) particles containing rapidly labeled RNA were isolated from interphase cells of the cellular slime mold Dictyostelium discoideum and characterized. The size of the isolated RNP particles was small (10S to 50S) in comparison with that of nuclear RNP particles found in higher eukaryotes. These small RNP particles do not seem to be artifacts due to degradation during the preparation of nuclear extracts. The rapidly labeled RNA of the nuclear RNP particles was heterogeneous in size and a considerable amount contained polyadenylic acid sequences. Synthesis of RNA in the nuclear RNP particles was resistant to a relatively high concentration of actinomycin D. The protein component of the RNP particle consists of at least four proteins with molecular weights of 80,000, 66,000, 60,000, and 42,000. Thus it is suggested that almost all of the nuclear RNP particles containing rapidly labeled RNA in interphase cells are RNP complexes consisting of Heterogeneous nuclear RNA and several protein species.