Isolation and characterization of the predominant protein in nuclear ribonucleoprotein particles from rat liver

The predominant protein of the nuclear ribonucleoprotein particles of rat liver was isolated by polyacrylamide gel electrophoresis. The polypeptide represented 35% to 40% of the total mass of the protein moiety. Its molecular weight was estimated to be 38 000 and its NH2-terminal residue was found to be threonine. The amino acid composition is unique in having a high content of glycyl residues (20%) and NG-dimethylarginine (14% of total arginyl residues).     

Rapid purification of native C protein from nuclear ribonucleoprotein particles

A rapid three step procedure is described for the purification of C protein from HeLa 40 S hnRNP particles. The procedure takes advantage of the salt resistant RNA binding of C protein, the size of the C protein-RNA complex, and the strong binding of C protein to an anion-exchange resin. Typically 120 micrograms of C protein is obtained from 4.0 X 10(9) cells with greater than 95% electrophoretic purity. Proteins C1 and C2 copurify in the ratio of 3.5 Cl to 1 C2. The purified C protein participates in hnRNP particle reconstitution and on this basis is judged to be native. The purified C protein binds to a gel filtration matrix at 0.5 M NaCl but at higher salt concentrations it elutes before the marker protein, apoferritin (Mr = 443,000). An abbreviated two step purification procedure utilizing anion-exchange chromatography is also described. This procedure results in relatively pure C protein, as well as a useful separation of the other hnRNP proteins.     

Isolation and characterisation of subribosomal ribonucleoprotein particles from radish seeds and seedlings

Ribonucleoprotein (RNP) particles were isolated from the postribosomal supernatant of radish seeds and seedlings. Most of them sediment at about 20 S and have a buoyant density in CsCl of 1.38 g · cm⁻³. A minor fraction appears as a shoulder on the heavy side of the 20 S peak; it bands at 1.41 – 1.43 g · cm⁻³. Two types of RNA families have been identified in the particulate fraction : heterogenous RNA (18-4 S) and RNA comigrating with 4 S tRNA. Poly(A) has been found in this RNA but it accounts for less than 10% of total stored poly(A). The polypeptide moiety of the particles was analysed and compared with ribosomal and soluble proteins. It was shown that the 20 S RNP gradually disappears during germination. These results indicate that the post-ribosomal supernatant of seeds contains some mRNA (or nuclear precursor) associated with proteins and a large amount of another type of RNP in which tRNA is associated with proteins.     

Size heterogeneity of the structural units of brain nuclear ribonucleoprotein particles

Nuclear ribonucleoprotein particles were examined by electron microscopy after negative staining. The results confirmed the polymeric nature of the particles. The constitutive units were distributed into 4 size classes (diameters varying from 100 to 300 Å, approximately) indicating that the monoparticle population was heterogenous. When examined on ultrathin sections, the particles had a fibrillar appearance.     

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.     

Nuclear ribonucleoprotein particles from adenovirus infected Hela cells

Heterogenous nuclear RNA-protein complexes (hnRNP) from adenovirus-2 (Ad-2) infected Hela cells contain most of the virus-specific RNA which is labeled in the nucleus during periods lasting from 45 seconds to 2 hours. Moreover, the percentage of RNA which is Ad-2 specific as monitored by filter hybridization increases progressively from early to late period where it accounts for as much as 50–60% of the labeled RNA. The Ad-2 sequences are found in heterogenous complexes sedimenting between 30 and 200 S the density of which in CsCl (p1.39) as well as in metrizamide (p1.29) seems to be the same as that of the bulk particles. A more detailed analysis with restriction fragments shows that all regions of the Ad-2 genome are represented in these particles.     

Structure of complexes between a major protein of heterogeneous nuclear ribonucleoprotein particles and polyribonucleotides

We have investigated the structure of complexes formed between a series of poly(A)n (n = 30 to 480) and HD40 (helix-destabilizing protein, molecular weight of 40,000), the major protein component of 30 S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from the brine shrimp Artemia salina. Protein HD40 is similar to corresponding hnRNP proteins from higher eukaryotes and the complexes it forms with single-stranded nucleic acids are strikingly similar to the native "beads-on-a-string" structure of hnRNP. Using analytical ultracentrifugation and electron microscopy we find: (1) complexes formed between HD40 and long ribohomopolymers also have a beads-on-a-string structure, showing that the ability to form this structure is an inherent property of HD40, and is not dependent on any structural features of natural RNA; (2) complexes between HD40 and poly(A)160 form disks that are about 3 nm high by 18 nm in diameter and contain 20 HD40 molecules; (3) complexes of HD40 with poly(A)n with fewer than 160 nucleotides form sectors of a disk: 40 nucleotides give rise to a quarter of a disk, 80 nucleotides, half a disk, etc. The molecular weights increase with the size of poly(A)n at the rate of 5300 per nucleotide, a stoichiometry of eight nucleotides per HD40; (4) as the size of the poly(A)n increases beyond 160 nucleotides, the additional nucleoprotein elements may either initiate the formation of a second disk adjacent to the first or stack on top of the first disk to form a 6 nm high helix with a diameter of 18 nm. Based on these results, we propose that the existence of lateral protein-protein interactions that produce the basic 3 nm X 18 nm disk, combined with the marginal stability of the helix result in (a) interruptions of the helix that give rise to the beads-on-a-string appearance of the complexes, and (b) inherent heterogeneity of individual "beads" which may contain one or more turns of the helix. From measurements of HD40 complexes with coliphage MS2 RNA, phi X174 viral DNA as well as with the homopolymers, a bead is estimated to contain an average of approximately 300 nucleotides; approximately 1 X 8 turns of the helix.     

Inhibition of exogenous RNA-dependent protein synthesis by a low-molecular-weight RNA from nuclear ribonucleoprotein particles of adenovirus-infected HeLa cells

Low-molecular-weight RNA (4S to > 5.5S) isolated from nuclear ribonucleo-protein particles of adenovirus-infected HeLa cells inhibited cell-free protein synthesis directed by polyribosomal RNA from rabbit reticulocytes by more than 80%. In a reconstituted system inhibitory RNA did not prevent the binding of Met-tRNA_f-GTP-IF ternary complex to 40S subunits; however, it repressed the formation of 80S from 40S-mRNA complex and 60S subunits. In binding assays in which authentic IF-M2A and IF-M2B were present, the inhibitor competed with messenger molecules for binding site(s) in IF-M2B. The inhibitory RNA appears to be a 5.5S RNA.     

General organization of protein in HeLa 40S nuclear ribonucleoprotein particles

The majority of the protein mass of HeLa 40S heterogeneous nuclear ribonucleoprotein monoparticles is composed of multiple copies of six proteins that resolve in SDS gels as three groups of doublet bands (A1, A2; B1, B2; and C1, C2) (Beyer, A. L., M. E. Christensen, B. W. Walker, and W. M. LeStourgeon. 1977. Cell. 11: 127-138). We report here that when 40S monoparticles are exposed briefly to ribonuclease, proteins A1, C1, and C2 are solubilized coincidentally with the loss of most premessenger RNA sequences. The remaining proteins exist as tetramers of (A2)3(B1) or pentamers of (A2)3(B1)(B2). The tetramers may reassociate in highly specific ways to form either of two different structures. In 0.1 M salt approximately 12 tetramers (derived from three or four monoparticles) reassemble to form highly regular structures, which may possess dodecahedral symmetry. These structures sediment at 43S, are 20-22 nm in width, and have a mass near 2.3 million. These structures possess 450-500 bases of slowly labeled RNA, which migrates in gels as fragments 200-220 bases in length. In 9 mM salt the tetramers reassociate to form 2.0 M salt-insoluble helical filaments of indeterminant length with a pitch near 60 nm and diameter near 18 nm. If 40S monoparticles are treated briefly with nuclease-free proteases, the same proteins solubilized by nuclease (A1, C1, and C2) are preferentially cleaved. This protein cleavage is associated with the dissociation of most of the heterogeneous nuclear RNA. Proteins A2 and B1 again reassemble to form uniform, globular particles, but these sediment slightly slower than intact monoparticles. These findings indicate that proteins A1, C1, and C2 and most of the premessenger sequences occupy a peripheral position in intact monoparticles and that their homotypic and heterotypic associations are dependent on protein-RNA interactions. Protein cross-linking studies demonstrate that trimers of A1, A2, and C1 exist as the most easily stabilized homotypic association in 40S particles. This supports the 3:1 ratio (via densitometry) of the A and C proteins to the B proteins and indicates that 40S monoparticles are composed of three or four repeating units, each containing 3(A1),3(A2),1(B1),1(B2),3(C1), and 1(C2).     

Characterization of preribosomal ribonucleoprotein particles from Tetrahymena pyriformis.

Ribonucleoprotein particles present in extracts of nuclei prepared from Tetrahymena pyriformis labelled for 1, 2.5, 5 and 10 min with [3H]uridine during exponential growth were analysed by sedimentation through linear 10--30% sucrose gradients. After 1 min of labelling, the early ribosomal RNA precursor (36-S) is found to be associated with slowly sedimenting particles which form a broad peak centred at approximately 50 S. Other kinds of particles sedimenting at 80 S, 66 S, 60 S and 44 S are observed when labelling is carried out for longer periods (2.5, 5 and 10 min). The 80-S particle contains 29-S and 18-S RNA species together with traces of 36-S RNA; the 60-S and 44-S particles contain 26-S and 17-S RNAs respectively. Similar results were obtained when [Me-3H]methionine was used for labelling in place of [3H]uridine. Methylation of the RNA present in slowly sedimenting nuclear components (30-70-S) is rapid, reaching a plateau at 5 min while that of the faster sedimenting (70--90-S) components is still increasing after 10 min. Only three types of ribonucleoprotein particles (80-S, 66-S, and 44-S) were observed when the cells were labelled after prolonged starvation. A scheme of ribosome biogenesis based on these results is presented.     

Evidence for the existence of a structural RNA component in the nuclear ribonucleoprotein particles containing heterogeneous RNA

The structural organisation of nuclear ribonucleoprotein particles carrying the HnRNA has been investigated. Experiments are presented which indicate the existence in the RNP particles of two different RNA species: (1) the rapidly labelled HnRNA and (2) stable, low molecular weight RNA, probably located in the interior of the protein moiety, which may serve a structural role for the arrangement of the proteins within the RNP particle.