Inhibition of cell-free protein synthesis by low-molecular-weight nuclear polyadenylate-containing ribonucleic acid species isolated from the lactating guinea pig.

1. Poly(A)-containing RNA was isolated from the nuclei of mammary gland, liver and brain of lactating guinea pigs. 2. Total nuclear poly(A)-containing RNA from mammary gland inhibited mRNA-directed protein synthesis by a wheat-germ cell-free system. It also inhibited the endogenous activity of the wheat-germ and other cell-free systems. It did not inhibit a wheat-germ cell-free system directed by poly(U). 3. Total nuclear poly(A)-containing RNA from liver and brain did not inhibit the mRNA-directed wheat-germ system. 4. Fractionation of the nuclear poly(A)-containing RNA revealed inhibitory activity in the less than 10 S fraction from mammary gland as well as that from liver and brain. 5. The mechanism of protein-synthesis inhibition appeared to be at the level of elongation. 6. The inhibitory activity could be reversed in a wheat-germ system by increasing the amount of S-30 supernatant. 7. The mechanism of inhibition of protein synthesis is discussed in relation to other RNA species known to inhibit such systems.     

Pre-proparathyroid hormone: fidelity of the translation of parathyroid messenger RNA by extracts of wheat germ.

Pre-proparathyroid hormone is the major protein synthesized in wheat-germ extracts in response to addition of an 8-15S fraction of parathyroid RNA. The accuracy of the translation of the mRNA from parathyroid tissue was examined by analysis of the carboxyl-terminal tryptic peptide and the amino-terminal amino acid of the protein, by analysis of the size distribution of the mRNA, and by translation of the mRNA in a second cell-free extract. When 8-15S RNA was fractionated on a sucrose gradient containing formamide, RNA that supported the synthesis of pre-proparathyroid hormone was present in a single symmetrical peak, suggesting that it was homogeneous. Analyses by paper chromatography and electrophoresis of the proline-containing tryptic peptides of pre-proparathyroid hormone indicate that they are identical with the corresponding proline-containing peptides of parathyroid hormone. Because the COOH-terminal tryptic peptide of parathyroid hormone contains proline, the data indicate that the COOH termini of pre-proparathyroid hormone and parathyroid hormone are identical. Methionine from initiator [35S]Met-tRNAfMet was rapidly incorporated into pre-proparathyroid hormone by the wheat-germ extract, and a single-step Edman degradation selectively removed almost all of the initiator [35S]methionine present in pre-proparathyroid hormone. Translation of the 8-15S RNA in a cell-free extract from Krebs-II ascites cells resulted in a protein that comigrated with pre-proparathyroid hormone on sodium dodecyl sulfate-acrylamide gel electrophoresis. These data support the conclusion that the wheat-germ system accurately translates the mRNA for parathyroid hormone, and they strengthen the contention that pre-proparathyroid hormone is the initial biosynthetic product.     

Fractionation of constituents of ribonucleoproteins containing heterogeneous nuclear ribonucleic acid.

A method of fractionation of hnRNP constituents adaptable to large-scale preparation is presented. It is based on differential resistance to salt dissociation of the two classes of units of hnRNP, the 30--50S monoparticles and the heterogeneous complexes. The monoparticle proteins were released from hnRNP by 0.4 M NaCl. They were separated from the salt-resistant RNP corresponding to the heterogeneous complexes in three steps: chromatography on DEAE-cellulose, high-speed centrifugation, and Bio-Gel chromatography. The latter chromatography permitted a first fractionation of monoparticle proteins according to molecular weight. Such fractions may serve for purification of individual proteins of molecular weight below 80 000. After the two first steps, two fractions of salt-resistant RNP were obtained. In addition to heterogeneous RNA up to 30 S, small nuclear RNAs were detected which represented 6% of total RNA. The protein pattern was complex, and no clear-cut segregation of groups of proteins could be observed between the two fractions. They were both highly enriched in phosphoproteins as compared to nomoparticle proteins. In another fraction corresponding to the void volume of Bio-Gel chromatography, one-third of the RNA was small nuclear RNA. It is suggested that this fraction contains snRNP in addition to free proteins of molecular weight above 80 000 and to salt-resistant RNP similar to those described above but of small size.     

Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels.

Poly A containing RNA isolated from embryonic chick calvaria was transferred from 6% formaldehyde 0.75% agarose gels to diazobenzyloxymethyl paper and the paper then hybridized to either nick translated pro alpha 1 collagen cDNA clones, pCg1 or pCg54, or to the nick translated pro alpha 2 collagen cDNA clone, pCg45. From the mobilities of the bands hybridizing most strongly to each, pro alpha 2 collagen mRNA was shown to be slightly larger than pro alpha 1 mRNA; they are 5100 and 4900 nucleotides long respectively. pCg54 also hybridized weakly to two bands of lower mobility, corresponding to RNAs 6.4 and 5.6 kb long. Neither pCg54 nor pCg45 hybridized to type II procollagen mRNA in poly A containing RNA isolated from embryonic chick sterna.     

Characterization and messenger activity of poly(A)-containing RNA from Chlorella.

Poly(A)-containing RNA was isolated by cellulose column chromatography from total RNA extracted from Chlorella fusca var. vacuolata 211/8p. RNA retained by the column was identified as poly(A)-containing RNA because it contained ribonuclease-resistant tracts, 25 to 55 nucleotides in length, from which not less than 80% of base was found to be adenine after acid hydrolysis. The base composition of poly(A)-containing RNA differed from that of RNA (largely ribosomal) which did not adsorb to cellulose, having a higher adenine content and a lower guanine content. Poly(A)-containing RNA was polydisperse including molecules with mobilities from 10S to 40S with a mean of about 20S. In an in vitro system derived from wheat-germ, protein synthesis was stimulated by adding poly(A)-containing RNA from Chlorella. Optimum conditions were established in this system with respect to the amount of poly(A)-containing RNA added and the concentration of KCl and Mg-2+. It is proposed that, in Chlorella, poly(A)-containing RNA includes cytoplasmic mRNA as has been shown for some other eucaryotic organisms.     

Macromolecular and ultrastructural organization of the mitotic chromosome scaffold

Using electron microscopy (EM), we have examined three structural domains of the mitotic chromosome scaffold of mouse erythroleukemia (MEL) Friend cells with different morphologic organization: centromeric, intermediate, and telomeric. The intermediate, most extensive, domain exhibited a specific fibrogranular structure representing tightly packed granular bodies with diameters between 20 and 60 nm. The chromosome scaffold contained three main components: proteins (81%), RNA (12%), and DNA (7%). The residual DNA extracted from the scaffold represented short fragments, 300 bp on average, belonging to the class of tandemly arranged repetitive DNA. In situ hybridization experiments confirmed its typical centromeric location. Scaffold RNA represented three fractions: a major RNA fraction with an electrophoretic mobility corresponding to that of 5S RNA and two minor fractions with electrophoretic mobilities somewhat lower than that of 18S RNA. Scaffold RNA was localized mainly in the centromeric region. We show that the newly synthesized protein component of the chromosome scaffolds migrates slowly to the chromosomes, reaching a maximum specific radioactivity 12 h from the onset of the chase period.     

Phylogeny of the 5S ribosomal RNA from Synechococcus lividus II: the cyanobacterial/chloroplast 5S RNAs form a common structural class

The complete nucleotide sequence of the 5S ribosomal RNA from the cyanobacterium Synechococcus lividus II has been determined. The sequence is (sequence in text) This 5S RNA has the cyanobacterial- and chloroplast-specific nucleotide insertion between positions 30 and 31 (using the numbering system of the generalized eubacterial 5S RNA) and the chloroplast-specific nucleotide-deletion signature between positions 34 and 39. The 5S RNA of S. lividus II has 27 base differences compared with the 5S RNA of the related strain S. lividus III. This large difference may reflect an ancient divergence between these two organisms. The electrophoretic mobilities on nondenaturing polyacrylamide gels of renatured 5S RNAs from S. lividus II, S. lividus III, and spinach chloroplasts are identical, but differ considerably from that of Escherichia coli 5S RNA. This most likely reflects differences in higher-order structure between the 5S RNA of E. coli and these cyanobacterial and chloroplast 5S RNAs.     

Parathyroid mRNA directs the synthesis of pre-proparathyroid hormone and proparathyroid hormone in the Krebs ascites cell-free system.

Translation in a cell-free extract of Krebs II ascites cells of a mRNA fraction prepared from bovine parathyroid glands results in the synthesis of two radioactive products that appear identical to pre-proparathyroid hormone (Pre-ProPTH) (M.W. ～ 14,000), the suspected earliest biosynthetic precursor of parathyroid hormone (PTH) (M.W. 9,500), and to proparathyroid hormone (ProPTH) (M.W. 10,200), the immediate biosynthetic precursor of PTH. The two products of synthesis in the ascites extract co-electrophoresed on both urea-acetate and urea-SDS acrylamide gels with Pre-ProPTH obtained from cell-free translation of parathyroid RNA in extracts of wheat-germ and with ProPTH isolated from parathyroid slices. Both products were precipitated with an antiserum to PTH. Partial analysis of the amino acid sequence of [³⁵S]methionine-labeled Pre-ProPTH synthesized by the ascites extract indicates that a substantial fraction of the product is lacking the two N-terminal methionines present in the Pre-ProPTH synthesized by the wheat-germ system. The results indicate that, ($\text{i}$), unlike the wheat-germ, ascites extracts contain enzymes that remove the initiator methionine from Pre-ProPTH and convert Pre-ProPTH into ProPTH (no ProPTH was observed in the wheat-germ system) and ($\text{ii}$) the cleavage processes appear to occur in association with synthesis, inasmuch as neither removal of NH₂-terminal methionine nor formation of ProPTH was observed upon incubation of Pre-ProPTH isolated from either the wheat-germ system or from the ascites system when put back into the ascites system.     

Wheat-germ agglutinin is synthesized as a glycosylated precursor

The biosynthesis and processing of wheat-germ agglutinin (WGA) were studied in developing wheat (Triticum aestivum L. cv. Marshall) embryos using pulse-chase labeling, subcellular fractionation and immunocytochemistry. A substantial amount of newly synthesized WGA was organelle-associated. Isolation of WGA on affinity columns of immobilized N-acetylglucosamine indicated that it was present in a dimeric form. When extracts from embryos pulse-labeled with [³⁵S]cysteine were fractionated on an isopycnic sucrose gradient, radioactivity incorporated into WGA was detected at a position coincident with the endoplasmic reticulum (ER) marker enzyme NADH-cytochromec reductase. The WGA in the ER could be slowly chased into the soluble, vacuolar fraction, with a half-life of approx. 8 h. Immunolocalization studies demonstrated the accumulation and distribution of WGA throughout the vacuoles.Four forms of the WGA monomer were characterized using immunoaffinity purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In-vitro translation of polyadenylated RNA isolated from developing wheat embryos produced a polypeptide with M_r 21 000. In-vivo labeling of embryos with radioactive amino acids resulted in the formation of a polypeptide of M_r 23 000 and the mature monomer of M_r 18000. When [³H]mannose was used in labeling studies, only the polypeptide of M_r 23 000 was detected. In-vivo labeling in the presence of tunicamycin yielded an additional polypeptide of M_r 20 000. These results indicate that WGA is cotranslationally processed by the removal of a signal peptide and the addition of a glycan, presumably at the carboxy-terminus (N.V. Raikhel and T.A. Wilkins, 1987, Proc. Natl. Acad. Sci. USA 84, 6745–6749). The glycosylated precursor of WGA is post-translationally processed to the mature form by the removal of a carboxyl-terminal glycopeptide.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - M_r relative molecular mass - poly(A)⁺RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - WGA wheat-germ agglutinin     

CHARACTERIZATION OF RAT BRAIN RIBONUCLEIC ACIDS BY AGAR GEL ELECTROPHORESIS

Abstract— —The characteristics of total and rapidly-labelled RNAs of rat brain were studied by agar gel electrophoresis. The bulk (more than 90 per cent) of total, nuclear and cytoplasmic brain RNA was represented by the 28 S, 18 S and 4 S RNA components. The 28 S/18 S RNA mass ratio in cytoplasmic RNA was 2·55. Lower values for this ratio were obtained with total and nuclear RNAs. Five minor RNA components were detected in total brain RNA with mobilities in agar gel corresponding to 24 S, 22 S, 14 S, 9 S and 6 S. Two broad rapidly labelled RNA components were detected in total and nuclear (but not in cytoplasmic) brain RNA with mobilities corresponding to about 45 S and 31 S. These fractions were of nuclear origin and resembled ribosomal precursor RNAs of other animal tissues. In cytoplasmic RNA the radioactivity and ultraviolet profiles coincided at all labelling times down to 1 hr. The G + C/A + U ratio of brain RNA was 1·50 for total RNA, 1·39 for nuclear RNA and 1·59 for cytoplasmic RNA. The G + C/A + U ratio of 1 hr-labelled total brain RNA (determined by ³²P-distribution) was 0·94. This ratio rose to 1·31 at 24 hr labelling. The possible significance of these results for the elucidation of ribosomal and messenger RNA metabolism in brain is discussed.     

A simple procedure for the isolation and purification of protamine messenger ribonucleic acid from trout testis.

Preparation of milligram quantities of purified poly(A)+ (polyadenylated) protamine mRNA from trout testis tissue was accomplished by a simple procedure using gentle conditions. This involves chromatography of the total nucleic acids isolated by dissociation of polyribosomes with 25 mM-EDTA to release messenger ribonucleoprotein particles and deproteinization of the total postmitochondrial supernatant with 0.5% sodium dodecyl sulphate in 0.25 M-NaCl by binding it to a DEAE-cellulose column. Total RNA was bound under these conditions, and low-molecular-weight RNA, lacking 18S and 28S RNA, could be eluted with 0.5 M-NaCl and chromatographed on oligo(dT)-cellulose columns to select for poly(A)+ RNA. Further purification of both the unbound poly(A)- RNA and the bound poly(A)+ mRNA on sucrose density gradients showed that both 18S and 28S rRNA were absent, being removed during the DEAE-cellulose chromatography step. Poly(A)- RNA sedimented in the 4S region whereas the bound poly(A)+ RNA fraction showed a main peak at 6S [poly(A+) protamine mRNA] and a shoulder in the 3-4S region. Analysis of the main peak and the shoulder on a second gradient showed that most of the main peak sedimented at 6S, whereas the shoulder sedimented slower than 4S. The identity of the poly(A)+ protamine mRNA was established by the following criteria: (1) purified protamine mRNA migrated as a set of four bands on urea/polyacrylamide-gel electrophoresis; (2) analysis of the polypeptides synthesized in the wheat-germ extract by starch-gel electrophoresis showed a single band of radioactivity which co-migrated exactly with the carrier trout testis protamine standard; and (3) chromatography of the polypeptide products on CM-cellulose (CM-52) showed the presence of three or four radioactively labelled protamine components that were co-eluted with the unlabelled trout testis protamine components added as carrier. The availability of large quantities of purified protamine mRNA should now permit a more thorough analysis of its physical and chemical properties.     

A novel antibody which precipitates 7.5S RNA is isolated from a patient with autoimmune disease

We have isolated a nobel antibody from a patient with autoimmune disease which reacts with the ribonucleoprotein complex containing 7.5S RNA. The 7.5S RNA consists of two species having slightly different electrophoretic mobilities. Fingerprinting analysis of these two species demonstrates that nucleotide sequences of the RNAs are very similar to each other. Nucleotide composition of the 7.5S RNA is found to be; A/U/G/C=20/18/32/30, indicating that the ratio of GC content of the RNA(62%) is relatively high. The RNA contains a pseudouridylic acid residue as a modified nucleotide. Immunofluorescence pattern stained with the antibody suggests that the ribonucleoprotein complex containing 7.5S RNA is located both in the nucleolus and the cytoplasm.     

Translation of embryonic-chick tendon procollagen messenger ribonucleic acid in two cell-free protein-synthesizing systems

Embryonic-chick tendon poly(A)-containing RNA was translated in the wheat-germ and mRNA-dependent rabbit reticulocyte-lysate systems. The ability of each system to synthesize polypeptides similar to pro-alpha chains of collagen was tested on the bases of electrophoretic mobility and susceptibility to highly purified bacterial collagenase. Very small amounts of polypeptides in the size range of pro-alpha chains were synthesized in the wheat-germ system, whereas efficient synthesis of two polypeptides similar to pro-alpha1 and pro-alpha2 chains was achieved in the reticulocyte lysate. The collagenous nature of the major high-molecular-weight products synthesized was demonstrated by their susceptibility to collagenase and ability to act as a substrate for purified collagen proline hydroxylase. Determinations of the relative amounts of these translation products suggest that the 2:1 ratio of pro-alpha1 and pro-alpha2 chains found in type I procollagen is reflected in proportional amounts of translatable mRNA for pro-alpha1 and pro-alpha2 chains. Comparisons of the electrophoretic mobilities of hydroxylated and unhydroxylated reticulocyte-lysate translation products were made with appropriate standards of hydroxylated and unhydroxylated procollagen polypeptides. The results suggest that, in common with a number of secreted proteins, procollagen is synthesized as pre-pro molecules consistent with the ;Signal Hypothesis'.     

Polypeptide composition of the purified Photosystem II pigment-protein complex from spinach

The Photosystem II pigment-protein complex, the chlorophyll α-protein comprising the reaction center of Photosystem II, was prepared from EDTA-treated spinach chloroplasts by digitonin extraction, sucrose-gradient centrifugation, DEAE-cellulose column chromatography, and isoelectrofocussing on Ampholine.The dissociated pigment-protein complex exhibits two polypeptide subunits that migrate in SDS-polyacrylamide gel with electrophoretic mobilities corresponding to molecular weights of approximately 43 000 and 27 000. The chlorophyll was always found in the free pigment zone at the completion of the electrophoresis. Heat-treatment of the sample (100°C, 90 s) for electrophoresis caused association of the two polypeptides into large aggregates. It is concluded that these two polypeptides, 43 000 and 27 000, are valid structural or functional components of Photosystem II pigment-protein complex.     

Cytoplasmic nonpolysomal messenger ribonucleoprotein containing actin messenger RNA in chicken embryonic muscles.

Cytoplasmic nonpolysomal mRNAs have been isolated in the form of 16-40S ribonucleoprotein particles from the postribosomal supernatant of 14-day-old chick embryonic muscles. An 8-20S RNA fraction isolated from these particles directs the synthesis of actin in a wheat germ embryo S-30 system, as judged by copurification of the products with chicken muscle actin by repeated cycles of G- to F-actin transformation; mobilities of the purified product on sodium dodecyl sulfate-polyacrylamide gels and urea gels; and analysis of the CNBr-cleaved peptides. The 16-40S particles have a buoyant density of 1.4 g/cm3 which corresponds to an RNA/protein ratio of 1:3. They do not contain detectable levels of ribosomal subunits, as judged by the absence of typical ribosomal proteins in the range of 15,000-30,000. They contain at least eight distinct polypeptide species in the molecular weight range of 44,000-100,000, including a prominent 44,000 species. The presence of these particles suggests that they may have a role in the regulation of translation in developing muscles.     

Isolation and characterization of nuclear ribonucleoprotein complexes from Drosophila melanogaster

The isolation of total nuclear ribonucleoprotein particles fromDrosophila melanogaster embryos, using a pH 8.0, 01 M NaCl extraction of purified nuclei, is described. When the extract is fractionated on isokinetic sucrose gradients, at least six major classes of nuclear ribonucleoprotein complexes, differing in RNA and protein content as well as sedimentation behavior, are observed. The two largest complexes are preribosomal complexes. The remaining four major classes of RNPs sediment at roughly 6S, 8S, 12S and 30S. A minor class at 17S is also observed. The 30S fraction is 200–250 Å in width and appears to be analogous to the mammalian monoparticle. It is composed primarily of polypeptides at about 36 000 and 37 000 daltons, along with 1–2 kilobase RNA fragments. The 6S, 8S and 12S complexes contain a few discrete small nuclear RNAs from 80–600 bases in length, along with a small number of polypeptides, about 50 000, 52 000, 56 000 and 75 000 daltons. These novel complexes are of the order of a 100 Å in width (60–120 Å range).     

The conformation of 16S RNA in the 30S ribosomal subunit from Escherichia coli.

The digestion of E. coli 16S RNA with a single-strand-specific nuclease produced two fractions separable by gel filtration. One fraction was small oligonucleotides, the other, comprising 67.5% of the total RNA, was highly structured double helical fragments of mol. wt. 7,600. There are thus about 44 helical loops of average size corresponding to 12 base pairs in each 16S RNA. 10% of the RNA could be digested from native 30S subunits. Nuclease attack was primarily in the intraloop single-stranded region but two major sites of attack were located in the interloop single-stranded regions. Nuclease digestion of unfolded subunits produced three classes of fragments, two of which, comprising 80% of the total RNA, were identical to fragments from 16S RNA. The third, consisting of 20% RNA, together with an equal weight of peotein, was a resistant core (sedimentation coefficient 7S).     

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.     

Phylogeny of the 5S ribosomal RNA fromSynechococcus lividus II: The cyanobacterial/chloroplast 5S RNAs form a common structural class

Summary The complete nucleotide sequence of the 5S ribosomal RNA from the cyanobacteriumSynechococcus lividus II has been determined. The sequence is 5-UGCCUAGUGUUUAUGGCGCG-GUGGAACCACGCUGAUCCAUCCCGAACUC-AGAGGUGAAACAUCGCAGCGGUGAAGAU-AGUUGGAGGGUAGCCUCCUGCAAAAAUA-GCUCAAUGCUAGGCA_OH-3. This 5S RNA has the cyanobacterial- and chloroplast-specific nucleotide insertion between positions 30 and 31 (using the numbering system of the generalized eubacterial 5S RNA) and the chloroplast-specific nucleotide-deletion signature between positions 34 and 39. The 5S RNA ofS. lividus II has 27 base differences compared with the 5S RNA of the related strainS. lividus III. This large difference may reflect an ancient divergence between these two organisms. The electrophoretic mobilities on nondenaturing polyacrylamide gels of renatured 5S RNAs fromS. lividus II,S. lividus III, and spinach chloroplasts are identical, but differ considerably from that ofEscherichia coli 5S RNA. This most likely reflects differences in higher-order structure between the 5S RNA ofE. coli and these cyanobacterial and chloroplast 5S RNAs.