Highly purified ceruloplasmin messenger RNA from rat liver

Summary Highly purified ceruloplasmin mRNA was isolated from rat liver polyribosomes. The molecular weight of ceruloplasmin mRNA is in a range from 1.05 to 1.25 · 10⁶ daltons which is large enough to code for a putative precursor of ceruloplasmin (∼700 amino acids). Ceruloplasmin mRNA contains 3′-terminal poly(A) the length of which varies from 38 to 165 nucleotides. The 5′-end of ceruloplasmin mRNA is blocked with confronting m⁷G residue which is a component of cap I (m⁷G^5′ppp^5′X^mpAp). The addition of ceruloplasmin mRNA to wheat-germ cell-free system programmed the synthesis of a product that was largely precipitated by anti-ceruloplasmin immunoglobulins. The translation product was homogeneous in polyacrylamide gel-sodium dodecylsulfate electrophoresis. Cell-free translation of ceruloplasmin mRNA was sensitive to inhibition by cap analogue.     

Isolation and partial purification of ceruloplasmin messenger RNA from rat liver

Partially purified ceruloplasmin mRNA was isolated using indirect immunoprecipitation of rat liver polysomes and poly(U)-Sepharose chromatography of polysomal RNA. This RNA programmed the synthesis of ceruloplasmin polypeptides in a cell-free system from mitochondria. Immunochemical analysis of the translation products revealed a 40-fold enrichment of the ceruloplasmin mRNA activity. The purified ceruloplasmin mRNA migrated as a major homogeneous component with an apparent molecular weight about 1×10⁶ daltons in polyacrylamide gels containing sodium dodecyl sulfate. The immunoprecipitated products of the cell-free translation had molecular weights in the range 4.5–5.4×10⁴ daltons as estimated by gel-electrophoresis under denaturating conditions. These values approach the weight of the half-molecule of native ceruloplasmin.     

Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells

A variety of methylated oligonucleotides were derived from mouse L cell messenger RNA and heterogeneous nuclear RNA by digestion with specific ribonucleases, and the cap-containing oligonucleotides separated from those containing internal m⁶A by chromatography on diborylaminoethyl-cellulose. Cap-containing sequences of the type m⁷GpppX^mpG, m⁷GpppX^mpY^(m)pG, m⁷GpppX^mpY^(m) pNpG and m⁷GpppX^mpY^(m)p(Np)_{> 1}G have distinctive non-random compositions of the 2′-O-methylated constituent X^m; yet sequences of a particular type and composition occur with a remarkably similar frequency in mRNA and hnRNA². For example, approximately 20% of the cap sequences in both hnRNA and mRNA are m⁷Gppp(m⁶)A^mG, whereas less than 1% are m⁷GpppU^mpG. The high degree of similarity in cap sequences is consistent with the previously postulated precursor-product relationship between hnRNA caps and mRNA caps.The composition of the Y position in capped hnRNA molecules was determined to be (29% G, 20% A, 51% Py), which differs considerably from the composition of Y^m in the cap II forms of mRNA (8% G^m, 11% A^m, 81% Py). Given the precursor-product relationship between hnRNA caps and mRNA caps, this result provides strong evidence that only a restricted subclass of mRNA molecules receive the secondary methylation at position Y.In both hnRNA and mRNA the internal m⁶A occurs in well-defined sequences of the type: $\text{-N}{}_1\text{-(}\text{G}\text{A}\text{)-m}{}^6\text{A-C-N}{}_2\text{-}$, the 5′ nearest-neighbor of m⁶A being G in about three-quarters of the molecules and A in about one-quarter of the molecules. The nucleotide N¹ is a purine about 90% of the time and the nucleotide N² is rarely a G. These same sequences are present in large (> 50 S), as well as small (14 S to 50 S) hnRNA. These results raise the possibility that the internal m⁶A, like caps, may be conserved during the processing of large hnRNA into mRNA. Two models based on this idea are discussed.     

Identification of a 110 000 molecular weight protein associated with heterogeneous nuclear RNA and messenger RNA in rat liver cells

The composition of proteins associated with heterogeneous nuclear RNA (hnRNA) and polyribosomal messenger RNA (mRNA) in rat liver cells has been studied using sodium dodecylsulfate (SDS)-plate gel electrophoresis. The nuclear RNP was isolated as the 30–40 S monomer by means of several different procedures including extraction of nuclei at 0 and 25 °C and ultrasonic treatment. These preparations were shown to contain the same set of specific proteins when analysed electrophoretically. Dissociation of free polyribosomes was accomplished in the presence of either EDTA or puromycin at high ionic strength and the mRNP separated on columns of oligo(dT) cellulose. Two to three proteins with identical molecular weights were identified in the SDS band patterns of both hnRNP and mRNP; in particular a 110000 D double-band was most conspicuous in both band patterns.     

Frequency distribution of messenger sequences within polysomal mRNA and nuclear RNA from rat liver.

DNA complementary to polysomal poly(A)-containing mRNA (cDNA) of male rat liver was used to study the diversity of messenger sequences in the nucleus and in polysomes. 1. Hybridization of cDNA against an excess of its own polysomal mRNA template revealed that about 10,000 different mRNA species are expressed in the liver tissue. They are distributed in a wide frequency range derived from approximately 0.5% of the total genome. 2. Hybridization of the cDNA against total nuclear RNA shows that messenger sequences comprise less than 1% of the mass of total nuclear RNA. Messenger sequences have a different frequency distribution in nucleus and cytoplasm. 3. In hybridizations using cDNA, which had been fractionated into sequences representing abundant and scarce polysomal mRNA molecules, it was found that although abundant cytoplasmic messenger sequences are also abundant in the nucleus, they exist in a significantly lower frequency range in the nuclear compartment.     

Repetitious and unique sequences in the heterogeneous nuclear and cytoplasmic messenger RNA of mammalian and insect cells

Initiation of DNA synthesis has been followed in mouse myeloma cells grown in suspension culture. In cells labeled with ³H-thymidine for short times, label first appears in short fragments of DNA which can be chased into bulk DNA (>50 S) upon further incubation in unlabeled thymidine. In a 15 min pulse, DNA fragments with a sedimentation coefficient of 30 S tend to accumulate. Our results support the contention that DNA synthesis is discontinuous in myeloma cells.However, a search for RNA associated with nascent DNA in the myeloma system was unsuccessful. Newly synthesized DNA was isolated on a benzoylated naphthoylated DEAE cellulose column. After heat denaturation, this fraction was centrifuged to equilibrium in a Cs₂SO₄ density gradient. The nascent DNA displays no shift in density greater than the density of the bulk DNA. When cells were pulse labeled with ³H-uridine and the nascent DNA fraction analyzed on Cs₂SO₄ density gradients, no ³H-labeled RNA was found associated with the DNA peak or at intermediate densities that would be indicative of a RNA-DNA molecule, covalently linked. Unless scission of the RNA primers occurs immediately after the initiation of DNA synthesis, our results indicate that DNA synthesis commences without RNA primers in myeloma cells.     

ADP-ribosylation of proteins associated with heterogeneous nuclear RNA in rat liver nuclei

Purified rat liver nuclei were incubated in vitro in the presence of [adenylate-32P]nicotinamide adenine dinucleotide. The label was rapidly incorporated into trichloroacetic acid-insoluble material and also detected in particles carrying heterogeneous nuclear RNA. The particles were isolated by density gradient centrifugation, and their size determined to be 30-40 S from parallel experiments using nuclei labelled with [3H]uridine 5'-triphosphate under similar conditions. Treatment of the 30-40 S-particles with enzymes of different specificities showed that the label was tightly bound to proteins, not incorporated into nuclei acids and not utilized in phosphorylation of proteins. The label was detached by phosphodiesterase I from snake venom and identified as ADP-ribose and adenosine 5'-phosphate present at a ratio of 7.5 to 1 using thin layer chromatography on poly(ethyleneimine)-cellulose. Radioactively labelled (ADP-ribosylated) proteins were visualized by autoradiography following SDS-polyacrylamide gel electrophoresis. They included several major species of the ribonucleoprotein with molecular weights of 36000, 39000 and 42000, and a limited number of high molecular weight polypeptides.     

Nuclear precursors for ceruloplasmin-coding messenger RNA from rat liver

The distribution of the sequences coding for ceruloplasmin (CP) in rat liver heterogeneous nuclear RNA (hnRNA) was studied using highly specific CP cDNA as a hybridization probe. The content of CP-coding sequences in poly(A)-containing and poly(A)-free subfractions of hnRNA was shown to be respectively 1 and 27 equivalents of CP mRNA molecule per one hepatocyte. The gel electrophoresis of hnRNA under strongly denaturing conditions with the subsequent transfer of RNA to diazobenzyloxymethyl paper and hybridization with [32P]-cDNA probe showed that CP mRNA sequences were of multiple molecular weight distribution. In particular, 9.0, 6.6, 2.4 and 1.6 megadalton fractions of non-polyadenylate hnRNA carried CP-coding sequences while the only hand that hybridized to CP cDNA was detected in polyadenylated hnRNA. This band was of a molecular weight 1.1-1.2 megadaltons corresponding to that of cytoplasmic CP mRNA. The hybridization of high molecular weight hnRNA with full-length CP cDNA followed by the determination of the size of cDNA fragments protected against SI nuclease demonstrated that coding sequences of CP pre-mRNA are interrupted by intervening sequences.     

The distribution of rapidly hybridizing RNA sequences in heterogeneous nuclear RNA and mRNA from hela cells

Both HnRNA and mRNA contain sequences derived from DNA which is reiterated many times in the cell genome. The degree of reiteration for sites binding HnRNA appears to be greater than for those DNA sites which bind mRNA.     

Comparison of proteins bound to heterogeneous nuclear RNA and messenger RNA in HeLa cells.

Ribonucleoprotein particles containing either heterogeneous nuclear RNA or polyribosomal messenger RNA were isolated from growing HeLa cells in order to compare their respective protein components. The major obstacle to analysing the proteins bound to HeLa cell mRNA proved to be the cosedimentation of a large fraction of the mRNP² particles with ribosomal subunits following puromycin or EDTA disassembly of polyribosomes. This was circumvented by oligo(dT)-cellulose chromatography, in which essentially all of the ribosomal subunits passed through the column without retention, while approximately 80% of the pulse-labeled, poly(A)-containing mRNP became bound and could be eluted with formamide. Polyacrylamide gel electrophoresis of the non-bound fraction (ribosomal subunits) revealed polypeptides between 15,000 and 55,000 molecular weight, with no detectable components greater than 55,000. The oligo-(dT)-bound mRNP contained a much simpler protein complement, consisting of three major components having molecular weights of 120,000, 76,000 and 52,000.In the case of the nuclear ribonucleoprotein particles that contain heterogeneous nuclear RNA, oligo(dT)-cellulose chromatography revealed two classes of particles. The first contained 10 to 20% of the hnRNA, did not bind to oligo(dT)-cellulose in 0.25 m-NaCl, 10 mm-sodium phosphate buffer, pH 7.0 (4 °C), and contained primarily a single polypeptide component having an estimated molecular weight of 40,000 (“informofers”). A second population of hnRNP particles comprised approximately 80% of the hnRNA, displayed strong binding to oligo(dT)-cellulose at 0.25 m-NaCl, and contained a very complex population of proteins, having molecular weights between 40,000 and 180,000, the same as unfractionated hnRNP. The results indicate that, at the resolution of gel electrophoresis and at the sensitivity of Coomassie blue dye, the proteins bound to HeLa cell hnRNA are qualitatively distinct from those bound to polyribosomal mRNA and, in addition, that the hnRNP proteins are the more complex of the two. These results are discussed in relation to the possible nucleotide sequence elements in hnRNA and mRNA to which these specific proteins are bound.     

Isolation of a nuclear ribonucleoprotein fraction from chick oviduct containing ovalbumin messenger RNA sequences

A method was developed for the isolation of a ribonucleoprotein fraction from chick oviduct nuclei that contains 70% of the pulse-labeled RNA. These fractions also contain about 1% of the nuclear DNA and have an average RNA to DNA ratio of about 4:1. The major nuclear RNP proteins of 32,000 M_r are present along with many additional proteins including histories. However, polysomal proteins and major oviduct cytoplasmic proteins are absent. Nuclei from fully stimulated chick oviduct contain about 3000 copies of ovalbumin messenger RNA sequences of which about 200 are in the RNP complexes: these complexes have sedimentation coefficients of 30 to 350 S and are resistant to disruption by EDTA.The level of ovalbumin mRNA sequences in these complexes reflects the overall rate of synthesis of this RNA. Withdrawal of estrogen leads to a parallel decline of nuclear estrogen receptors and ovalbumin mRNA sequences in the RNP complexes and a subsequent loss of cytoplasmic ovalbumin mRNA about three hours later. The 300-fold decrease in the level of ovalbumin mRNA sequences in these complexes and the eightfold decrease in stability of cytoplasmic ovalbumin mRNA account for the 2500-fold decrease in the level of cytoplasmic ovalbumin mRNA observed during withdrawal. Upon stimulation with estrogen, the kinetics of reappearance of ovalbumin mRNA sequences in the RNP complexes apparently accounts for the accumulation of cytoplasmic ovalbumin mRNA. Thus the nuclear RNP has some of the properties expected of nascent RNP complexes.The levels of ovalbumin and conalbumin mRNA sequences increase in the nuclear RNP with markedly different kinetics: conalbumin mRNA sequences reach half maximum by 1.5 hours, whereas ovalbumin mRNA sequences in these complexes reach half maximum at about eight hours. In the analysis in the accompanying Appendix, we show that the immediate increase of conalbumin mRNA sequences in the nuclear RNP may be accounted for by interaction of the hormone receptor complex with a single regulatory site, whereas the delayed increase of ovalbumin mRNA sequences in the RNP may be due to a requirement for interaction of the hormone receptor complex with multiple regulatory sites.     

Purification of messenger RNA coding for glycine methyltransferase from rat liver

Rat liver messenger RNA coding for glycine methyltransferase was associated preferentially with free polysomes. The mRNA was purified about 1000-fold over the total poly(A)-containing RNA by specific immunoadsorption of polysomes to protein A-Sepharose followed by oligo(dT)-cellulose column chromatography. Sodium dodecyl sulfate-gel electrophoresis of the in vitro translation products in a rabbit reticulocyte lysate system revealed only one major band which migrated to the position of the purified glycine methyltransferase subunit. The result shows that the mRNA isolated is nearly homogeneous and suggests that no precursor form of the enzyme existed. The mRNA sedimented at the position slightly smaller than 18 S rRNA in a sucrose density-gradient centrifugation and was shown to contain about 1,300 nucleotides by the Northern blot hybridization analysis with a cDNA probe.     

Isolation of rat liver albumin messenger RNA.

Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.     

The detection of messenger ribonucleic acid sequences in heterogeneous nuclear ribonucleic acid fractions of the oestrogen-stimulated rat uterus.

Active xanthine oxidase was labelled specifically with 33S in the cyanide-labile site of the molybdenum centre. The Very Rapid molybdenum (V) e.p.r. signal, generated from this, shows strong coupling of 33S to molybdenum, providing unambiguous evidence that, at least in the signal-giving species, this sulphur atom is a ligand of molybdenum. The structure of the signal-giving species is discussed.