Duck Erythroblasts Research Articles

Disruption of prosomes by some bivalent metal ions results in the loss of their multicatalytic proteinase activity and cancels the nuclease resistance of prosomal RNA.

Prosomes are ribonucleoprotein particles constituted by a variable set of about 20 proteins found associated with untranslated mRNA. In addition, they contain a small RNA, the presence of which has been an issue of controversy for a long time. The intact particles have a multicatalytic proteinase (MCP) activity and are very stable; we have never observed autodigestion of the particle by its intrinsic proteinase activity. Surprisingly it was found that Zn2+ and Cu2+ ions at concentrations of 0.1-1 mM disrupt the prosome particles isolated from HeLa cells and duck erythroblasts and abolish instantaneously its MCP activity, without altering the two-dimensional electrophoretic pattern of the constituent proteins. Fe2+, however, seems to induce autodegradation rather than dissociation of the prosome constituents. Most interestingly, protein or oligopeptide substrates protect the particle and its proteinase activity from disruption by Zn2+ or Cu2+. Nuclease-digestion assays reveal that the prosomal RNA, which is largely resistant in the intact particle, becomes digestible after dissociation of prosomes by Zn2+. These data give, for the first time, unambiguous proof of the presence of an RNA in the particle. Furthermore, they demonstrate a structure-function relationship between the complex and its enzyme activity, which seems to be based on the particle as an entity and not on the single constituent proteins.

The protein of M(r) 21,000 constituting the prosome-like particle of duck erythroblasts is homologous to apoferritin.

In duck erythroblasts, two major populations of untranslated messenger (m) RNP can be separated by sucrose gradient centrifugation in low ionic strength. One of these contains globin mRNA associated to protein factors, among them the prosomes. The other, sedimenting in the 35S zone, contains non-globin mRNA. From this '35S' mRNP, a new RNP particle called the prosome-like particle was isolated and characterized [Akhayat, O., Infante, A. A., Infante, D., Martins de Sa, C., Grossi de Sa, M.-F. & Scherrer, K. (1987) Eur. J. Biochem. 170, 23-33]. The PLP is a multimer of a protein of M(r) 21,000, and contains small RNA species. The particle is tightly associated with repressed mRNA and inhibits in vitro protein synthesis. We show here that the protein of M(r) 21,000, constituting the prosome-like particle, is apoferritin. Different approaches confirm the RNP character of this particle and provide evidence that some of its RNA species are tRNA. The hypothesis is discussed as to whether (apo-)ferritin might serve other functions in addition to iron storage.

Structure and RNA content of the prosomes

Duck erythroblast, prosomes were analysed by small angle neutron scattering (SANS), dynamic light scattering and (cryo-)electron microscopy. A molecular weight of ≈ 720,000 ± 50,000, a radius of gyration of 64 ± 2 Å and a hydrodynamic radius of ≈ 86 Å were obtained. Electron micrographs show a hollow cylinder-like particle with a diameter of 120 Å, n height of 170 Å and a diameter of 40 Å for the cavity, built of four discs, the two outer ones being more pronounced than those in the center. Results from SANS indicate less then 5% of RNA in the purified prosomes, but nuclease protection assays confirm its presence.

The major RNA in prosomes of HeLa cells and duck erythroblasts is tRNA(Lys,3).

Two-dimensional gel electrophoresis of HeLa cell prosomal RNAs, 3'-end labeled by RNA ligase, revealed one prominent spot. Determination of a partial sequence at the 3'-end indicated full homology to the 18 nucleotides at the 3'-end of tRNA(Lys,3) from rabbit, the bovine and the human species. An oligonucleotide complementary to the 3'-end of tRNA(Lys,3) hybridized on Northern blots with prosomal RNA from both HeLa cells and duck erythroblasts. In two-dimensional PAGE, the major pRNA of HeLa cells co-migrated with bovine tRNA(Lys,3). Reconstitution of the CCA 3'-end of RNA from both human and duck prosomes, by tRNA-nucleotidyl-transferase, confirmed the tRNA character of this type of RNA. Furthermore, it revealed at least one additional tRNA band about 85 nt long among the prosomal RNA from both species. Finally, confirming an original property of prosomal RNA, we show that in vitro synthesized tRNA(Lys,3) hybridizes stably to duck globin mRNA, and to poly(A)(+)- and poly(A)(-)-RNA from HeLa cells.

The sea urchin multicatalytic protease: purification, biochemical analysis, subcellular distribution, and relationship to snRNPs.

We have purified and extensively characterized a 19-S particle from sea urchin eggs. This particle is the sea urchin homologue of the "prosome", a particle originally identified in duck erythroblasts. We now show that these sea urchin prosomes contain multiple proteolytic activities. As shown for analogous particles from other cells, these particles hydrolyze synthetic substrates containing neutral hydrophobic or basic amino acids at the carboxy terminus of the synthetic peptides. They contain 16-20 small proteins ranging in molecular weight from 20,000 to 32,000. Peptide mapping shows that most of the polypeptides are unique, however, three exist in two isoelectric forms. We have investigated the possible function of the sea urchin multicatalytic proteases (MCPs) by determining their subcellular distribution, their relationship to egg snRNPs, and their possible role in translational repression. There are almost as many MCPs (2 x 10(8] as ribosomes (6.6 x 10(8] or mRNPs (1.8 x 10(7] per egg. This suggests that like ribosomes, the MCPs are stored in the egg for use during later development. We find that a substantial proportion of egg MCPs move into nuclei by the late blastula stage. Using a specific antibody against one of the sea urchin MCP proteins and antibodies against U1-U6, La, and Ro RNPs, we show that the sea urchin particle is distinct from these RNPs, although the anti-U1-U6 RNP antibody cross-reacts with a single MCP protein. In addition, the sea urchin MCP appears to be associated with a large structure in the cytoplasm of unfertilized eggs and is released under the same conditions that activate egg mRNPs in vitro.

The association of prosomes with some of the intermediate filament networks of the animal cell.

The small RNP complexes of defined morphology and biochemical composition termed prosomes, first isolated from the cytoplasm associated with repressed mRNA (Martins de Sa, C., M.-F. Grossi de Sa, O. Akhayat, F. Broders, and K. Scherrer. J. Mol. Biol. 1986. 187:47-493), were found also in the nucleus (Grossi de Sa, M.-F., C. Martins de Sa, F. Harper, O. Coux, O. Akhayat, P. Gounon, J. K. Pal, Y. Florentin, and K. Scherrer. 1988. J. Cell Sci. 89:151-165). Immunofluorescence, immunoelectron microscopy, and immunochemical studies using mAbs directed against some of the prosomal proteins of duck erythroblasts indicate that in the cytoplasm of HeLa and PtK cells, prosome antigens are associated with the intermediate filament network of the cytokeratin type.

A new type of prosome‐like particle, composed of small cytoplasmic RNA and multimers of a 21‐kDa protein, inhibits protein synthesisin vitro

A large fraction of the translationally repressed non-globin messenger RNA in duck erythroblasts is present in non-polyribosomal free mRNP structures which sediment in the 30-40-S range ('35 S'). In 0.5 M KCl, they form core complexes which show a pronounced peak at about 32 S containing mRNA and a discrete spherical RNP particle with a diameter of about 12 nm and the typical morphology of a prosome [H.-P. Schmid et al. (1984) EMBO J. 3, 29-34]. Buoyant density measurements and chromatography on oligo(dT)-cellulose indicate that this particle is bound to mRNA; it can be released from the mRNA by treatment of the free mRNP fraction with SDS. This prosome-like particle inhibits the translation of mRNA in vitro. It is composed primarily of multimers of a single 21-kDa protein and at least one species of RNA of about 80-100 nucleotides. It is resistant to dissociation by 2 M CS2SO4 and 1% SDS; the 21-kDa protein is not attacked by proteinase K unless the particle is extracted with phenol prior to treatment with the protease. The small RNA moiety of the particle hybridizes to the poly(A)-rich mRNA derived from the free mRNPs, as well as to polyribosomal mRNA. These data indicate that prosomes may serve to regulate mRNA translation; they show furthermore that prosome-like particles (about 600 kDa mass) may be built of up to 25 molecules of a single specific protein, rather than of the entire set of about 20 prosomal proteins previously identified.

Sea urchin prosome: characterization and changes during development.

A cytoplasmic particle displaying properties in common with a structure present in duck erythroblasts, termed the prosome, has been isolated from eggs and embryos of two species of sea urchin. This particle was partially purified by sedimentation in sucrose gradients containing 0.5 M KCl, and some of its physical properties and its behavior during early development were determined. The prosome sediments between 16 and 19 S and has a buoyant density of 1.30 g/cm3 in Cs2SO4 gradients. Biochemically, the particle is characterized as 20-25 polypeptides of molecular size 24-35 kDa with about 10 small RNAs. A monoclonal antibody directed against the 27-kDa protein of duck erythroblast prosome recognizes a 27-kDa protein of the sea urchin prosome. We have used this protein, as representative of the prosome, to immunologically determine the level and the subcellular localization of the particle during development. Immunoblotting and cellular fractionation studies show that the 27-kDa prosome polypeptide is present almost entirely in the postribosomal supernatant of unfertilized egg lysates. After fertilization and during early development, the total amount of 27-kDa protein per embryo remains constant, but the amount in the postribosomal supernatant decreases; there is a concomitant increase in the level of the 27-kDa protein in a rapidly sedimenting, particulate fraction containing nuclei. Immunofluorescence studies further show that the 27-kDa protein is located mainly in the cytoplasm of eggs and two-cell embryos. The subcellular location of the prosome, therefore, appears to change during development. In vivo labeling experiments have failed to detect the synthesis of either the prosome proteins or RNAs in eggs and embryos up to 48 hr of development, suggesting that this cytoplasmic particle is not synthesized de novo in early embryogenesis and thus is metabolically stable. The prosome is thus a normal cellular constituent of the sea urchin and is most likely synthesized during oogenesis and stored in the unfertilized egg.

Prosomes: Ubiquity and inter-species structural variation

The “prosomes”, a novel type of ubiquitous ribonucleoprotein particle of extraordinary stability and of defined electron microscopical structure, have been characterized in several cell types and species. Identified as a 19 S sub-component of free mRNA-protein complexes, including globin and other repressed mRNA, in the cytoplasm of duck, mouse and HeLa cells, they were previously found to inhibit protein synthesis in vitro. In all cells studied, electron microscopy shows an identical, seemingly ring-like but rather raspberry-shaped particle of 12 nm diameter, resistant to EDTA and 1% (w/v) Sarkosyl. Two-dimensional electrophoretic analysis of prosomal proteins shows a characteristic pattern in the 19,000 to 35,000 M r range of pI 4 to 7, with an additional 56,000 M r component specific to avian species. The prosomes found in globin mRNA-protein complexes contain about 25 protein components, 16 of which have identical molecular weight and pI values in duck and mouse, and which are also found in the prosomes of the heterogeneous free mRNPs of HeLa cells. Seral and monoclonal antibodies raised in mice against the prosomes of duck erythroblasts cross-react with some of the proteins of the mouse and HeLa cell particles. Prosomes isolated from duck and mouse globin mRNP, both contain small cytoplasmic RNAs of 70 to 90 nucleotides, which represent about 15% of the particle mass. The molecular weight and the 3′-terminal oligonucleotide of each one of these small cytoplasmic RNAs are identical in the two animal species; fingerprints of their oligonucleotides generated by RNase T 1 show that more than 80% of spots are identical. In contrast, the prosomes of HeLa cells, associated with a large population of repressed mRNA, contain at least 12 small cytoplasmic RNA species. All prosomal RNAs tested so far hybridize to mRNA. The data available indicate that prosomes constitute a novel class of ubiquitous cellular ribonucleoprotein complexes, present in the nucleus and cytoplasm that, in its structural variations shown here, reflects function and species.

Differential repression of specific mRNA in erythroblast cytoplasm: a possible role for free mRNP proteins.

Two types of in vivo untranslated 'free' mRNA-protein particles (mRNP) were isolated from duck erythroblast cytoplasm and characterised. Both types, namely the highly purified globin mRNA-specific '20S' mRNP and the '35S' mRNP containing a heterogenous non-globin mRNA population, are not translatable in rabbit reticulocyte lysates, but yield active mRNA upon deproteinisation. In vivo, 90% of globin mRNA is translated, but the majority of mRNA types are found in the inactive mRNP fraction, including fully repressed mRNA species. Searching for the factors controlling differential mRNA repression, we characterised and compared the protein composition of globin and '35S' mRNP using two dimensional gel electrophoresis, in vivo labelling with [35S]methionine and in vivo phosphorylation. The major proteins ubiquitously bound to globin or any other mRNA in the polyribosomes (e.g., the 73 K mol. wt. poly(A) binding protein) were not detected in purified inactive mRNP. In the latter some polypeptides appear to be associated with only one of the two inactive mRNA types while some others are common to both mRNPs. Furthermore, different rates of synthesis and phosphorylation characterize the protein populations of the two types of repressed mRNP. The specificity in composition and metabolism of the populations of polypeptides associated with different subpopulations of inactive cytoplasmic mRNA, as shown here, argues in favour of a role of mRNP proteins in mRNA recognition and selective translational repression, possibly in association with the ScRNA previously found as components of the free mRNP and able to inhibit protein synthesis.

The translation of the messenger for the poly(A)-binding protein-associated with translated mRNA is suppressed: A case of cytoplasmic repression in duck erythroblasts

In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the M r 73 000 poly(A)-binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.

Transcriptional and post-transcriptional regulation in duck erythroblasts

In order to study the relative importance of transcriptional and post-transcriptional regulation in duck erythroblasts, unique sequence pre-mRNA and mRNA complexity was assayed at four levels. Hybridization and Cr 0t analysis of total nuclear RNA with nick-translated unique sequence genomic DNA showed that of the order of 10% of the duck genome is transcribed; about one-half of the spectrum of RNA sequence formed decays within 40 min in the nucleus. The full-sequence complexity of cytoplasmic mRNA amounts to less than 0.15% of that of the genomic DNA. To assay for cytoplasmic controls, messenger RNA was isolated from polyribosomes and from repressed mRNP free of ribosomes; these two compartments contain 88 and 12%, respectively, of the polyadenylated mRNA. Both mRNA populations were either transcribed into cDNA by RNA-dependent DNA-polymerase, or translated in the nuclease-treated reticulocyte lysate. The kinetic complexity of the polyribosomal cDNA hybridizing back to its template shows the presence of about 200 mRNA species in three abundancy classes. The α- and β-globin mRNAs (70% in mass of polyribosomal mRNA) are represented by about 3800 copies/cell, 7 other mRNA types by about 90 copies/cell, and 202 mRNA by 2 copies/cell. The repressed free cytoplasmic mRNP contains 1400 different mRNAs, also in three abundancy classes. The α- and β-globin mRNAs (44% in mass) are represented by about 285 copies, there are 19 mRNA types with 7 copies, and 1380 mRNAs represented by less than 1 copy per cell. These data indicate that about 1200 mRNA species are not represented at all in polyribosomal mRNA. Heterologous cross-hybridization of cDNA transcribed from free mRNP message with polyribosomal mRNA suggests that the intermediate frequency mRNA classes are very differently represented in the two compartments. The qualitative difference of these mRNA populations is also demonstrated by the analysis of the products of in vitro translation by two-dimensional electrophoresis. Among the almost fully repressed mRNA is found, e.g., the mRNA for the polyribosomal poly(A)-binding protein. In conclusion the transcribed unique sequence DNA is of a complexity more than 100 times larger than that of total cytoplasmic mRNA; at an estimated mRNA/IVS ratio in pre-mRNA of 1:5 this amounts to a selection for transport to the cytoplasm of at best 1 mRNA molecule in 20. The repressed mRNA population in the cytoplasm is at least sevenfold more complex than the translated one. Individual equilibria of activity exist for each mRNA which allow almost full translation, and thus expression of some genes, but condition also full repression at post-transcriptional and cytoplasmic levels of a majority of the activated genes. These data show the higher importance of post-transcriptional controls compared to transcriptional regulation (selection of only 1 in 10) in a highly differentiated cell and are discussed in relation to the Cascade Regulation Scheme.

The nuclear matrix of duck erythroblasts is associated with globin mRNA coding sequences but not with the major proteins of 40S nuclear RNP

A residual protein matrix has been prepared from avian erythroblast nuclei by extensive extraction with salines and detergent and subsequent digestion with high concentrations of RNase and DNase. Ultrastructural examination reveals considerable internal structure, the most prominent feature being the remains of the nucleoli embedded in a network of fibres of fairly uniform diameter of 50 Å. The proteins which make up this structure have been examined by two-dimensional electrophoresis and are shown to consist of a characteristic set of about 30, mainly acidic components, including four prominent species of 43 000, 52 000, 66 000 and 68 000 molecular weight (MW). In parallel preparations of the nuclear matrix digested with DNase alone, much of the nuclear RNA is found associated with the residual structure, including globin-coding sequences. These results correlate well with the ultrastructural appearance of DNase-digested matrix preparations which show that superimposed on the 50 Å fibrous network is a 200–300 Å granular component, the combined fibrillo-granular structure resembling the interchromatin RNP previously identified in situ. However, the proteins of the DNase-digested matrix seen by two-dimensional electrophoresis are indistinguishable from the proteins of matrix preparations digested with both DNase and RNase. Furthermore, two-dimensional comparison between the proteins of the DNase-digested matrix and purified 40S nuclear RNP particles shows that the bulk of the proteins found associated with nuclear RNA in vitro are extracted during matrix preparation, and only two, with MWs of 43 000 and 73 000, remain. The latter species co-migrates with the poly(A)-binding protein.

Distribution of low-molecular-weight RNAs in avian erythroblast nuclear ribonucleoprotein complexes associated with pre-messenger RNA.

Duck erythroblast nuclei, isolated under conditions designed to preserve lysosome integrity, were extracted to yield a bulk population of nuclear ribonucleoprotein (nRNP) particles which, when fractionated on sucrose gradients, was resolved into two classes of RNA‐protein complexes containing low‐molecular‐weight RNA. The classical pre‐messenger ribonucleoprotein complex (pre‐mRNP), which had been previously characterized in this laboratory and was shown to contain rapidly‐labeled pre‐messenger RNA (pre‐mRNA) sequences, was observed to contain approximately eight discrete species of nuclear RNA between 90 and 230 nucleotides in length (major species of Mr 80000 and 55000, and minor species of Mr 61 000, 58000, 51000, 38000, 36000 and 32000). The size and relative distribution of these RNA sequences indicated that they were the small nuclear RNA species previously described in the literature.In addition, two smaller ribonucleoprotein complexes with sedimentation coefficients of 17 S and 12 S were characterized and shown to be composed of characteristic, although not mutually exclusive, sets of low‐Mr RNA species. Comparison of these RNA species in the two smaller nRNP complexes with those found in the 40‐S pre‐mRNP particles demonstrated that all but three of the major low‐Mr RNA bands found in the small nRNP were found in the pre‐mRNP. The additional RNA species unique to the small nRNP were major ones of MI 150000 and 110000, minor ones of Mr 48000, 34000 and 27000 and three major bands of Mr approximately 25000 as well.Further comparison revealed that while each particle was composed of a characteristic subset of polypeptides, several common proteins were observed in both small nRNP and pre‐mRNP. The common RNA and polypeptide components observed in these two classes of nuclear ribo‐ nucleoprotein suggested that these small nRNPs may represent either integral parts of intact physiological pre‐mRNP or nuclear structures which are closely associated with pre‐mRNP in vivo.No difference was observed in either the RNA or protein composition of small nRNP or pre‐mRNP when prepared in the presence of the protease inhibitor phenylmethylsulfonyl fluoride, indicating that endogenous protease activity was not a problem in the preparation of nRNP from duck erythroblasts. CsCl gradient analysis of the two fractionated small nRNPs revealed buoyant densities of 1.39 and 1.40 g/ml for the 17‐S and 12‐S particles respectively. From these densities, an approximate RNA: protein ratio of 1 : 4, similar to that previously determined for the 40‐S pre‐mRNP complex, was estimated. Isoelectric focusing of the small nRNP on a pH gradient between pH 4 and pH 6 and electron microscopy studies reaffirmed the particulate nature of these smaller ribonucleoprotein complexes. In addition, ultrastructural analysis of small nRNP particles indicated an average diameter of 15 nm, compared with an average diameter of 35 ‐ 40 nm for the bulk of the 40‐S pre‐mRNP particles.The poly(A)‐protein complex of pre‐RNA, sedimenting at a position of 15 S on the sucrose gradients, was bound to oligo(dT)‐cellulose and subsequently eluted from the column in buffer containing 50 % formamide. Six major proteins were associated with this poly(A) sequence, including the 73 000‐Mr poly(A)‐binding protein typically seen associated with the poly(A) sequence of mRNA and pre‐mRNA. In contrast, neither the 17‐S nor the 12‐S nRNP was bound to the oligo(dT)‐ cellulose column indicating that these nRNPs did not contain poly(A) sequences and were, there‐ fore, nRNP complexes distinctly different from the previously described poly(A)‐protein complex of pre‐mRNA.

Potential role of mRNP proteins in cytoplasmic control of gene expression in duck erythroblasts.

In the present report, we try to summarize recent results concerning our studies on messenger ribonucleoprotein particles isolated from the cytoplasm of duck erythroblasts. A flow scheme of mRNA through the cell, slightly modified from a recent review (10) is proposed as a theoretical framework for analyzing the relationship between the different mRNP characterized.

Identification and Characterization of the Translationally Repressed Cytoplasmic Globin Messenger‐Ribonucleoprotein Particles from Duck Erythroblasts

Globin messenger ribonucleoprotein (mRNP) particles which have been isolated from duck erythroblast post-polyribosomal supernatant are translationally inactive in vivo and in vitro but contain translatable mRNA active after deproteinisation. They were characterized following purification by successive sucrose gradient sedimentation in a buffer containing 0.05 M KCl. The complex, which sediments homogeneously at about 20 S, has a density of 1.39 g/cm3 and thus consists of four parts protein to one part RNA; 40% of this RNA is globin mRNA and no other mRNA could be detected. Sedimentation of the purified globin mRNP on sucrose gradients in 0.5 M KCl produced four components while polyacrylamide gel electrophoresis in non-denaturing conditions and in the presence of EDTA resulted in the separation of three components. Hybridization to globin cDNA and translation in vitro of the RNA extracted from these subparticles revealed the existence of two core particles containing globin mRNA with nominal sedimentation coefficients of 13 S and 16 S. Analysis of the protein components of the isolated sub-complexes by dodecyl sulfate and bidimensional gel electrophoresis indicated a very characteristic protein composition for each of these complexes. The 16-S and 13-S globin mRNPs differed essentially by the presence in the 13-S mRNP only of a group of major polypeptides. Of the other two sub-complexes, one consisted of 90% small RNA in the 4-S range; the second sedimented ahead of the globin mRNP core particles at about 19S and consisted of a very characteristic set of about 14 polypeptides. The polyribosomal 73000-Mr poly(A)-binding protein was not detected in the purified free globin mRNP although the mRNA in the untranslatable particle is polyadenylated. The presence in the cytoplasm of duck erythroblasts of two forms of untranslated globin messenger ribonucleoprotein particles, distinct in their protein composition from polyribosomal globin mRNP, suggests that they may have a specific role in the regulation of translation of globin mRNA.

Two low molecular weight nuclear RNAs, isolated from avian erythroblast nuclear ribonucleoprotein complexes, hybridize to duck pre-messenger and globin messenger RNA

Low molecular weight RNAs (1mw RNA) extracted from isolated small nuclear ribonucleoprotein complexes (snRNP) of purified duck erythroblast nuclei were enzymatically labeled in vitro at the 5′ terminus using ( 32P)-ATP and T 4 polynucleotide kinase (1). Hybridization of these labeled 1mw RNA species to purified fractions of duck nuclear and cytoplasmic RNA revealed 2 principal RNA species of approximately 27,000 and 58,000 molecular weight which hydrogen-bonded to nuclear pre-messenger RNA (both poly(A) + and poly(A) −) and to purified cytoplasmic globin messenger RNA.

Characterization of pre-messenger-RNA-containing nuclear ribonucleoprotein particles from avian erythroblasts.

Ribonucleoprotein particles have been isolated from duck erythroblast nuclei using a procedure designed to produce maximal cytoplasmic dispersion with minimal release of endogenous hydrolytic enzymes. The RNA extracted from the purified nuclear ribonucleoprotein fraction is shown to contain globin messenger RNA sequences at a concentration comparable to that present in total nuclear RNA. The polypeptide composition of this fraction revealed by electrophoresis in two dimensions is complex, consisting of at least 65 acidic species and 21 basic species. Several lines of evidence suggest that these are authentic components of nuclear ribonucleoprotein. The so-called 'core' proteins of nuclear ribonucleoprotein which were previously shown to migrate as a single band on low-pH urea gels, and as six bands on sodium dodecyl sulphate gels are here shown to be considerably more complex being resolved by two-dimensional electrophoresis into a group of 15 basic and 6 more and less neutral polypeptides. Isoelectric focusing of nuclear ribonucleoprotein under non-denaturing conditions suggests that these latter species are not uniformly distributed along the pre-messenger RNA molecule.

Globin mRNA Precursor

The globin mRNA sequences present in duck erythroblast nuclei appear under non-denaturing conditions to be associated with heterogeneous nuclear RNA (hnRNA) molecules of various sizes. Under denaturing conditions, however, the bulk of the globin mRNA sequences associated with hnRNA are released as molecules of size close to that of the active globin mRNA. To find out whether hydrogen-bonded structures occur in situ or arise after RNA extraction, nuclei were treated with aminomethyltrioxalen and exposed to ultraviolet light. This treatment generates covalent links between opposite strands of double-stranded nuclei acids, which were visualised by electron microscopy. It appears that, after cross-linking, a fraction of the globin mRNA sequences present in nuclei is associated with high-molecular-weight hnRNA molecules by a link found associated with a band of 0.9 x 10(6) molecular weight approximately. It is suggested that within the erythroblast nucleus, globin mRNA sequences are associated by hydrogen bonds with RNA of high molecular weight. These structures may represent intermediate steps in globin mRNA processing.

Characterization and Kinetics of Synthesis of 15S -Globin RNA, a Putative Precursor of -Globin mRNA

Several authors (for review, see Darnell et al. 1973) have proposed that mRNA of eukaryotic cells is derived by processing from heterogeneous nuclear RNA (hnRNA), a class of RNA with a high rate of turnover (Soeiro et al. 1968; Brandhorst and McConkey 1974), sedimenting between 20S and 100S (Scherrer et al. 1963; Georgiev et al. 1963; Warner et al. 1966). A number of reports have dealt with the detection of sequences specific for a particular mRNA in high-molecular-weight hnRNA. Polysome-derived globin mRNAs, with a chain length of 600–700 nucleotides, sediment at about 9S (Chantrenne et al. 1967). It has been reported that globin-specific sequences could be detected in duck erythroblast nuclear RNA and mouse fetal liver RNA sedimenting above 30S (Imaizumi et al. 1973; Williamson et al. 1973). In addition, Macnaughton et al. (1974) identified globin-specific sequences in duck erythroblast nuclear RNA sedimenting at 14S. On the other hand, Spohr...