Synthesis of delta crystallin from embryonic chick lens messenger ribonucleoprotein complex

A single, major 21 S messenger ribonucleoprotein (mRNP complex) was isolated and purified by sucrose gradient centrifugation after EDTA treatment of high salt washed polysomes from 15 day embryonic chick lenses. A 17 S mRNA was released from the 21 S mRNP. The 21 S mRNP complex coded for a 50 000 molecular weight protein identical to the subunit of delta crystallin. Similar results were obtained with the 17 S mRNA released from the 21 S mRNP complex.     

Possible involvement of messenger RNA-associated proteins in protein synthesis

Two distinct forms of globin messenger RNA were isolated from mouse spleen cells infected with Friend erythroleukemia virus: polyribosomal messenger ribonucleoprotein particles (15S mRNP), and their corresponding protein-free mRNAs obtained by chemical deproteinization. The translation efficiencies of both messenger forms were assayed in a Krebs II ascites cell-free system. Selective removal of RNA-binding proteins from the ascites cell lysate did not affect globin synthesis when the mRNA was supplied as 15S mRNP; deproteinized mRNA however was not translated. Only in the presence of two fractions of RNA-binding proteins was the protein-free mRNA translated. Some of the RNA-binding proteins have the same molecular weights and isoelectric points as the principal proteins of 15S mRNP.     

The relationship between polyribosomal and latent membrane-bound messenger ribonucleoprotein particles in Artemia embryos

Polysomal messenger ribonucleoprotein (mRNP) particles from developing Artemia cysts were isolated, characterized and compared with latent membrane-bound mRNP particles isolated from dormant cysts. The polyribosomal mRNP particles sedimented between 25-35 S in a sucrose gradient and had a buoyant density of 1.33 g/cm3 in Cs2So4. Latent particles had a higher sedimentation coefficient and lower buoyant density. The poly(A) + RNA in the two kinds of particles was comparable in size, 10-20 S. The protein composition of the particles, as determined by electrophoresis, was different. Polyribosomal particles contained 9 major and 6 minor proteins; a 72 k poly(A)-associated protein was present. Latent particles were characterized by a complex protein pattern ranging in apparent mol. wt between 14,000-140,000. Some proteins with similar molecular weight and isoelectric point were probably common to both kinds of particles.     

Prosomes and their multicatalytic proteinase activity.

Prosomes were first described as being mRNA-associated RNP (ribonucleoprotein) particles and subcomponents of repressed mRNPs (messenger ribonucleoprotein). We show here that prosomes isolated from translationally inactive mRNP have a protease activity identical to that described by others for the multicatalytic proteinase complex (MCP, 'proteasome'). By RNase or non-ionic detergent treatment, the MCP activity associated with repressed non-globin mRNP from avian erythroblasts, sedimenting at 35 S, could be quantitatively shifted on sucrose gradients to the 19-S sedimentation zone characteristic of prosomes, which were identified by monoclonal antibodies. The presence of small RNA in the enzymatic complex was shown by immunoprecipitation of the protease activity out of dissociated mRNP using a mixture of anti-prosome monoclonal antibodies; a set of small RNAs 80-120 nucleotides long was isolated from the immunoprecipitate. Furthermore, on CsCl gradients, colocalisation of the MCP activity with prosomal proteins and prosomal RNA was found, and no difference in the prosomal RNA pattern was observed whether the particles were fixed or not prior to centrifugation. These data indicate that the MCP activity is a property of prosomes, shown to be in part RNP and subcomplexes of in vivo untranslated mRNP. A hypothesis for the role of the prosome-MCP particles in maintaining homeostasis of specific protein levels is proposed.     

Non-polysomal poly(A)-containing messenger ribonucleoproteins of cryptobiotic gastrulae of Artemia salina

Non-polysomal poly(A)-containing messenger ribonucleoprotein (mRNP) of Artemia salina has been isolated by thermal chromatography on oligo(dT)-cellulose in moderate (250 mM) and low (50 mM NaCl and 5 mM MgCl2) ionic strength. The purified particles sedimented between 5 S and 30 S and banded at a density of 1.38-1.40 g/cm3 and 1.26-1.27 g/cm3 in CsCl and sucrose isopycnic centrifugation, respectively. The translatability of the mRNP in a cell-free system depended on the conditions of isolation. The protein composition of the free mRNP is independent of the conditions used in oligo(dT)-cellulose chromatography. The proteins have Mr of 87,000, 76,000, 65,000, 50,000, 45,000, 38,000 and 23,500. A specific set of proteins is associated wtih different ribonucleoproteins, although some proteins are present on multiple particles. The main 17 +/- 2-S particle is composed of proteins with Mr of 87,000, 76,000, 45,000 and 38,000. Approximately the same proteins were present on free mRNP and mRNP isolated from non-polysomal mRNP-ribosome complexes. Poly(A)-binding proteins have Mr of 38,000 and 23,500. The 38,000-Mr protein comprised at least 60% of the total mRNP protein. Poly(A)-binding proteins with Mr of 38,000 and 76,000 are also present in a free state in the cytoplasm. A relation between the main poly(A)-binding mRNP protein and the helix-destabilizing protein HD40 [Marvil, D. K., Nowak, L., and Szer, W. (1980) J. Biol. Chem. 255, 6466-6472] is discussed.     

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.     

In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p

Many messenger RNA export proteins have been identified; yet the spatial and temporal activities of these proteins and how they determine directionality of messenger ribonucleoprotein (mRNP) complex export from the nucleus remain largely undefined. Here, the bacteriophage PP7 RNA-labeling system was used in Saccharomyces cerevisiae to follow single-particle mRNP export events with high spatial precision and temporal resolution. These data reveal that mRNP export, consisting of nuclear docking, transport, and cytoplasmic release from a nuclear pore complex (NPC), is fast (∼200 ms) and that upon arrival in the cytoplasm, mRNPs are frequently confined near the nuclear envelope. Mex67p functions as the principal mRNP export receptor in budding yeast. In a mex67-5 mutant, delayed cytoplasmic release from NPCs and retrograde transport of mRNPs was observed. This proves an essential role for Mex67p in cytoplasmic mRNP release and directionality of transport.     

Cytoplasmic Poly(ADP-Ribose) Polymerase Associated with Free Messenger Ribonucleoprotein Particles in Rat Brain

Poly(ADP-ribose) polymerase associated with free cytoplasmic messenger ribonucleoprotein particles (free mRNP particles) carrying messenger RNA has been characterized in rat brain. There were first-order kinetics for NAD with an apparent Km for NAD of 90.5 +/- 0.70 microM and Vmax of 19.7 +/- 2.8 pmol ADP-ribose incorporated min-1 mg protein-1. Five poly(ADP-ribose) protein acceptors were identified in the Mr 37,000-120,000 range. It is hypothesized that ADP-ribosylation of specific free mRNP proteins might play a role in the derepression and translation of the silent mRNAs of free mRNP particles.     

Characterization of 20-S and 40-S non-polysomal cytoplasmic messenger ribonucleoprotein particles from rat liver

Two populations of free messenger ribonucleoprotein (mRNP) particles, sedimenting at 20 S and 40 S respectively, were isolated from a rat liver postpolysomal supernatant. After treatment with 0.5 M KCl and recentrifugation through a sucrose layer, the mRNP particles were characterized with respect to their low-molecular-weight RNA and protein components. 40-S and 20-S particles show very different RNA patterns. Four distinct low-molecular-weight RNA species of approximately 105, 139, 187 and 256 nucleotides were found as components of the 40-S mRNPs. The 20-S mRNP particles contain one major low-Mr RNA species of approximately 243 nucleotides and a characteristic pattern of low-Mr RNAs similar to the one found in nuclear ribonucleoprotein particles. In contrast to the low-Mr RNAs found in nuclear RNP particles most of the low-Mr RNA species present in 20-S and 40-S mRNP particles are rapidly labeled after [3H]orotate administration. Whereas the low-Mr RNA composition of 20-S and 40-S mRNP particles is very different, the protein patterns of both mRNP complexes are very similar. Six major polypeptides with the following molecular weights of 117000, 79800, 76700, 53800, 43900, 36300 and several minor ones were found in both 20-S and 40-S mRNPs. In a cell-free system from wheat germs neither 20-S nor 40-S mRNP particles stimulated the incorporation of [3H]leucine into proteins. However, phenol-extracted RNA from 20-S and 40-S mRNPs stimulated total protein synthesis 16-fold and 3-fold, respectively. Furthermore, the RNA from both mRNP pools directed the synthesis of albumin in vitro.     

Statistical modeling of mRNP transport in dendrites: A comparative analysis of β-actin and Arc mRNP dynamics

Localization of messenger RNA (mRNA) in dendrites is crucial for regulating gene expression during long-term memory formation. mRNA binds to RNA-binding proteins (RBPs) to form messenger ribonucleoprotein (mRNP) complexes that are transported by motor proteins along microtubules to their target synapses. However, the dynamics by which mRNPs find their target locations in the dendrite have not been well understood. Here, we investigated the motion of endogenous β-actin and Arc mRNPs in dissociated mouse hippocampal neurons using the MS2 and PP7 stem-loop systems, respectively. By evaluating the statistical properties of mRNP movement, we found that the aging Lévy walk model effectively describes both β-actin and Arc mRNP transport in proximal dendrites. A critical difference between β-actin and Arc mRNPs was the aging time, the time lag between transport initiation and measurement initiation. The longer mean aging time of β-actin mRNP (~100 s) compared with that of Arc mRNP (~30 s) reflects the longer half-life of constitutively expressed β-actin mRNP. Furthermore, our model also permitted us to estimate the ratio of newly generated and pre-existing β-actin mRNPs in the dendrites. This study offers a robust theoretical framework for mRNP transport, which provides insight into how mRNPs locate their targets in neurons.     

In vitro translation studies of the cytoplasmic nonpolysomal particles containing messenger RNA.

We analyzed the translational capacity of different kinds of free cytoplasmic messenger ribonucleoprotein complexes (free mRNP) in a Hela cell cell free system. Native free mRNP are not translated although free mRNP washed with 0.5 M KC1 can direct polypeptide synthesis. Furthermore, the 0.5 M KC1 wash possesses a factor which inhibits the translation of 0.5 M KC1 washed free mRNP as well as globin mRNA naked mRNA from plasmocytoma, or Hela cells. We also demonstrated that native free mRNP are able to form a complex with ribosomal subunits in the presence of initiation factors. This indicates that inhibition of translation by the 0.5 M KC1 wash occurs either at some point after initiation complex formation or at the elongation step.     

Identification of the substrates of the casein kinase II associated with non-polysomal messenger ribonucleoproteins of A. salina cryptobiotic embryos

The association of a protein kinase with cytoplasmic non-polysomal messenger ribonucleoproteins is demonstrated by chromatography on oligo(dT)-cellulose and sucrose gradient centrifugation. The cAMP-independent enzyme is inhibited by caffeine and poly(L)-glutamic acid and is classified as a casein kinase II. Among the exogenous proteins initiation factor eIF2 is the best substrate and is 7.8 times more efficiently phosphorylated than casein. Endogenous mRNP protein substrates have a Mr of 125000, 65000, 38000, 26000 and 23500. The main phosphate acceptor is the Mr38000 poly(A)-binding protein. Dephosphorylation of the poly(A)-binding protein by protein phosphatases decreases its RNA binding property. The effect of phosphorylation-dephosphorylation of mRNP proteins on the initiation of protein synthesis is discussed.Abbreviations PMSF phenylmethanesulfonyl fluoride - mRNP messenger ribonucleoprotein - iRNA small inhibitor RNA - eIF2 eukaryotic initiation factor 2     

Characterization of mouse reticulocyte free globin mRNP

Globin messenger ribonucleoprotein particles (free and polysomal) from mouse reticulocyte lysates were characterized for their mRNA composition, translational activity as well as the proteins in direct contact with them. In contrast to the homogeneous single-peak distribution of rabbit and duck reticulocyte free mRNPs, mouse free mRNP particles were heterogeneously dispersed on the sucrose density gradient into two major domains called region I and region II. Region I appeared enriched with alpha-globin mRNP and region II with beta-globin mRNP. mRNP from both regions was translationally active. Examination of lysates prepared from beta-thalassemic mice revealed a reduction of translatable beta minor mRNP within region I, supporting the hypothesis of a compensatory recruitment of beta minor free mRNP into polysomes in beta-thalassemic mice.     

Identification of a 60-kDa phosphoprotein that binds stored messenger RNA of Xenopus oocytes

Rapidly labelled, polyadenylated RNA is contained in three distinct fractions isolated from homogenized amphibian oocytes: (a) in ribonucleoprotein particles that are associated with a fibrillar matrix, the complexes sedimenting at greater than 1500S; (b) in ribonucleoprotein particles that sediment at 20-120S and have the characteristics of stored (maternal) messenger ribonucleoprotein (mRNP) and (c) in polyribosomes that sediment at 120-360S. We have compared the RNA and protein components of the first two of these RNP fractions. The polyadenylated RNA extracted from the two RNP fractions differs in that the RNA from fibril-associated RNP contains a much higher content of repeat sequences than does the RNA from mRNP. In other words, the RNA from fibril-associated RNP is largely unprocessed and may constitute a premessenger state, which for convenience is referred to as premessenger RNP (pre-mRNP). RNA-binding experiments demonstrate that the polypeptide most tightly bound in pre-mRNP is a 54-kDa component (p54), whereas the polypeptide most tightly bound in mRNP is a 60-kDa component (p60). Antibodies raised against p60 are used to show that this polypeptide is a common major component of pre-mRNP and mRNP and that it is also located in oocyte nuclei. However the state of p60 is modified between the premessenger and stored message levels: the polypeptide in mRNP is heavily phosphorylated whereas the equivalent polypeptide in pre-mRNP is completely unphosphorylated. The relative roles of the presence of repeat sequences and phosphorylation of mRNA-associated protein in blocking translation are discussed.     

Proteins associated with the messenger ribonucleoprotein particle for the estrogen-regulated apolipoprotein II mRNA.

The stability of the mRNA for apolipoprotein (apo) II is regulated by estrogen [Gordon et al. (1988) J. Biol. Chem. 263, 2625-2631]. On the hypothesis tha estrogen regulation of apoII mRNA stability is mediated through mRNA-protein interaction, we have examined the messenger ribonucleoprotein particle (mRNP) for apoII mRNA following release from chicken liver polyribosomes. Polyribosomes containing undegraded apoII mRNA were obtained when tissue was homogenized without detergent, and polyribosomes were isolated following simultaneous addition of detergent and magnesium to a 20000g supernatant. ApoII mRNP released by EDTA sedimented at 12-18 S in sucrose gradients, and banded at rho = 1.4 g/mL in CsCl isopycnic centrifugation, indicative of a 3:1 ratio of protein to mRNA. A fraction in which apoII mRNP was enriched to 40-50% of total mRNP was prepared by successive size fractionation steps on sucrose gradients. Proteins associated with sucrose gradient enriched apoII mRNP were examined by iodination of UV-cross-linked proteins followed by SDS-polyacrylamide gel electrophoresis. Comparisons of proteins in highly enriched apoII mRNP to proteins in mRNP from non-estrogen-treated rooster liver did not reveal any differences. This result suggests that the major proteins associated with apoII mRNA are mRNP proteins also associated with the bulk of liver mRNAs.     

Messenger ribonucleoprotein complexes isolated by oligodeoxythymidylate-cellulose chromatography from Neurospora crassa polysomes.

Messenger ribonucleoprotein (mRNP) complexes were isolated from ethylenediaminetetraacetic acid-dissociated polysomes of Neurospora crassa. Approximately 15% of the [3H]uridine incorporated into polysomal ribonucleic acid (RNA) during a 15-min pulse was eluted from oligodeoxythymidylate-cellulose as an mRNP complex. The isolated mRNP complexes exhibited sedimentation coefficients ranging from 15S to greater than 60S. RNA isolated from these mRNP complexes sedimented in sucrose gradients between 4S and 40S, with broad peaks at 15S and 24S. The buoyant density of mRNP complexes eluted with 25% formamide was 1.42 to 1.44 g/cm3, whereas for mRNP complexes eluted with 50% formamide it was 1.48 to 1.50 g/cm3. Six polypeptides, with molecular weights of 14,000, 19,000, 24,000, 31,000, 44,000, and 66,000, were associated with mRNP complexes eluted with 25% formamide. The mRNP complexes eluted with 50% formamide had one associated polypeptide, of molecular weight 27,000.     

Messenger ribonucleoprotein particles in silkmoth oogenesis

Silkmoth oocytes contain significant amounts of nontranslating cytoplasmic messenger RNA apparently stored until fertilization. The physical state of this mRNA was examined by bouyant density centrifugation on cesium chloride gradients. Messenger elements were isolated either by oligo(dT) cellulose chromatography or by separation of ooplasm on sucrose gradients. After CsCl density gradient centifugation the mRNA particles banded in a region (1.42–1.48 g/c³) which would indicate a substantial protein content. Electron microscope examination of mRNP fractions revealed particles ranging in size from 180–250 Å.     

Lipoxygenase mRNA in rabbit reticulocytes. Its isolation, characterization and translational repression

The synthesis of the erythroid lipoxygenase, an enzyme which is of importance for the degradation of mitochondria during the maturation of reticulocytes to erythrocytes, was studied in reticulocytes from bone marrow and in density-separated fractions from peripheral blood of anemic rabbits. Lipoxygenase mRNA was enriched to about 75% by digestion of polysomes with protease K, poly(U)-Sepharose chromatography and repeated sucrose gradient centrifugation. From sucrose gradient centrifugation, electrophoresis and electron microscopy a molecular weight of about 10(6) was calculated. Synthesis of lipoxygenase is absent in erythroblasts, in very young reticulocytes obtained from bone marrow, or in the lightest fractions of reticulocytes from the peripheral blood. More mature blood reticulocytes show a considerable synthesis of the enzyme. The induction of the synthesis of the lipoxygenase seems to be initiated when reticulocytes have reached the peripheral blood. It is shown that lipoxygenase mRNA is present in reticulocytes as a translationally inactive free cytoplasmic messenger ribonucleoprotein (mRNP) particle. After deproteinization isolated mRNA obtained from masked mRNP codes for authentic lipoxygenase in a cell-free protein-synthesizing system of reticulocytes.