The Size and Distribution of Cytochrome c Oxidase Subunit IV mRNA between Free and Membrane‐Bound Polyribosomes

Self Cite

The biosynthesis of al-antitrypsin, a secretory glycoprotein, has been studied in vitro in a cell-free translation system from wheat germ and in vivo with hepatocytes. When messenger RNA from rat liver polysomes was translated in a heterologous cell-free system a translation product was immunoprecipitated by anti-(al-antitrypsin) which had a smaller molecular weight (42 000) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis than the authentic glycoprotein (54000) isolated from rat serum. An intact amino terminus of the al-antitrypsin synthesized in vitvo could be demonstrated by repetitive Edman degradation.When hepatocytes, labeled with [35S]methionine, were analyzed with respect to newly synthesized ccl-antitrypsin, a protein with a molecular weight of 49 000 was immunoprecipitated from a high-speed supernatant. When the hepatocytes were treated with tunicamycin, an inhibitor of protein glycosylation, a protein of a molecular weight of 40000 was immunoprecipitated from the cells as well as from the medium. Interestingly, an al-antitrypsin form of an even higher molecular weight (54000) was immunoprecipitated from the hepatocyte monolayer medium. Several conclusions can be drawn from these findings. First, al-antitrypsin is synthesized as a largermolecular-weight precursor with an extra amino-terminal sequence of about 20 amino acids. Second, the liver form of al-antitrypsin is different from the El-antitrypsin which has been secreted, and third, the carbohydrate moieties of rat liver ccl-antitrypsin do not appear to represent an absolute requirement for the secretion process.

Section: Discussionmentioning

confidence: 99%

Section: Preparation and Fractionation Of Po1ysomesmentioning

confidence: 99%

See 1 more Smart Citation

Synthesis of α₁‐Antitrypsin in Rat‐Liver Hepatocytes and in a Cell‐Free System

Geiger

Northemann

Schmelzer

et al. 1982

Self Cite

“…KCl treatment of free mRNPs mRNPs were treated with 0.5 M KC1 as described elsewhere [6], with the following modifications. An equal volume of 1.0 M KCl in buffer B (10 mM Tris/HCl, pH 7.5, 5 mM MgC12) was added to the mRNPs fraction.…”

Section: Isolation Of Free Cytoplasmic Mrnpsmentioning

confidence: 99%

Small cytoplasmic RNAs from human placental free mRNPs

Lorberboum

Digweed

Erdmann

et al. 1986

A new family of small cytoplasmic RNA species (scRNAs) was found to be associated with human termplacental free messenger ribonucleoprotein particles (mRNPs). Placental scRNAs strongly inhibit translation of both homologous and heterologous mRNAs in a cell-free rabbit reticulocyte system. scRNAs could be resolved into at least four different RNA species. One of the RNA molecules, scRNA species 1, was the most potent protein synthesis inhibitor found among the placental scRNAs. The nucleotide sequence of the scRNA species 1 was determined. In spite of its short length, scRNA species 1 still exhibited a very strong inhbitory effect on the in vitro protein synthesis. scRNAs were found to be complexed with proteins in the form of scRNPs. Proteins of these complexes enhanced the inhibitory effect of scRNAs on in vitro translation. Experiments provided evidence that inhibition of in vitro protein synthesis by the scRNAs is not dependent upon mRNA concentration. However, inhibition can be overcome by increasing the ratio lysate/scRNAs, thus suggesting that scRN As act on some essential component of the cell-free system. The degree of inhibition is decreased when scRNAs are added after the start of translation, suggesting that scRNAs (or scRNPs) interfere with the initiation stage of translation, probably acting on an initiation factor(s). Placental scRNAs are unique in their size, being smaller than other known scRNAs. Their association with free cytoplasmic repressed mRNPs in human placenta suggests that scRNAs play a role in the regulation of mRNP metabolism and, consequently, in the control of mRNA translation.A considerable portion of cytoplasmic mRNAs in various eukaryotic cells is found outside the polyribosomes as proteinbound complexes [l]. A growing body of evidence, using mRNA-specific probes [2 -41, indicates that, depending upon the requirements of the cell, potentially functional mRNA can be stored as a repressed mRNA in the form of free cytoplasmic messenger ribonucleoprotein particles (mRNPs) until needed.Evidence that these free cytoplasmic mRNPs are inactive in protein synthesis stems from unsuccessful attempts to translate these particles in in vitro cell-free systems from a variety of eukaryotic sources [5 -81. It has been proposed that the masking of translational activity of the mRNA in the free cytoplasmic mRNP particles may be due to translational repressor protein(s) [9, 101. Nevertheless, until the present no one has demonstrated, either in vivo or in vitro, the involvement of specific protein(s) in this process. Others have suggested that this repressed state of the mRNPs is due to translational control RNA [6, 13 -151. Free cytoplasmic mRNP complexes have been isolated by us from human term placenta and have been characterized [16]. These placental free cytoplasmic mRNPs are not translated in a cell-free system,

“…On the other hand, depending on the conditions of isolation translationally active [7-91 as well as inactive forms of nonpolysomal cytoplasmic mRNPs [5,7,8,10-131 have been described. It has been proposed that the masking of translational activity of free cytoplasmic RNP particles may be due to a translational repressor protein [lo, 121 or translational control low-molecular-weight RNA [8,11,14,15].In previous studies we have isolated 40-S free nonpolysomal RNP particles from rat liver, which were not translatable. Even the deproteinized RNP particles showed only weak incorporation of [3H]leucine into proteins in a cell-free wheat germ system, indicating that the mRNA is in a repressed form or that there are other molecules which inhibit the translation of the mRNP RNA.…”

mentioning

confidence: 99%

Inhibition of Cell‐Free Protein Synthesis by Low‐Molecular‐Weight RNAs from Free Cytoplasmic Ribonucleoprotein Particles

Kühn¹,

Villringer²,

Falk³

et al. 1982

Self Cite

Free cytoplasmic messenger ribonucleoprotein (mRNP) particles from rat liver were treated with EDTA and separated into two populations of RNP particles with sedimentation maxima of 20 S and 35 S respectively. The 20-S and 35-S RNP particles, treated with 0.5 M KCI, have protein-to-RNA ratios of 0.31 : 1 and 5.7: 1 respectively. Whereas 20-S and 35-S RNP particles exhibit a similar protein complement of seven major polypeptides, the low-molecular-weight RNA components of the two particle populations are different. A characteristic set of distinct low-molecular-weight RNAs is found for 20-S and 35-S RNP particles.When the individual low-molecular-weight RNAs of 20-S and 35-S RNP particles isolated from preparative polyacrylamide gels were assayed for their capability to inhibit protein synthesis in vitro, several potent translational inhibitory RNAs were detected. In particular, the low-molecular-weight RNAs of 147, 203 and 263 nucleotides in length associated with the 35-S RNP particles turned out to be strong inhibitors of protein synthesis.It is now generally accepted that mRNA in the cytoplasm of eukaryotic cells is always found to be complexed with proteins (for reviews see [l -31). Two types of mRNA . protein complexes, designated as polysomal and free cytoplasmic ribonucleoprotein particles, have been described. The polysoma1 mRNPs are translatable in cell-free systems [4-61. On the other hand, depending on the conditions of isolation translationally active [7-91 as well as inactive forms of nonpolysomal cytoplasmic mRNPs [5,7,8,10-131 have been described. It has been proposed that the masking of translational activity of free cytoplasmic RNP particles may be due to a translational repressor protein [lo, 121 or translational control low-molecular-weight RNA [8,11,14,15].In previous studies we have isolated 40-S free nonpolysomal RNP particles from rat liver, which were not translatable. Even the deproteinized RNP particles showed only weak incorporation of [3H]leucine into proteins in a cell-free wheat germ system, indicating that the mRNA is in a repressed form or that there are other molecules which inhibit the translation of the mRNP RNA. The latter possibility was tested by adding RNA from 40-S free mRNP particles to a cell-free translation system programmed with polysomal RNA. A strong inhibition of translation was observed in vitro. It was our working hypothesis that the observed inhibition of translation was due to the presence of low-molecular-weight RNAs in mRNP RNA.In the present paper we present evidence that several lowmolecular-weight RNAs can be isolated from previously described 40-S free mRNP particles after EDTA treatment and further fractionation. Some of them were shown to be strong inhibitors of cell-free protein synthesis. The isolation of 40-S RNP particles from a postpolysomal rat liver supernatant has been previously described in detail [ll]. 30 ml pooled fractions of the 40-S RNP particles in buffer A (50 mM Tris/HCl, pH 7.5, 0.25 M sucrose, 25 mM KCI, 5 mM MgC12 and 0.15 mg heparin...