Protein synthesis directed by polyadenylated and non-polyadenylated RNA isolated from membrane-bound and free polysomes of Tetrahymena pyriformis

Free and membrane-bound polysomes were prepared from the protozoa Tetrahymena pyriformis using a procedure which gives good recovery and practically no cross-contamination. Polysomes are intact as analysed by sedimentation analysis. Poly(A)-rich RNA and poly(A)-free RNA, isolated from both populations of polysomes, show similar electrophoretic patterns. These RNAs were translated in the rabbit reticulocyte lysate cell-free system and the translation products were analysed by one-dimensional and two-dimensional gel electrophoresis. The most striking differences were found in the two-dimensional electrophoretic analysis namely: (a) a group of polypeptides (10) is synthesized mainly on membrane-bound polysomes, (b) a second abundant group is synthesized mainly in free polysomes (c) and a third class of polypeptides is synthesized on both kinds of polysomes. Poly(A)-free RNAs, isolated from free polysomes, are also able to promote synthesis of some polypeptides. The results are discussed taking into account the fact that T. pyriformis is a non-secretory cell.     

Site of tubulin synthesis in Tetrahymena pyriformis

Free and membrane-bound polysomes were isolated from the protozoa Tetrahymena pyriformis, and the contribution of these two types of polysomes to tubulin synthesis were studied using immunoprecipitation of the 35S-translational products in a rabbit reticulocyte lysate. One-dimensional electrophoretic analysis shows that tubulin is synthesized by polyadenylated RNA isolated from free and membrane-bound polysomes. Non-polyadenylated RNAs of free polysomes are also able to direct tubulin synthesis. Two-dimensional electrophoretic analysis using O'Farrell's system confirms these results and also reveals the existence of the alpha- and beta-tubulin subunits.     

Cell-Free Translation of Free and Membrane-Bound Polysomes and Polyadenylated mRNA from Rabbit Brain Following Administration of d-Lysergic Acid Diethylamide In Vivo

Abstract: Free and membrane-bound polysomes and polyadenylated mRNA isolated from rabbit brain were translated in an mRNA-dependent rabbit reticulocyte lysate system. Electrophoretic analysis of the cell-free translation products demonstrated that although most of the nascent proteins were common to both free and membrane-bound brain polysomes, qualitative and quantitative differences were observed. Compared with the results obtained with purified polyadenylated mRNA, the addition of intact polysomes to the cell-free translation assay was more efficient and produced higher molecular weight products. Analysis of the translation products of free and membrane-bound polysomes revealed the appearance of 74K protein following either LSD administration or hyperthermia induced by elevated temperature treatment. The presence of this 74K protein was verified by analysis of the translation products by two-dimensional gel electrophoresis.     

In-vitro translation of different mRNA-containing fractions of Chlamydomonas chloroplasts

Starting from isolated chloroplasts of the Chlamydomonas reinhardii cw 15 mutant, several mRNA-containing chloroplast subfractions, i.e. thylakoid-bound polysomes, detached polysomes or isolated RNA, were prepared and incubated in homologous and heterologous translation systems. In the reticulocyte lysate these fractions gave rise to strikingly different product patterns. A most prominent difference concerned the in-vivo rapidly labelled 32,000-dalton thylakoid polypeptide. Neither this membrane protein nor its 34,000-dalton precursor was formed when membrane-containing or free polysomes were translated, while the 34,000-dalton precursor was a main product of the RNA isolated from the same membranes. The influence of thylakoid membranes during translation was also observed in homologous translation systems with lysed chloroplasts supplemented with ATP. Membrane and soluble fractions, when translated separately, yielded product patterns which differed from each other, although the RNAs extracted from the respective fractions gave the same product patterns when translated in reticulocyte lysate; the latter included a soluble protein, the large subunit of ribulose-1,5-bisphosphate carboxylase, and a membrane protein, the 34,000-dalton precursor of the 32,000-dalton membrane protein, as major labelled translation products. These results point to a regulatory role of thylakoid membranes in the expression of chloroplast mRNA and argue against compartmentation of the chloroplast mRNAs between the soluble and membrane fractions.Abbreviation SDS sodium dodecyl sulfate     

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. I. Functional tests on rat liver microsomal subfractions

A preparation of rat liver microsomes containing 70% of the total cellular endoplasmic reticulum (ER) membranes was subfractionated by isopycnic density centrifugation. Twelve subfractions of different ribosome content ranging in density from 1.06 to 1.29 were obtained and analyzed with respect to marker enzymes, RNA, and protein content, as well as the capacity of these membranes to bind 80S ribosomes in vitro. After removal of native polysomes from these microsomal subfractions by puromycin in a buffer of high ionic strength their capacity to rebind 80S ribosomes approached levels found in the corresponding native membranes before ribosome stripping. This indicates that in vitro rebinding of ribosomes occurs to the same sites occupied in the cell by membrane-bound polysomes. Microsomes in the microsomal subfractions were also tested for their capacity to effect the translocation of nascent secretory proteins into the microsomal lumen utilizing a rabbit reticulocyte translation system programmed with mRNA coding for the precursor of human placental lactogen. Membranes from microsomes with the higher isopycnic density and a high ribosome content showed the highest translocation activity, whereas membranes derived from smooth microsomes had only a very low translocation activity. These results indicate the membranes of the rough and smooth portions of the endoplasmic reticulum are functionally differentiated so that sites for ribosome binding and the translocation of nascent polypeptides are segregated to the rough domain of the organelle.     

Extraction from free ribosomes of a factor mediating ribosome detachment from rough microsomes.

A salt extract of rabbit reticulocyte free monosomes or polysomes contains a factor with an activity that detaches membrane bound monosomes but not polysomes from dog pancreas rough microsomes. It is proposed that this activity, referred to as detachment factor, functions in the dissociation of membrane bound ribosomes from the microsomal membrane after each round of translation. In addition to free ribosomes, the factor is also present in a ribosome-free, high speed supernatant, the cell fractionation equivalent of the cytosol. The factor can be extracted from free ribosomes of a variety of tissues and species, and is able to function on ribosome membrane junctions homologous as well as heterologous with respect to its source.     

Cell-Free Synthesis of the D2-Cell Adhesion Molecule: Evidence for Three Primary Translation Products

The D2-cell adhesion molecule (D2-CAM) is a membrane glycoprotein that is involved in cell-cell adhesion in the nervous system. To study the biosynthesis of D2-CAM we have translated free and membrane-bound polysomes from rat brain in vitro in the rabbit reticulocyte lysate system. D2-CAM was exclusively synthesized on membrane-bound polysomes. The primary translation products of D2-CAM were three polypeptides of apparent molecular weights 187,000, 134,000, and 112,000. No interconversion between these polypeptides was detected. In contrast to previous suggestions, we conclude that all three D2-CAM polypeptides are primary translation products. When translating polysomes from embryonic and postnatal rat brain, we found that the relative amounts of the three polypeptides synthesized varied with age. Their molecular weights, however, were not age-dependent.     

Site of synthesis of the alpha and beta subunits of the Na,K-ATPase in brine shrimp nauplii

Developing nauplii (embryos) of the brine shrimp Artemia salina are an excellent model system for studying the biogenesis of the sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase). The nauplii exhibit a burst of Na,K-ATPase synthesis between 6 and 32 h of development (Peterson, G. L., Churchill, L., Fisher, J. A., and Hokin, L. E. (1982) J. Exp. Zool. 221, 295-308). We have now determined the sites of synthesis of the alpha and beta subunits of the Na,K-ATPase in developing A. salina nauplii. Membrane-bound and free polysomes were isolated from nauplii, and RNA was extracted from the polysomes. The polysomal RNA was translated in vitro in a rabbit reticulocyte lysate, and the translation products were immunoprecipitated by anti-subunit antisera. The immunoprecipitated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. Our data show that the alpha subunit precursor is synthesized on membrane-bound polysomes and the beta subunit precursor is synthesized on free polysomes. In addition, the alpha subunit precursor appears as two separate peptides on sodium dodecyl sulfate-polyacrylamide gels, which suggests that the two alpha subunit forms seen in mature brine shrimp Na,K-ATPase are products of two distinct messenger RNAs. The beta subunit precursor appears as a single discrete band, unlike the mature beta subunit, which appears as a diffuse band.     

Circular polysomes predominate on the rough endoplasmic reticulum of somatotropes and mammotropes in the rat anterior pituitary

We have studied the shape and size distribution of membrane-bound polysomes in somatotropes and mammotropes, which are the sources, respectively, of growth hormone and of prolactin in the rat pituitary. The observations were made in conventional electron micrographs of these cells in situ, where occasional surface or en face views of the rough endoplasmic reticulum allow the polysomes to be seen as rows of ribosomes arranged in distinctive patterns on the membranes. It is possible by this means to characterize the shape and number of ribosomes for the total population of bound polysomes in the respective cell types. The great majority of membrane-bound polysomes in these two cell types (81% in somatotropes, 78% in mammotropes) have an approximately circular shape and contain an average of 6.8 (somatotropes) or 6.5 (mammotropes) ribosomes, which is an appropriate size for translation of the polypeptide hormones produced by these cells. About 17% of the membrane-bound polysomes in somatotropes and 20% in mammotropes have a spiral shape, resembling somewhat the letter "G," and contain about eight to nine ribosomes in both cell types. The preponderance of circular polysomes on the rough endoplasmic reticulum of somatotropes and mammotropes suggests the possibility that ribosomes (or the 40S ribosomal subunit) may recycle on the polysome after the translation of growth hormone or of prolactin.     

Translation of messenger RNA fractions extracted from free and membrane bound rat forebrain ribosomes in a rabbit reticulocyte cell-free system

Messenger RNA fractions were obtained from free and membrane bound rat forebrain ribosomes by alkaline phenol extractions. These RNA fractions stimulated protein synthesis in a cell-free rabbit reticulocyte system partially dependent on the addition of exogenous mRNA. The polypeptide products of protein synthesis with RNA fractions derived from free and membrane bound brain ribosomes and reticulocyte ribosomes were compared by polyacrylamide gel electrophoresis and found to have different distributions.     

Immunological detection of the messenger RNA cap-binding protein

The 24-kilodalton messenger RNA cap-binding protein (CBP) was purified from the rabbit reticulocyte postribosomal supernatant fraction using an affinity resin consisting of the p-aminophenyl gamma-ester of m7GTP coupled to Sepharose. The affinity-purified CBP was used to raise a goat antiserum. Anti-CBP antibodies were purified by adsorption to CBP coupled to either Controlled-Pore Glass or diazobenzyloxymethyl paper. The affinity-purified antibodies reacted specifically with only the 24-kilodalton polypeptide in whole reticulocyte lysate and in initiation factors prepared from the same source. During a conventional (nonaffinity) purification of CBP from a high salt extract of the ribosomal pellet, immunological reactivity paralleled the ability to reverse cap analogue inhibition of translation, indicating that the 24-kilodalton polypeptide present in the postribosomal supernatant fraction is immunologically cross-reactive with the CBP purified from ribosomes. Fractionation of whole reticulocyte lysate by sucrose gradient sedimentation followed by immunoblotting revealed that CBP was present in the supernatant fraction and the region of the gradient corresponding to ribosomal subunits but not in mono- or polysomes. The CBP to ribosome ratio was found to be approximately 0.02, assuming that the m7GTP-Sepharose retains all of the protein. This is considerably lower than that of other initiation factors and suggests that CBP may be the limiting polypeptide factor involved in the initiation of protein synthesis. The antibodies also inhibited the translation of a capped messenger RNA (globin). Inhibition of the translation of an uncapped RNA (satellite tobacco necrosis virus) was also observed, but to a lesser degree than with globin mRNA.     

In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate.

The translation of poliovirus RNA in rabbit reticulocyte lysate was examined. Translation of poliovirus RNA in this cell-free system resulted in an electrophoretic profile of poliovirus-specific proteins distinct from that observed in vivo or after translation in poliovirus-infected HeLa cell extract. A group of proteins derived from the P3 region of the polyprotein was identified by immunoprecipitation, time course, and N-formyl-[35S]methionine labeling studies to be the product of the initiation of protein synthesis at an internal site(s) located within the 3'-proximal RNA sequences. Utilization of this internal initiation site(s) on poliovirus RNA was abolished when reticulocyte lysate was supplemented with poliovirus-infected HeLa cell extract. Authentic P1-1a was also synthesized in reticulocyte lysate, indicating that correct 5'-proximal initiation of translation occurs in that system. We conclude that the deficiency of a component(s) of the reticulocyte lysate necessary for 5'-proximal initiation of poliovirus protein synthesis resulted in the ability of ribosomes to initiate translation on internal sequences. This aberrant initiation could be corrected by factors present in the HeLa cell extract. Apparently, under certain conditions, ribosomes are capable of recognizing internal sequences as authentic initiation sites.     

Incomplete polypeptides are formed in vitro by premature chain termination

The mechanism of incomplete polypeptides formation during protein synthesis was studied in the wheat germ cell-free system programmed with brome mosaic virus RNA 4. The synthesis of coat protein, the complete product of RNA 4 translation, was accompanied by the appearance of polypeptides of lower molecular mass. It was shown that incomplete products are formed by translation of different lengths of RNA 4, always from the first 5' AUG codon, and were due neither to proteolysis of coat protein nor to the translation of nucleolytic fragments of mRNA. The molecular masses of incomplete products were determined and the nucleotide sequence of RNA 4 was examined in the regions where wheat germ ribosomes stop translating. It was found that they contained, on average, a slightly higher guanosine content than the total coding part of RNA 4. Translation of RNA 4 in the reticulocyte lysate resulted in a marked diminution of incomplete polypeptides. Addition of high-speed supernatant from reticulocyte lysate prevented the formation of incomplete products during translation of RNA 4 in the wheat germ system. This suggests that reticulocyte lysate contains some factor(s) which facilitate the movement of ribosomes beyond the regions where the elongation is retarded.     

Heating RNA before cell-free translation gives no selective increases among the translation products of RNA from rat heart and mammary gland, rabbit reticulocyte membranes, or trout liver

A recent study has reported that the heating of a population of guanidinium-extracted mRNAs prior to translation causes a selective increase in the translation of certain mRNAs. To determine if this phenomenon is a general property of mRNAs, we carried out a comparison of the translation products obtained when phenol-extracted rat heart and mammary gland RNAs, rabbit reticulocyte membrane RNAs, and trout liver RNAs were translated in the reticulocyte translation system, with and without a prior heat treatment. Our results show that no selective increase in the translation of mRNAs was observed for any of these samples. Among the 14 RNA preparations examined, one total mammary RNA preparation did display a twofold increase in the translation of all mRNAs after heat treatment. It is shown that the heat enhancement of translational activity observed for this sample was due to the reversible formation of intermolecular aggregates with a contaminant that can be removed by chromatography on oligo(dT)-cellulose. Since heat treatment did not selectively enhance the activity of any mRNA in these samples, our results show that the current practice of translating phenol-extracted RNAs without a prior heat treatment should be satisfactory for the translation of most mRNA populations.     

Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver

We investigated the site of synthesis of two abundant proteins in clofibrate-induced rat hepatic peroxisomes. RNA was extracted from free and membrane-bound polysomes, heated to improve translational efficiency, and translated in the mRNA-dependent, reticulocyte-lysate- cell-free, protein-synthesizing system. The peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase-beta-hydroxyacyl-CoA dehydrogenase 35S- translation products were isolated immunochemically, analyzed by SDS PAGE and fluorography, and quantitated by densitometric scanning. The RNAs coding for these two peroxisomal proteins were found predominantly on free polysomes, and the translation products co-migrated with the mature proteins. As in normal rat liver, preproalbumin and catalase were synthesized mainly by membrane-bound and by free polysomes, respectively. mRNAs for a number of minor 35S-translation products also retained by the anti-peroxisomal immunoadsorbent were similarly found on free polysomes. These results, together with previous data, allow the generalization that the content proteins of rat liver peroxisomes are synthesized on free polysomes, and the data imply a posttranslational packaging mechanism for these major content proteins.     

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.