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Inhibition of cell-free protein synthesis by low-molecular-weight nuclear polyadenylate-containing ribonucleic acid species isolated from the lactating guinea pig.

ABSTRACT: 1. Poly(A)-containing RNA was isolated from the nuclei of mammary gland, liver and brain of lactating guinea pigs. 2. Total nuclear poly(A)-containing RNA from mammary gland inhibited mRNA-directed protein synthesis by a wheat-germ cell-free system. It also inhibited the endogenous activity of the wheat-germ and other cell-free systems. It did not inhibit a wheat-germ cell-free system directed by poly(U). 3. Total nuclear poly(A)-containing RNA from liver and brain did not inhibit the mRNA-directed wheat-germ system. 4. Fractionation of the nuclear poly(A)-containing RNA revealed inhibitory activity in the less than 10 S fraction from mammary gland as well as that from liver and brain. 5. The mechanism of protein-synthesis inhibition appeared to be at the level of elongation. 6. The inhibitory activity could be reversed in a wheat-germ system by increasing the amount of S-30 supernatant. 7. The mechanism of inhibition of protein synthesis is discussed in relation to other RNA species known to inhibit such systems.

SUBMITTER: Bathurst IC

PROVIDER: S-EPMC1161609 | biostudies-other | 1980 Feb

REPOSITORIES: biostudies-other

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Project description:1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87--93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.

Project description:1. RNA isolated from the post-nuclear supernatant of the lactating guinea-pig mammary gland was fractionated with oligo(dT)-cellulose into three populations; those that bound at ;low salt' [long poly(A) tracts, 78-32 nucleotides]; those that bound at ;high salt' [shorter poly(A) tracts, 48-21 nucleotides]; and those that did not bind [no poly(A) or short poly(A) tracts, <20 nucleotides]. Nuclear RNA was fractionated into two populations, those that bound in ;low salt' and those that did not bind. All the post-nuclear RNA fractions directed the synthesis of milk proteins in a Krebs II ascites cell-free system. 2. (3)H-labelled DNA complementary to the post-nuclear poly-(A)-containing RNA population (low-salt fraction) was fractionated into abundant (milk-protein mRNA), moderately abundant and scarce sequences. This complementary DNA was then used to investigate the distribution of the mRNA sequences in the different RNA populations. This showed that all sequences were present in polyadenylated and non-polyadenylated fractions, but that major quantitative differences were apparent. The abundant milk-protein mRNA sequences predominated in the ;low-salt' post-nuclear poly(A)-containing RNA fraction, whereas the moderately abundant sequences predominated in the non-polyadenylated post-nuclear RNA fraction. In total cellular RNA, those sequences deemed initially to be moderately abundant within the ;low-salt' poly(A)-containing RNA population were present at a concentration very similar to those of the abundant milk-protein mRNA (approx. 6x10(5) copies of each sequence/cell). Similarly, analysis of the nuclear RNA populations showed that the ;abundant' and so-called ;moderately abundant' sequences were present in essentially identical concentrations (2x10(3) copies of each sequence/cell). The majority of these (90-95%) were non-polyadenylated. 3. The results are discussed in terms of the post-transcriptional mechanisms involved in the regulation of gene expression in the lactating guinea-pig mammary gland.

Project description:1. The major milk proteins synthesized by the lactating mammary gland of the guinea pig were identified and designated as caseins A, B and C and alpha-lactalbumin, with estimated mol.wts. of 28000, 25500, 20500 and 14500 respectively. 2. Antisera to the total casein fraction and to alpha-lactalbumin were prepared from rabbits. The milk proteins were also iodinated with either 131I or 125I. 3. A poly(A)-rich RNA fraction was isolated from lactating guinea-pig mammary glands. Isolation was by affinity chromatography on oligo(dT)-cellulose. 4. Examination of this RNA fraction by electrophoresis on polyacrylamide gels containing formamide indicated three major species 1, 2 and 3, with estimated wol.wts. of 5.4 X 10(5) and 3.3 X 10(5), and the apparent absence of rRNA species. 5. The poly(A)-rich RNA stimulated protein synthesis in heterologous cell-free systems based on wheat germ, Krebs II ascites-tumour cells, and the latter supplemented with an initiation factor-3 fraction from rabbit reticulocyte ribosomes. 6. Between 80 and 90% of the protein synthesis directed by the mRNA was for milk proteins. 7. Analysis of the proteins immunoprecipitated by the alpha-lactalbumin antiserum showed in the wheat-germ system that the product was a protein with a molecular weight greater than that of alpha-lactalbumin, whereas in the ascites-tumour-cell systems both this protein and alpha-lactalbumin were found. When the larger protein was treated with CNBr and the resulting peptides were examined, it was shown that the extra peptide was at the N-terminus. This and other evidence is adduced for the initial translation product of alpha-lactalbumin being a precursor with an addition of about ten amino acids at the N-terminus. 8. Similar analysis of the casein immlnospecific proteins produced under the direction of mRNA indicated that the products had a molecular weight that was apparently a littel smaller than that of the caseins synthesized in vivo. This was not consistent with higher-molecular weight casein precursors. 9. Possible explanations for the results obtained are discussed, especially in terms of the physiological significance of the pre-alpha-lactalbumin as a secretory protein.

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Inhibition of cell-free protein synthesis by low-molecular-weight nuclear polyadenylate-containing ribonucleic acid species isolated from the lactating guinea pig.

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