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The existence of non-ribosomal rapidly-labelled ribonucleoprotein particles has been examined by isopycnic and sucrose gradient centrifugation in nuclear and cytoplasmic subcellular fractions of sheep thyroid tissue. These particles have been described in the saline extract (pH 8.0) of purified nuclei, in EDTA-treated polyribosomes and in postribosomal regions of deoxycholatetreated postmitochondrial supernatant. They have been identified as a special class of ribonucleoproteins rich in proteins by the following criteria: low buoyant densities in CsCl gradients (from 1.36 to 1.41 g/ml, with a predominance at 1.41), DNA-like base composition of their constitutive RNA, increase or decrease of their buoyant densities after trypsin or ribonuclease digestion. Although the specific nature of the constitutive protein(s) is not yet determined, it appears that both nuclei-associated and cytoplasmic (postribosomal and polyribosomal) rapidlylabelled ribonucleoproteins are complexes of messenger-like RNA and protein in a proportion, of about 15 to 30% RNA, 85 to 70% protein by weight. RNA of cytoplasmic postribosomal ribonucleoprotein particles is heterogeneous and composed of species ranging from about 6S to 22S. It stimulates protein synthesis in the E. coli cell-free system whereas the corresponding ribonucleoproteins are poorly stimulatory. The relationships between these particles and their possible role in messenger RNA function are discussed. [ABSTRACT FROM AUTHOR]

The messenger character of nuclear and polysomal rapidly-labeled thyroid RNA has been established using stimulation of amino acid incorporation into trichloracetic­insoluble material in the Escherichia coli system, mononucleotide composition of the early-labeled material and tenacity of adsorption on columns of methylated serum albumin adsorbed on Kieselguhr (MAK columns). A relation between the difficulty to extract nuclear or cytoplasmic thyroid RNA by phenol at 0°, their rapidity of labeling and their capacity to stimulate protein synthesis in vitro has been demonstrated. 80% of the nRNA labeled for 15 min was tenaciously bound to MAK columns. After elution with a buffer containing sodium dodecylsulphate at 35–70°, it showed a DNA-like base composition and sedimented in 4 discrete size classes: 30 S, 22 S, 13 S and 7 S. The remaining 20% has been identified as preribosomal RNA. After a 9 h-period of labeling, the total nRNA fraction still contained labeled preribosomal RNA, rRNA and DNA-like RNA. Most of the early-labeled polysomal RNA has been identified as mRNA by tenacity of adsorption on MAK columns and base composition. Even after a 1 h-pulse the labeled rRNA accounted only for 30–40% of the total RNA. Absence of preribosomal RNA in polysomes as shown by MAK column chromatography was considered as a good index of the absence of contamination of polysomes by nuclear material. Polysomes pulse-labeled with [32P]orthophosphate and [14C]-protein hydrolysate were dissociated by ethylenediaminetetraacetic acid and fixed with formaldehyde. The buoyant densities of the nucleoprotein particles were determined by banding in CsCl gradients. Under these conditions, the bulk of the material in ribosomes banded at characteristic densities (d = 1.57 and 1.52) but most of RNA with high specific radioactivity banded at a density approximately 1.38. This material contained [32P]RNA and [14C]protein, and was identified as an association of mRNA and protein by base composition of its RNA: (G + C)/ (A + U) = 0.83. Purification of pulse-labeled nuclear mRNA was carried out by the hot-phenol fractionation procedure of Georgiev et al. [9,10]. Specific radioactivity, specific stimulating activity of protein synthesis in vitro and base composition of the five fractions obtained were determined. The RNAs extracted from nuclei between 55–65° and 65–85° were considerably enriched in mRNA. Sucrose-density gradient sedimentation showed substantial polydispersity of the stable RNA of all the fractions. However, the main component of the RNA extracted by phenol between 65° and 85° culminated in a metabolically homogeneous 16 S peak. A definite correlation between pulse-labeled nRNA isolated by the hot-phenol procedure and pulse-labeled polysomal RNA was noticed. As compared with 18 S and 28 S rRNA, the fractions of stable polysomal thyroid RNA which corresponded in sedimentation to the rapidly-labeled RNA (30–40 S, 22 S, 13 S and 7 S) definitely stimulated polypeptide synthesis in vitro. The size of the monocistronic mRNA which codes for the peptide chains of thyroglobulin is discussed. If a. molecular weight of 73,000 for the two different chains is assumed, their amino acid sequence might be encoded in the 13 S polysomal RNA. The functional significance of the 30–40 S and 22 S mRNA and the possible importance of polysomal messenger nucleoprotein particles are also discussed. [ABSTRACT FROM AUTHOR]

Nuclear and cytoplasmic RNA from sheep thyroid slices in vitro labeled with [3H]uridine or [32P]phosphate from 5 min up to 9 h, have been obtained by different phenol extraction procedures. Early-labeled RNA was only extracted by phenol containing 0.1 or 1% (w/v) sodium dodecylsulfate (SDS) at 0° or 60°, whereas stable nuclear or polysomal RNA was easily extracted by phenol at 0°. Time course of incorporation of label into subcellular particles has shown the early labeling of nuclear RNA and the transfer of radioactivity into the cytoplasm. Rapidly-labeled RNA extracted from nuclei by phenol-1% (w/v) SDS and analyzed by sedimentation was shown to sediment in three discrete classes 50–30 S, 22 S and 13 S, different from ribosomal RNA. After 9 h of incubation the 22 S and 13 S labeled RNAs were still present in detectable amounts in the nuclei, indicating a somewhat low turnover rate for these components. The kinetics of incorporation of label into polysomes was studied in thyroid slices incubated from 15 rain up to 5 h with [3H]uridine or [32P]phosphate. RNA was dissociated from polysomes by dodecylsulfate treatment and centrifuged on sucrose gradient. As for nuclei, the rapidly-labeled RNAs sediment in discrete size classes, 22 S and 13 S. After a long incubation time (5 h) the label was predominant in 28 S and 18 S ribosomal RNA but 28 S RNA was not completely labeled. Up to 30 min labeling a high molecular weight, early-labeled RNA was present in purified polysomes. Its radioactivity declines after 60 rain labeling. A part of the rapidly labeled polysomal RNA can be reversibly separated from rRNP by the action of EDTA (1.25 to 5 μmoles EDTA/mg RNP). It sediments in a broad zone (12–30 S) of high specific radioactivity. Increasing Mg++ concentration results in the reassociation of rapidly-labeled RNA to ribosomal sub-particles. Buoyant densities of the polysomal RNP particles obtained by EDTA treatment of 32P-labeled polysomes and stabilized by means of formaldehyde fixation, were determined by banding in CsCl gradients. The bulk of the stable material banded at characteristic densities (d = 1.55 and 1.48), but most of the rapidly-labeled RNA banded at d = 1.38. The specific radioactivity of this material was the same as that of the 12–30 S RNA isolated by sucrose gradient centrifugation from the same EDTA­treated polysomes. Preliminary double labeling experiments and computation of the RNA to protein ratio calculated from the RNA and protein density values indicate this material to be early-labeled nucleoprotein particles with a high protein content. [ABSTRACT FROM AUTHOR]

Cytoplasmic giant-size ribonucleoprotein particles has been purified from the deoxycholate treated 15000 x g supernatant of sheep thyroid slices incubated in the presence of [³H] uridine for 0.5 to 8 hours. After treatment of unclarified polyribosomes with 10mM EDTA, 400 to 1000 S ribonucleoprotein particles have been isolated free of ribosomal subparticles by differential sedimentation. They are heterogenous in size and buoyant density in CaCl gradients (from 138 to 1.52 g/ml, with maxima at 152, 146, and 1.42). Polyribosome-associated 400 to 1000 S ribonucleoprotein particles deprived of free ribosomal subparticles contain messenger-like and ribosomal RNA as shown by the rate of labeling and sucrose gradient centrifugation, polyacrylamide gel electrophoresis and base composition. Particle proteins obtained by 3 M LiCl-6 M urea treatment separate in polyacrylamide gel electrophoresis into 3 to 4 basic components which disclose the same electrophoretic mobility as some ribosomal proteins. These 3 to 4 protein bands are distinct from total thyroid soluble proteins and from histones. Similar protein species are present in ribonucleoprotein particles diffusing spontaneously from thyroid nuclei in vitro. The artifactual formation of these structures during EDTA exposure or slice incubation has been rendered unlikely by appropriate controls. Although tentative, the possible physiological significance of these particles is briefly discussed. [ABSTRACT FROM AUTHOR]