Reversible dissociation of nuclear ribonucleoprotein particles containing mRNA into RNA and protein

Samarina, O. P.; Molnár, J.; Lukanidin, E. M.; Брусков, В. И.; Krichevskaya, A. A.; Georgiev, G.P.

doi:10.1016/0022-2836(67)90359-2

Cited by 49 publications

(20 citation statements)

References 5 publications

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“…RNAs extracted from polysomes showed a slightly wider distribution of radioactivity through the gradient, covering sedimentation rates from 30 S to 40 S, but with a distribution in the region below 188, very similar to pulse labelled RNA from nuclear extracts. The nuclear particles were resistant to deoxyribonuclease (see also [17] but were attacked by ribonuclease and by pronase. Lastly we showed that phenol-extracted RNAs from either undigested nuclei or nuclear particles were not affected by deoxyribonuclease or pronase treatment but ribonuclease completely destroyed them.…”

Section: Sixes Of Rnas Obtained From Nuclear Particlesmentioning

confidence: 99%

Particles of Nuclear Origin Carrying Rapidly Labelled RNA

Köhler

Arends

1968

Eur J Biochem

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Nuclei of KB cells have been isolated and incubated at 37" in pH 8.0 saline. The experia) Isolated nuclei release well defined nucleoprotein particles, which contain RNAs not deb) These particles carry rapidly labelled RNA. c) In cytoplasm they cosediment with the long known smaller ribosomal particles. d) Chase experiments suggest that nuclear particles are precursors of cytoplasmic particles. ments described in this paper provide evidence that: tectable in untreated nuclei.In higher organisms the bulk of cellular RNA, e. g. 25 S and 18 S ribosomal RNAs, messenger-and transfer RNAs, are synthesized within the nucleus on the chromosomes [l]. The center of activity of these RNAs, namely the polysomes, are on the other hand exclusively located in the cytoplasm. Ribosomal and messenger RNAs of the same sizes as those obtained from polysomes are not found in the nucleus. I n contrast, nuclei bf mammalian cells contain types of RNA having a relatively high sedimentation rate (> 30 S), which is supposed to reflect a high molecular weight and a great chain length. Such molecules, however, are not found in the cytoplasm [ 2 ] . Previous research has accumulated evidence that RNA has to pass the nuclear membrane in order to get to the cytoplasm. It is then apparently on this passage that such RNA is cut to the required sizes [3,4].In the cytoplasm newly synthesized rapidly labelled RNAs, do not appear as free molecules, but are bound to small particles, e . g. either pre-ribosomal particles or informosomes [3,5-81. The synthesis and/or the passage of the M e r e n t RNA species differs strongly. The smaller 45 S subunits of the ribosomes are labelled faster with radioactive precursors than the larger 60 S subunits; it is obvious that the two enter the cytoplasm independently, and that the 45 S particle is transferred a t a higher rate.This could imply that it is synthesized a t higher rates also.From our own work [6] and from experiments of Girard and collaborators [9], it is further evident that messenger-RNA is the very first to leave the nucleus.Unusual Abbreviations. niRNA, messenger RNA; tRNA. transfer RNA.Note. The sedimentation coefficients of the particles and RNA species discussed here are approximate and were not dotermincd. The values are adopted from the publications cited. They are used to name the hitherto nameless particles and RNA species.

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Section: Sixes Of Rnas Obtained From Nuclear Particlesmentioning

confidence: 99%

Particles of Nuclear Origin Carrying Rapidly Labelled RNA

Köhler

Arends

1968

Eur J Biochem

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“…La presence dans les noyaux de cellules thyroidiennes de RNA rapidement marquB tres lourd (30- [64,65]. Ces particules seraient constitubes par l'association de proteines globulaires et de mRNA dont la taille serait en rapport avec celle des particules.…”

Section: Rna ~O~~s O R N~q U~~ Launclassified

Acides ribonucléiques messagers de la glande thyroïde de mouton

Cartouzou

Aquaron

Manté

et al. 1968

European Journal of Biochemistry

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The messenger character of nuclear and polysomal rapidly-labeled thyroid RNA has been established using stimulation of amino acid incorporation into trichloracetic-insoluble material in the Escherichia coli system, mononucleotide composition of the early-labeled material and tenacity of adsorption on columns of methylated serumalbumin adsorbed on Kieselguhr (MAK columns).A relation between t,he difficulty to extract nuclear or cytoplasmic thyroid RNA by phenol at O", their rapidity of labeling and their capacity to stimulate protein synthesis in vitro has been demonstrated. 800/, 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 X, 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 [l4CC]-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 a t charac- 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 85-85" 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 18s and 2 8 s rRNA, the fractions of stable polysomal thyroid RNA which corresponded in sedimentation to the rapidly-labeled RNA (30-40 S, 22 X, 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' poly...

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“…According to their interpretation, the slightly alkaline pH facilitates the dissociation of these polysome-like structures from the chromatin, thereby allowing their extraction from the nuclei [22]. A similar rationale was followed in this study by using a 0.3 M salt concentration during the third extraction.…”

Section: Discussionmentioning

confidence: 81%

Nuclear RNA Fractions Obtained by Sequential Extraction of Rat Liver Nuclei with Salt Solutions and their Relationship to Polysome‐Associated RNA

Drews¹

1969

European Journal of Biochemistry

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Pulse-labeled RNA from rat liver nuclei was fractionated by sequential treatment of purified nuclei with buffered salt solutions of Werent ionic strength in the presence of ribonuclease inhibitor prepared from rat liver cytoplasm. Two fractions of pulse-labeled nuclear RNA were successively extracted from rat liver nuclei with 0.14 M NaC1, 0.001 M MgCl, a t pH 7.0 and pH 8.0, respectively. The labeled material isolated a t pH 7.0 sediments between 6-16 S while the pulse-labeled RNA extracted a t pH 8.0 sediments in the range of 10-34 S. Both fractions are characterized by a high content in AMP. With respect to [3aP]nucleotide composition, sedimentation characteristics and hybridizability, these fractions correspond closely to non-ribosomal RNA associated with polysomes. When a third extraction is carried out a t pH 8.0 in 0.3 M NaCl another rapidly labeled RNA component is obtained. This RNA sediments above 34 S, turns over more rapidly than the RNA species extracted in 0.14 M NaC1, and contains less AMP. As judged by [32P]base-composition, sedimentation and hybridizability, this RNA component is not present in polysomes. The pulse-labeled RNA remaining in the nuclei after three salt extractions comprises mainly ribosomal or ribosomal precursor RNA as well as DNA-like RNA species which show little or no nucleotide sequence homology with polysome-associated RNA.Since nuclear RNA species clearly different from polysome-associated cytoplasmic RNA are released from nuclei only at a relatively high ionic strength and are retained a t a physiologic salt concentration it is suggested that RNA species not designated to function in cytoplasmic protein synthesis are a priori bound more tightly to nuclear structures than those types of RNA which serve as vehicles for the nucleo-cytoplasmic transfer of genetic information.Recent reports from a number of laboratories have indicated that only a fraction of non-ribosomal RNA synthesized in the nuclei of mammalian cells is eventually released into the cytoplasm [l-41. A substantial portion of newly-formed DNA-like RNA appears to be subjected to a rapid turnover in the nucleus itself [5,6]. The experimental evidence which gave rise to such concepts is indirect: it was shown that the kinetics of labeling of DNA-like RNA from nuclei, nucleoplasm, cytoplasm and from purified polysomes were clearly different and virtually excluded a simple precursor product relationship between these RNA species [l, 5,7]. Moreover, hybridization-competition experiments demonstrated that cytoplasmic or microsomal RNA from various mammalian cell species can compete with the respective labeled nuclear RNA for DNA binding sites only to a very limited degree [2,3,8].A higher degree of nucleotide sequence homology between nuclear RNA and microsomal RNA than in normal liver was observed in regenerating liver [8,9]. 18.I n a number of Morris hepatomas this homology appeared to be nearly complete [8]. On the basis of these results, it was postulated that the stringency of the selection mechanism operating at...

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Reversible dissociation of nuclear ribonucleoprotein particles containing mRNA into RNA and protein

Cited by 49 publications

References 5 publications

Particles of Nuclear Origin Carrying Rapidly Labelled RNA

Particles of Nuclear Origin Carrying Rapidly Labelled RNA

Acides ribonucléiques messagers de la glande thyroïde de mouton

Nuclear RNA Fractions Obtained by Sequential Extraction of Rat Liver Nuclei with Salt Solutions and their Relationship to Polysome‐Associated RNA

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