Effects of Valine Deprivation on the Biosynthesis of Ribosomal and Messenger RNA in a Mammalian Cell Line (L 5178 Y)

Tiollais, Pierre; Galibert, Francis; Boiron, M

doi:10.1111/j.1432-1033.1971.tb01211.x

Cited by 32 publications

(11 citation statements)

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“…The methods of cell culture, labeling, extraction of RNA, and analysis by polyacrylamide gel electrophoresis, have been described (18). RESULTS Coelectrophoresis (in 1.9% polyacrylamide gel) of RNA from two LY cell cultures, one labeled with [3H]uridine for 5 min and the other labeled with [14C] uridine for 60 min shows that the "45S fraction" consists of two peaks of RNA, separated by a distance of 2 mm (Fig.…”

Section: Methodsmentioning

confidence: 99%

Evidence for the Existence of Several Molecular Species in the “45S Fraction” of Mammalian Ribosomal Precursor RNA

Tiollais

Galibert

Boiron

1971

Proc. Natl. Acad. Sci. U.S.A.

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Analysis of RNA (after 5 or 60 min of labeling with ['Hiuridine) from L5178Y cells by electrophoresis in 1.7% polyacrylamide gels demonstrates that the ribosomal RNA "45S fraction" is not homogeneous but consists of at least three molecular species, conventionally designated 47S RNA, 46S RNA, and 45S RNA.Alternatives to the classical scheme for the biosynthesis of ribosomal RNA in mammalian cells are discussed.The 28S and 18S ribosomal RNA in eucarvotic cells are believed to be derived from a common precursor. This precursor would contain the sequences of one 28S molecule and one 18S molecule, associated with a nonribosomal sequence that is not conserved during the conversion of this precursor into mature RNA (1, 2). Therefore, the biosynthesis of ribosomal RNA in eucaryotes appears to differ completely from the biosynthesis of 23S and 16S bacterial ribosomal RNA, which are synthesized independently (2). This concept arose with the discovery in L and, Hela cells of a polynucleotide called "45S RNA", which appeared from kinetic studies and actinomycin D "chase" experiments to be the precursor of both 18S and 32S RNA, the latter giving rise to the 28S RNA (3, 4).The possibility that the 45S RNA fraction may consist of two distinct molecular species of similar molecular weight, one being the precursor of 28S RNA and the other the precursor of 18S RNA, seems to have been excluded by the following two observations. (a) The number of methyl residues contained in a 45S molecule has been measured and found to be equal to the sum of the methyl residues contained in a 28S and an 18S molecule (5, 6). Furthermore, most ribosomal RNA methylation occurs on the 45S molecule (7,8) (6). (c) The analogy of the base compositions, the partial sequences, and the methylation patterns, although excellent for the 45S, 32S, and 28S RNA, are not satisfactory for the 45S and 18S RNA (5, 10). Finally, if the arguments in favor of the existence of a common precursor appear stronger than those suggesting separate origins, it is mostly because the "45S fraction" appears as a single molecular species. In this paper we report the analysis of "45S fraction" by gel electrophoresis; we find that this fraction is heterogeneous and contains several molecular species. MATERIALS AND METHODSThe cells utilized were the L5178Y line, which grows in suspension without shaking (16), and the HeLa-type S3 line, which grows in spinner culture (17). The methods of cell culture, labeling, extraction of RNA, and analysis by polyacrylamide gel electrophoresis, have been described (18). RESULTS Coelectrophoresis (in 1.9% polyacrylamide gel) of RNA from two LY cell cultures, one labeled with [3H]uridine for 5 min and the other labeled with [14C] uridine for 60 min shows that the "45S fraction" consists of two peaks of RNA, separated by a distance of 2 mm (Fig. 1). The 5-min peak (3H) migrates slightly slower than the peak in the preparation labeled for 60 min (14C), and corresponds to a shoulder in the 60-min peak. Therefore, the use of two different labelin...

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Section: Methodsmentioning

confidence: 99%

Evidence for the Existence of Several Molecular Species in the “45S Fraction” of Mammalian Ribosomal Precursor RNA

Tiollais

Galibert

Boiron

1971

Proc. Natl. Acad. Sci. U.S.A.

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“…Total cellular RNA was extracted by the hot phenol/dodecylsulfate procedure of Scherrer and Darnell [33], modified as previously reported [34]. RNA analysis was performed by electrophoresis through a 1.7 % acrylamide/0.5 % agarose composite gel.…”

Section: Rna Extraction and Analysismentioning

confidence: 99%

Characterization of Proteins from Nucleolar Preribosomes of Mouse Leukemia Cells by Two‐Dimensional Polyacrylamide Gel Electrophoresis

Auger-Buendia

Longuet

1978

European Journal of Biochemistry

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Proteins of isolated 80-S and 60-S nucleolar preribosomal particles were characterized by means of two-dimensional polyacrylamide gel electrophoresis, in the lymphocytic mouse leukemia cells L5178Y. Their identification and metabolic relationships with ribosomal subunit proteins were investigated using co-electrophoresis of unlabeled polysomal proteins with labeled proteins of either nucleolar preribosomes or ribosomal subunits. The large and small ribosomal subunits contain 40 and 31 proteins, respectively. The nucleolar 80-S preribosomes were analysed after 2 and 5 h of incubation with tritiated valine and leucine and were shown to contain about 55 proteins. Most of them were identical to cytoplasmic ribosomal subunit proteins. The nucleolar 60-S preribosomes contain all the proteins which are common to 80-S preribosomes and large ribosomal subunits, and one additional protein (L10). The ribosomal proteins which were absent from nucleolar particles were found to be labeled in the cytoplasmic ribosomes after the same incubation period. Thus, in addition to the association of the bulk of ribosomal proteins with 45-S RNA within the 80-S preribosomes, results indicate that a group of ribosomal proteins and particularly from the small subunits, become associated at later stages of the maturation process of mammalian ribosomes. It was further shown that a set of 10 proteins, different from ribosomal polypeptides, were present in nucleolar preribosomal particles. Several of them were associated with polyribosomes in the cytoplasm, whereas the others were unique to the nucleolus.Ribosome assembly in the nucleolus of eucaryotic cells and the maturation process of ribosomal precursor ribonucleoprotein particles are under continuing study [l-71. Although much is known about ribosomal RNA synthesis and maturation [8][9][10], investigations of the biosynthetic pathway of the ribosomal proteins remains incomplete. It has been shown that ribosomal proteins are synthesized on cytoplasmic polysomes [l 1 -131 and rapidly transported to the nucleolus where they associate with newly transcribed ribosomal immature RNA [7]. No important pool of ribosomal structural proteins could be found in the cytoplasm and in the nucleolus of eucaryotic cells [14-161. Experiments where protein synthesis was inhibited or reduced [I7 -201 demonstrated the apparent feed-back control of proteins on ribosomal RNA transcription and processing.The characterization of nucleolar preribosomal proteins has been first performed by unidimensional polyacrylamide gel electrophoresis [1 ,21-251 or alternatively by fingerprints of trypsin digestion products [26]. Bidimensional polyacrylamide gel electrophoresis provides a high degree of resolution in the separation of ribosomal proteins [27,28] and nucleolar proteins [29], and has been used for analysing the proteins from the 60-S preribosomal particles [30] and mixtures of nucleoltlr preribosomal particles [15,31]. However, a detailed composition of the 80-S preribosomal particle, which appears as the most nas...

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“…As for the 45-S RNA, all characteristic oligonucleotides from either 18-S RNA or 28-S RNA can be recognized in both 47-S and 41-S RNA. Since 47-S, 45-S and 41-S RNA contain an equal amount of all the oligonucleotides characteristic of 18-S and 28-S RNA, one can concluded that 18-S and 28-S RNA have a common unique precursor, the 47-S RNA giving rise to the 45-S RNA as previously established by kinetic and pulse-chase experiment [7] then to the 41-S RNA, which is split into two parts, one containing the 18-S and the other the 28-S RNA. The only alternative of this scheme would be the existence, in equal amounts, of two distinct RNA series both with sedimentations of 47 S, 45 S and 41 S which would give rise independently to the 18-S and 28-S RNA.…”

Section: -S Rna Fingerprintmentioning

confidence: 55%

Fingerprinting Studies of the Maturation of Ribosomal RNA in Mammalian Cells

Galibert¹,

Tiollais²,

Me³

1975

European Journal of Biochemistry

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32P-labelled ribosomal RNA of L 5178 Y cells (a mouse cell line) was digested with TI ribonuclease and fingerprinted by electrophoresis at pH 3.5 on cellulose acetate and homochromatography on DEAE-cellulose thin-layer plate. From this, it can be concluded that 18-S and 28-S RNA have different and characteristic fingerprints and that the number, the size and the frequency of the large T, oligonucleotides demonstrate that the guanylic residues are randomly interspaced along the molecule.Using a double-labelling technique with 32P-labelled 45-S RNA and ''C-labelled 1 8 4 RNA or 28-S RNA, long T, oligonucleotides of the 45-S RNA can be divided into three classes: (a) those which are lost during the transition, (b) those which are present in the 18-S RNA and (c) those which are present in the 28-S RNA. These results provide direct evidence for the existence of one common precursor for the two mature ribosomal RNAs.The comparison of the fingerprints of T,-ribonuclease-digested 47-S, 45-S and 41-S RNA shows that the 47-S and 41-S RNA have a characteristic ribosomal pattern. Finally the size, the number and the mobility of the oligonucleotides present in the different RNA precursors but absent from the mature RNA demonstrate that the non-conserved RNA pieces do not have a monotonous and repetitive sequence.The processing of the 45-S RNA into the two ribosomal RNA species, namely 18-S and 28-S RNA is well documented and generally accepted [l-31. Nevertheless, neither chemical data nor direct evidence have yet conclusively demonstrated that a single molecule of 45-S RNA could contain the sequences of both 18-S and 28-S ribosomal RNA [4-61. This seems important to verify since the 45-S ribosomal RNA which was demonstrated to be the precursor of both ribosomal RNA species is actually a mixture of several molecular species, namely 47-S, 46-S and 45-S RNA 173. Numerous studies have shown that, while the size of ribosomal RNA has been strongly conserved through evolution, the size of the precursors has not, so that large differences in size can be found between evolutionarily divergent species (for review, see [8]). As previously shown, roughly 10% of the primary product of transcription is lost during the maturation of the ribosomal RNA in bacteria, while the nonconserved sequence accounts for 20% in Xerzopus and up to 45% in mammals. This difference might well have some significance in terms of evolution and Enzyme. TI ribonuclease (EC 3.1.4.8) Eur. J. Biochem. 55 (1975) post-transcriptional regulation. It would therefore be interesting to obtain information about the primary structure of the RNA pieces lost during maturation. We have made use of the fingerprinting technique, including homochromatography, to show that the 47-S RNA contains the sequence of both 18-S and 28-S ribosomal RNA. We have also been able to identify among the large 45-S RNA T, oligonucleotides those present in the 18-S and 28-S RNA and those lost during maturation.

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Effects of Valine Deprivation on the Biosynthesis of Ribosomal and Messenger RNA in a Mammalian Cell Line (L 5178 Y)

Cited by 32 publications

References 26 publications

Evidence for the Existence of Several Molecular Species in the “45S Fraction” of Mammalian Ribosomal Precursor RNA

Evidence for the Existence of Several Molecular Species in the “45S Fraction” of Mammalian Ribosomal Precursor RNA

Characterization of Proteins from Nucleolar Preribosomes of Mouse Leukemia Cells by Two‐Dimensional Polyacrylamide Gel Electrophoresis

Fingerprinting Studies of the Maturation of Ribosomal RNA in Mammalian Cells

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