Fingerprinting Studies of the Maturation of Ribosomal RNA in Mammalian Cells

Abstract

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.

Intranuclear maturation pathways of rat liver ribosomal ribonucleic acids

“…This interpretation is supported further by the observed variations in size and secondary structure of transcribed spacer segments in the molecules from the 45S pre-rRNA peak (see Plate Ib-le). The difference among 45S pre-rRNA components probably extends to primary structure, as demonstrated by the distinct 'fingerprints' obtained from the '45S' and '47S' constituents of the primary pre-rRNA from a mouse cell line (Galibert et al, 1975). Our electron-microscopic observations indicate that the heterogeneity among 45S pre-rRNA molecules reflects differences in the large transcribed spacer segment, the remaining parts of the molecule being remarkably constant in their secondary-structure pattern.…”

Nucleotide sequence neighbouring a late modified guanylic residue within the 28S ribosomal RNA of several eukaryotic cells

“…The 45S and 28S RNAs were extracted (2) and purified on 5 % -30 % sucrose gradient. After hydrolysis with T1 RNase, the RNAs were subjected to two-dimensional fractionation (2,(5)(6). The composition of spot A was determined after hydrolysis with pancreactic RNase followed by DEAE-cellulose paper electrophoresis and potassium hydroxide hydrolysis followed by ionophoresis at pH 3.5 on Whatman 3MM paper (7) as well as by partial snake venom phosphodiesterase (SVP) and spleen phosphodiesterase (SPD) hydrolyses followed by two-dimensional fractionation (3,8).…”

“…RNA labelled with 14C enabled to detect among the large T1 oligonucleotides those belonging to 18S RNA, those recovered in 28S RNA, and finally those lost during the maturation process. The same technique, in addition, enabled us to find that a single large T1 oligonucleotide characteristic of 28S and 41S RNAs did not exist in the 45S precursor (2). Two hypotheses may be considered to explain the appearance of this oligonucleotide, termed A, during the maturation of ribosomal RNAs.…”