Quantitative alterations in the nucleolar and nucleoplasmic ribosomal ribonucleic acids in regenerating rat liver

A quantitative analysis of the nuclear pre-rRNA (precursor to rRNA) and rRNA in normal and 12h-regenerating rat liver was carried out, and the absolute amounts of the identified pre-rRNA and rRNA species in the nucleolus and nucleoplasm were determined. Characteristic changes in the pre-rRNA and rRNA pool sizes in regenerating liver are found which reveal alternations in both pre-rRNA processing and nucleocytoplasmic transition of ribosomes.     

Intranuclear maturation pathways of rat liver ribosomal ribonucleic acids.

The maturation of pre-rRNA (precursor to rRNA)in liver nuclei is studied by agar/ureagel electrophoresis, kinetics of labelling in vivo with [14C] orotate and electron-microscopic observation of secondary structure of RNA molecules. (1) Processing starts from primary pre-rRNA molecules with average mol. wt. 4.6X10(6)(45S) containing the segments of both 28S and 18S rRNA. These molecules form a heterogeneous peak on electrophoresis. The 28S rRNA segment is homogeneous in its secondary structure. However, the large transcribed spacer segment (presumably at the 5'-end) is heterogeneous in size and secondary structure. A minor early labelled RNA component with mol.wt. about 5.8X10(6) is reproducibly found, but its role as a pre-rRNA species remains to be determined. (2) The following intermediate pre-rRNA species are identified: 3.25X10(6) mol.wt.(41S), a precursor common to both mature rRNA species ; 2.60X10(6)(36S) and 2.15X10(6)(32S) precursors to 28S rRNA; 1.05X10(6) (21S) precursor to 18S rRNA. The pre-rRNA molecules in rat liver are identical in size and secondary structure with those observed in other mammalian cells. These results suggest that the endonuclease-cleavage sites along the pre-rRNA chain are identical in all mammalian cells. (3) Labelling kinetics and the simultaneous existence of both 36S and 21S pre-rRNA reveal that processing of primary pre-rRNA in adult rat liver occurs simultaneously by at least two major pathways: (i) 45S leads to 41S leads to 32S+21S leads to 28S+18S rRNA and (ii) 45S leads to 41S leads to 36S+18S leads to 32S leads to 28S rRNA. The two pathways differ by the temporal sequence of endonuclease attack along the 41 S pre-rRNA chain. A minor fraction (mol.wt.2.9X10(6), 39S) is identified as most likely originating by a direct split of 28S rRNA from 45S pre-rRNA. These results show that in liver considerable flexibility exists in the order of cleavage of pre-rRNA molecules during processing.     

Agar gel chromatography of rat liver ribosomal ribonucleic acids.

Ribosomal RNA (28s rRNA) of rat liver is selectively retained in 2 or 4% squashed agar gels equilibrated at 24–25°C with a sodium dodecyl sulfate-Tris-EDTA buffer containing 0.7 m NaCl. The absorbed polynucleotide could be recovered by elution with 0.1 m NaCl in the same buffer. Agar-gel electrophoresis and nucleotide composition indicate that the separation is close to quantitative. Column beds of 100–200 ml were used in the range of 3–6 mg of RNA. The procedure is simple, rapid and reproducible and gives excellent separation of 28 and 18s rat liver rRNA.     

Differential stability of 28s and 18s rat liver ribosomal ribonucleic acids

Rat liver ribosomal RNA (rRNA) free from nuclease contaminants was isolated by a modification of the phenol technique. The 28s and 18s rRNA species were separated by preparative agar-gel electrophoresis. The two rRNA species were heated at different temperatures under various conditions and the amount of undegraded rRNA was determined by analytical agar-gel electrophoresis. The 18s rRNA remained unaltered after heating for up to 10min. at 90 degrees in water, acetate buffer, pH5.0, or phosphate buffer, pH7.0. Under similar or milder conditions 28s rRNA was partially degraded, giving rise to a well-delimited 6s peak and a heterogeneous material located in the zone between 28s and 6s. The dependence of degradation of 28s rRNA on the temperature and the ionic strength of the medium was studied. The greatest extent of degradation of 28s rRNA was observed on heating at 90 degrees in water. It is suggested that the instability of rat liver 28s rRNA is due to two factors: the presence of hidden breaks in the polymer chain and a higher susceptibility of some phosphodiester bonds to thermal hydrolysis.     

Fidelity of ribosomal ribonucleic acid synthesis by nucleoli and nucleolar chromatin.

Isolated nucleoli, nucleolar chromatin, and nucleolar DNA were used as templates for DNA synthesis in appropriately supplemented systems in which RNA polymerases other than RNA polymerase I were blocked by alpha-amanitin. With the aid of nucleotide analysis, DNA-RNA hybridization, and homochromatography fingerprinting, it was found that isolated nucleoli and nucleolar chromatin serve primarily as templates for synthesis of rRNA. However, the products formed with purified nucleolar DNA as a template do not contain the specific rRNA oligonucleotides nor are they appreciably hybridized to the rDNA region on cesium chloride gradients. These results indicate that whole nucleoli and nucleolar chromatin contain control mechanisms that restrict readouts by RNA polymerase I of nucleolar DNA to rDNA.     

The circular dichroism of ribosomal ribonucleic acids.

1. The c.d. (circular dichroism) of Drosophila melanogaster rRNA (42% G+C) and of G+C-rich fragments (78% G+C) obtained by partial hydrolysis of rabbit L-rRNA (the largest RNA species isolated from the large subribosomal particle) were measured and found to differ substantially. 2. To interpret these spectra a relation between c.d. of bihelical RNA and % G+C was derived, namely delta epsilonfG = AFG2+bfG+c, where deltaepsilonfG is the c.d. of RNA characterized by a mole fraction, fG, of guanine nucleotides and a, b and c are constants. 3. A frame of reference was established by studying the c.d. of a range of rRNA species, including S-rRNA (the RNA species isolated from the smaller subribosomal particle) and L-rRNA of Escherichia coli. 4. It was found for the rRNA species studied that 0.60+/-0.05 of residues appear to form bihelical secondary structure. 5. A higher helical content, 0.66+/-0.05, was found for the G+C-rich fragment of L-rRNA. The difference in the c.d. of rabbit L-rRNA and of D. melanogaster rRNA is attributable to the dependence of c.d. of the bihelical parts on %G+C. 6. The minimum in c.d. at 295 nm increases with increasing %G+C. The c.d. of rRNA was compared with that of the parent subparticle in this region of the spectrum, where high precision may be attained.