Mariana D. Dabeva

Simple agar--urea-gel electrophoretic fractionation of high molecular weight ribonucleic acids.

Abstract A simple technique for the fractionation of RNA by electrophoresis in agar-urea gels is described. It is shown that under optimal conditions the presence of urea decreases markedly the band width of RNA fractions and improves the resolving power of the method. A linear relationship between log MW and mobility is established for ribosomal RNA in the molecular weight range from 0.5 × 10 6 to 4.7 × 10 6 daltons.

Ribosome Biogenesis and Nucleolar Ultrastructure in Neuronal and Oligodendroglial Rat Brain Cells

Abstract: The absolute amounts of precursor to ribosomal RNA (pre‐rRNA) and ribosomal RNA (rRNA) in isolated rat brain neuronal and oligodendroglial nuclei were determined. The amount of the major pre‐rRNA and rRNA species in neuronal nuclei was about twofold higher than in oligodendroglial nuclei. The relative rate of pre‐rRNA synthesis in vivo was 2.3‐ to 2.7‐fold higher in neuronal as compared with oligodendroglial nuclei. This corresponds to a 2.7‐fold higher activity of the “template‐bound” RNA polymerase I in isolated neuronal nuclei, whereas the activity of the “free” enzyme in both neuronal and glial nuclei was almost identical. The higher transcription rates of rRNA genes correlated with the markedly more prominent fibrillar component in neuronal nucleoli. The turnover times of the major pre‐rRNA and rRNA species in neuronal and oligodendroglial nuclei were similar, except for 45S pre‐rRNA, which turned over at an 1.5‐fold slower rate in neuronal nuclei. The relative rates of processing of pre‐rRNA and of nucleocytoplasmic transport of rRNA in neuronal cells were 2.7‐fold higher than in oligodendroglial cells and corresponded to the differences in rRNA gene transcription rates. The established ribosome formation features correlated with an abundant (neurons) or exceedingly scarce (oligodendrocytes) nucleolar granular component. The turnover rate of cytoplasmic ribosomes in rat brain neurons was twofold slower than in oligodendrocytes, largely because of the about fivefold higher amount of ribosomes in the cytoplasm of neurons. We conclude that ribosome formation and turnover in neuronal and oligodendroglial cells are adapted to the protein synthetic levels in these two types of brain cells.

Different Rates of Synthesis and Turnover of Ribosomal RNA in Rat Brain and Liver

Abstract: The kinetics of in vivo labeling of cellular free UMP and nucleolar, nucleoplasmic, and cytoplasmic rRNA with [14C]orotate in rat brain and liver were investigated. Evaluation of the experimental data shows: (a) The rate of nucleolar precursors of ribosomal RNA (pre‐rRNA) synthesis and the deduced rate of ribosome formation in brain is about fivefold lower than in liver and corresponds to 220–260 ribosomes/min/nucleus. (b) The lower rate of in vivo pre‐rRNA synthesis is correlated with a lower activity of RNA polymerase I in isolated brain nuclei, (c) The half‐lives of nucleolar rRNA in brain and liver are 210 and 60 min, respectively, thus showing a slower rate of processing of pre‐rRNA in brain nucleoli. (d) The nucleo‐cytoplasmic transport of ribosomes in brain is also markedly slower than in liver and reflects the lower rates of synthesis and processing of pre‐rRNA. (e) Cytoplasmic ribosomes in brain and liver turn over with half‐lives of about 6 and 4 days, respectively. It is concluded that the markedly lower rate of ribosome biogenesis in brain is specified mainly at the level of transcription of rRNA genes.

Ribosomal RNA precursor transcription in rat liver is not dependent on continuous synthesis of proteins

The effect of inhibition of protein synthesis by cycloheximide on rRNA precursor transcription in rat liver was analyzed. Two doses of the drug were studied: low, 5 mg/kg, and high, 20 mg/kg. Both doses of cycloheximide cause rapid, complete and continuous inhibition of protein synthesis. The low dose of the antibiotic does not alter the rRNA precursor transcription for at least 4 h, while the high dose, which is lethal to rats, leads gradually to suppression of rRNA precursor synthesis. It is shown that the high dose of cycloheximide causes profound changes in the metabolism of the free nucleotides and drastic inhibition of [14C]orotate and [32P]orthophosphate uptake into the pool of free nucleotides. It is supposed that the strong side-effects of cycloheximide, rather than the cessation of protein synthesis, are responsible for the observed inhibition of rRNA precursor synthesis. It is concluded that rRNA precursor transcription is not regulated by rapidly turning-over protein(s).

Turnover of ribosomal proteins in regenerating rat liver after partial hepatectomy

The rates of synthesis and degradation of ribosomal proteins, prelabelled with [14C]bicarbonate, were determined as an index of the rate of ribosome turnover in regenerating rat liver. The half-life of ribosomes is about 178 and 75 hr in regenerating and normal liver, respectively. The comparison of turnover rates of ribosomal proteins with the corrected values of rRNA, based on re-utilization of nucleotides, suggests that ribosomes are metabolized as a unit in vivo. There is at least 70% overestimation for ribosome half-life when orotate-labelled RNA is used for turnover determinations. The absolute rate of synthesis is estimated as 3925 and 1081 ribosomes/min per cell in 24 hr regenerating and normal rat liver, respectively.

Activated ribosomal RNA synthesis in regenerated rat liver upon inhibition of protein synthesis

Cycloheximide (Cyh), administered at a dose of 5 mg/kg body wt blocks protein synthesis in normal rat liver (NRL) and regenerating rat liver (RRL). The rate of synthesis of 45S pre-rRNA in RRL, studied after RNA labelling in vivo is activated 2.8 times. Pre-r RNA synthesis in RRL is more sensitive to the stopped translation, but never falls down to the level in NRL. The major contribution to the rDNA transcription activation in RRL comes from the 20.-fold increase in the number of pol I molecules engaged in the transcription, the elongation rate being 1.4-fold accelerated. Cyh quenches partially the enhanced rDNA transcription in RRL: the number of pol I molecules and their elongation rate are about 1.7-fold and 1.5-fold higher, respectively, than the corresponding values in NRL after Cyh treatment. The results show that two different mechanisms control the number and the rate of initiation and elongation of RNA polymerase I in rat liver; one of them depends on continuous protein synthesis and can be inactivated by Cyh, the other is Cyh resistant.

Ribosomal RNA synthesis in liver of adrenalectomized rats after partial hepatectomy

The ribosome formation in four experimental groups: normal, adrenalectomized, partially hepatectomized and adrenalectomized - partially hepatectomized rats was studied. Ribosomal RNA was labelled for different intervals and the transfer of the radioactivity from 45 S pre-rRNA through the nucleolar pre-rRNA and rRNA pools into cytoplasmic 28S and 18S rRNA was followed. The results show that there are at least two ways of positive control of rRNA synthesis, one of them being glucocorticoid-dependent. The acceleration of the pre-rRNA processing through the shortest maturation pathway in regenerating liver is reduced in the absence of the hormone. Glucocorticoids do not influence nucleo-cytoplasmic rRNA transport.

CONTROL OF RIBOSOMAL RNA PROCESSING IN EUKARYOTES

ABSTRACT Processing of pre-rRNA plays a major role in eukaryotes. The control mechanisms involved seem to result in the fine adaptation of ribosome biogenesis to efficiency of translation. Experimental evidence on some of these control mechanisms is presented. Identification of 5′-terminal triphosphates of adenosine and guanosine in 37 S primary pre-rRNA of S.cerevisiae shows that it is the primary transcript of rRNA genes. These results suggest that processing of primary pre-rRNA can start only after transcription of rRNA genes is completed. Experiments on D-galactosamine block of RNA synthesis show that processing and nucleo-cytoplasmic transfer of rRNA in liver is independent of continuous transcription. Analysis of labelling kinetics data for liver pre-rRNA reveals considerable flexibility in the sequence of nuclease attacks on pre-rRNA chains and the simultaneous operation of several processing pathways. The cleavages leading to mature 28 S and 18 S rRNA appear to be under more stringent control than those generating intermediate pre-rRNA. Block of protein synthesis with low doses of cycloheximide does not inhibit transcription, but alters the processing pathways of pre-rRNA and creates a dichotomy in the output of large and small ribosomes. The role of the supply of ribosomal proteins in the control of pre-rRNA processing is emphasized.