Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture

Contractile activity of neonatal cardiac myocytes stimulated hypertrophic growth as compared with nonbeating cells that were depolarized with 50 mM KCl. Growth of contracting myocytes was associated with an increased rRNA content as measured by the total RNA/DNA ratio. The fractional rates of rRNA synthesis (K8) and rRNA degradation were determined in contracting and nonbeating myocytes to assess their relative contributions in increasing rRNA content during growth. The values for K8 were calculated from the specific radioactivity of 3'-[3H]UMP in 18 and 28 S rRNA after purification by hybridization to cloned rDNA. The cellular [3H]UTP pool served as the precursor for rRNA synthesis in myocytes that were labeled with 50 microM [3H]uridine. K8 values for 18 and 28 S rRNA in contracting myocytes were accelerated by 59 and 53%, respectively, after 3 days as compared with nonbeating myocytes. Calculations of the rate of cellular rRNA synthesis, which took into account the increased content of myocyte rRNA, revealed that synthesis of both 18 and 28 S rRNA was accelerated 2-fold after 2 days of contraction. The derived values for degradation of 18 and 28 S rRNA were increased marginally in contracting myocytes, but cellular rRNA degradation rates averaged 57% higher. The difference between cellular rates of rRNA synthesis and degradation in contracting myocytes accounted for the 30% increase in rRNA content. These data demonstrated that increased rRNA content in contracting myocytes resulted from acceleration of the fractional rate of rRNA synthesis.     

Stimulation of ribosomal RNA synthesis during hypertrophic growth of cultured heart cells by phorbol ester

Primary cultures of neonatal cardiac myocytes were used to determine the effects of tumor-promoting phorbol esters on ribosomal RNA (rRNA) synthesis during myocyte growth. Treatment of myocytes with phorbol-12,13-dibutyrate (PDBu) increased protein accumulation by 25% and RNA content by 20%. Rates of rRNA synthesis were measured to assess the mechanism by which rRNA accumulated during myocyte growth. Rates of rRNA synthesis were determined from the incorporation of [³H]uridine into UMP of purified rRNA and the specific radioactivity of the cellular UTP pool. After 24h of PDBu treatment, cellular rates of 18S and 28S rRNA synthesis were accelerated by 67% and 64%, respectively. The increased rate of rRNA synthesis accounted for the net increase in myocyte rRNA content after PDBu treatment.     

RNA transcription and translation in sea urchin oocytes and eggs

The steady-state concentrations and absolute rates of synthesis of ribosomal RNA (rRNA) molecules were measured in oocytes, eggs, embryos, and larvae of the Hawaiian sea urchin Tripneustes gratilla. The steady-state concentration per genome of the RNA precursor sequences measured by hybridization to a cloned rDNA fragment was approximately 100- to 300-fold greater in the RNA obtained from oocytes and eggs than in the RNA extracted from embryos and larvae. Since the rate of processing of the rRNA precursor at different stages is not greatly different, the rates of rRNA synthesis must be considerably greater in oocytes than in embryo cells. The absolute rate of RNA synthesis in oocytes and embryos was determined from the incorporation of [³H]guanosine into cellular GTP pools and into both precursor and mature rRNA species. The data indicate an approximately 40-fold higher rate of rRNA synthesis in oocytes than that measured in embryos or previously in larvae (J. Griffith and T. Humphreys, 1979, Biochemistry18, 2178–2185). Together these results indicate that the ribosomal genes are transcribed much more rapidly during sea urchin oogenesis than during embryogenesis or larval stages.     

Estimation of rRNA synthesis and degradation rates in senescing wheat leaves

Changes in cytoplasmic and chloroplast rRNA content and rates of rRNA synthesis and degradation of detached wheat leaves were determined. It was found that rRNA loss is proportionally higher in chloroplasts than in cytoplasm. Rates of synthesis were measured by incorporation of large amounts of [3H]orotic acid into rRNA. This approach overcame size differences between pyrimidine pools of cells under different physiological status. Furthermore, these pools reached nearly the same specific radioactivity as that of the administered solution. Rates of degradation were estimated either as the difference between synthesis and net variation of rRNA or by disappearance of radioactivity from 32P-labeled rRNA. Results indicated a decrease in the net rRNA synthesis capacity of leaves after 48 h of detachment. However, the fractional rates of rRNA synthesis were maintained in both cytoplasm and chloroplasts. Ribosomal RNA degradation rates were 2.5-fold higher in chloroplast than in cytoplasm. The observed chloroplast rRNA loss is due to an increased degradation rate which is 15-fold higher than the synthesis rate 48 h after detachment.     

Ribonucleic acid labelling and nucleotide pools during compensatory renal hypertrophy

During the first 48h of compensatory renal hypertrophy induced by unilateral nephrectomy, RNA content per cell increased by 20-40%. During this period, rates of RNA synthesis derived from the rates of labelling of UTP and RNA after a single injection of [5-(3)H]uridine showed no change in the rate of RNA synthesis (3.1nmol of UTP incorporated into RNA/min per mg of RNA). ATP and ADP pools were not changed. The rate of RNA synthesis was considerably in excess of the increment of total RNA appearing in the kidneys. With [5-(3)H]uridine as label, only continuous infusion for 24h could produce an increase (60%) in the specific radioactivity of renal rRNA in mice with contralateral nephrectomies. With a single injection of [methyl-(3)H]methionine used to identify methyl groups inserted into newly synthesized rRNA, the specific radioactivity of this rRNA was unchanged 5h after contralateral nephrectomy, increased by 60% at 9-48h, and returned to normal values at 120h. Most RNA synthesized in both nephrectomized and sham-nephrectomized mice has a short half-life. Since total cellular RNA content increases in compensatory hypertrophy despite unchanged rates of rRNA synthesis, the accretion of RNA might involve conservation of ribosomal precursor RNA or a change in rate of degradation of mature rRNA.