Unusual metabolism of 5 S RNA in HeLa cells

5 S ribosomal RNA is found initially in the cytoplasmic soluble fraction soon after its synthesis. After a lag of half an hour, the 5 S RNA becomes associated with nucleoprotein in the nucleus, where some of it later becomes incorporated into the large ribosomal sub-unit. In exponentially growing HeLa cells, 5 S RNA is made in amounts approximately four times greater than required for synthesis of ribosomal sub-units.     

RNA metabolism in nuclei isolated from HeLa cells.

Nuclei with low cytoplasmic contamination, capable of synthesizing RNA for an extended period of time, were prepared from HeLa cells. Besides elongating RNA chains already initiated in vivo, the nuclear preparation initiates the synthesis of new RNA chains. This was shown by labelling the newly synthesized RNA with [gamma-32P]GTP and by detecting the presence of labelled guanosine tetraphosphate among the alkaline hydrolysis products of synthesized RNA. By synthesizing RNA in the presence of each of the four gamma-32P-labelled nucleoside triphosphates, it was possible to conclude that RNA chain synthesis starts predominantly with a purine base. Both nucleolar and nucleoplasmic RNAs are made. The nuclear preparation methylates the nucleolar RNA by utilizing S-adenosyl-L-methionine as a methyl-group donor.     

Inactivation of splicing factors in HeLa cells subjected to heat shock

The nuclear extracts from HeLa cells subjected to heat shock at 43 or 46 degrees C for 2 h were unable to splice pre-mRNA in vitro. Analysis of snRNPs in the extracts revealed that the U4.U5.U6 small nuclear ribonucleoprotein particle (snRNP) complex was disrupted at both temperatures while U1 and U2 snRNPs remained unaffected at 43 degrees C but were disrupted to certain extent during heat shock at 46 degrees C. During splicing reaction, the extract from cells heat shocked at 43 degrees C formed intermediate splicing complexes alpha and beta but was unable to form a functional spliceosome, complex gamma. Addition of fractions from a normal nuclear extract restored splicing activity only in the extract from cells subjected to heat shock at 43 degrees C. Using this complementation assay, we have partially purified the factor(s) inactivated at this temperature. The purified factor(s) was essentially devoid of snRNAs and snRNPs and resistant to micrococcal nuclease, indicating that the factor(s) inactivated by in vivo heat shock at 43 degrees C is a protein. We have also subjected the nuclear extracts from normal HeLa cells to in vitro heat treatment at 43 or 46 degrees C. The results indicate that during in vitro heat treatment of the extracts the damage to splicing machinery is more extensive than that during in vivo heat shock. These experiments also suggest that the factor(s) inactivated by heat shock at 43 degrees C is different from previously identified thermolabile splicing factors.     

Effect of 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole on ribonucleotide metabolism and accumulation of mitochondrial RNA and low-molecular-weight cytoplasmic RNA in HeLa cells

These results provide additional information on the selective inhibition of RNA synthesis by 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole (DRB). DRB only slightly inhibited the poly(A⁺) RNA and ribosomal RNA in the mitochondria (maximal inhibition was ~25%) but severely inhibited the poly(A⁺) RNA in the postmitochondrial supernatant (~95%) and the poly(A⁺) RNA associated with the cytoplasmic membranes (~80%). Separation of the cytoplasmic low-molecular-weight RNAs showed that DRB inhibited the 5.8 S rRNA, a product of RNA polymerase I, by ~95% while there was only a slight inhibition of the 4 S RNAs (~20%) and 5 S RNA (<5%), products of RNA polymerase III. DRB severely inhibited the appearance in the cytoplasm of 28 S rRNA (~95%) and 18 S rRNA (~80%). These results, along with other recent reports (31–34), may suggest that DRB most severely inhibits RNAs that are extensively processed and/or transcribed from genes that contain extensive intervening sequences. These experiments also indicate that the mechanism of DRB inhibition does not involve alterations in ribonucleotide metabolism. DRB did not affect the phosphorylation of any ribonucleotides to triphosphates or the cellular conversion of [³H]uridine to UTP. Also, the size of the UTP and ATP pools in DRB-treated cells was equal to or greater than those in control cells through a period of 240 min. Significant amounts of DRB triphosphate could not be detected in DRB-treated cells suggesting that this may not be the inhibitory form of DRB. Measurements of the specific activity of the UTP pool allowed direct measurements of the accumulation of picomoles of the individual RNAs in the presence of DRB.     

Effects of heat shock on amino Acid metabolism of cowpea cells

When cowpea (Vigna unguiculata) cells maintained at 26°C are transferred to 42°C, rapid accumulation of γ-aminobutyrate (>10-fold) is induced. Several other amino acids (including β-alanine, alanine, and proline) are also accumulated, but less extensively than γ-aminobutyrate. Total free amino acid levels are increased approximately 1.5-fold after 24 hours at 42°C. Heat shock also leads to release of amino acids into the medium, indicating heat shock damage to the integrity of the plasmalemma. Some of the changes in metabolic rates associated with heat shock were estimated by monitoring the ¹⁵N labeling kinetics of free intracellular, extracellular and protein-bound amino acids of cultures supplied with ¹⁵NH₄⁺, and analyzing the labeling data by computer simulation. Preliminary computer simulation models of nitrogen flux suggest that heat shock induces an increase in the γ-aminobutyrate synthesis rate from 12.5 nanomoles per hour per gram fresh weight in control cells maintained at 26°C, to as high as 800 nanomoles per hour per gram fresh weight within the first 2 hours of heat shock. This 64-fold increase in the γ-aminobutyrate synthesis rate greatly exceeds the expected (Q₁₀) change of metabolic rate of 2.5- to 3-fold due to a 16°C increase in temperature. We suggest that this metabolic response may in part involve an activation of glutamate decarboxylase in vivo, perhaps mediated by a transient cytoplasmic acidification. Proline appears to be synthesized from glutamate and not from ornithine in cowpea cells. Proline became severalfold more heavily labeled than ornithine, citrulline and arginine in both control and heat-shocked cultures. Proline synthesis rate was increased 2.7-fold by heat shock. Alanine, β-alanine, valine, leucine, and isoleucine synthesis rates were increased 1.6-, 3.5-, 2.0-, 5.0-, and 6.0-fold, respectively, by heat shock. In contrast, the phenylalanine synthesis rate was decreased by 50% in response to heat shock. The differential effects of heat stress on metabolic rates lead to flux and pool size redistributions throughout the entire network of amino acid metabolism.     

Effect of heat shock on the metabolism of glutathione in maize roots

High performance liquid chromatography analyses revealed that glutathione (GSH) and cysteine are two of the major low molecular weight thiol compounds in maize root extracts. Treatment of maize roots to heat shock temperatures of 40°C resulted in a decrease of cysteine levels and an increase of GSH levels. Pulse labeling of maize roots with [³⁵S]cysteine showed that the rate of incorporation of ³⁵S into GSH or glutathione disulfide (GSSG) in heat shocked tissues was twice that in nonheat shocked tissues. In addition, extracts from heat shocked maize, barley, and soybean tissues contained an unidentified low molecular weight compound that increased from 1.2- to 8-fold within 2 hours of heat shock treatment depending on the tissue and plant involved. Our results indicate that during heat shock there is an increase in the activity of the GSH synthetizing capacity in maize root cells. The elevated synthesis of GSH may be related to the cells capacity to cope with heat stress conditions.     

Translational regulation of the synthesis of a major heat shock protein in HeLa cells

The synthesis of a major heat shock protein (HSP 70) was measured in HeLa cells incubated at 42.5 degrees C and then transferred to 37 degrees C or 30 degrees C. After 90 min, synthesis of HSP 70 decreased by 54 and 85%, respectively, whereas HSP 70 mRNA was reduced at most by 20%. Therefore, the reduced synthesis of HSP 70 could not be accounted for by mRNA turnover. HSP 70 was associated with large polyribosomes (6-10 ribosomes) in cells kept at 42.5 degrees C, but with medium or small polyribosomes in cells transferred to 37 degrees C or 30 degrees C (5-6 or 2-3 ribosomes, respectively). Addition of puromycin to these cells resulted in the release of all ribosomes from HSP 70 mRNA, indicating that they were translationally active. The regulation of HSP 70 synthesis was investigated in cell-free systems prepared from heat-shocked or control cells and incubated at 30 degrees C and 42 degrees C. After 5 min at 42 degrees C, the cell-free system from heat-shocked cells synthesized protein at 3 times the rate of the control cell-free system. This difference was in large part due to synthesis of HSP 70. Addition of HSP mRNA to the control cell-free system stimulated protein synthesis at 42 degrees C, but not at 30 degrees C. These findings suggest that translation of HSP 70 mRNA is specifically promoted at high temperature and repressed during recovery from heat shock by regulatory mechanisms active at the level of initiation.     

The effects of ‘Step-down’ on RNA metabolism in HeLa cells

HeLa cells were subjected to ‘step-down’ conditions, and measurements were made of the high and low salt RNA polymerase activity, phosphorylation of uridine, incorporation of precursors into both RNA and protein and their respective acid-soluble pools, at different cell densities. It was found that ‘step-down’ conditions induced increased activity in both types of polymerase, decreased phosphorylation of uridine and reduced the incorporation of radioactive precursors into both the amino acid and nucleotide pools.     

RNA synthesis in permeable HeLa cells

The equilibria and kinetics are reported for the partial reactions of the catalytic cycle of the Ca²⁺ ionophore X537A in phospholipid vesicles. The analysis is based on the study of the behavior of the ionophore's intrinsic fluorescence in fluorescence lifetime, stopped-flow, temperature, and conventional steady-state fluorescence experiments. Binding to dimyristoyl phosphatidylcholine vesicles gives rise to an enhancement of the fluorescence. At the pH of study (7.4) this involves the singly negatively charged form (X^?). Complexation of the membrane-bound form (X_m^?) by monovalent (M⁺) or divalent (M²⁺) cations to give 1:1 (M-X)_m and (M-X)_m⁺ complexes, respectively, gives rise to a further fluorescence enhancement. No evidence could be found for stoichiometries other than 1:1 in the equilibrium experiments. The fluorescence of X537A in the presence of phosphatidic acid vesicles or phosphatidylcholine/ phosphatidylethanolamine or phosphatidylcholine/cholesterol mixtures is much smaller than for pure phosphatidylcholine. Fluorescence lifetime experiments show that this is due to a reduction in binding rather than a reduction of the quantum yield of the bound species. Fluorescence decay profiles from the above-mentioned membranes showed two exponential components indicating that there were two fluorescent species. The shorter-lived species had a lifetime of 3–5 ns and accounted for 80–90% of the membrane-bound ionophore. The longerlived species (9–13 ns) was estimated to account for the remaining 10–20%. This species enjoys a higher degree of hydrophobic shielding than the shorter-lived species. Possible interpretations in terms of the ionophore orientation in the membrane are discussed. Temperature-jump experiments show that the binding rate of the ionophore is fast. The binding and dissociation rate constants were ca. 2 × 10⁷m (PC)^?1 s^?1 and 2 × 10³ s^?1, respectively. Stopped-flow experiments gave evidence for a slower “insertion” process with a ca. 10-ms half-time. Analysis shows that this process is capable of transport of (K-X) across the membrane with a rate constant ≤ 69^?1. In the presence of divalent cations a slower process involving transport of M²⁺-ionophore complexes across the membrane can be observed. The dependence of the rate on the total ionophore concentration indicates that the transported species is a neutral (M-X₂) complex. The lower limit for the rate constant for transport of the (Ca-X₂) complex is 35 s^?1. The divalent cation specificity of the overall reaction was shown to be Mg²⁺ ? Ca² < Sr²⁺ < Ba²⁺. The rates of the overall transport at low ionophore concentration are limited by the equilibrium constant for formation of the (M-X₂)_m complex from the (M-X)_m⁺ complex.