Low molecular weight RNA of Drosophila cells which is induced by heat shock--II. Structural properties

Heat shock-induced small cytoplasmic RNA (HSI scRNA) from Drosophila culture cell was larger in size after dimethylchloroacetal treatment than in 7 M urea. Judging from its thermal denaturation profile and the nuclease S1 digestion experiment it was supposed that HSI scRNA is double-stranded. Nucleotide sequence analysis of the scRNA also suggested that it is double-stranded RNA with the blunt-ends.     

Tetrahymena cells secrete a low-molecular weight factor which inhibits RNA synthesis in vivo and in vitro.

Medium which has been dialyzed against a dense population of Tetrahymena cells contains a factor which reduces the rate of RNA synthesis in other Tetrahymena cells inoculated on that medium. Furthermore, this extracellular factor inhibits the ribosomal RNA synthesis in vitro when added to an RNA synthesizing r-chromatin preparation.     

No heat shock protein synthesis is required for induced thermostabilization of translational machinery.

For Tetrahymena thermophila cells to survive at 43 degrees C, a normally lethal temperature, they require a pretreatment which either elicits the synthesis of heat shock proteins or one which brings about a change in the translational machinery of the cell such that is is not inactivated when transferred to 43 degrees C. In this report I present evidence showing that the latter modification can occur in the complete absence of protein synthesis, indicating that heat shock protein production is not required for the induced thermostabilization of the translational machinery.     

Synthesis of low molecular weight RNA components A, C and D by polymerase II in alpha-amanitin-resistant hamster cells.

In an attempt to establish which RNA polymerase catalyzes the synthesis of the low molecular weight RNA components A, C and D, Ama 1 cells (mutant Chinese hamster cells) were used in experiments with addition of alpha-amanitin. Ama 1 cells contain an altered RNA polymerase II which is 800 times more resistant towards inhibition by alpha-amanitin than the wild type enzyme. Alpha-amanitin (up to 200 microgram/ml) added to these cells does not affect the synthesis of the low molecular weight RNAs A, C and D. These data together with our previous data showing that alpha-amanitin (0.5 - 5.0 microgram/ml) preferentially inhibits the synthesis of A, C and D in normal cells indicate that RNA polymerase II catalyzes the synthesis of the low molecular weight RNA components A, C and D.     

Glutamine is a powerful effector of heat shock protein expression in Drosophila Kc cells.

We have investigated the effects of extracellular anions on the regulation of expression of the heat shock response in Drosophila Kc cells incubated in defined balanced salt solutions. Widely varying chloride concentrations had no effect on normal or heat shock protein (hsp) expression. Increasing glutamate concentrations from zero to 15 mM increased hsp expression more than 100-fold while affecting expression of non-heat-shock proteins minimally. Glutamine was 20-100-fold more potent than glutamate in supporting hsp expression, while other amino acids were less effective or supported no detectable hsp synthesis in heat shock. Inhibition of glutamine synthetase with methionine-sulfoximine resulted in very low hsp expression with glutamate and normal high level expression with glutamine, confirming the importance of glutamine. The absence of glucose and treatment with 2-deoxyglucose did not change the requirement for adequate glutamine for hsp expression. Cells heat shocked under conditions which gave very low hsp expression resumed growth when returned to normal medium as well as cells which expressed normal levels of hsps. Measurements of free amino acid levels in cells heat shocked in the presence and absence of glutamine showed a correlation between glutamine levels and amount of hsp expression. We conclude that a physiological process regulated by glutamine or a glutamine metabolite is important for normal hsp expression in heat shock conditions in Drosophila.     

Low molecular weight nuclear RNA in human lymphocytes : A comparison of PHA-treated and control cells

Seven species of low molecular weight nuclear (LMN) RNA were identified in human peripheral lymphocytes. These were designated as A, B, C, D, D′, E and F. The same 7 species of LMN RNA were obtained from resting lymphocytes and from lymphocytes exposed to PHA for 16 and 60 h, respectively. Thus, no detectable qualitative changes were seen in the spectrum of LMN RNAs during PHA-induced lymphocyte transformation. However, the amount of species A, D-D′, E and F per nucleus in fully transformed cells was greater than in untreated lymphocytes. This increase had not yet occurred after 16 h treatment with PHA. ³H-Uridine was incorporated into all species of LMN RNA of resting and PHA-treated lymphocytes. Furthermore, all species of LMN RNA except C (5S RNA) were methylated in both resting and transformed cells. The ³H and ¹⁴C specific activities of LMN RNAs following an 8 h exposure to ³H-uridine and ¹⁴C-methyl methionine were higher in PHA-treated cells than in untreated lymphocytes. For several species of LMN RNA (A, D-D′, E, F) the highest ³H and ¹⁴C spec. act. were observed after 16 h exposure to PHA. The possibility that quantitative alterations in the synthesis and methylation of LMN RNAs may occur during lymphocyte transformation is discussed.     

Effect of X rays on protein synthesis and RNA synthesis in adenovirus-infected cells.

Ionizing radiation exerts a deteriorating effect on the rate of protein and RNA synthesis in HeLa cells. This effect is higher in cells infected with adenovirus, thus pointing to a higher radiosensitivity of viral syntheses compared with the cellular ones.     

Zinc-induced synthesis of low molecular weight zinc-binding protein by human lymphocytes

Incubation of human peripheral blood lymphocytes with zinc transferrin (with or without phytohemagglutinin) induces the synthesis of protein that elutes from a Sephadex G-75 column at aV _e/V _o value corresponding to a molecular weight of 6600. Synthesis depends on the concentration of zinc transferrin in the medium and is sensitive to actinomycin D. Detectable synthesis occurs 5h after initiation of lymphocyte culture and plateaus at 24–30h. Polyacrylamide gel electrophoresis of the zinc-induced protein showed two closely moving bands, both of which show immunologic identity to rat liver metallothionein. Partial characterization of this protein yielded the following results: absorbance maximum at 220 nm; zinc content of 5.8 mol/6600 daltons; sulfhydryl content of 20.2 mol/6600 daltons. Additionally, synthesis of zinc-induced protein is altered in both chronic lymphocytic leukemic and acute lymphoblastic leukemic lymphocytes.     

Down regulation of extramacrochaetae mRNA by a Drosophila neural RNA binding protein Rbp9 which is homologous to human Hu proteins.

Rbp9 is an RNA binding protein expressed mainly in the central nervous system of adult Drosophilamelanogaster. Rbp9 shares a high degree of sequence similarity with human neural proteins referred to as Hu antigens. Hu antigens bind to U-rich mRNA destabilizing elements with a high affinity and, thus, have been implicated as regulators of mRNA stability. Using in vitro RNA binding assays, we found that Rbp9 binds strongly to poly U sequences. We then employed a Selex system to identify a consensus Rbp9 binding site (UUUXUUUU). Information obtained from the Selex results allowed the detection of two repeats of the Rbp9 consensus binding sequence in the 3' untranslated region of extramacrochaetae mRNA. UV crosslinking experiments demonstrated that Rbp9 interactsspecifically with emc mRNA. The requirement of Rbp9 protein in the down regulation of emc mRNA was confirmed by northern (RNA) analysis, which revealed that the level of emc mRNA increased 10-fold in rbp9 mutant flies. Taken together with the in vitro RNA binding results, the genetic evidence obtained strongly supports the hypothesis that Rbp9 functions as a regulator of RNA stability.     

Inhibition of protein synthesis sensitizes thermotolerant cells to heat shock induced apoptosis

Hyperthermia is a potent inducer of apoptosis in many cell lines. A brief exposure to mildly elevated temperatures elicits a transient state of augmented resistance to subsequent thermal stress. Here we show that a hyperthermic treatment of 43°C for 1 h is sufficient to induce apoptosis in the cell line HL-60. This observation is based on morphologic evaluation and on comet assay results (an extremely sensitive method of detecting and quantifying apoptotic DNA fragmentation in individual cells). The thermotolerance phenomenon was also verified in the same manner by giving the cells a brief 30 min sub-lethal heat conditioning treatment at 43°C followed by a 6 h incubation time prior to the administration of a lethal heat load (43°C for 1 h). We observed a dramatic decrease in resultant apoptoses in the thermotolerized cells in comparison to unconditioned cells. We assessed the necessity of de novo protein synthesis in the protective phenomenon. When the conditioned cells were given a cycloheximide treatment prior to heat conditioning we saw a sensitization of the conditioned cells to secondary thermal injury. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis of low molecular weight RNA components in cells with a temperature-sensitive polymerase II

AF 8 cells are a mutant cell line of baby hamster kidney cells with a temperature-sensitive polymerase II activity. When these cells grow at the non-permissive temperature (40 degrees C) the syntheseis of low molecular weight RNA components D, C and A is preferentially inhibited, whereas the synthesis of rRNA, tRNA, 5 S RNA and component L is affected only a little or not at all. These results indicate that polymerase II catalyzes the synthesis of components D, C and A.