Concentrations of individual RNA sequences in polyadenylated nuclear and cytoplasmic RNA populations of Drosophila cells.

Steady state concentrations of individual RNA sequences in poly(A) nuclear and cytoplasmic RNA populations of Drosophila Kc cells were determined using cloned cDNA fragments. These cDNAs represent poly(A) RNA sequences of different abundance in the cytoplasm of Kc cells, but their steady state concentrations in poly(A) hnRNA was always lower. Of ten different sequences analysed, eight showed some four-fold lower concentration in hnRNA mRNA, two were underrepresented in hnRNA relative to the others. The obvious clustering of mRNA/hnRNA ratios is discussed in relation to sequence complexity and turnover rates of these RNA populations.     

Drosophila cells and ecdysterone: a model system for gene regulation

When Drosophila cell lines are exposed to physiological doses of the steroid molting hormone, ecdysterone, they enter mitotic arrest and differentiate morphologically. These responses are accompanied by specific changes in gene expression. Several enzyme activities (acetylcholinesterase, beta-galactosidase, dopa decarboxylase, and catalase) are induced and the synthesis of a cytoplasmic actin and the four small heat-shock proteins is initiated. Several of these ecdysterone inducible genes have been physically isolated and characterized, in several cases by DNA sequencing. Current studies focus on introducing cloned ecdysterone inducible genes into responsive cells by DNA mediated transfection. Once it is clear that these introduced genes acquire the normal pattern of hormone-regulated gene expression in the cell, in vitro mutagenesis can be used before transfection to modify their structure. Transient expression, then, can become a functional assay to define regions of DNA flanking the coding region of inducible genes that are needed for proper gene expression and regulation in cultured cells.     

Diversity of sequences in total and polyadenylated nuclear RNA from Drosophila cells.

Complementary DNA was synthesized using polyadenylated nuclear RNA of cultured Drosophila cells as template. The kinetics of hybridization of this cDNA with nuclear RNA indicated that the complexity of this RNA population is five to ten times greater than that of cytoplasmic mRNA. The same difference in the fraction of DNA represented was obtained when nuclear and cytoplasmic RNA were hybridized with labeled unique sequence DNA. The fraction of the DNA sequences represented in total number of polyadenylated nuclear RNA is much higher than that represented in cytoplasmic RNA.     

The nuclear experience of CPEB: Implications for RNA processing and translational control

CPEB is a sequence-specific RNA binding protein that promotes polyadenylation-induced translation in early development, during cell cycle progression and cellular senescence, and following neuronal synapse stimulation. It controls polyadenylation and translation through other interacting molecules, most notably the poly(A) polymerase Gld2, the deadenylating enzyme PARN, and the eIF4E-binding protein Maskin. Here, we report that CPEB shuttles between the nucleus and cytoplasm and that its export occurs via the CRM1-dependent pathway. In the nucleus of Xenopus oocytes, CPEB associates with lampbrush chromosomes and several proteins involved in nuclear RNA processing. CPEB also interacts with Maskin in the nucleus as well as with CPE-containing mRNAs. Although the CPE does not regulate mRNA export, it influences the degree to which mRNAs are translationally repressed in the cytoplasm. Moreover, CPEB directly or indirectly mediates the alternative splicing of at least one pre-mRNA in mouse embryo fibroblasts as well as certain mouse tissues. We propose that CPEB, together with Maskin, binds mRNA in the nucleus to ensure tight translational repression upon export to the cytoplasm. In addition, we propose that nuclear CPEB regulates specific pre-mRNA alternative splicing.     

Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates.

1. A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E. coli cells. 2. Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E. coli cells. 3. Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+. Other divalent cations could not replace Mn2+. 4. Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal. 5. The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C. 6. Further characterization of this activity indicated that it is RNase III. 7. To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E. coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C). 8. This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E. coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III.     

Mitochondrial ribosomal RNA genes of yeast: Their mutations and a common nuclear suppressor

Summary Due to the absence of repetition of the rRNA genes in S. cerevisiae mitochondria, isolation of ribosomal mutants at the level of the rRNA genes is relatively easy in this system. We describe here a novel thermosensitive mutation, ts1297, localized by rho^- deletion mapping in (or very close to) the sequence corresponding to the small ribosomal RNA (15S) gene. Defective mutations of the small rRNA have not been reported so far.In the mutant, the amount of 15S rRNA and of the small ribosomal subunit, 37S, is reduced. The quantity of the large ribosomal RNA (21S), directly extracted from mitochondria, appears normal. However, the large ribosomal subunit, 50S, seems to be fragile and could be recovered only in the presence of Ca²⁺ in place of Mg²⁺. The 50S particles seem to be completely degraded under normal conditions of extraction with Mg²⁺.The thermosensitive phenotype of the ts1297 mutant is suppressed by a nuclear mutation SU₁₀₁. The SU₁₀₁ mutation had been originally isolated as a suppressor of another mitochondrial mutation, ts902, which is located within the 21S rRNA gene.These results suggest that the mitochondrial mutations ts1297 and ts902 are both involved in the interaction of the large and small ribosomal subunits.