Developmental expression and 5S rRNA-binding activity of Xenopus laevis ribosomal protein L5.

Ribosomal protein L5 binds specifically to 5S rRNA to form a complex that is a precursor to 60S subunit assembly in vivo. Analyses in yeast cells, mammalian cells, and Xenopus embryos have shown that the accumulation of L5 is not coordinated with the expression of other ribosomal proteins. In this study, the primary structure and developmental expression of Xenopus ribosomal protein L5 were examined to determine the basis for its distinct regulation. These analyses showed that L5 expression could either coincide with 5S rRNA synthesis and ribosome assembly or be controlled independently of these events at different stages of Xenopus development. L5 synthesis during oogenesis was uncoupled from the accumulation of 5S rRNa but coincided with subunit assembly. In early embryos, the inefficient translation of L5 mRNA resulted in the accumulation of a stable L5-5S rRNA complex before ribosome assembly at later stages of development. Additional results demonstrated that L5 protein synthesized in vitro bound specifically to 5S rRNA.     

Purification and characterization of ribosomal protein S6 kinase I from Xenopus eggs

Ribosomal protein S6 kinase I has been purified from unfertilized Xenopus eggs to near homogeneity as a Mr = 90,000 protein. S6 kinase I is phosphorylated when activated in vivo and can be phosphorylated by mitogen-activated protein kinase in vitro. The purified enzyme is inactivated upon treatment with protein phosphatase 2A. Immunological data and analysis of substrate specificity demonstrate that S6 kinase I is related to, but distinct from, the previously characterized S6 kinase II. Both enzymes are members of the ribosomal protein S6 kinase (rsk) gene family.     

Translational regulation of the expression of ribosomal protein genes in Xenopus laevis

The mRNAs coding for ribosomal proteins (rp-mRNA) are subjected to translational control during Xenopus oogenesis and embryogenesis, and also during nutritional changes in Xenopus cultured cells. This regulation, which appears to respond to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA engaged on polysomes, each translated rp-mRNA molecule always remaining fully loaded with ribosomes. All rp-mRNAs analyzed up to now show this translational behavior, and also share some structural features in their untranslated portions. In particular they all have rather short 5' untranslated regions, similar to each other, and always start at the very 5' end with a stretch of several pyrimidines. Fusion to a reporter-coding sequence of the 5' untranslated region of r-protein S19 has shown that this is involved in the translational regulation.     

Nucleotide sequence and 40 S subunit assembly of Xenopus laevis ribosomal protein S22

We have isolated and determined the nucleotide sequence of a cDNA encoding Xenopus laevis ribosomal protein S22. A synthetic S22 mRNA derived from this cDNA directs the synthesis of an in vitro translation product that is indistinguishable from S22 purified from Xenopus ovarian ribosomes. In vitro translated S22 is assembled into 40 S subunits when microinjected into the cytoplasm of oocytes. Analysis of the derived amino acid sequence indicates that Xenopus S22 is homologous to Escherichia coli ribosomal protein S10.     

Analysis of the mechanisms controlling the expression of the L1 ribosomal protein gene in Xenopus laevis

Conclusions We have shown that the accumulation of mature L1 mRNA is regulated, by reducing the efficiency of the splicing reaction, at the level of nuclear RNA stability. The present analysis is directed to the identification of the factors that are responsible for the block of splicing and for the specific cleavage of the precursor RNA. The results obtained so far demonstrated that a protein of 40 K interacts specifically with introns 2 and 3.     

The effect of insulin on intracellular ph and ribosomal protein S6 phosphorylation in oocytes of Xenopus laevis

Both insulin and progesterone are capable of stimulating germinal vesicle breakdown (GVBD) of large, Stage VI oocytes of Xenopus laevis. Numerous studies have shown an increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation prior to GVBD in oocytes treated with progesterone. In this study the effect of insulin and progesterone on pHi and S6 phosphorylation was compared. Both hormones increased pHi and S6 phosphorylation to similar levels and the time course of pHi change was the same for both hormones. Half-maximal effects of insulin were observed at 7 X 10(-8) M concentrations. In the presence of 1 nM cholera toxin, the ability of progesterone to induce these two responses was inhibited while the action of insulin was unaffected. However, GVBD induced by either hormone was blocked by cholera toxin. In small, Stage IV oocytes that do not undergo GVBD in response to either progesterone or insulin, a partial increase in pHi without S6 phosphorylation occurred in response to progesterone but both events occurred in response to insulin. These results suggest that the inability of Stage IV oocytes to undergo GVBD in response to hormone is not due to a failure to increase pHi or phosphorylate S6. The results in this paper also indicate that these events are regulated differently by insulin and progesterone in Xenopus oocytes.     

Isolation of the ribosomal protein gene S6 from Schizosaccharomyces pombe

Summary We screened a Schizosaccharomyces pombe genomic library using the ribosomal protein gene SI0 from Saccharomyces cerevisiae as a probe. Hybrid-selected translation of the positive clones revealed a ribosomal protein of S. pombe which is probably equivalent to the ribosomal protein SI0 from S. cerevisiae.     

The phosphorylation of ribosomal protein S6 from progesterone-stimulated Xenopus laevis oocytes. Kinetic studies and phosphopeptide analysis

Xenopus laevis oocytes were prelabeled with [32P]orthophosphate overnight before maturation was induced by progesterone stimulation. The phosphorylation status of ribosomal protein S6 from control oocytes and the temporal changes in S6 phosphorylation after progesterone treatment were analyzed by two-dimensional gel electrophoresis. S6 protein was separated in up to five distinct S6 species, which differed in their degree of phosphorylation. 32P labeling of S6, as judged from the shift of radioactivity into more highly phosphorylated S6 derivatives, continuously increased in progesterone-stimulated oocytes even at later times when germinal vesicle breakdown was completed. S6 protein of unstimulated oocytes was labeled to a lower degree. Trypsin cleavage of total S6 protein, isolated from control and maturing oocytes, gave rise to different complex phosphopeptide patterns reflecting the existence of various multiply phosphorylated S6 derivatives in both samples. Two of the more highly phosphorylated S6 derivatives showed considerable differences between the phosphopeptide elution profiles of control and stimulated oocytes indicating that dissimilar sites had been modified under both physiological conditions. Only phosphoserine was detected in the phosphoamino acid analysis of individual S6 derivatives.     

Purification and characterization of a protein kinase from Xenopus eggs highly specific for ribosomal protein S6

In Xenopus oocytes ribosomal protein S6 becomes phosphorylated on serine residues in response to hormones or growth factors and following microinjection of the tyrosine-specific protein kinases associated with Rous sarcoma virus or Abelson murine leukemia virus. To begin characterization of the enzymes responsible for S6 phosphorylation in this system, we have undertaken the purification of S6 protein kinases from unfertilized Xenopus eggs. DEAE-Sephacel chromatography of crude extracts revealed two peaks of S6 kinase activity, and the peak eluting at 160 mM NaCl was chosen for further purification. Successive chromatography on Mono S, Sephacryl S-200, Mono Q, and heparin-Sepharose resulted in purification of the enzyme to a single protein migrating at Mr = 92,000 on polyacrylamide gels. The final preparation was purified about 500-fold from the DEAE-Sephacel peak with a recovery of 10%. Apparent Km values of the enzyme for ATP and 40 S subunits were 28 and 5 microM, respectively, and the specific activity with 330 microM ATP and 5.6 microM 40 S subunits was 300 nmol/min/mg. The enzyme was inhibited by beta-glycerophosphate, sodium fluoride, potassium phosphate, ADP, heparin, quercetin, and spermine. The availability of a purified S6 protein kinase should facilitate elucidation of the molecular mechanism of S6 phosphorylation during growth stimulation.     

Isolation and characterization of cloned cDNAs that code for human ribosomal protein S6

Ribosomal protein (rp) S6 is the major substrate of protein kinases in eukaryotic ribosomes. To facilitate the identification of cloned cDNAs for human rpS6, we used published amino acid (aa) sequence data for rat liver rpS6 and yeast (Saccharomyces carlsbergensis) rpS10 to design mixed oligodeoxynucleotide probes. Screening of several human cDNA libraries with these probes permitted the isolation of plasmids which encompass the entire coding sequence of rpS6 (249 aa residues), 27 bp of the 5'-untranslated leader and all 39 bp of the 3'-untranslated region. A comparison of the predicted human rpS6 amino acid sequence and the yeast rpS10 amino acid sequence shows highly conserved areas separated by regions of divergence.     

Complementarity of conserved sequence elements present in 28S ribosomal RNA and in ribosomal protein genes of Xenopus laevis and Xenopus tropicalis

The sequence analysis of the L1 ribosomal protein (r-protein) gene of Xenopus laevis has revealed a strong homology in four out of the nine introns of the gene; this homology region spans 60 nucleotides (nt) with 80% homology [Loreni et al., EMBO J. 4 (1985) 3483-3488]. We have extended our analysis to X. tropicalis, a species which is closely related to X. laevis. Partial sequencing of the isolated L1 gene has revealed that these 60-nt homology regions are also present in at least two introns of the X. tropicalis L1 gene. Computer analysis has revealed that perfect nt sequence complementarity exists between 13 nt of this intron region and the 28S ribosomal RNA in a region which is conserved in all eukaryotes, suggesting a possible base-pairing interaction between these two sequences.