Applying genetics to the splicing problem.
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Genes, Fungal↗
Biomedical subjects
J R Warner
Publications and source records attributed to J R Warner.
Phosphorylation of the Saccharomyces cerevisiae equivalent of ribosomal protein S6 has no detectable effect on growth.
The phosphorylation of mammalian ribosomal protein S6 is affected by a variety of agents, including growth factors and tumor promoters, as well as by expressed oncogenes. Its potential role in the regulation of protein synthesis has been the object of much study. We have developed strains of Saccharomyces cerevisiae in which the phosphorylatable serines of the equivalent ribosomal protein (S10) were converted to alanines by site-directed mutagenesis. The S10 of such cells is not phosphorylated. Comparison of these cells with the parental cells, whose genomes differ by only six nucleotides, revealed no differences in the lag phase or logarithmic phase of a growth cycle, in growth on different carbon sources, in sporulation, or in sensitivity to heat shock. We conclude that in S. cerevisiae the phosphorylation of ribosomal protein S10 may play no role in regulating the synthesis of proteins. This conclusion leads one to ask whether certain protein phosphorylations are simply the adventitious, if easily observable, result of the imperfect specificity of one or another protein kinase.
Amino Acid Sequence↗
Autogenous regulation of splicing of the transcript of a yeast ribosomal protein gene.
The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.
Escherichia coli↗
An RNA polymerase I enhancer in Saccharomyces cerevisiae.
By the use of an artificial gene coding for rRNA (rDNA gene), we found that transcription of the major precursor rRNA in Saccharomyces cerevisiae cells is stimulated 15-fold by a positive control element located 2 kilobases upstream of the transcription initiation site. Analysis of in vitro runon transcripts suggests that this promoter element increases the frequency of initiation by RNA polymerase I molecules. A 190-base-pair fragment encompassing the promoter element can stimulate transcription on a centromere plasmid in either orientation, upstream or downstream of the transcription initiation site, suggesting that it is an enhancer element. Integration of artificial rDNA genes into a nonribosomal locus in the genome demonstrates that the rDNA enhancer functions either 5' or 3' to an rRNA transcription unit, suggesting it may operate in both directions within the rDNA tandem array. This is the first observation in S. cerevisiae of the stimulation of transcription by an element placed downstream. Finally, enhancer activity is dependent upon sequences that lie at both boundaries of the 190-base-pair fragment. In particular, a 5-base-pair deletion at the extreme 3' boundary of the 190-base-pair fragment greatly reduces the activation of transcription and implicates a set of inverted repeats.
Base Sequence↗
Molecular inventory control in ribosome biosynthesis.
The eukaryotic cell coordinates the accumulation of each ribosomal protein with every other ribosomal protein, with ribosomal RNA and with the needs of the cell. To do so it regulates the transcription, processing, translation and lifetime of the mRNA for ribosomal proteins. When all else fails, it rapidly degrades any excess ribosomal protein which is synthesized.
Animals↗
Phosphorylation of the yeast equivalent of ribosomal protein S6 is not essential for growth.
The yeast equivalent of ribosomal protein S6, known as S10, can be modified by the addition of two phosphates. The two adjacent serines that are likely to be subject to phosphorylation were deduced by comparison with the known sites of phosphorylation on rat liver S6. Using oligonucleotide mutagenesis, we altered the gene for S10 to replace these two serines with alanines. This mutant gene was introduced into a diploid yeast cell heterozygous for each of the two S10 genes. After sporulation, we obtained colonies in which the mutant gene was the only intact S10 gene. Although the ribosomes of these cells contained a full complement of S10, no phosphorylation of S10 was detected. These cells grow exponentially with a doubling time about 50% greater than that of control cells. We conclude that the phosphorylation of S10 is not essential for growth. However, the mutant gene in such cells is very unstable, frequently reverting to wild type, presumably by interaction with the disrupted host genes. We suggest that at some stage of the growth cycle there is strong selection for S10 that can be phosphorylated.
Fungal Proteins↗
Saccharomyces cerevisiae coordinates accumulation of yeast ribosomal proteins by modulating mRNA splicing, translational initiation, and protein turnover.
The rate of accumulation of each ribosomal protein is carefully regulated by the yeast cell to provide the equimolar ratio necessary for the assembly of the ribosome. The mechanisms responsible for this regulation have been examined by introducing into the yeast cell extra copies of seven individual ribosomal protein genes carried on autonomously replicating plasmids. In each case studied the plasmid-borne gene was transcribed to the same degree as the genomic gene. Nevertheless, the cell maintained a balanced accumulation of ribosomal proteins, using a variety of methods other than transcription. (i) Several ribosomal proteins were synthesized in substantial excess. However, the excess ribosomal protein was rapidly degraded. (ii) The excess mRNA for two of the ribosomal protein genes was translated inefficiently. We provide evidence that this was due to inefficient initiation of translation. (iii) The transcripts derived from two of the ribosomal protein genes were spliced inefficiently, leading to an accumulation of precursor RNA. We present a model which proposes the autogenous regulation of mRNA splicing as a eucaryotic parallel of the autogenous regulation of mRNA translation in procaryotes. Finally, the accumulation of each ribosomal protein was regulated independently. In no instance did the presence of excess copies of the gene for one ribosomal protein affect the synthesis of another ribosomal protein.
Cloning, Molecular↗
A comparison of yeast ribosomal protein gene DNA sequences.
The DNA sequences of eight yeast ribosomal protein genes have been compared for the purpose of identifying homologous regions which may be involved in the coordinate regulation of ribosomal protein synthesis. A 12 bp homology was identified in the 5' DNA sequence preceding the structural gene for 6 out of 8 yeast ribosomal protein genes. In each case the homologous sequence was found at a position approximately 300 bp preceding the transcription start of the ribosomal protein gene. This homology was not identified in any non-ribosomal protein gene examined. Additional homologies between ribosomal protein genes were identified in the transcribed regions, including the untranslated 5' and 3' DNA regions flanking the coding regions.
Amino Acid Sequence↗
A yeast ribosomal protein gene whose intron is in the 5' leader.
The complete gene encoding yeast ribosomal protein 29, a component of the 60 S ribosomal subunit of Saccharomyces cerevisiae has been isolated and its nucleotide sequence determined. The coordinates of transcription initiation and termination of the rp29 gene have been mapped. Transcription appears to start at several sites, spanning nearly 30 nucleotides. The gene is transcribed into a precursor RNA molecule of 1110 to 1085 nucleotides from which an intron of 458 nucleotides is spliced out. The splice junction has been determined by comparing the nucleotide sequences of the rp29 gene with those of a homologous cDNA clone. Interestingly, the intron of the rp29 gene resides in the 5' untranslated region of the rp29 mRNA, the first instance where this has been observed in yeast. The single open reading frame in the rp29 gene encodes a very basic protein of 155 amino acid residues.
Amino Acid Sequence↗
DNA sequence analysis on the IBM-PC.
We have developed, for the IBM-PC microcomputer, a menu driven, interactive set of programs which provide the functions routinely used for DNA sequence data analyses.
Amino Acid Sequence↗
The major promoter element of rRNA transcription in yeast lies 2 kb upstream.
Conventional genetic analysis of the transcription of rDNA in yeast is precluded because the genes are highly reiterated. As an alternative strategy to determine which sequences modulate transcription of pre-rRNA, a series of artificial rRNA genes containing a fragment of DNA from E. coli bacteriophage T7 were introduced into the yeast Saccharomyces cerevisiae. Correct transcription of the artificial genes was observed. Three regions of ribosomal spacer are found to affect transcription of rRNA. Sequences within 210 bp of the 5' terminus of 35S rRNA support low levels of transcription, but at multiple initiation points. Sequences from -210 to -2230 direct correct initiation and increase somewhat the efficiency of transcription. Most striking is that sequences from -2230 to -2420 stimulate transcription 15-fold. The function of this major promoter element is absolutely orientation-dependent but relatively independent of position. Its activity is blocked when an rRNA transcription termination sequence is placed between it and the site of initiation.
Base Composition↗
Cycloheximide resistance in yeast: the gene and its protein.
Mutations in the yeast gene CYH2 can lead to resistance to cycloheximide, an inhibitor of eukaryotic protein synthesis. The gene product of CYH2 is ribosomal protein L29, a component of the 60S ribosomal subunit. We have cloned the wild-type and resistance alleles of CYH2 and determined their nucleotide sequence. Transcription of CYH2 appears to initiate and terminate at multiple sites, as judged by S1 nuclease analysis. The gene is transcribed into an RNA molecule of about 1082 nucleotides, containing an intervening sequence of 510 nucleotides. The splice junction of the intron resides within a codon near the 5' end of the gene. In confirmation of peptide analysis by Stocklein et al. (1) we find that resistance to cycloheximide is due to a transversion mutation resulting in the replacement of a glutamine by glutamic acid in position 37 of L29.
Alleles↗
Messenger RNA for ribosomal proteins in yeast.
The concentrations and mean lifetimes of the messenger RNAs for four ribosomal proteins of the yeast, Saccharomyces cerevisiae, have been determined by the method of approach to equilibrium labeling, using cloned genes as hybridization probes. In cells growing in a minimal medium with glucose as a carbon source, there are roughly equimolar amounts of the four mRNAs, whose half-lives are very similar: 14 +/- 2 minutes, approximately 10% of a generation time. These results lead to an analysis of the economy of mRNA for ribosomal proteins that suggests that the mRNAs may be substantially underutilized.
Fungal Proteins↗
Mild temperature shock alters the transcription of a discrete class of Saccharomyces cerevisiae genes.
In Saccharomyces cerevisiae the synthesis of ribosomal proteins declines temporarily after a culture has been subjected to a mild temperature shock, i.e., a shift from 23 to 36 degrees C, each of which support growth. Using cloned genes for several S. cerevisiae ribosomal proteins, we found that the changes in the synthesis of ribosomal proteins parallel the changes in the concentration of mRNA of each. The disappearance and reappearance of the mRNA is due to a brief but severe inhibition of the transcription of each of the ribosomal protein genes, although the total transcription of mRNA in the cells is relatively unaffected by the temperature shock. The precisely coordinated response of these genes, which are scattered throughout the genome, suggests that either they or the enzyme which transcribes them has unique properties. In certain S. cerevisiae mutants, the synthesis of ribosomal proteins never recovers from a temperature shift. Yet both the decline and the resumption of transcription of these genes during the 30 min after the temperature shift are indistinguishable from those in wild-type cells. The failure of the mutant cells to grow at the restrictive temperature appears to be due to their inability to process the RNA transcribed from genes which have introns (Rosbash et al., Cell 24:679-686, 1981), a large proportion of which appear to be ribosomal protein genes.
RNA, Messenger↗
Molecular cloning and analysis of yeast gene for cycloheximide resistance and ribosomal protein L29.
A cosmid clone bank of yeast DNA has been used to isolate the cycloheximide resistance gene cyh2 of Saccharomyces cerevisiae. A cosmid carrying this gene was identified by cross hybridization to another cloned gene, tsm437. The two genes, which are tightly linked genetically are both present on a 31 kb segment of cloned DNA. The cyh2 gene encodes ribosomal protein L29, a component of the large subunit. Blot hybridization analysis reveals that this gene is present as a single copy in the yeast genome, unlike many other yeast ribosomal protein genes which appear to be duplicated. The cyh2 gene also appears to contain an intervening sequence, a characteristic common to most yeast ribosomal protein genes that have been cloned.
Alleles↗
Ribosomal proteins are synthesized preferentially in cells commencing growth.
Mouse 3T3 cells, in stationary phase because of serum deprivation, have only half the ribosome content of growing cells. Furthermore, the proportion of protein synthesis devoted to ribosomal proteins is only half that in growing cells. On addition of serum the synthesis of each ribosomal protein increases threefold, demonstrating the coordination of the synthesis of the ribosomal proteins. Half that increase is due to a general increase in total protein synthesis; half is due to a differential increase in ribosomal protein synthesis. The latter is abolished by a concentration of actinomycin D which blocks only ribosomal RNA transcription. The results are discussed with reference to a general hypothesis of growth regulation proposed by Stanners et al. (1979).
Animals↗
Yeast use translational control to compensate for extra copies of a ribosomal protein gene.
The efficient assembly of ribosomes requires a balanced synthesis of ribosomal RNA and each ribosomal protein. In an attempt to establish the mechanisms responsible for such balanced synthesis we have altered the gene dosage for one of the components by introducing into yeast an autonomously replicating plasmid containing the gene tcm1, which codes for ribosomal protein L3. The plasmid is maintained at 5-10 copies per cell by selection for expression of its URA3 gene. The plasmid-containing cells transcribe 7.5 times as much L3 mRNA as control cells, maintain 3.5 times as much L3 mRNA as control cells and synthesize no more than 1.2 times as much L3 protein as control cells. We conclude that the balanced synthesis of ribosomal proteins is maintained by modulating both the efficiency of translation and the lifetime of their mRNAs.
DNA, Recombinant↗
Survival of mononuclear phagocytes depends on a lineage-specific growth factor that the differentiated cells selectively destroy.
CSF-1 is a hemopoietic growth factor that specifically causes the proliferation and differentiation of mononuclear phagocytic cells. Receptors for CSF-1 occur exclusively on cells of the mononuclear phagocytic series (precursor leads to monoblast leads to promonocyte leads to monocyte leads to macrophage). Studies of the actions of CSF-1 on freshly explanted macrophages have been complicated by contamination of the primary cell isolates with CSF-1-producing cells and by the heterogeneity of the proliferative responses of individual macrophages. A method is described for the production of a highly purified and homogeneous population of adherent bone marrow-derived macrophages (BMMs) that are devoid of CSF-1-producing cells. The method may also be used to obtain nonadherent precursors of the mononuclear phagocytic series. Studies of CSF-1 action and degradation in cultures of BMMs have revealed several new findings. First, CSF-1 is required for both the survival (without proliferation) and the proliferation of BMMs. Second, CSF-1 is degraded by BMMs in a concentration-dependent manner, over the range of concentrations that stimulates both cell survival and proliferation. Third, the rate of CSF-1 degradation is saturable (or approximately 7 X 10(4) molecules per cell per hour) at CSF-1 concentrations that cause maximum proliferation (or approximately 0.4 nM). Under these conditions, BMMs are greatly enlarged and contain numerous phase-lucent vacuoles. Thus macrophages specifically require CSF-1 for both survival and proliferation, yet selectively and rapidly degrade it. This apparent dichotomy may have important implications for the role of CSF-1 in macrophage homeostasis in vivo.
Animals↗