Jonathan R. Warner

The economics of ribosome biosynthesis in yeast

In a rapidly growing yeast cell, 60% of total transcription is devoted to ribosomal RNA, and 50% of RNA polymerase II transcription and 90% of mRNA splicing are devoted to ribosomal proteins (RPs). Coordinate regulation of the approximately 150 rRNA genes and 137 RP genes that make such prodigious use of resources is essential for the economy of the cell. This is entrusted to a number of signal transduction pathways that can abruptly induce or silence the ribosomal genes, leading to major implications for the expression of other genes as well.

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.

How Common Are Extraribosomal Functions of Ribosomal Proteins

Ribosomal proteins are ubiquitous, abundant, and RNA binding: prime candidates for recruitment to extraribosomal functions. Indeed, they participate in balancing the synthesis of the RNA and protein components of the ribosome itself. An exciting new story is that ribosomal proteins are sentinels for the self-evaluation of cellular health. Perturbation of ribosome synthesis frees ribosomal proteins to interface with the p53 system, leading to cell-cycle arrest or to apoptosis. Yet in only a few cases can we clearly identify the recruitment of ribosomal proteins for other extraribosomal functions. Is this due to a lack of imaginative evolution by cells and viruses, or to a lack of imaginative experiments by molecular biologists?

Rapidly labeled HeLa cell nuclear RNA: I. Identification by zone sedimentation of a heterogeneous fraction separate from ribosomal precursor RNA

The rapidly labeled RNA from HeLa cells has been further studied. The previously described 45 s ribosomal precursor RNA can be separated in zone sedimentation experiments from a heterogeneous 20 to 80 s rapidly labeled species. As much as 50% of the [3H]uridine incorporated into RNA in four to five minutes is found in this heterogeneous fraction. The majority of the rapidly labeled RNA which decays when cells are treated with actinomycin comes from this RNA class.

Ribosomal RNA synthesis in Saccharomyces cerevisiae

Ribosomal RNA biosynthesis in metabolically active spheroplasts of the yeast Saccharomyces cerevisiae was selected as a model system for the study of nucleo-cytoplasmic interactions. Examination of the total cellular RNA synthesized during short exposures to labeled nucleosides demonstrated the existence of three large, short-lived RNA species of 35 s, 27 s and 20 s, in addition to the mature ribosomal RNAs, 25 s and 18 s. Little or no RNA larger than 35 s is found. The kinetics of synthesis of these RNA molecules was pursued by continuous labeling and pulse-chase experiments, using [methyl-3H]methionine to label only ribosomal species. These results demonstrated the following precursor-product relationship: In the presence of cycloheximide the terminal processing steps leading to the mature ribosomal RNAs are severely inhibited, indicating that the maturation of ribosomal precursor RNA requires continuous protein synthesis. RNA denaturation studies using dimethyl sulfoxide showed the precursors to be single polyribonucleotide chains larger than the mature rRNAs, rather than aggregates of smaller molecules, or conformational isomers of the ribosomal species. Molecular sizes of these RNAs were estimated by electrophoretic mobility in acrylamide gels. The 35 s precursor is 2.5 × 106 daltons. Its cleavage appears to be conservative, producing intermediate molecules of 1.6 × 106 and 0.8 × 106 daltons. The final maturation steps yield mature rRNAs of 1.3 × 106 and 0.7 × 106 daltons with the concomitant loss of about 20% of the original precursor molecule. In addition, it was concluded that the small RNA species, 5.8 s (6 × 104 daltons) found non-covalently bound to the 25 s RNA, is generated during the final cleavage of the 27 s precursor molecule. In contrast, 5 s RNA is synthesized independently. These results support the contention that all eukaryotes synthesize ribosomes similarly and that yeast may be a useful organism for the investigation of intracellular communication, particularly across the nuclear membrane.

Proposed Uniform Nomenclature for Mammalian Ribosomal Proteins

SummaryThe numbering systems for mammalian ribosomal proteins used in several laboratories have been correlated and a proposal for a standard system is presented.

Continued functioning of the secretory pathway is essential for ribosome synthesis.

To explore the regulatory elements that maintain the balanced synthesis of the components of the ribosome, we isolated a temperature-sensitive (ts) mutant of Saccharomyces cerevisiae in which transcription both of rRNA and of ribosomal protein genes is defective at the nonpermissive temperature. Temperature sensitivity for growth is recessive and segregates 2:2. A gene that complements the ts phenotype was cloned from a genomic DNA library. Sequence analysis revealed that this gene is SLY1, encoding a protein essential for protein and vesicle transport between the endoplasmic reticulum and the Golgi apparatus. In the strain carrying our ts allele of SLY1, accumulation of the carboxypeptidase Y precursor was detected at the nonpermissive temperature, indicating that the secretory pathway is defective. To ask whether the effect of the ts allele on ribosome synthesis was specific for sly1 or was a general result of the inactivation of the secretion pathway, we assayed the levels of mRNA for several ribosomal proteins in cells carrying ts alleles of sec1, sec7, sec11, sec14, sec18, sec53, or sec63, representing all stages of secretion. In each case, the mRNA levels were severely depressed, suggesting that this is a common feature in mutants of protein secretion. For the mutants tested, transcription of rRNA was also substantially reduced. Furthermore, treatment of a sensitive strain with brefeldin A at a concentration sufficient to block the secretion pathway also led to a decrease of the level of ribosomal protein mRNA, with kinetics suggesting that the effect of a secretion defect is manifest within 15 to 30 min. We conclude that the continued function of the entire secretion pathway is essential for the maintenance of ribosome synthesis. The apparent coupling of membrane synthesis and ribosome synthesis suggest the existence of a regulatory network that connects the production of the various structural elements of the cell.

Central role of Ifh1p-Fhl1p interaction in the synthesis of yeast ribosomal proteins

The 138 genes encoding the 79 ribosomal proteins (RPs) of Saccharomyces cerevisiae form the tightest cluster of coordinately regulated genes in nearly all transcriptome experiments. The basis for this observation remains unknown. We now provide evidence that two factors, Fhl1p and Ifh1p, are key players in the transcription of RP genes. Both are found at transcribing RP genes in vivo. Ifh1p, but not Fhl1p, leaves the RP genes when transcription is repressed. The occupancy of the RP genes by Ifh1p depends on its interaction with the phospho‐peptide recognizing forkhead‐associated domain of Fhl1p. Disruption of this interaction is severely deleterious to ribosome synthesis and cell growth. Loss of functional Fhl1p leads to cells that have only 20% the normal amount of RNA and that synthesize ribosomes at only 5–10% the normal rate. Homeostatic mechanisms within the cell respond by reducing the transcription of rRNA to match the output of RPs, and by reducing the global transcription of mRNA to match the capacity of the translational apparatus.

The Saccharomyces cerevisiae TIF6 Gene Encoding Translation Initiation Factor 6 Is Required for 60S Ribosomal Subunit Biogenesis

ABSTRACT Eukaryotic translation initiation factor 6 (eIF6), a monomeric protein of about 26 kDa, can bind to the 60S ribosomal subunit and prevent its association with the 40S ribosomal subunit. InSaccharomyces cerevisiae, eIF6 is encoded by a single-copy essential gene. To understand the function of eIF6 in yeast cells, we constructed a conditional mutant haploid yeast strain in which a functional but a rapidly degradable form of eIF6 fusion protein was synthesized from a repressible GAL10 promoter. Depletion of eIF6 from yeast cells resulted in a selective reduction in the level of 60S ribosomal subunits, causing a stoichiometric imbalance in 60S-to-40S subunit ratio and inhibition of the rate of in vivo protein synthesis. Further analysis indicated that eIF6 is not required for the stability of 60S ribosomal subunits. Rather, eIF6-depleted cells showed defective pre-rRNA processing, resulting in accumulation of 35S pre-rRNA precursor, formation of a 23S aberrant pre-rRNA, decreased 20S pre-rRNA levels, and accumulation of 27SB pre-rRNA. The defect in the processing of 27S pre-rRNA resulted in the reduced formation of mature 25S and 5.8S rRNAs relative to 18S rRNA, which may account for the selective deficit of 60S ribosomal subunits in these cells. Cell fractionation as well as indirect immunofluorescence studies showed that c-Myc or hemagglutinin epitope-tagged eIF6 was distributed throughout the cytoplasm and the nuclei of yeast cells.

Coordinate control of syntheses of ribosomal ribonucleic acid and ribosomal proteins during nutritional shift-up in Saccharomyces cerevisiae.

We investigated the regulation of ribosome synthesis in Saccharomyces cerevisiae growing at different rates and in response to a growth stimulus. The ribosome content and the rates of synthesis of ribosomal ribonucleic acid and of ribosomal proteins were compared in cultures growing in minimal medium with either glucose or ethanol as a carbon source. The results demonstrated that ribosome content is proportional to growth rate. Moreover, these steady-state concentrations are regulated at the level of synthesis of ribosomal precursor ribonucleic acid and of ribosomal proteins. When cultures growing on ethanol were enriched with glucose, the rate of ribosomal ribonucleic acid synthesis, measured by pulsing cells with [methyl-3H]methionine, increased by 40% within 5 min, doubled within 15 min, and reached a steady state characteristic of the new growth medium by 30 min. Labeling with [3H]leucine reveal a coordinate increase in the rate of synthesis of 30 or more ribosomal proteins as compared with that of total cellular proteins. Their synthesis was stimulated approximately 2.5-fold within 15 min and nearly 4-fold within 60 min. The data suggest that S. cerevisiae responds to a growth stimulus by preferential stimulation of the synthesis of ribosomal ribonucleic acid and ribosomal proteins.