Regulation Of Ribosomal Protein Genes Research Articles

Repression of rRNA synthesis due to a secretory defect requires the C-terminal silencing domain of Rap1p in Saccharomyces cerevisiae

A secretory defect causes specific transcriptional repression of both ribosomal protein and ribosomal RNA genes, suggesting the coupling of plasma membrane and ribosome syntheses. We previously reported that the rap1-17 allele, which produced C-terminally truncated Rap1p, derepressed transcription of ribosomal protein genes when the secretory pathway was blocked. In this paper, we demonstrate that the rap1-17 mutation also leads to significant attenuation of transcriptional repression of rRNA genes due to a secretory defect. In contrast, the rap1-2 temperature-sensitive allele containing a unique missense mutation in the middle of the coding sequence has only a weak effect on repression. These results suggest that the C-terminal silencing domain of Rap1p is required for transcriptional repression of rDNA in response to a secretory defect. We also demonstrated that transcriptional regulation of ribosomal protein genes in response to nitrogen limitation was not affected by the rap1-17 allele, suggesting that the mechanism of nitrogen response is distinct from that of the secretory response.

Isolation and molecular characterisation of the gene encoding the cytoplasmic ribosomal protein S28 in Prunus persica [L.] Batsch

RT-PCR was performed on peach (Prunus persica [L.] Batsch) RNA to isolate cDNAs corresponding to transcripts which are differentially expressed in leaves borne on basal and apical shoots. A gene was identified which was more highly expressed in the leaves of basal shoots, and codes for the cytoplasmic protein S28 present in the small ribosomal subunit. The 5' leader regions of RPS28 mRNAs were found to harbour 8-11 pyrimidine tracts, which suggested similarities to regulatory stretches that control the translation of mRNAs for ribosomal proteins in animals. The peach S28 is encoded by two intron-containing genes, which are both transcribed in mitotically active tissues such as developing leaves and roots. In situ hybridisation to shoot vegetative apices and the measurement of nucleus/nucleolus ratios indicated that RPS28 expression was confined to areas undergoing active cell division. The mature RPS28 mRNA was detected as a single species in actively dividing tissues such as apical tips, developing leaves, vegetative buds, stamens, developing fruits and roots. In contrast, accumulation of a precursor RNA, in the presence of the mature product, was found in fully expanded leaves and subtending stems, while only the precursor species was detected in several late-stage tissues. This phenomenon suggested that expression of the mature RNA is controlled at the level of splicing and turnover of the precursor RNA. This is similar to the mode of regulation of ribosomal protein genes in animals.

Nutrient availability and the RAS/cyclic AMP pathway both induce expression of ribosomal protein genes in Saccharomyces cerevisiae but by different mechanisms.

By differential hybridization, we identified a number of genes in Saccharomyces cerevisiae that are activated by addition of cyclic AMP (cAMP) to cAMP-depleted cells. A majority, but not all, of these genes encode ribosomal proteins. While expression of these genes is also induced by addition of the appropriate nutrient to cells starved for a nitrogen source or for a sulfur source, the pathway for nutrient activation of ribosomal protein gene transcription is distinct from that of cAMP activation: (i) cAMP-mediated transcriptional activation was blocked by prior addition of an inhibitor of protein synthesis whereas nutrient-mediated activation was not, and (ii) cAMP-mediated induction of expression occurred through transcriptional activation whereas nutrient-mediated induction was predominantly a posttranscriptional response. Transcriptional activation of the ribosomal protein gene RPL16A by cAMP is mediated through a upstream activation sequence element consisting of a pair of RAP1 binding sites and sequences between them, suggesting that RAP1 participates in the cAMP activation process. Since RAP1 protein decays during starvation for cAMP, regulation of ribosomal protein genes under these conditions may directly relate to RAP1 protein availability. These results define additional critical targets of the cAMP-dependent protein kinase, suggest a mechanism to couple ribosome production to the metabolic activity of the cell, and emphasize that nutrient regulation is independent of the RAS/cAMP pathway.

Ribosomal Protein S11 Genes from Arabidopsis and Soybean

Ribosomes found in the eukaryotic cytosol are composed of four rRNAs and more than 70 different ribosomal proteins. Hariharan and Peny (1990) have demonstrated that several mouse ribosomal protein genes are transcribed at nearly equal rates, that these unrelated mouse genes have a remarkably similar promoter architecture, and that in some instances these genes appear to bind common trans-acting factors. Although a number of cDNA clones encoding plant cytosolic ribosomal proteins have been isolated and used to explore gene expression, the structure of very few plant ribosomal protein genes has been examined and little sequence information has been determined outside transcribed regions. The leve1 of ribosomal protein mRNA is typically proportional to the cellular growth rate and consequently is high in plant meristems (Lebrun and Freyssinet, 1991). The abundance of these transcripts can also be induced by a variety of treatments that lead to cellular proliferation (Gantt and Key, 1985). Clearly, expression of plant ribosomal protein genes is influenced by a large number of factors and virtually nothing is known about the mechanisms controlling the expression of these genes. To begin a search for cis-acting elements that might be important for ribosomal protein gene regulation, we determined the sequences of one of two or more genes (rpsll) encoding ribosomal protein S11 in Arabidopsis thaliana (Gantt and Thompson, 1990; Lu et al., 1993) and a homologous soybean gene (Table I). The 5‘ ends of the A. thaliana rpsll transcripts were mapped by primer extension and RNase protection analyses; results from both procedures suggest that transcripts are initiated at several closely spaced sites. We also observed two different primer extension products when soybean S l l RNAs were examined. Little sequence similarity is found when A. thaliana and soybean rpsll genes are compared. In the 5’ untranslated region, the sequence TTTGCCTACAA starts at positions 42 and 62 in the A. thaliana and soybean genes, respectively. In the 5’ flanking region, the sequence AAAAAGTAAAA is found at -185 (A. thaliana) and -239 (soybean). The sequence TTAGGGTTTT is also found in both genes: at -10 in A. thaliana and +16 in soybean. This sequence is notable because it is also found in the A. thaliana gene encoding ribosomal protein 515 (at +52), and a similar sequence has been noted in the promoter region

Transcription regulation of ribosomal protein genes at different growth rates in continuous cultures of Kluyveromyces yeasts.

We have investigated the relationship between the growth rate of two Kluyveromyces strains that differ in their maximum growth rate, namely K. lactis (mumax = 0.5 h-1) and K. marxianus (mumax = 1.1 h-1), and the transcription rate of ribosomal protein (rp) genes in these strains. The growth rate of either strain was varied by culturing the cells in a chemostat under conditions of glucose limitation at different dilution rates. Although the steady-state levels of transcription of the rp-genes of both Kluyveromyces strains were tightly coupled to the cellular growth rate, no clear relationship between the level of rp-gene transcription and the amount of in vitro binding of the RAP1- and ABF1-like proteins to the promoters of these rp-genes was observed. Upon a sudden increase in the growth rate of a steady-state culture, the transcription of rp-genes of K. lactis showed a different response from that in K. marxianus. Whereas a substantial overexpression of the K. lactis rp-genes was found during at least 4-5 h, the level of expression of the K. marxianus rp-genes was almost immediately adjusted to the new growth rate.

Coordiante expression of ribosomal protein genes inNeurospora Crassaand identification of conserved upstream sequences

The relative levels of rRNAs and ribosomal proteins are coordinately regulated by growth rate and carbon nutrition in Neurospora crassa. However, little is known about the mechanisms involved. To investigate the transcriptional regulation of ribosomal protein genes in N. crassa, we cloned and sequenced a ribosomal protein gene (crp-3). The inferred crp-3 protein sequence shares 89% and 83% homology at its N-terminus with the yeast rp51 and the human S17 ribosomal proteins respectively. The crp-3 gene contains two introns, neither of which are conserved in position with the RP51 or the S17 genes. The crp-3 gene is present in a single copy and was mapped by RFLP analysis to the right arm of linkage group IV, near the cot-1 locus. Sequence comparisons of the upstream regions of the three sequenced crp genes revealed several common features. These include a 'Taq box' (consensus: ARTTYGACTT) at -39, a CG repeat (consensus: CCCRCCRRR) at -65, and a major transcription initiation site embedded in a purine rich region flanked by an upstream pyrimidine rich sequence. Using four N.crassa ribosomal protein genes as probes, we demonstrated that the levels of the four ribosomal protein mRNAs were closely coordinated during a nutritional downshift from sucrose to quinic acid.

Post transcriptional regulation of ribosomal protein genes in xenopus and yeast

Post transcriptional regulation of ribosomal protein genes in xenopus and yeast

Sequence and regulatory responses of a ribosomal protein gene from the fission yeast Schizosaccharomyces pombe.

We have determined the nucleotide sequence and mapped the 5' and 3' termini of a ribosomal protein gene. The gene is transcribed into a RNA molecule of about 770 nt and appears to initiate at multiple sites, as judged by SI nuclease analysis. Gene dosage experiments with a plasmid born gene leads to a proportional increase of the messenger RNA, but not to an overproduction of the protein, suggesting a posttranscriptional control mechanism. However, the heat shock response of this gene indicates that there is also a potential for transcriptional control. Comparison of the 5' flanking region of this gene with the ribosomal protein gene S 6 from Schizosaccharomyces pombe and with ribosomal protein genes from Saccharomyces cerevisiae revealed homologous sequences, which may be involved in the regulation of ribosomal protein genes.

Posttranscriptional regulation and assembly into ribosomes of a Saccharomyces cerevisiae ribosomal protein-beta-galactosidase fusion.

To study the regulation of ribosomal protein genes, we constructed a 'lacZ fusion of the Saccharomyces cerevisiae RP51A gene, containing the first 64 codons of RP51A. In a strain lacking an intact RP51A gene (cells are viable due to the presence of an active RP51B gene), beta-galactosidase activity is 10-fold greater than in a strain containing RP51A. RP51A-lacZ mRNA levels are equal in the two strains, indicating that regulation is posttranscriptional. In the absence of the RP51A gene, the fusion protein is predominantly cytoplasmic and associated with polysomes, whereas in the presence of RP51A, the fusion protein is predominantly nuclear, and none is associated with polysomes. Deletions were made in the RP51A-coding portion of the fusion gene. The most extensively deleted gene, containing only the first seven RP51A codons fused to lacZ, produced a high level of beta-galactosidase activity in both the presence and the absence of the RP51A gene. In both cases, little or none of this shorter fusion protein was found associated with polysomes. Thus, a regulatory site (or sites) lies in the protein-coding region of RP51A. We suggest that posttranscriptional regulation of the rp51 fusion protein is related to assembly of the protein into ribosomes.

Posttranscriptional regulation and assembly into ribosomes of a Saccharomyces cerevisiae ribosomal protein-beta-galactosidase fusion.

To study the regulation of ribosomal protein genes, we constructed a 'lacZ fusion of the Saccharomyces cerevisiae RP51A gene, containing the first 64 codons of RP51A. In a strain lacking an intact RP51A gene (cells are viable due to the presence of an active RP51B gene), beta-galactosidase activity is 10-fold greater than in a strain containing RP51A. RP51A-lacZ mRNA levels are equal in the two strains, indicating that regulation is posttranscriptional. In the absence of the RP51A gene, the fusion protein is predominantly cytoplasmic and associated with polysomes, whereas in the presence of RP51A, the fusion protein is predominantly nuclear, and none is associated with polysomes. Deletions were made in the RP51A-coding portion of the fusion gene. The most extensively deleted gene, containing only the first seven RP51A codons fused to lacZ, produced a high level of beta-galactosidase activity in both the presence and the absence of the RP51A gene. In both cases, little or none of this shorter fusion protein was found associated with polysomes. Thus, a regulatory site (or sites) lies in the protein-coding region of RP51A. We suggest that posttranscriptional regulation of the rp51 fusion protein is related to assembly of the protein into ribosomes.