TOR signaling regulates ribosome and tRNA synthesis via LAMMER/Clk and GSK-3 family kinases.

Target of rapamycin (TOR)-dependent signaling and the control of cell growth is deregulated in many cancers. However, the signaling molecules downstream of TOR that coordinately regulate the synthesis of ribosomes and tRNAs are not well defined. Here, we show in yeast that conserved kinases of the LAMMER/Cdc-like and GSK-3 families function downstream of TOR complex 1 to repress ribosome and tRNA synthesis in response to nutrient limitation and other types of cellular stress. As a part of this response, we found that the LAMMER kinase Kns1 is differentially expressed and hyperphosphorylated and accumulates in the nucleus after rapamycin treatment, whereupon it primes the phosphorylation of the RNA polymerase III subunit Rpc53 by a specific GSK-3 family member, Mck1. In cooperation with another polymerase subunit, Rpc11, this phosphorylation of Rpc53 modifies the function of the enzyme and together with dephosphorylation of the Maf1 repressor inhibits the growth-promoting activity of RNA polymerase III transcription.

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?

Sequence context for transcription and translation of the Arabidopsis RPL23aA and RPL23aB paralogs.

The 80S cytoplasmic ribosome is responsible for translating the transcriptome into the proteome. Demand for ribosome production depends on growth rate, and both the ribosomal RNA (rRNA) and ribosomal protein (RP) components must respond coordinately and rapidly to positive and negative growth stimuli to prevent deleterious effects of excess or insufficient subunits. The 81 RPs of the Arabidopsis 80S ribosome are encoded by multigene families that often exhibit overlapping patterns of transcript accumulation; however, only one isoform of each RP family (with the exception of a small number of acidic RPs) assembles into a single ribosome. Here we dissected the regulatory regions (RRs) of both members of the RPL23a family (RPL23aA and RPL23aB) to identify salient cis-acting elements involved in transcriptional, posttranscriptional, and translational regulation of expression. Full length and truncated RRs of RPL23a paralogs were cloned upstream of a GUS reporter gene and expressed in Arabidopsis transgenic plants. High level expression in mitotically active tissues, driven by RPL23aA and RPL23aB RRs, required TATA-box, telo-box, and site II motif elements. First and second introns were found to play a minor role in posttranscriptional regulation of paralogs, and conserved transcript features (e.g., UTR base composition) may be involved in enhancing translational efficiency. Overall, our results indicate that RPL23a expression is governed by a complex network of multiple regulatory layers.

Fine-structure analysis of ribosomal protein gene transcription.

The ribosomal protein genes of Saccharomyces cerevisiae, responsible for nearly 40% of the polymerase II transcription initiation events, are characterized by the constitutive tight binding of the transcription factor Rap1. Rap1 binds at many places in the yeast genome, including glycolytic enzyme genes, the silent MAT loci, and telomeres, its specificity arising from specific cofactors recruited at the appropriate genes. At the ribosomal protein genes two such cofactors have recently been identified as Fhl1 and Ifh1. We have now characterized the interaction of these factors at a bidirectional ribosomal protein promoter by replacing the Rap1 sites with LexA operator sites. LexA-Gal4(AD) drives active transcription at this modified promoter, although not always at the correct initiation site. Tethering Rap1 to the promoter neither drives transcription nor recruits Fhl1 or Ifh1, showing that Rap1 function requires direct DNA binding. Tethering Fhl1 also fails to activate transcription, even though it does recruit Ifh1, suggesting that Fhl1 does more than simply provide a platform for Ifh1. Tethering Ifh1 to the promoter leads to low-level transcription, at the correct initiation sites. Remarkably, activation by tethered LexA-Gal4(AD) is strongly reduced when TOR kinase is inhibited by rapamycin. Thus, TOR can act independently of Fhl1/Ifh1 at ribosomal protein promoters. We also show that, in our strain background, the response of ribosomal protein promoters to TOR inhibition is independent of the Ifh1-related protein Crf1, indicating that the role of this corepressor is strain specific. Fine-structure chromatin mapping of several ribosomal protein promoters revealed that histones are essentially absent from the Rap1 sites, while Fhl1 and Ifh1 are coincident with each other but distinct from Rap1.

Eukaryotic cells producing ribosomes deficient in Rpl1 are hypersensitive to defects in the ubiquitin-proteasome system.

It has recently become clear that the misassembly of ribosomes in eukaryotic cells can have deleterious effects that go far beyond a simple shortage of ribosomes. In this work we find that cells deficient in ribosomal protein L1 (Rpl1; Rpl10a in mammals) produce ribosomes lacking Rpl1 that are exported to the cytoplasm and that can be incorporated into polyribosomes. The presence of such defective ribosomes leads to slow growth and appears to render the cells hypersensitive to lesions in the ubiquitin-proteasome system. Several genes that were reasonable candidates for degradation of 60S subunits lacking Rpl1 fail to do so, suggesting that key players in the surveillance of ribosomal subunits remain to be found. Interestingly, in spite of rendering the cells hypersensitive to the proteasome inhibitor MG132, shortage of Rpl1 partially suppresses the stress-invoked temporary repression of ribosome synthesis caused by MG132.

Why Dom34 stimulates growth of cells with defects of 40S ribosomal subunit biosynthesis.

A set of genome-wide screens for proteins whose absence exacerbates growth defects due to pseudo-haploinsufficiency of ribosomal proteins in Saccharomyces cerevisiae identified Dom34 as being particularly important for cell growth when there is a deficit of 40S ribosomal subunits. In contrast, strains with a deficit of 60S ribosomal proteins were largely insensitive to the loss of Dom34. The slow growth of cells lacking Dom34 and haploinsufficient for a protein of the 40S subunit is caused by a severe shortage of 40S subunits available for translation initiation due to a combination of three effects: (i) the natural deficiency of 40S subunits due to defective synthesis, (ii) the sequestration of 40S subunits due to the large accumulation of free 60S subunits, and (iii) the accumulation of ribosomes "stuck" in a distinct 80S form, insensitive to the Mg(2+) concentration, and at least temporarily unavailable for further translation. Our data suggest that these stuck ribosomes have neither mRNA nor tRNA. We postulate, based on our results and on previously published work, that the stuck ribosomes arise because of the lack of Dom34, which normally resolves a ribosome stalled due to insufficient tRNAs, to structural problems with its mRNA, or to a defect in the ribosome itself.

Yeast ribosomes: variety is the spice of life.

In the budding yeast Saccharomyces cerevisiae, 59 of the 79 cytoplasmic ribosomal proteins are encoded by two genes, stemming from an ancient genome duplication event. Komili et al. (2007) now report that these paralogous genes are not functionally equivalent, suggesting the possible existence of a "ribosome code."

The two ribosomal protein L23A genes are differentially transcribed in Arabidopsis thaliana.

Arabidopsis thaliana ribosomal protein (r-protein) L23A (RPL23A) is a member of the conserved L23/L25 family of primary ribosomal RNA (rRNA) binding proteins. The 2 AtRPL23A isoforms, RPL23A-1 and RPL23A-2, are 94% identical at the amino acid level, yet RPL23A-1 and RPL23A-2 share only approximately 40-50% primary sequence identity within the 5' regulatory regions. While the RPL23A-1 and -2 5' regulatory regions share many similar predicted motifs, the arrangement and number of these motifs differs between the 2 genes. Differences in regulation between RPL23A-1 and -2 have been investigated via reverse transcription-PCR (RT-PCR) expression profiles. Overall, transcript abundance for RPL23A-1 and -2 varied slightly in specific tissues and under some abiotic stresses. The highest transcript abundance for both RPL23A genes was detected in mitotically active tissues such as bud, flower and elongating carpel, as well as in root and stem while the lowest transcript levels were found in mature leaf and bract. Hormone-treated seedlings showed increased RPL23A-1 and -2 transcript levels following IAA and BAP treatment while ABA treatment resulted in a transient lowering of transcript levels. Expression patterns differed between RPL23A-1 and -2 in cold-, wound-, and copper-stressed seedlings. In all tissues examined, RPL23A-2 transcript levels were consistently lower than those of RPL23A-1. This report shows differential transcriptional regulation of the 2 RPL23A genes, which should no longer be identified as "housekeeping" genes.