Translational control by 80S formation and 60S availability: The central role of eIF6, a rate limiting factor in cell cycle progression and tumorigenesis

Ribosome biogenesis and translation can be simplified as the processes of generating ribosomes and their use for decoding mRNA into a protein. Ribosome biogenesis has been efficiently studied in unicellular organisms like the budding yeast, allowing us a deep and basic knowledge of this process in growing cells. Translation has been modeled in vitro and in unicellular organisms. These studies have given us an important insight into the mechanisms and evolutionarily conserved aspects of ribosome biology. However, we advocate the need of the direct study of these processes in multicellular organisms. Analysis of ribosome biogenesis and translation in vivo in Metazoa and mammalian models is emerging and unveils the unexpected consequences of perturbed ribosome biogenesis and translation. Here, we will describe how one factor, eIF6, plays a crucial role both in the generation of the large ribosomal subunit and its availability for translation. From there, we will make specific conclusions on the physiological relevance of eIF6 in 80S formation, cell cycle progression and disease, raising the point that the control of gene expression may occur at the unexpected level of the large ribosomal subunit. In the future, the modulation of eIF6 binding to the 60S may be pharmacologically exploited to reduce the growth of cancer cells or ameliorate the phenotype of SDS syndrome.     

The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M_r 25,550), designated TIF6, has been cloned and expressed in Escherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.     

Coordination of gene expression with growth rate: A feedback or a feed-forward strategy?

In the budding yeast, a large fraction of genes is coordinately regulated with growth rate. We argue that this correlation does not reflect a direct feedback from growth rate to gene expression. Rather, what appears to be a response to growth rate is dominated by environmental sensing. External parameters, such as nutrition or temperature, feed-forward to define gene expression pattern that is tuned to the evolutionary-predicted growth rate. While such a feed-forward strategy requires fine-tuning of signaling mechanisms, and is limited in the range of environments that can be monitored, it enables advanced preparation to physiological changes that predictably occur following environmental switching. The capacity to anticipate and prepare for changing conditions was probably a major selection force during yeast evolution.     

Coordination of Ribosomal Protein and Ribosomal RNA Gene Expression in Response to TOR Signaling

Cells grow in response to nutrients or growth factors, whose presence is detected and communicated by elaborate signaling pathways. Protein kinases play crucial roles in processes such as cell cycle progression and gene expression, and misregulation of such pathways has been correlated with various diseased states. Signals intended to promote cell growth converge on ribosome biogenesis, as the ability to produce cellular proteins is intimately tied to cell growth. Part of the response to growth signals is therefore the coordinate expression of genes encoding ribosomal RNA (rRNA) and ribosomal proteins (RP). A key player in regulating cell growth is the Target of Rapamycin (TOR) kinase, one of the gatekeepers that prevent cell cycle progression from G1 to S under conditions of nutritional stress. TOR is structurally and functionally conserved in all eukaryotes. Under favorable growth conditions, TOR is active and cells maintain a robust rate of ribosome biogenesis, translation initiation and nutrient import. Under stress conditions, TOR signaling is suppressed, leading to cell cycle arrest, while the failure of TOR to respond appropriately to environmental or nutritional signals leads to uncontrolled cell growth. Emerging evidence from Saccharomyces cerevisiae indicates that High Mobility Group (HMGB) proteins, non-sequence-specific chromosomal proteins, participate in mediating responses to growth signals. As HMGB proteins are distinguished by their ability to alter DNA topology, they frequently function in the assembly of higher-order nucleoprotein complexes. We review here recent evidence, which suggests that HMGB proteins may function to coordinate TOR-dependent regulation of rRNA and RP gene expression.Key Words: Rapamycin, TORC1, HMO1, high mobility group, yeast, RP gene, rDNA.     

The human mitochondrial ADP/ATP carriers: kinetic properties and biogenesis of wild-type and mutant proteins in the yeast S. cerevisiae

The mitochondrial adenine nucleotide carrier, or Ancp, plays a key role in the maintenance of the energetic fluxes in eukaryotic cells. Human disorders have been found associated to unusual human ANC gene (HANC) expression but also to direct inactivation of the protein, either by autoantibody binding or by mutation. However, the individual biochemical properties of the three HAncp isoforms have not yet been deciphered. To do so, the three HANC ORF were expressed in yeast under the control of the regulatory sequences of ScANC2. Each of the three HANC was able to restore growth on a nonfermentable carbon source of a yeast mutant strain lacking its three endogenous ANC. Their ADP/ATP exchange properties could then be measured for the first time in isolated mitochondria. HANC3 was the most efficient to restore yeast growth, and HAnc3p presented the highest V(M) (80 nmol ADP min(-1) mg protein(-1)) and K(ADP)(M)(8.4 microM). HAnc1p and HAnc2p presented similar kinetic constants (V(M) approximately 30-40 nmol ADP min(-(1) mg protein(-1) and K(ADP)(M) approximately 2.5-3.7 microM), whose values were consistent with HANC1's and HANC2's lower capacity to restore yeast growth. However, the HANC genes restored growth at a lower level than ScANC2, indicating that HAncp amount may be limiting in vivo. To optimize the HAncp production, we investigated their biogenesis into mitochondria by mutagenesis of two charged amino acids in the N-terminus of HAnc1p. Severe effects were observed with the D3A and D3K mutations that precluded yeast growth. On the contrary, the K10A mutation increased yeast growth complementation and nucleotide exchange rate as compared to the wild type. These results point to the importance of the N-terminal region of HAnc1p for its biogenesis and transport activity in yeast mitochondria.     

Ribosomal 18 S RNA Processing by the IGF-I-responsive WDR3 Protein Is Integrated with p53 Function in Cancer Cell Proliferation

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G₁ phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.     

Nitrate regulation of metabolism and growth.

Recent research shows that signals derived from nitrate are involved in triggering widespread changes in gene expression, resulting in a reprogramming of nitrogen and carbon metabolism to facilitate the uptake and assimilation of nitrate, and to initiate accompanying changes in carbon metabolism. These nitrate-derived signals interact with signals generated further downstream in nitrogen metabolism, and in carbon metabolism. Signals derived from internal and external nitrate also adjust root growth and architecture to the physiological state of the plant, and the distribution of nitrate in the environment.