mRNA translation is thought to be the most energy-consuming process in the cell. Translation and energy metabolism are dysregulated in a variety of diseases including cancer, diabetes, and heart disease. However, the mechanisms that coordinate translation and energy metabolism in mammals remain largely unknown. The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) stimulates mRNA translation and other anabolic processes. We demonstrate that mTORC1 controls mitochondrial activity and biogenesis by selectively promoting translation of nucleus-encoded mitochondria-related mRNAs via inhibition of the eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Stimulating the translation of nucleus-encoded mitochondria-related mRNAs engenders an increase in ATP production capacity, a required energy source for translation. These findings establish a feed-forward loop that links mRNA translation to oxidative phosphorylation, thereby providing a key mechanism linking aberrant mTOR signaling to conditions of abnormal cellular energy metabolism such as neoplasia and insulin resistance.

Cell growth and proliferation require coordinated ribosomal biogenesis and translation. Eukaryotic Initiation Factors (eIF) control translation at the rate-limiting step of initiation 1,2 . So far, only two eIFs connect extracellular stimuli to global translation rates 3 ; eIF4E acts in the eIF4F complex and regulates binding of capped mRNA to 40S subunits, downstream of growth factors 4 ; eIF2 controls loading of the ternary complex on the 40S subunit and is inhibited upon stress stimuli [5][6] . No eIFs have been found to link extracellular stimuli to the activity of the large 60S ribosomal subunit. eIF6 binds 60S ribosomes precluding ribosome joining in vitro [7][8][9] . However studies in yeasts showed that eIF6 is required for ribosome biogenesis rather than translation [10][11][12][13] . We show that mammalian eIF6 is required for efficient initiation of translation, in vivo. eIF6 null embryos are lethal at preimplantation. Heterozygous mice have 50% reduction of eIF6 levels in all tissues, and show reduced mass of hepatic and adipose tissues due to a lower number of cells and to impaired G1/S cell cycle progression. eIF6 +/− cells retain sufficient nucleolar eIF6 and normal ribosome biogenesis. The liver of eIF6 +/− mice displays an increase of 80S in polysomal profiles, indicating a defect in initiation of translation. Consistently, isolated hepatocytes have impaired insulin-stimulated translation. Heterozygous mouse embryonic fibroblasts (MEFs) recapitulate the organism phenotype and have normal ribosome biogenesis, reduced insulin-stimulated translation, and delayed G1/S phase progression. Furthermore, eIF6 +/− cells resist to oncogene-induced transformation. Thus, eIF6 is the first eIF associated with the large 60S subunit that regulates translation in response to extracellular signals.The eIF6 gene was deleted by homologous recombination using embryonic stem (ES) cell technology ( Supplementary Fig. 1a). The portion of the gene containing the first two exons and the first two introns was substituted by a cassette containing the neomycin resistance gene. The presence of the neomycin resistance cassette did not affect expression of wt eIF6 and of adjacent genes ( Supplementary Fig. 2). Germline transmission was achieved and intercrossingCorrespondence and requests for materials should be addressed to stefano.biffo@hsr.it. * These authors contributed equally Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. Table 1). The lethality of eIF6 −/− embryos is consistent with the early expression of the protein in the blastocysts ( Supplementary Fig. 1d).Heterozygous eIF6 +/− mice were viable and indistinguishable from wt counterparts up to 30 days after birth. At three months of age, heterozygous mice, independently from gender and genetic background, weighted less than their wt littermates (Fig. 1a). The head-anus length of eIF6 +/− and wt mice was identical, suggesting that the reduction of body mass in eIF6 +/− mice could be due to smaller size of specific...

The diversity of MTOR-regulated mRNA translation remains unresolved. Whereas ribosome-profiling suggested that MTOR almost exclusively stimulates translation of the TOP (terminal oligopyrimidine motif) and TOP-like mRNAs, polysome-profiling indicated that MTOR also modulates translation of mRNAs without the 5′ TOP motif (non-TOP mRNAs). We demonstrate that in ribosome-profiling studies, detection of MTOR-dependent changes in non-TOP mRNA translation was obscured by low sensitivity and methodology biases. Transcription start site profiling using nano-cap analysis of gene expression (nanoCAGE) revealed that not only do many MTOR-sensitive mRNAs lack the 5′ TOP motif but that 5′ UTR features distinguish two functionally and translationally distinct subsets of MTOR-sensitive mRNAs: (1) mRNAs with short 5′ UTRs enriched for mitochondrial functions, which require EIF4E but are less EIF4A1-sensitive; and (2) long 5′ UTR mRNAs encoding proliferation- and survival-promoting proteins, which are both EIF4E- and EIF4A1-sensitive. Selective inhibition of translation of mRNAs harboring long 5′ UTRs via EIF4A1 suppression leads to sustained expression of proteins involved in respiration but concomitant loss of those protecting mitochondrial structural integrity, resulting in apoptosis. Conversely, simultaneous suppression of translation of both long and short 5′ UTR mRNAs by MTOR inhibitors results in metabolic dormancy and a predominantly cytostatic effect. Thus, 5′ UTR features define different modes of MTOR-sensitive translation of functionally distinct subsets of mRNAs, which may explain the diverse impact of MTOR and EIF4A inhibitors on neoplastic cells.

AbstractmRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed "anota" algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

SUMMARY Earlier, we reported that S6K1−/− mice have reduced body fat mass, elevated rates of lipolysis, severely decreased adipocyte size, and are resistant to high-fat-diet (HFD)-induced obesity. Here we report that adipocytes of S6K1−/− mice on a HFD, have the capacity to increase in size to a degree comparable to that of wild-type (WT) mice, but not in number, indicating an unexpected lesion in adipogenesis. Tracing this lesion revealed that S6K1 is dispensable for terminal adipocyte differentiation, but is involved in the commitment of embryonic stem cells (ESCs) to early adipocyte progenitors. We further show that absence of S6K1 attenuates the upregulation of transcription factors critical for commitment to adipogenesis. These results led to the conclusion that a lack of S6K1 impairs the generation of de novo adipocytes when mice are challenged with a HFD, consistent with a reduction in early adipocyte progenitors.

Active-site mTOR inhibitors (asTORi) hold great promise for targeting dysregulated mTOR signaling in cancer. Because of the multifaceted nature of mTORC1 signaling, identification of reliable biomarkers for the sensitivity of tumors to asTORi is imperative for their clinical implementation. Here, we show that cancer cells acquire resistance to asTORi by downregulating eukaryotic translation initiation factor (eIF4E)-binding proteins (4E-BPs-EIF4EBP1, EIF4EBP2). Loss of 4E-BPs or overexpression of eIF4E renders neoplastic growth and translation of tumor-promoting mRNAs refractory to mTOR inhibition. Conversely, moderate depletion of eIF4E augments the anti-neoplastic effects of asTORi. The anti-proliferative effect of asTORi in vitro and in vivo is therefore significantly influenced by perturbations in eIF4E/4E-BP stoichiometry, whereby an increase in the eIF4E/4E-BP ratio dramatically limits the sensitivity of cancer cells to asTORi. We propose that the eIF4E/4E-BP ratio, rather than their individual protein levels or solely their phosphorylation status, should be considered as a paramount predictive marker for forecasting the clinical therapeutic response to mTOR inhibitors. Cancer Res; 72(24); 6468-76. Ó2012 AACR.

General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.• Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Platinum anticancer drugs have been used for three decades despite their serious side effects and the emerging of resistance phenomena. Recently, a phosphine copper(I) complex, [Cu(thp)4][PF6] (CP), gained special attention because of its strong antiproliferative effects. CP killed human colon cancer cells more efficiently than cisplatin and oxaliplatin and it overcame platinum drug resistance. CP preferentially reduced cancer cell viability whereas non-tumour cells were poorly affected. Colon cancer cells died via a programmed cell death whose transduction pathways were characterized by the absence of hallmarks of apoptosis. The inhibition of 26S proteasome activities induced by CP caused intracellular accumulation of polyubiquitinated proteins and the functional suppression of the ubiquitin–proteasome pathway thus triggering endoplasmic reticulum stress. These data, providing a mechanistic characterization of CP-induced cancer cell death, shed light on the signaling pathways involved in paraptosis thus offering a new tool to overcome apoptosis-resistance in colon cancer cells.