Most of the mammalian genome is transcribed. This generates a vast repertoire of transcripts that includes protein-coding messenger RNAs, long non-coding RNAs (lncRNAs) and repetitive sequences, such as SINEs (short interspersed nuclear elements). A large percentage of ncRNAs are nuclear-enriched with unknown function. Antisense lncRNAs may form sense-antisense pairs by pairing with a protein-coding gene on the opposite strand to regulate epigenetic silencing, transcription and mRNA stability. Here we identify a nuclear-enriched lncRNA antisense to mouse ubiquitin carboxy-terminal hydrolase L1 (Uchl1), a gene involved in brain function and neurodegenerative diseases. Antisense Uchl1 increases UCHL1 protein synthesis at a post-transcriptional level, hereby identifying a new functional class of lncRNAs. Antisense Uchl1 activity depends on the presence of a 5' overlapping sequence and an embedded inverted SINEB2 element. These features are shared by other natural antisense transcripts and can confer regulatory activity to an artificial antisense to green fluorescent protein. Antisense Uchl1 function is under the control of stress signalling pathways, as mTORC1 inhibition by rapamycin causes an increase in UCHL1 protein that is associated to the shuttling of antisense Uchl1 RNA from the nucleus to the cytoplasm. Antisense Uchl1 RNA is then required for the association of the overlapping sense protein-coding mRNA to active polysomes for translation. These data reveal another layer of gene expression control at the post-transcriptional level.

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...

Signaling through the mammalian target of rapamycin (mTOR) controls cell size and growth as well as other functions, and it is a potential therapeutic target for graft rejection, certain cancers, and disorders characterized by inappropriate cell or tissue growth. mTOR signaling is positively regulated by hormones or growth factors and amino acids. mTOR signaling regulates the phosphorylation of several proteins, the best characterized being ones that control mRNA translation. Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) undergoes phosphorylation at multiple sites. Here we show that amino acids regulate the N-terminal phosphorylation sites in 4E-BP1 through the RAIP motif in a rapamycin-insensitive manner. Several criteria indicate this reflects a rapamycin-insensitive output from mTOR. In contrast, the insulin-stimulated phosphorylation of the C-terminal site Ser64/65 is generally sensitive to rapamycin, as is phosphorylation of another well-characterized target for mTOR signaling, S6K1. Our data imply that it is unlikely that mTOR directly phosphorylates Thr69/70 in 4E-BP1. Although 4E-BP1 and S6K1 bind the mTOR partner, raptor, our data indicate that the outputs from mTOR to 4E-BP1 and S6K1 are distinct. In cells, efficient phosphorylation of 4E-BP1 requires it to be able to bind to eIF4E, whereas phosphorylation of 4E-BP1 by mTOR in vitro shows no such preference. These data have important implications for understanding signaling downstream of mTOR and the development of new strategies to impair mTOR signaling.

In mammalian cells, amino acids affect the phosphorylation state and function of several proteins involved in mRNA translation that are regulated via the rapamycin-sensitive mTOR (mammalian target of rapamycin) pathway. These include ribosomal protein S6 kinase, S6K1, and eukaryotic initiation factor 4E-binding protein, 4E-BP1. Amino acids, especially branched-chain amino acids, such as leucine, promote phosphorylation of 4E-BP1 and S6K1, and permit insulin to further increase their phosphorylation. However, it is not clear whether these effects are exerted by extracellular or intracellular amino acids. Inhibition of protein synthesis is expected to increase the intracellular level of amino acids, whereas inhibiting proteolysis has the opposite effect. We show in the present study that inhibition of protein synthesis by any of several protein synthesis inhibitors tested allows insulin to regulate 4E-BP1 or S6K1 in amino-acid-deprived cells, as does the addition of amino acids to the medium. In particular, insulin activates S6K1 and promotes initiation factor complex assembly in amino-acid-deprived cells treated with protein synthesis inhibitors, but cannot do so in the absence of these compounds. Their effects occur at concentrations commensurate with their inhibition of protein synthesis and are not due to activation of stress-activated kinase cascades. Inhibition of protein breakdown (autophagy) impairs the ability of insulin to regulate 4E-BP1 or S6K1 under such conditions. These and other data presented in the current study are consistent with the idea that it is intracellular amino acid levels that regulate mTOR signalling.

The translational repressor protein eIF4E-binding protein 1 (4E-BP1, also termed PHAS-I) is regulated by phosphorylation through the rapamycin-sensitive mTOR (mammalian target of rapamycin) pathway. Recent studies have identified two regulatory motifs in 4E-BP1, an mTOR-signaling (TOS) motif in the C terminus of 4E-BP1 and an RAIP motif (named after its sequence) in the N terminus. Other recent work has shown that the protein raptor binds to mTOR and 4E-BP1. We show that raptor binds to full-length 4E-BP1 or a Cterminal fragment containing the TOS motif but not to an N-terminal fragment containing the RAIP motif. Mutation of several residues within the TOS motif abrogates binding to raptor, indicating that the TOS motif is required for this interaction. 4E-BP1 undergoes phosphorylation at multiple sites in intact cells. The effects of removal or mutation of the RAIP and TOS motifs differ. The RAIP motif is absolutely required for phosphorylation of sites in the N and C termini of 4E-BP1, whereas the TOS motif primarily affects phosphorylation of Ser-64/65, Thr-69/70, and also the rapamycin-insensitive site Ser-101. Phosphorylation of N-terminal sites that are dependent upon the RAIP motif is sensitive to rapamycin. The RAIP motif thus promotes the mTOR-dependent phosphorylation of multiple sites in 4E-BP1 independently of the 4E-BP1/raptor interaction.Eukaryotic initiation factor (eIF)-4E 1 plays a key role in mRNA translation in mammalian cells and in its regulation (1). eIF4E binds the 5Ј-cap structure of the mRNA, which contains the 7-methyl-GTP (m 7 GTP) moiety. It also interacts with the scaffold protein eIF4G and thereby recruits 40 S ribosomal subunits to the mRNA (2). A large body of evidence shows that eIF4E plays key roles in cell proliferation (1) and cell survival/ apoptosis (3). For example, overexpression of eIF4E can transform cells (4,5), and many human tumor cells express high levels of this protein (6). Nuclear eIF4E seems to be important in the export of certain mRNAs to the cytoplasm such as that for cyclin D1 (7), which is important in cell cycle progression.The transforming abilities of eIF4E may be linked to this effect (8,9).The availability of eIF4E for binding to eIF4G is regulated by its interaction with heat-stable proteins termed eIF4E-binding proteins (4E-BPs). 4E-BP1 is by far the best understood of these. Phosphorylation of 4E-BP1 leads to its release from eIF4E, allowing eIF4E to form initiation complexes with eIF4G (1). Expression of 4E-BP1 reverses the transforming ability of increased levels of eIF4E (10). Phosphorylation of 4E-BP1 induced by insulin or other agents is blocked by the immunosuppressant rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR) (11). 4E-BP1 undergoes phosphorylation at up to seven sites in intact cells; phosphorylation is complex, hierarchical, and, at some sites, is required for the subsequent modification of others (12-15).Regulatory motifs have been identified in the N and C termini of 4E-BP1. The N terminus contains the so-c...