Stefano Biffo

MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F

MicroRNAs (miRNAs) play an important role in gene regulatory networks in animals. Yet, the mechanistic details of their function in translation inhibition or messenger RNA (mRNA) destabilization remain controversial. To directly examine the earliest events in this process, we have developed an in vitro translation system using mouse Krebs-2 ascites cell–free extract that exhibits an authentic miRNA response. We show here that translation initiation, specifically the 5′ cap recognition process, is repressed by endogenous let-7 miRNAs within the first 15 minutes of mRNA exposure to the extract when no destabilization of the transcript is observed. Our results indicate that inhibition of translation initiation is the earliest molecular event effected by miRNAs. Other mechanisms, such as mRNA degradation, may subsequently consolidate mRNA silencing.

Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat

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.

Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly

The assembly of 80S ribosomes requires joining of the 40S and 60S subunits, which is triggered by the formation of an initiation complex on the 40S subunit. This event is rate-limiting for translation, and depends on external stimuli and the status of the cell. Here we show that 60S subunits are activated by release of eIF6 (also termed p27BBP). In the cytoplasm, eIF6 is bound to free 60S but not to 80S. Furthermore, eIF6 interacts in the cytoplasm with RACK1, a receptor for activated protein kinase C (PKC). RACK1 is a major component of translating ribosomes, which harbour significant amounts of PKC. Loading 60S subunits with eIF6 caused a dose-dependent translational block and impairment of 80S formation, which were reversed by expression of RACK1 and stimulation of PKC in vivo and in vitro. PKC stimulation led to eIF6 phosphorylation, and mutation of a serine residue in the carboxy terminus of eIF6 impaired RACK1/PKC-mediated translational rescue. We propose that eIF6 release regulates subunit joining, and that RACK1 provides a physical and functional link between PKC signalling and ribosome activation.

Deregulation of the PI3K and KRAS signaling pathways in human cancer cells determines their response to everolimus

Personalized cancer medicine is based on the concept that targeted therapies are effective on subsets of patients whose tumors carry specific molecular alterations. Several mammalian target of rapamycin (mTOR) inhibitors are in preclinical or clinical trials for cancers, but the molecular basis of sensitivity or resistance to these inhibitors among patients is largely unknown. Here we have identified oncogenic variants of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) and KRAS as determinants of response to the mTOR inhibitor everolimus. Human cancer cells carrying alterations in the PI3K pathway were responsive to everolimus, both in vitro and in vivo, except when KRAS mutations occurred concomitantly or were exogenously introduced. In human cancer cells with mutations in both PIK3CA and KRAS, genetic ablation of mutant KRAS reinstated response to the drug. Consistent with these data, PIK3CA mutant cells, but not KRAS mutant cells, displayed everolimus-sensitive translation. Importantly, in a cohort of metastatic cancer patients, the presence of oncogenic KRAS mutations was associated with lack of benefit after everolimus therapy. Thus, our results demonstrate that alterations in the KRAS and PIK3CA genes may represent biomarkers to optimize treatment of patients with mTOR inhibitors.

Eukaryotic Initiation Factor 6 is rate-limiting in translation, growth and transformation

Cell growth and proliferation require coordinated ribosomal biogenesis and translation. Eukaryotic initiation factors (eIFs) control translation at the rate-limiting step of initiation. So far, only two eIFs connect extracellular stimuli to global translation rates: eIF4E acts in the eIF4F complex and regulates binding of capped messenger RNA to 40S subunits, downstream of growth factors, and eIF2 controls loading of the ternary complex on the 40S subunit and is inhibited on stress stimuli. 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. However, studies in yeasts showed that eIF6 is required for ribosome biogenesis rather than translation. Here 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 recapitulate the organism phenotype and have normal ribosome biogenesis, reduced insulin-stimulated translation, and delayed G1/S phase progression. Furthermore, eIF6+/- cells are resistant to oncogene-induced transformation. Thus, eIF6 is the first eIF associated with the large 60S subunit that regulates translation in response to extracellular signals.

Reduced AKT/mTOR signaling and protein synthesis dysregulation in a Rett syndrome animal model

Rett syndrome (RTT) is a neurodevelopmental disorder with no efficient treatment that is caused in the majority of cases by mutations in the gene methyl-CpG binding-protein 2 (MECP2). RTT becomes manifest after a period of apparently normal development and causes growth deceleration, severe psychomotor impairment and mental retardation. Effective animal models for RTT are available and show morphofunctional abnormalities of synaptic connectivity. However, the molecular consequences of MeCP2 disruption leading to neuronal and synaptic alterations are not known. Protein synthesis regulation via the mammalian target of the rapamycin (mTOR) pathway is crucial for synaptic organization, and its disruption is involved in a number of neurodevelopmental diseases. We investigated the phosphorylation of the ribosomal protein (rp) S6, whose activation is highly dependent from mTOR activity. Immunohistochemistry showed that rpS6 phosphorylation is severely affected in neurons across the cortical areas of Mecp2 mutants and that this alteration precedes the severe symptomatic phase of the disease. Moreover, we found a severe defect of the initiation of protein synthesis in the brain of presymptomatic Mecp2 mutant that was not restricted to a specific subset of transcripts. Finally, we provide evidence for a general dysfunction of the Akt/mTOR, but not extracellular-regulated kinase, signaling associated with the disease progression in mutant brains. Our results indicate that defects in the AKT/mTOR pathway are responsible for the altered translational control in Mecp2 mutant neurons and disclosed a novel putative biomarker of the pathological process. Importantly, this study provides a novel context of therapeutic interventions that can be designed to successfully restrain or ameliorate the development of RTT.

B‐50/GAP43 Expression Correlates with Process Outgrowth in the Embryonic Mouse Nervous System

The hypothesis that B‐50/GAP43, a membrane‐associated phosphoprotein, is involved in process outgrowth has been tested by studying the developmental pattern of expression of B‐50/GAP43 mRNA and protein during mouse neuroembryogenesis. B‐50/GAP43 mRNA is first detectable at embryonic day 8.5 (E8.5) in the presumptive acoustico‐facialis ganglion. Subsequently, both B‐50/GAP43 mRNA and protein were co‐expressed in a series of neural structures: in the ventral neural tube (from E9.5) and dorsal root ganglia (from E10.5), in the marginal layer of the neuroepithelium surrounding the brain vesicles and in the cranial ganglia (from E9.5), in the autonomic nervous system (from E10.5), in the olfactory neuroepithelium and in the mesenteric nervous system (from E11.5), in a continuum of brain regions (from E12.5) and in the retina (from E13.5). Immunoreactive fibers were always seen arising from these regions when they expressed B‐50/GAP43 mRNA. The spatial and temporal pattern of B‐50/GAP43 expression demonstrates that this protein is absent from neuroblasts and consistently appears in neurons committed to fiber outgrowth. The expression of the protein in immature neurons is independent of their embryological origin. Our detailed study of B‐50/GAP43 expression during mouse neuroembryogenesis supports the view that this protein is involved in a process common to all neurons elaborating fibers.

Cellular localization of carnosine-like and anserine-like immunoreactivities in rodent and avian central nervous system.

Aminoacylhistidine dipeptides are present in the nervous tissue of many species. The olfactory mucosa and bulb of many vertebrates are rich in carnosine (beta-alanyl-L-histidine). Two related dipeptides homocarnosine (gamma-aminobutyryl-L-histidine) and anserine (beta-alanyl-N-methyl-L-histidine) are present in the CNS of mammals and birds, respectively. This manuscript describes the production, characterization and use in immunolocalization studies of antisera directed against carnosine and anserine. The anserine antiserum is highly specific for anserine while the carnosine antiserum cross-reacts with all three dipeptides. The differential specificity of the antisera, coupled with chemical characterization of the dipeptide composition of various brain regions, has permitted assignment of the cellular localization of the various dipeptides. Immunocytochemical localization of anserine has not been previously reported. Carnosine immunoreactivity in the olfactory system is restricted to the mature neurons in the olfactory mucosa, their axons and synaptic terminations in the glomerular layer of the olfactory bulb. Similar reactivity is seen in the accessory olfactory system. Astrocytes and cerebellar Bergmann glia seem to account for all the non-olfactory carnosine-like immunoreactive staining in the rodent brain. In contrast, in the avian CNS where anserine is chemically abundant, anserine-like immunoreactivity is widespread and apparently exclusively associated with glial cells. Thus, the olfactory receptor neurons appear to be the only neuronal population that expresses carnosine. Elsewhere in the CNS the aminoacylhistidine dipeptides are associated with various populations of glia.

Isolation of a Novel β4 Integrin-binding Protein (p27BBP) Highly Expressed in Epithelial Cells

The integrin β4 has a long cytodomain necessary for hemidesmosome formation. A yeast two-hybrid screen using β4 cytodomain uncovered a protein called p27BBP that represents a β4 interactor. Both in yeast and in vitro, p27BBP binds the two NH2-terminal fibronectin type III modules of β4, a region required for signaling and hemidesmosome formation. Sequence analysis of p27BBP revealed that p27BBP was not previously known and has no homology with any isolated mammalian protein, but 85% identical to a yeast gene product of unknown function. Expression studies by Northern analysis and in situ hybridization showed that, in vivo, p27BBP mRNA is highly expressed in epithelia and proliferating embryonic epithelial cells. An antibody raised against p27BBP COOH-terminal domain showed that all β4-containing epithelial cell lines expressed p27BBP. The p27BBP protein is insoluble and present in the intermediate filament pool. Furthermore, subcellular fractionation indicated the presence of p27BBP both in the cytoplasm and in the nucleus. Confocal analysis of cultured cells showed that part of p27BBP immunoreactivity was both nuclear and in the membrane closely apposed to β4. These results suggest that the p27BBP is an in vivointeractor of β4, possibly linking β4 to the intermediate filament cytoskeleton.

Eukaryotic initiation factor 6 mediates a continuum between 60S ribosome biogenesis and translation

Eukaryotic ribosome biogenesis and translation are linked processes that limit the rate of cell growth. Although ribosome biogenesis and translation are mainly controlled by distinct factors, eukaryotic initiation factor 6 (eIF6) has been found to regulate both processes. eIF6 is a necessary protein with a unique anti‐association activity, which prevents the interaction of 40S ribosomal subunits with 60S subunits through its binding to 60S ribosomes. In the nucleolus, eIF6 is a component of the pre‐ribosomal particles and is required for the biogenesis of 60S subunits, whereas in the cytoplasm it mediates translation downstream from growth factors. The translational activity of eIF6 could be due to its anti‐association properties, which are regulated by post‐translational modifications; whether this anti‐association activity is required for the biogenesis and nuclear export of ribosomes is unknown. eIF6 is necessary for tissue‐specific growth and oncogene‐driven transformation, and could be a new rate‐limiting step for the initiation of translation.