International Symposium: Found in translation: Exploring the role of protein synthesis in stress and disease
Madrid, 16 y 17 de junio de 2014
Madrid, June 16-170, 2014
RESÚMENES/ ABSTRACTS
Translational control of cancer, metabolism and autism, Nahum Sonenberg
A new function for CPEB1 coordinates alternative 3' UTR processing with translational regulation in cell cycle and cancer, Raúl Méndez
New angles on eukaryotic RNA-binding proteins, Matthias Hentze
Post-transcriptional gene regulation by UNR: from dosage compensation to cancer progression, Fátima Gebauer
Targeting Translational Control in Diseases of the Brain, Eric Klann
Translation under (viral) stress, Iván Ventoso
RNA-binding proteins controlling internal initiation of translation in RNA viruses, Encarnación Martínez-Salas
MicroRNAs are major regulators of gene expression which are dysregulated in human disease, but how do they work? , MartinBushell
NMD, translation termination, and the development of therapeutic nonsense suppression, Allan Jacobson
Global translational landscape of cellular differentiation in fission yeast, Juan Mata
Stories about the ribosomal A site: Entrance and checking point for tRNAs, Mikel Valle
The role of adenosinedeamination in tRNAs in eukaryotic translational control, Lluis Ribas de Pouplana
Relationships between translational control, protein misfolding and cell death, Randal J. Kaufman
eIF2alpha phosphorylation:A keystone in the cellular response to stress, Juan José Berlanga
Translation Makes an Impact: Tailor-made Protein Expression for Metabolism, Cancer and Disease, Davide Ruggero
Translational control of cancer, metabolism and autism, Nahum Sonenberg
A key component of translation initiation is the mRNA 5’-cap-binding protein, eIF4E. eIF4E overexpression transforms rodent and human cells and causes cancer in mice. eIF4E levels are elevated in several types of cancers. Phosphorylation of eIF4E on Ser209 is required for efficient transformation by eIF4E, and promotes metastasis in mice, by facilitating the epithelial-mesenchymal transition (EMT).
Important downstream targets of the PTEN/Akt/mTOR signaling pathway, which is strongly implicated in cancer etiology, are the 4E-BPs, which bind to eIF4E and inhibit cap-dependent translation. 4E-BPs impair the assembly of the eIF4F pre-initiation complex by competing with eIF4G for binding to eIF4E. We demonstrated that the 4E-BPs are critical targets of mTOR in the control of energy homeostasis, via translational control of mitochondria-related mRNAs
Mutations in the mTOR pathway are associated with autism spectrum disorder (ASD). Mice lacking 4E-BP2 exhibit many autism-like symptoms, including poor social interaction, altered communication and repetitive behaviors. In the absence of Eif4ebp2, translation of neuroligins mRNAs, which encode adhesion molecules, is enhanced. This engenders `hyperconnectivity' that underlies the symptoms of ASD.
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A new function for CPEB1 coordinates alternative 3' UTR processing with translational regulation in cell cycle and cancer, Raúl Méndez
CPEB (Cytoplasmic Polyadenilation Element Binding protein) is an RNA-binding protein that recognizes maternal mRNAs in the cytoplasm of Xenopus L. oocytes and directs their poly(A) tail elongation and translational activation during meiotic progression. CPEB has been shown to regulate the translation of hundreds of mRNAs in both somatic and germ cells and to drive events as diverse as learning and memory, cell cycle progression and tumor development. Now, we have found that the cytoplasm and the translational regulation is only part of the life of a protein that moonlights as a nuclear factor responsible for the pre-mRNA processing of the same mRNAs that, later, is going to regulate at the translational level. Thus, CPEB is a nucleocytoplasmic shuttling protein that recognizes the same cis-acting element in the cytoplasmic mature mRNA as in the nuclear pre-mRNA, recruiting the cleavage and polyadenylation machinery that mediates both the cytoplasmic polyadenylation and the nuclear pre-mRNA cleavage and polyadenylation at specific polyadenylation sites.This is a new function for CPEB, where hundreds of mRNAs are regulated by alternative processing in the nucleus in a coordinated manner and associated with cell cycle and tumor development. A global model for the regulation of gene expression by the CPEB family of proteins in cell cycle and cell differentiation will be presented.
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New angles on eukaryotic RNA-binding proteins, Matthias Hentze
RNA-binding proteins (RBP) exert a broad range of posttranscriptional functions. We determined the repertoire of RBPs active in proliferating human Huh7 cells and the yeast Saccharomyces cerevisiae. Collectively, the datasets define 287 proteins as the RNA-binding proteome that is conserved from yeast to man. At 678 RBPs (FDR<1%) more than 10% of the total yeast proteome has RNA-binding properties. Surprisingly, only 56% of yeast and 81% of the conserved RBPs have functions assigned to RNA biology and/or structural motifs known to convey RNA-binding. Thus, for one fifth of conserved RBPs (termed ‘enigmRBPs’) the physiological role of RNA-binding remains to be determined. Amongst these enigmatic RBPs are cellular adaptors, redox effectors and enzymes of central metabolic pathways, including the majority of glycolysis. While some enigmRBPs may moonlight as regulators of RNA expression, we propose that the orthodox, already known functions of some others may be subject to regulation by ligand “effector RNAs”.
We also developed RBDmap to identify the RNA-binding domains (RBDs) of native RBPs in cultured cells. RBDmap validates an unexpected variety of RBPs and uncovers their RNA-binding architectures. To our surprise, RBDs overlap with the interaction domains of multimeric proteins or coincide with catalytic centres, further invoking effector roles of RNA in the control of protein function. We also note that lysine acetylation and arginine methylation are prevalent amongst RNA-binding sites, suggesting modes of metabolic and signal-stimulated regulation of RBPs.
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Post-transcriptional gene regulation by UNR: from dosage compensation to cancer progression, Fátima Gebauer
Upstream of N-Ras (UNR) is a conserved RNA binding protein that regulates mRNA translation and stability. In the fruit fly Drosophila melanogaster, UNR is involved in the regulation of dosage compensation, a process that balances the expression of X-chromosomal genes in males (XY) and females (XX). UNR performs sex-specific, opposite roles in the formation of the dosage compensation complex (DCC) by using distinct RNA regulatory mechanisms. In females, UNR inhibits the translation of the transcript encoding the limiting DCC subunit MSL2, while in males UNR promotes the interaction of two DCC components, the lncRNA roX2 and the RNA helicase MLE. During my talk, I will address our current view of the molecular mechanism of msl-2 translational repression, and will explain how molecular and genetics studies in Drosophila have led us to investigate the role of UNR in human cancer progression.
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Targeting Translational Control in Diseases of the Brain, Eric Klann
A requirement for de novo protein synthesis is one of the hallmarks of long-lasting synaptic plasticity and the consolidation long-term memory. Recent studies, including several from our laboratory, have identified signaling cascades, including the mTORC1 signaling pathway, that couple neurotransmitter and neurotrophin receptors to the translation regulatory machinery during synaptic plasticity and memory. Interestingly, mutations in negative upstream regulators and downstream effectors of mTORC1 are associated with certain types of developmental disability and autism. Synaptic plasticity and memory phenotypes in genetically engineered mice that display altered mTORC1 signaling and cap-dependent translation will be discussed. These studies have revealed interesting links between mTORC1 signaling, synaptic plasticity, memory, and behavior. These studies also have provided insight into the molecular basis of certain types of developmental disability, autism, and schizophrenia. Supported by NIH Grants NS034007 and NS047384, DoD CDRMP Award W81XWH-11-1-0389, the Simons Foundation, the Alzheimer's Association, and the FRAXA Research Foundation.
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Translation under (viral) stress, Iván Ventoso
Mammalian cells respond to viral stress through the phosphorylation of translation initiation factor eIF2, a response aimed to prevent the synthesis of viral proteins in infected cells. During the adaptation to vertebrate hosts, Alphaviruses acquired resistance to eIF2 phosphorylation by creating stable stem loops (DLPs) in their mRNA that stalled the 43S ribosome on initiation codon, allowing the efficient translation of viral mRNA in mammalian cells. By combining genetic, biochemical and structural approaches, we found that viral DLPs are trapping around the RNA extensions that are projected from the body of 40S ribosome. The stability of DLPs entangled in rRNA extensions was so high that resisted the unwinding activity of eIF4A helicase, thus allowing the slow down of 43S ribosome. These novel findings reveal new insights into the scanning process, and illustrate how some viruses have exploited the topology of ribosome to promote a non-canonical translation initiation.
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RNA-binding proteins controlling internal initiation of translation in RNA viruses, Encarnación Martínez-Salas
Internal ribosome entry site (IRES) elements govern protein synthesis in various RNA viruses. This process requires the interaction of the IRES element with host factors. Novel factors IRES-binding factors were identified in a riboproteomic approach carried out with two unrelated IRES elements, hepatitis C (HCV) and foot-and-mouth disease virus (FMDV). One of these factors is Gemin5, a predominantly cytoplasmic protein involved in the assembly of the SMN complex, and thus, in the biogenesis of the small nuclear ribonucleoproteins. In addition, Gemin5 acts as a negative regulator of translation. Interestingly, the C-terminal region of Gemin5 bears two non-canonical bipartite RNA-binding sites (RBS). While RBS1 exhibits
greatter affinity for RNA than RBS2, it does not affect IRES-dependent translation in Gemin5-depleted cells. In solution, the RBS1 three-dimensional structure behaves as an ensemble of flexible conformations rather than having a defined tertiary structure. However, expression of the polypeptide bearing the low RNA-binding affinity RBS2, repressed IRES-dependent translation. Comparison of the RNA-binding capacity and translation control properties of constructs expressed in mammalian cells to the Gemin5 proteolysis products observed in infected cells reveals that non-repressive products accumulated during infection while the repressor polypeptide is not stable. Taken together, our results define the low affinity RNA-binding site as the minimal element of Gemin5 being able to repress internal initiation of translation.
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MicroRNAs are major regulators of gene expression which are dysregulated in human disease, but how do they work?, Martin Bushell
MicroRNAs (miRNAs) are non-coding 21-25 nucleotide RNA molecules, which in metazoans base-pair imperfectly with regions in target mRNAs (generally within the 3’UTR) and repress the synthesis of the corresponding proteins. MiRNA control is dysregulated in a large array of human diseases, both by generic mechanisms perturbing the basic function of this control mechanism and by more subtle changes in miRNA expression patterns. However, the underlying mechanism by which these small RNA molecules control gene-expression still remains elusive.
Our recent data suggests that translational inhibition is the critical process for miRNA-mediated repression while mRNA deadenylation and mRNA degradation are secondary effects which are not required. Translational inhibition depends on miRNAs impairing the function of the eIF4F initiation complex. We define the RNA helicase eIF4A2 as the key factor of eIF4F through which miRNAs function. Importantly, here we show by MS of endogenously purified eIF4A2 and eIF4A1 complexes that cNOT1 (CCR4-NOT complex component) interacts strongly with only eIF4A2. We uncover a correlation between the presence of miRNA target sites in the 3’UTR of mRNAs and secondary structure in the 5’UTR, and show that mRNAs with unstructured 5’UTRs are refractory to miRNA repression. These data support a linear model for miRNA-mediated gene regulation in which translational repression via eIF4A2 is required first, followed by mRNA destabilisation.
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NMD, translation termination, and the development of therapeutic nonsense
suppression, Allan Jacobson
Nonsense mutations result in the occurrence of premature UAA, UAG, or UGA termination codons in the mRNA protein-coding region, leading to the production of a truncated polypeptide product and, frequently, to mRNA destabilization through NMD. A standard consequence of these combined effects is a severe depression of specific gene expression. Nonsense mutations have thus been implicated in numerous inherited diseases and several cancers, with at least 2400 different genetic disorders having one or more causative nonsense allele. Our studies of NMD in yeast led to the understanding that premature and normal translation termination differed mechanistically and this prompted us to consider the possibility that a single drug might be able to provide therapy for diseases caused by nonsense mutations by promoting selective nonsense codon readthrough at premature terminators. My talk will address our progress toward this goal.
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Global translational landscape of cellular differentiation in fission yeast, Juan Mata
Sexual development in the fission yeast Schizosaccharomyces pombe culminates in meiosis and sporulation. We used ribosome profiling to investigate the translational landscape of this developmental process. We show that the translation efficiency of hundreds of genes is regulated in complex patterns, often correlating with changes in RNA levels. Ribosome-protected fragments display a three-nucleotide periodicity that identifies translated sequences and their reading frame. Using this property, we identified 46 novel translated genes and found that 24% of non-coding RNAs are actively translated. We also detected 19 nested antisense genes, in which both DNA strands encode translated mRNAs. Finally, we identified 1,735 translated upstream ORFs in leader sequences. Our data show that fission yeast cells translate hundreds of small proteins that have not been previously annotated. Whether these proteins are stable and functional will be an important research topic in the future.
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Stories about the ribosomal A site: Entrance and checking point for tRNAs, Mikel Valle
Decoding during translation occurs at the A site from the small ribosomal subunit. In the last decade, data from X-ray crystalography and cryoEM have revealed the structural basis for monitoring codon-anticodon pairing, and hence, selection of the cognate aa-tRNA. Here, the flexibility of tRNAs play a central role, and tRNAs work as molecular springs during decoding. In bacteria, reponses to damages on mRNA and to amino acid starvation, trigger different mechanisms to rescue stalled ribosomes. Some of these responses will be discussed.
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The role of adenosinedeamination in tRNAs in eukaryotic translational control, Lluis Ribas de Pouplana
We have reported that the structure and codon composition of bacterial and eukaryotic
genomes was strongly influenced by the emergence of two distinct tRNA modification
enzymes whose function changed the codon recognition pattern of preexisting transfer RNAs.
We are now investigating the biological role of the eukaryotic enzyme responsible for this evolutionary phenomenon: adenosine deaminases acting on tRNAs (ADAT). We will report our latest results in our effort to understand how the function of this enzyme shapes the human proteome, and how variations in the levels of ADAT may be linked to cellular transformations and disease.
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Relationships between translational control, protein misfolding and cell death, Randal J. Kaufman
The unfolded protein response (UPR) is an adaptive response to resolve proteína misfolding in the endoplasmic reticulum (ER) that is signaled through three transmembrane sensors: IRE1, ATF6, and PERK. If protein misfolding is not resolved, cells initiate apoptosis, primarily through PERK mediated phosphorylation of eukaryotic initiation factor 2 (eIF2) on the alpha subunit to attenuate protein synthesis. Paradoxically, a number of mRNAs, including Atf4 mRNA, require eIF2a phosphorylation for efficient translation. ATF4 induces transcription of the C/EBP homologous protein Chop. We have shown that ATF4 and CHOP act together to induce genes encoding ER chaperones, autophagy and translational machinery. As a consequence, forced expression of ATF4 and CHOP is sufficient to increase protein synthesis that leads to ATP depletion and oxidative stress (1). We demonstrate that protein misfolding in the ER in the pancreatic beta cell, by expression of a mutant proinsulin or deletion of the molecular chaperone P58IPK/DNAJC3, is sufficient to cause beta cell failure and glucose intolerance. Importantly, antioxidant treatment reverses the process and normalizes glucose tolerance. These findings demonstrate that antioxidant feeding restores ER function in cells deleted of an ER molecular chaperone and implicate oxidative stress as a fundamental process in beta cell failure.
1)Han, J., et al. 2013 Endoplasmic reticulum (ER) stress-induced transcripcional regulation increases protein synthesis leading to cell death. Nature Cell Biol. 15(5): 481-90. PMC3692270.
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eIF2alpha phosphorylation:A keystone in the cellular response to stress, Juan José Berlanga
Translational control is crucial for the stress response in mammals, leading to protein synthesis reprogramming which allows the cells to adapt and survive. Phosphorylation of eIF2α is one of the best-characterized mechanisms of translation initiation control. There are four eIF2α kinases that specifically respond to distinct stresses, HRI, PKR, GCN2 and PERK. The work done by our laboratory in the last years has allowed to shed light into the involvement of these kinases in different aspects of the stress response: involvement of GCN2 in the cellular response against RNA viruses such as Sindbis virus or human immunodeficiency virus type 1 (HIV-1); GCN2 activity and eIF2α phosphorylation are essential in the regulation of cell cycle in the fission yeast Schizosaccharomyces pombe; and GCN2 activity is modulated by its interaction with cellular and viral proteins. Thus, eIF2α kinase activity seems to be a good therapeutic target for fighting environmental stress and disease.
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Translation Makes an Impact: Tailor-made Protein Expression for Metabolism, Cancer and Disease, Davide Ruggero
Our research is centered on understanding the molecular mechanisms by which impairments in accurate control of mRNA translation, cell growth, and overall cellular protein synthesis rates lead to cancer and human disease. We have uncovered that a common denominator of multiple oncogenic pathways (i.e. PI3K-AKT-mTOR, Ras, Myc) is their ability to directly control the core translation machinery of a cell, resulting in intrinsic regulation of the cancer cell gene expression program. Our work delineates how the “cancer translatome” can reprogram gene expression at the post-transcription level, by controlling the translation of specific subsets of mRNAs through unique cis-acting elements, during distinct steps of cancer progression. The immediate impact of these studies has been the design of a new generation of compounds to target the aberrant translation machinery, presently in clinical trials. I will discuss our most recent findings revealing that the Myc oncogene directs a feed-forward metabolic circuit that integrates the production of two of the most abundant macromolecules—proteins and nucleotides—in cancer. Remarkably, this is achieved through translational control of a single rate-limiting enzyme in the nucleotide biosynthesis pathway, PRPS2. These studies identify a translationally-anchored anabolic circuit critical for cancer cell survival and an unexpected vulnerability for "undruggable" oncogenes, such as Myc. In addition, I will discuss our work examining the in vivo requirement for a threshold of eIF4E activity in organismal development and for translating the mammalian genome. Strikingly, we find that eIF4E is normally maintained in excess levels required for normal development, but becomes selectively rate limiting for adaptation to cellular stress conditions; a fundamental hallmark underlying the ability of cancer cells to survive and proliferate. Together, these research efforts define how alterations in the translational machinery in cancer reprogram the translation of individual mRNAs and how novel 5'UTR sequence elements tightly control protein production in normal and transformed cells.