The lab is engaged with various research topics, all related to regulation of mRNA translation in mammalian cells in health and disease.
Regulation of gene expression at the level of mRNA translation into proteins is fundamental to growth, differentiation and development of multi-cellular organisms.
We are interested in:
- Molecular mechanisms regulating the translation of specific mRNAs.
- Molecular mechanisms that govern global rates of protein synthesis in response to physiological and stress signals.
REGULATION OF mRNA TRANSLATION IN THE BRAIN
Vanishing White Matter (VWM) Disease
We study a fatal neurodegenerative genetic disease caused by mutations in any of the five genes encoding eukaryotic translation initiation factor 2B (eIF2B) subunits. This disease is termed vanishing white matter (VWM) leukoencephalopathy. It affects myelin, the insulator of the nerve fibers in the brain. Without sufficient insulation, the brain’s nerves cannot function properly. VWM is a chronic-progressive disease, manifested by deterioration and loss of myelin in the CNS leading to neurological motor and cognitive associated symptoms which worsen upon physiological stress.
eIF2B is a major house-keeping complex. It governs the rate of global protein synthesis and attenuates it under stress conditions. VWM disease opens up the opportunity to address questions related to the regulation of protein synthesis during key events in myelin formation and maintenance.
Our initial findings demonstrated that eIF2B mutations render cells hyper-sensitive to ER stress (Kantor Hum. Gen. 2005; PLoS One, 2008), supporting the hypothesis that oligodendrocytes (the myelin producing cells which are typically highly sensitive to ER stress) are severely affected. To further study the molecular and cellular etiology of the clinical symptoms, we generated eIF2B5R132H/R132H mice, the first Knock-In (KI) mouse model for the disease. This mouse strain enabled analyses under a defined genetic background, during early post-natal development (pre-symptomatic) stages and during recovery from cuprizone-induced demyelination (Geva et al., Brain 2010; Marom et al., PLoS One 2011).
Primary cultures of glial cells isolated from the brains of eIF2B5R132H/R132H mice provided direct evidence for the involvement of astrocytic function in the disease and enabled the first demonstration of microglia involvement. eIF2B mutation lead to poor cerebral inflammatory response in response to LPS-induced systemic inflammation, illuminating the importance of environmental stress in VWM disease progression (Cabilly et al., PLoS One 2012).
Proteomics-level analysis of myelin formation and regeneration using eIF2B5R132H/R132H mice revealed the importance of mitochondrial function to myelin formation and regeneration (Gat-Viks et al., J. Neurochemistry, 2015). Further experiments are currently performed to discover the role of mitochondria and other cellular mechanisms in VWM pathology.
Our future goal is to transform our molecular insights regarding disease etiology into a therapeutic drug which will attenuate deterioration of clinical symptoms.
Oligodendrocyte – yellow; Astrocytes – green.
REGULATION OF mRNA TRANSLATION DURING THE CELL CYCLE
During the high-energy consuming process of mitosis, global translation is significantly reduced. We have shown that in addition to the initiation step, translation is also inhibited at the elongation step during the act of cell division. Attenuation of translation elongation is achieved by reduced delivery of charged tRNAs, leading to ribosomal stalling, thus protecting nascent mRNAs from degradation until exit from mitosis (Sivan et al., MCB 2007; JBC 2011). Despite the global inhibition, some mRNAs are preferentially translated during mitosis. Therefore, while some mitotic polysomes represent stalled ribosomes, others represent actively translating ribosomes. We are interested in understanding the molecular rules that determine which sub-classes of mRNAs are actively translated during a specific time point along the cell cycle. To identify the cell cycle translatome (i.e., the actively translated mRNAs at a given time point of interest), we developed a novel system-wide proteomic approach, termed PUromycin-associated Nascent CHain Proteome (PUNCH-P) (Aviner et al., Genes & Dev 2013). It enabled us to measure cell cycle-specific fluctuations in synthesis for over 5,000 proteins in mammalian cells and identify hundreds of proteins that are subject to cell-cycle related differential regulation at the level of translation. We aim to discover cis-elements harbored by distinct sub-populations of mRNA that are responsible for their active translation at specific phases of the cell cycle.
In addition, evidence from our and other laboratories suggests that mRNA- and ribosome- binding proteins can affect translation rate and specificity. Using a SILAC-based MS experimental design, we identified proteins that are differentially associated with polysomes during interphase and mitosis. Such proteins may govern translation during mitosis and determine spatial or temporal specificity. We aim to understand their potential role in cell-cycle- related translational regulation.
DEVELOPMENT OF NOVEL EXPERIMENTAL METHODOLOGIES
PUromycin-associated Nascent CHain Proteome (PUNCH-P)
PUNCH-P was developed in collaboration with Dr. Tamar Geiger. PUNCH-P is based on cell-free incorporation of biotinylated puromycin into newly-synthesized proteins followed by affinity purification and mass spectrometric (LC-MS/MS) analysis. It enables to generate a genome-wide snapshot of the cellular translatome. This simple and economical technique is broadly applicable to any cell type and tissue, rendering proteomic quantification of mRNA translation experimentally accessible for studying diverse biological questions (Aviner et al., Genes & Dev. 2013).
Fluorescent tRNA for translation monitoring (FtTM)
Fluorescent tRNA for translation monitoring (FtTM) was developed in collaboration with Prof. Marcelo Ehrlich and with Anima Cell Metrology. FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution. It employs quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). Fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions (Barhoom et al., NAR 2011).
Dicodon monitoring of protein synthesis (DiCoMPS)
DiCoMPS is based on FtTM technology and enables monitoring the rates of synthesis of specific proteins in single cells. It employs a specific tRNA isoacceptor pair, cognate to a dicodon that is highly enriched within mRNA encoding a specific protein of interest, relative to its occurrence within the entire transcriptome (Barhoom et al., NAR 2013).