RESUMEN
Membrane-less biomolecular compartmentalization is a core phenomenon involved in many physiological activities that occur ubiquitously in cells. Condensates, such as promyelocytic leukemia (PML) bodies, stress granules, and P-bodies (PBs), have been investigated to understand the process of membrane-less cellular compartmentalization. In budding yeast, PBs dispersed in the cytoplasm of exponentially growing cells rapidly accumulate in response to various stresses such as osmotic stress, glucose deficiency, and heat stress. In addition, cells start to accumulate PBs chronically in post-exponential phases. Specific protein-protein interactions are involved in accelerating PB accumulation in each circumstance, and discovering the regulatory mechanism for each is the key to understanding cellular condensation. Here, we demonstrate that Nst1 of budding yeast Saccharomyces cerevisiae is far more densely associated with PBs in post-exponentially growing phases from the diauxic shift to the stationary phase than during glucose deprivation of exponentially growing cells, while the PB marker Dcp2 exhibits a similar degree of condensation under these conditions. Similar to Edc3, ectopic Nst1 overexpression induces self-condensation and the condensation of other PB components, such as Dcp2 and Dhh1, which exhibit liquid-like properties. Altogether, these results suggest that Nst1 has the intrinsic potential for self-condensation and the condensation of other PB components, specifically in post-exponential phases.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Citoplasma , ARN Helicasas DEAD-box , Glucosa , Cuerpos de Procesamiento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: During skeletal muscle regeneration, satellite stem cells use distinct pathways to repair damaged myofibers or to self-renew by returning to quiescence. Cellular/mitotic quiescence employs mechanisms that promote a poised or primed state, including altered RNA turnover and translational repression. Here, we investigate the role of mRNP granule proteins Fragile X Mental Retardation Protein (Fmrp) and Decapping protein 1a (Dcp1a) in muscle stem cell quiescence and differentiation. METHODS: Using isolated single muscle fibers from adult mice, we established differential enrichment of mRNP granule proteins including Fmrp and Dcp1a in muscle stem cells vs. myofibers. We investigated muscle tissue homeostasis in adult Fmr1-/- mice, analyzing myofiber cross-sectional area in vivo and satellite cell proliferation ex vivo. We explored the molecular mechanisms of Dcp1a and Fmrp function in quiescence, proliferation and differentiation in a C2C12 culture model. Here, we used polysome profiling, imaging and RNA/protein expression analysis to establish the abundance and assembly status of mRNP granule proteins in different cellular states, and the phenotype of knockdown cells. RESULTS: Quiescent muscle satellite cells are enriched for puncta containing the translational repressor Fmrp, but not the mRNA decay factor Dcp1a. MuSC isolated from Fmr1-/- mice exhibit defective proliferation, and mature myofibers show reduced cross-sectional area, suggesting a role for Fmrp in muscle homeostasis. Expression and organization of Fmrp and Dcp1a varies during primary MuSC activation on myofibers, with Fmrp puncta prominent in quiescence, but Dcp1a puncta appearing during activation/proliferation. This reciprocal expression of Fmrp and Dcp1a puncta is recapitulated in a C2C12 culture model of quiescence and activation: consistent with its role as a translational repressor, Fmrp is enriched in non-translating mRNP complexes abundant in quiescent myoblasts; Dcp1a puncta are lost in quiescence, suggesting stabilized and repressed transcripts. The function of each protein differs during proliferation; whereas Fmrp knockdown led to decreased proliferation and lower cyclin expression, Dcp1a knockdown led to increased cell proliferation and higher cyclin expression. However, knockdown of either Fmrp or Dcp1a led to compromised differentiation. We also observed cross-regulation of decay versus storage mRNP granules; knockdown of Fmrp enhances accumulation of Dcp1a puncta, whereas knockdown of Dcp1a leads to increased Fmrp in puncta. CONCLUSIONS: Taken together, our results provide evidence that the balance of mRNA turnover versus utilization is specific for distinct cellular states.
Asunto(s)
Endorribonucleasas/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Mioblastos , Ribonucleoproteínas , Células Madre/citología , Transactivadores/genética , Animales , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas , Mioblastos/citologíaRESUMEN
Background: Local protein synthesis and mRNA metabolism mediated by mRNP granules in the dendrites and the postsynaptic compartment is essential for synaptic remodeling and plasticity in neuronal cells. Dysregulation of these processes caused by TDP-43 proteinopathy leads to neurodegenerative diseases, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Methods: Using biochemical analysis and imaging techniques, including super-resolution microscopy, we provide evidence, for the first time, for the postsynaptic localization of TDP-43 in mammalian synapses and we show that TDP-43 is a component of neuronal mRNP granules. Results: With activity stimulation and various molecular approaches, we further demonstrate activity-dependent mRNP granule dynamics involving disassembly of mRNP granules, release of mRNAs, activation of local protein translation, and the impairment of granule disassembly in cellular, animal and human models of TDP-43 proteinopathy. Conclusion: Our study elucidates the interplay between TDP-43 and neuronal mRNP granules in normal physiology and TDP-43 proteinopathy in the regulation of local protein translation and mRNA metabolism in the postsynaptic compartment.