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Structure-function analysis of the RNA-binding protein PGC-1a in skeletal muscle

Research Project
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01.07.2022
 - 30.06.2026

Plasticity of cells, tissues and organs relies on sensing of external and internal cues, integration of the engaged signaling pathways, and orchestration of a pleiotropic response. For example, contractile patterns, ambient temperature and oxygen levels, nutrient availability and composition, as well as other factors result in a massive remodeling of biochemical pathways, cellular metabolism and mechanical properties of skeletal muscle cells, most notably in the context of repeated bouts of exercise, ultimately leading to training adaptations. In turn, such adaptations of skeletal muscle trigger internal and external changes that contribute to the systemic effects of exercise with many health benefits, and strong preventative and therapeutic outcomes in a number of pathologies. Surprisingly, even though the physiological and clinical contributions of exercise-linked muscle plasticity are well recognized, the molecular mechanisms that control the corresponding biological programs are still poorly understood. In recent years, the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) has emerged as a central regulatory nexus of plasticity in various cell types. PGC-1α integrates upstream signaling pathways to coordinate the complex transcriptional networks encoding biological programs involved in mitochondrial function, cellular metabolism and more. In the case of endurance exercise-mediated plasticity of muscle cells, most, if not all of the adaptations are evoked by this stimulus. Accordingly, muscle-specific overexpression or knockout of PGC-1α elicits high endurance and pathologically sedentary phenotypes, respectively. Our proposal aims at elucidating the molecular underpinnings of the complex transcriptional control that is exerted by PGC-1α in a spatio-temporal manner. We have recently discovered that the binding of RNAs to PGC-1α contributes to full transcriptional activity, and is central for the inclusion of PGC-1α-containing multiprotein complexes in liquid-liquid phase separated nuclear condensates for the sequestration of transcription. We now plan to determine the list and common features of PGC-1α-bound RNAs, interrogate how RNA binding is brought about in a structure-function analysis, study the consequences on multiprotein complex formation, and ultimately investigate the physiological relevance in skeletal muscle cells in vitro and in vivo . To do so, novel animal models will be leveraged for single-end enhanced crosslinking and immunoprecipitation (seCLIP) experiments to identify PGC-1α-bound RNAs, structural information will be obtained by NMR spectroscopy, and validated using site-directed mutagenesis of key amino acids and nucleotides in PGC-1α and RNAs, respectively, to study PGC-1α-dependent liquid-liquid phase separation. Then, PGC-1α-containing multiprotein complexes will be co-purified to perform cryo-electron microscopy-based single particle structural analysis. Finally, the functional consequence of targeted mutagenesis of key residues will be assessed in muscle cells in culture and mouse muscle in vivo with state-of-the-art myotropic adeno-associated viral vectors (AAVMYO). The research plan thus a.) highly synergizes in terms of interdisciplinary approaches between the groups involved and b.) will result in hitherto unprecedented insights into molecular mechanisms that underlie complex spatio-temporal control of transcriptional networks with important implications for the understanding of cellular and tissue plasticity in health and disease.

Funding

Structure-function analysis of the RNA-binding protein PGC-1a in skeletal muscle

SNF Projekt (GrantsTool), 06.2023-05.2027 (48)
PI : Handschin, Christoph.
CI : Hiller, Sebastian.

Members (2)

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Christoph Handschin

Principal Investigator
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Sebastian Hiller

Co-Investigator