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In situ study of ribosome biogenesis, non-stop mRNA decay, and liquid-liquid phase separation by cryo-electron tomography
In situ study of ribosome biogenesis, non-stop mRNA decay, and liquid-liquid phase separation by cryo-electron tomography
The development of new methods in biochemistry and the advancement in microscopes have enabled scientists to disentangle complicated molecular mechanisms and decipher more protein complexes at an atomic level. However, detailed structural studies of proteins and macromolecular machineries require targeted extraction and purification, through which an information gap between the molecular architecture and its cellular context is created. Over the past years, cryo-electron tomography (cryo-ET), by which one can directly inspect and analyze macromolecules in the cell, has become a powerful tool to bridge the gap. In this dissertation, cryo-ET was applied in studying challenging biological questions that could not be comprehensively addressed by conventional biochemical analyses either due to the indispensability of their native environment or their low occurrence in cells. My first project examined ribosome biogenesis, which occurs in the nucleolus, a structure that is formed through liquid-liquid phase separation (LLPS) of various liquid-phase nucleolar compartments, but it has not been sufficiently substantiated in previous studies. In my study, distinct in situ pre-ribosomal structures were resolved and the spatiotemporal organization of ribosome biogenesis was revealed with liquid-phase nucleolar compartments for the first time, showing how this process is orchestrated through the surveillance of the nucleolar LLPS. Following an interesting discovery of a nuclear-exosome-associated preribosome class in my first project, I set out to explore non-stop mRNA decay (NSD), a mRNA surveillance mechanism in the cytoplasm, in my second project to gain more understanding of ribosome-associated quality control of mRNA. My study demonstrated it to be challenging to obtain in situ structures of NSD-related complexes together with ribosomes by cryo-ET. To improve this, I developed new strategies for in situ enrichment of NSD-related complexes at specific subcellular structures. The third project, again applied cryo-ET to analyze a special LLPS phenomenon generated by two intrinsically disordered proteins: SLP65 and CIN85, which are involved in B cell activation and protein translocation. Coupled with in vitro and in situ analyses, I provided a more comprehensive interpretation of SLP65-CIN85 LLPS to underpin the mechanism by which LLPS effectively regulates protein surveillance and translocation. In conclusion, this dissertation not only demonstrates the successful application of cryo-ET in studying intricate biological questions, but also provides valuable insights to future comprehensive cryo-ET studies integrated with conventional biochemistry.
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Hou, Zhen
2022
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Hou, Zhen (2022): In situ study of ribosome biogenesis, non-stop mRNA decay, and liquid-liquid phase separation by cryo-electron tomography. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The development of new methods in biochemistry and the advancement in microscopes have enabled scientists to disentangle complicated molecular mechanisms and decipher more protein complexes at an atomic level. However, detailed structural studies of proteins and macromolecular machineries require targeted extraction and purification, through which an information gap between the molecular architecture and its cellular context is created. Over the past years, cryo-electron tomography (cryo-ET), by which one can directly inspect and analyze macromolecules in the cell, has become a powerful tool to bridge the gap. In this dissertation, cryo-ET was applied in studying challenging biological questions that could not be comprehensively addressed by conventional biochemical analyses either due to the indispensability of their native environment or their low occurrence in cells. My first project examined ribosome biogenesis, which occurs in the nucleolus, a structure that is formed through liquid-liquid phase separation (LLPS) of various liquid-phase nucleolar compartments, but it has not been sufficiently substantiated in previous studies. In my study, distinct in situ pre-ribosomal structures were resolved and the spatiotemporal organization of ribosome biogenesis was revealed with liquid-phase nucleolar compartments for the first time, showing how this process is orchestrated through the surveillance of the nucleolar LLPS. Following an interesting discovery of a nuclear-exosome-associated preribosome class in my first project, I set out to explore non-stop mRNA decay (NSD), a mRNA surveillance mechanism in the cytoplasm, in my second project to gain more understanding of ribosome-associated quality control of mRNA. My study demonstrated it to be challenging to obtain in situ structures of NSD-related complexes together with ribosomes by cryo-ET. To improve this, I developed new strategies for in situ enrichment of NSD-related complexes at specific subcellular structures. The third project, again applied cryo-ET to analyze a special LLPS phenomenon generated by two intrinsically disordered proteins: SLP65 and CIN85, which are involved in B cell activation and protein translocation. Coupled with in vitro and in situ analyses, I provided a more comprehensive interpretation of SLP65-CIN85 LLPS to underpin the mechanism by which LLPS effectively regulates protein surveillance and translocation. In conclusion, this dissertation not only demonstrates the successful application of cryo-ET in studying intricate biological questions, but also provides valuable insights to future comprehensive cryo-ET studies integrated with conventional biochemistry.