Periodic Reporting for period 1 - RIBORESCUEPHAGY (How does autophagy rescue stalled ribosomes?)
Okres sprawozdawczy: 2022-10-01 do 2025-03-31
Cells rely on ribosomes to efficiently translate genetic information into functional proteins, a process essential for cellular survival and function. Ribosomes attached to the endoplasmic reticulum (ER) are particularly critical, as they synthesize approximately 40% of the proteome, including secreted and membrane-bound proteins. However, under stress or due to aberrant mRNA, ribosomal collisions occur, leading to stalled translation and the accumulation of incomplete and toxic polypeptides. These disruptions jeopardize ER function and overall protein homeostasis, presenting a significant challenge in eukaryotic cells.
Autophagy, a conserved cellular degradation pathway, plays a central role in recycling damaged or dysfunctional cellular components. While much is known about autophagy's role in general quality control, its involvement in resolving stalled ribosomes at the ER remains poorly understood. Recent discoveries in our lab have identified two key autophagy receptors, C53 and VCS, that mediate the clearance of harmful byproducts of ribosomal stalling. These findings suggest the existence of a novel quality control mechanism—RiboRescuePhagy—that rescues ER-bound ribosomes by selectively degrading stalled translation products.
The primary objective of this project is to unravel the molecular mechanisms and physiological significance of autophagy-mediated ribosomal rescue. Specifically, we aim to:
Characterize the role of C53 in degrading incomplete polypeptides that arise from ribosome stalling at the ER.
Investigate how VCS targets harmful RNAs generated during ribosome collisions and its dual function as an mRNA decapping regulator and autophagy receptor.
Elucidate the role of ufmylation, a specialized post-translational modification, in regulating these autophagy pathways.
This research integrates advanced genetic screens, structural biology, and biochemical approaches in both model plants (Arabidopsis and Marchantia) and human cells. By defining how autophagy maintains ER ribosome functionality, this project addresses a fundamental gap in our understanding of cellular quality control.
Expected Impact
The outcomes of this project will redefine our understanding of autophagy as a ribosome-associated quality control mechanism, with broad implications across eukaryotic biology. This knowledge is expected to inform therapeutic strategies for diseases linked to ER stress and ribosomal dysfunction, including neurodegenerative disorders and metabolic syndromes. Additionally, insights into RiboRescuePhagy could enhance biotechnological applications such as optimizing protein production in plants, a key tool for sustainable agriculture and pharmaceuticals.
Autophagy, a conserved cellular degradation pathway, plays a central role in recycling damaged or dysfunctional cellular components. While much is known about autophagy's role in general quality control, its involvement in resolving stalled ribosomes at the ER remains poorly understood. Recent discoveries in our lab have identified two key autophagy receptors, C53 and VCS, that mediate the clearance of harmful byproducts of ribosomal stalling. These findings suggest the existence of a novel quality control mechanism—RiboRescuePhagy—that rescues ER-bound ribosomes by selectively degrading stalled translation products.
The primary objective of this project is to unravel the molecular mechanisms and physiological significance of autophagy-mediated ribosomal rescue. Specifically, we aim to:
Characterize the role of C53 in degrading incomplete polypeptides that arise from ribosome stalling at the ER.
Investigate how VCS targets harmful RNAs generated during ribosome collisions and its dual function as an mRNA decapping regulator and autophagy receptor.
Elucidate the role of ufmylation, a specialized post-translational modification, in regulating these autophagy pathways.
This research integrates advanced genetic screens, structural biology, and biochemical approaches in both model plants (Arabidopsis and Marchantia) and human cells. By defining how autophagy maintains ER ribosome functionality, this project addresses a fundamental gap in our understanding of cellular quality control.
Expected Impact
The outcomes of this project will redefine our understanding of autophagy as a ribosome-associated quality control mechanism, with broad implications across eukaryotic biology. This knowledge is expected to inform therapeutic strategies for diseases linked to ER stress and ribosomal dysfunction, including neurodegenerative disorders and metabolic syndromes. Additionally, insights into RiboRescuePhagy could enhance biotechnological applications such as optimizing protein production in plants, a key tool for sustainable agriculture and pharmaceuticals.
Multiple ribosomes simultaneously move along the mRNAs to translate the genes into proteins. Cellular stress triggers collisions of ribosomes and disrupts protein synthesis. Eukaryotes have evolved multi-tiered quality control mechanisms that monitor ribosomes and rescue them on collision. While much is known about the rescue of cytosolic ribosomes, how the cell rescues stalled endoplasmic reticulum bound (ER-bound) ribosomes remains unknown. We recently discovered that the stalling of ER-bound ribosomes induces autophagy, a major cellular degradation pathway. We discovered two autophagy receptors that are induced upon stalling of ER- bound ribosomes and these proteins are conserved between plants and humans. We also showed that ufmylation, an elusive posttranslational modification system regulates ER-bound ribosome stalling-induced autophagy. These two discoveries indicate that autophagy plays a major role in the maintenance of a functional ER-bound ribosome population. Based on these discoveries, I hypothesize that autophagy rescues stalled ER-bound ribosomes by selectively degrading harmful polypeptides and RNAs that clog the ribosomes during collisions. Here, I propose to define and characterize this conserved quality control mechanism. I will establish a suite of complementary methods in the model plants Arabidopsis thaliana and Marchantia polymorpha to explore the physiological significance of autophagy-mediated ribosomal rescue (RiboRescuePhagy) in complex multicellular organisms. In parallel, I will carry out unbiased genetic screens in human cell lines to discover the molecular components that mediate RiboRescuePhagy. Finally, I will perform structure-function analysis of a key ufmylation enzyme to untangle the connection between ufmylation and autophagy. At the completion of this project, we will have defined a new quality control mechanism that rescues stalled ER-bound ribosomes to maintain cellular homeostasis in eukaryotes.
We are progressing as we planned. Thanks to the support from the ERC, we have published 5 major publications. We are currently working on three more publications that will be submitted within the time frame of the proposal.
Brief summary of the main discoveries so far:
UFMylation and ER Homeostasis
Through phylogenomic and ribosome profiling studies, we uncovered a novel mechanism where UFMylation regulates mRNA export to alleviate ER stress. This process re-compartmentalizes and preserves translation of key mRNAs, thus reducing the translational load on the ER and promoting cellular resilience.
UFMylation and Epigenetics
Our research identified a unique subset of 24-nucleotide small RNAs whose biogenesis depends on UFMylation. These small RNAs regulate CG-type DNA methylation, providing new insights into the role of UFMylation in epigenetic regulation. These findings highlight a previously unexplored intersection of RNA processing and epigenetics.
Ribosome Heterogeneity in Pollen Cells
Initial translation assays revealed that pollen cells possess distinct ribosomal subunits compared to vegetative cells, resulting in unique translational dynamics. We utilized CryoEM to resolve the structures of these specialized ribosomes, offering a mechanistic understanding of their functional divergence. Ongoing analyses aim to link these structural differences to translational regulation in pollen.
CryoET and Cellular Reprogramming
Adopting Marchantia polymorpha as a model, we optimized CryoET pipelines to study spore dormancy and reprogramming. For the first time in plants, we visualized ribosomal structures in their native context, identifying factors responsible for maintaining ribosomal hibernation during spore dormancy. This breakthrough establishes CryoET as a powerful tool for studying cellular processes in plants at high resolution.
3 people from the lab also obtained independent PI positions, thanks to the ERC support.
We are progressing as we planned. Thanks to the support from the ERC, we have published 5 major publications. We are currently working on three more publications that will be submitted within the time frame of the proposal.
Brief summary of the main discoveries so far:
UFMylation and ER Homeostasis
Through phylogenomic and ribosome profiling studies, we uncovered a novel mechanism where UFMylation regulates mRNA export to alleviate ER stress. This process re-compartmentalizes and preserves translation of key mRNAs, thus reducing the translational load on the ER and promoting cellular resilience.
UFMylation and Epigenetics
Our research identified a unique subset of 24-nucleotide small RNAs whose biogenesis depends on UFMylation. These small RNAs regulate CG-type DNA methylation, providing new insights into the role of UFMylation in epigenetic regulation. These findings highlight a previously unexplored intersection of RNA processing and epigenetics.
Ribosome Heterogeneity in Pollen Cells
Initial translation assays revealed that pollen cells possess distinct ribosomal subunits compared to vegetative cells, resulting in unique translational dynamics. We utilized CryoEM to resolve the structures of these specialized ribosomes, offering a mechanistic understanding of their functional divergence. Ongoing analyses aim to link these structural differences to translational regulation in pollen.
CryoET and Cellular Reprogramming
Adopting Marchantia polymorpha as a model, we optimized CryoET pipelines to study spore dormancy and reprogramming. For the first time in plants, we visualized ribosomal structures in their native context, identifying factors responsible for maintaining ribosomal hibernation during spore dormancy. This breakthrough establishes CryoET as a powerful tool for studying cellular processes in plants at high resolution.
3 people from the lab also obtained independent PI positions, thanks to the ERC support.