Award details

Understanding the crosstalk between spatially separated RNP granules during cellular stress responses

Reference	BB/V014110/1
Principal Investigator / Supervisor	Dr Tatyana Shelkovnikova
Co-Investigators / Co-Supervisors
Institution	University of Sheffield
Department	Neurosciences
Funding type	Research
Value (£)	253,251
Status	Current
Type	Research Grant
Start date	01/01/2022
End date	31/12/2024
Duration	36 months

Abstract

Phase separation is emerging as a key regulatory principle in eukaryotes, with the assembly and dissolution of membraneless organelles allowing the switch-like regulation of their RNA and protein components activities. Our overarching aim is to understand how RNP granules shape the cellular response to adverse conditions, uncovering how they specialise during stress to exert a specific response and mechanisms of their crosstalk under conditions of spatial segregation. Given their role in healthy responses and their deregulation in diseases such as viral infections and neurodegeneration, we will use stress granules and paraspeckles as prototypical RNP granules for cytoplasmic and nuclear membraneless organelles. First, we will determine how different stresses trigger the assembly of heterogeneous stress granules and paraspeckles with distinct functional flavours by establishing their structure, dynamics and composition. We will use high-throughput imaging and biochemical isolation of RNP granules coupled to proteomics to define the stress-specific properties of these RNP granules. Next and building on our recent evidence that stress granules can regulate paraspeckles assembly, we will identify the mechanisms responsible for this crosstalk by characterising the signalling pathways and specific proteins involved using both screening and targeted approaches informed by the proteomics. Finally, we will reveal how the assembly of aberrant stress granules impacts on paraspeckles and cellular functions in neurons using models of neurodegenerative diseases; and how the crosstalk between stress granules and paraspeckles contributes to antiviral signalling in infected cells, using viruses known to induce stress granules. Our study will establish guiding principles for how membraneless organelles specialise and communicate in healthy and pathological conditions, uncovering novel rules of cellular adaptation during stress.

Summary

Living organisms are constantly prompted to respond to the environment. This includes to changes in levels of nutrients, temperature, oxygen, invasion by pathogens and signals such as hormones. To pause and adapt, a key event is to limit protein synthesis, an energy hungry process. In addition, several signals are sent throughout the cell to communicate a state of emergency coordinating widespread changes. This allows for an overhaul of proteins in the cell to favour proteins that facilitate survival under the new conditions. According to textbooks, the main organising principle of a cell is the membrane with organelles such as the endoplasmic reticulum or mitochondria, wrapped in lipid bilayers. However, recent research is rethinking this model. Membraneless organelles allow the segregation of molecules, providing a new paradigm for cell biology. They form as a consequence of a change in the physical properties of their components, which now concentrate into specific regions of the cell. Because membraneless organelles can speed up reactions between their components or act as temporary storage, they are perfectly suited to contribute to rapid adaptation during stress. Proteins are encoded by RNA copies of genes called messenger RNA (mRNA). These mRNAs interact with a range of RNA binding proteins (RBPs) that control their fate. In response to stress and protein synthesis inhibition, mRNAs and RBPs bound to them, together with many other proteins, rapidly compartmentalise in the cytoplasm forming stress granules (SGs). Identified 35 years ago they are a paradigm for membraneless organelles. Several functions have been proposed for SGs. First, they help triage and store mRNAs to define which ones are needed to adapt to the new conditions and which are superfluous. Second, they are important for storing proteins that can send signals to trigger specific responses to the stress. Third, they are important in diseases; if anomalous they can contribute to diseases of the brain and they form part of our antiviral measures. Finally, our own findings suggest their assembly is important to trigger further waves of compartmentalisation, controlling the assembly of another membraneless organelle, the paraspeckle, in the nucleus. Despite this, major unsolved questions remain about how SGs function. They are part of a universal first line response to stress, yet it is apparent that SGs with distinct components and properties form depending on the nature of the stress. How and why specific components are selected, and how they drive specific functions, is currently poorly understood. Furthermore how SGs and other membraneless organelles like paraspeckles communicate, and the importance of these coordinated waves of compartmentalisation in normal and pathological conditions is unknown. Building on our expertise in studying SGs and paraspeckles, we now want to uncover how they contribute to cellular adaptation and specialised functions during stress. Our research program will comprehensively fingerprint SGs and paraspeckles under a range of different stresses to identify their components, interactions and functions. We will also define the molecular mechanisms by which SGs regulate the assembly of paraspeckles, uncovering how they communicate, and whether they regulate the assembly of other compartments. We will establish how SGs and paraspeckles contribute to the cellular defences against viruses and how the anomalous SGs associated with neurodegenerative diseases impact on paraspeckle-mediated responses in brain cells. Our current experience in isolating these organelles, and novel tools developed to image them are key for the success of these studies. Ultimately, the outcome of this work will advance our understanding of novel and fundamental aspects of cell biology and importantly relate this to pathological conditions.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Immunology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/V014528/1 Understanding the crosstalk between spatially separated RNP granules during cellular stress responses

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