Tailoring stress responses
When faced with environmental stress, cells respond by shutting down cellular processes such as translation and nucleocytoplasmic transport. At the same time, cells preserve cytoplasmic messenger RNAs in structures known as stress granules, and many cellular proteins are modified by the covalent addition of ubiquitin, which has long been presumed to reflect degradation of stress-damaged proteins (see the Perspective by Dormann). Maxwell et al. show that cells generate distinct patterns of ubiquitination in response to different stressors. Rather than reflecting the degradation of stress-damaged proteins, this ubiquitination primes cells to dismantle stress granules and reinitiate normal cellular activities once the stress is removed. Gwon et al. show that persistent stress granules are degraded by autophagy, whereas short-lived granules undergo a process of disassembly that is autophagy independent. The mechanism of this disassembly depends on the initiating stress.
Science, abc3593 and abf6548, this issue p. eabc3593 and p. eabf6548; see also abj2400, p. 1393
Structured Abstract
INTRODUCTION
Stress granules are dynamic structures composed of RNA and protein that arise in the cytoplasm in response to a variety of stressors. These structures form via liquid-liquid phase separation and are usually promptly disassembled after the initiating stress is relieved. Stress granules assemble when the sum of protein-protein, protein-RNA, and RNA-RNA interactions breaches a particular threshold known as the percolation threshold. When this threshold is crossed, individual protein and RNA molecules form a system-spanning network that separates itself from its milieu to form a distinct granule. When the network of interactions falls below the percolation threshold, the granule disassembles. For stress granules, G3BP1 and G3BP2 are the proteins that provide the largest contribution to establishing the percolation threshold for granule assembly.
RATIONALE
Whereas great progress has been made in understanding the molecular basis of stress granule assembly, little is known about the mechanisms that govern their elimination. This process is of particular interest given that many mutations that cause neurodegeneration lead to impaired clearance of stress granules. The present investigation into the mechanisms of stress granule disassembly was prompted by findings in an accompanying report that G3BP1 is ubiquitinated when stress granules assemble in response to heat shock, but not when cells are exposed to other types of granule-inducing stress.
RESULTS
We found that stress granule elimination in cultured human cells is accomplished via one of two possible pathways: autophagy-independent disassembly or autophagy-dependent degradation. The fate of stress granules depended on how long they remained assembled. Persistent stress granules, such as those that arise through chronic stress or disease mutations, were eliminated by autophagy-dependent degradation. In contrast, short-lived granules were rapidly disassembled in an autophagy-independent manner, which permits recycling of constituents. Moreover, the mechanism whereby stress granules are disassembled depended upon the nature of the initiating stress. We identified the molecular mechanism of disassembly of stress granules induced by heat stress and found that this ubiquitin-dependent mechanism is specific to heat shock and not other types of stress. In response to heat shock, polyubiquitination of G3BP1 enabled binding by FAF2, an endoplasmic reticulum (ER)–associated adaptor for the ubiquitin-dependent segregase p97/VCP. Subsequent extraction of G3BP1 from stress granules by VCP leads to their disassembly. Disease-causing mutations in VCP impaired this disassembly mechanism.
CONCLUSION
We found that the fate of stress granules and the mechanism of their elimination depends on the context in which they were formed and the duration of their assembly. In the setting of heat shock, ubiquitination and subsequent removal of G3BP1 reduce the driving forces within the stress granule network below the percolation threshold for phase separation. Furthermore, the localization of FAF2 in the ER membrane indicates that stress granules that arise in response to heat stress are disassembled at the ER, consistent with other heat shock–dependent stress responses such as the unfolded protein response and ER-associated degradation. Disease-causing mutations in VCP not only impair autophagy-dependent stress granule degradation, as previously reported, but also impair autophagy-independent disassembly. Thus, mutations in VCP represent a mechanistic link between neurodegeneration and aberrant phase transitions. This finding aligns with other disease-causing mutations (e.g., mutations in intrinsically disordered regions of RNA-binding proteins, hexanucleotide repeat expansions in C9ORF72) that similarly impinge on intracellular phase transitions.
Ubiquitinated G3BP1 interacts with the ER-associated protein FAF2, which engages the ubiquitin-dependent segregase VCP. Targeting G3BP1 enables the stress granule–specific interaction network to fall below the percolation threshold for phase separation, which causes disassembly.
” data-icon-position=”” data-hide-link-title=”0″>
Ubiquitinated G3BP1 interacts with the ER-associated protein FAF2, which engages the ubiquitin-dependent segregase VCP. Targeting G3BP1 enables the stress granule–specific interaction network to fall below the percolation threshold for phase separation, which causes disassembly.
Abstract
Stress granules are dynamic, reversible condensates composed of RNA and protein that assemble in eukaryotic cells in response to a variety of stressors and are normally disassembled after stress is removed. The composition and assembly of stress granules is well understood, but little is known about the mechanisms that govern disassembly. Impaired disassembly has been implicated in some diseases including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Using cultured human cells, we found that stress granule disassembly was context-dependent: Specifically in the setting of heat shock, disassembly required ubiquitination of G3BP1, the central protein within the stress granule RNA-protein network. We found that ubiquitinated G3BP1 interacted with the endoplasmic reticulum–associated protein FAF2, which engaged the ubiquitin-dependent segregase p97/VCP (valosin-containing protein). Thus, targeting of G3BP1 weakened the stress granule–specific interaction network, resulting in granule disassembly.