RBPs in Stress Granules

Stress granules (SGs) are critical to understanding ALS pathogenesis

A paradigm shift in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) occurred when mutations in RBPs, in particular Tar DNA-binding protein 43 (TARDP or TDP-43) and FUS/TLS, were found in patients with ALS. Most ALS patients display insoluble cytoplasmic inclusions of RBPs, most prominently TDP-43 or FUS. In addition, numerous RBPs harbor genetic mutations that fall within intrinsically disordered regions and change the phase separation properties of the mutant proteins to make them more aggregation prone. As a result, cells harboring such mutations display altered behavior of RNA granules, membrane-less organelles that regulate many aspects of RNA metabolism and protein homeostasis. Stress granules (SGs) are critical to understanding ALS pathogenesis, since many ALS-linked mutations affect SG biology (our review Nussbacher et al., Neuron 2019). Aberrant mRNA subcellular transport and localization are phenotypes of cellular stress also observed in ALS-mutant cells. Cellular stress directly impairs nucleocytoplasmic transport as demonstrated by a direct link between SG assembly and nuclear pore dysfunction. Defects in nucleocytoplasmic transport are increasingly seen as a central component of the molecular pathogenesis of ALS. Cellular stress also impairs axonal mRNA transport: neurite-localized and axonal SGs contain numerous transport-associated RBPs and limit localized mRNA translation. This is mirrored in neurons harboring mutations in TDP-43 and ANXA11, which show defects in subcellular and axonal transport of mRNA and RNA granules .

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Learn more about our ALS and Stress Granule Projects

To understand and identify ways to alter RNA granule biology, we published our landmark paper using proximity labeling to identify ~150 new proteins within SGs, making the surprising finding that granules are preassembled prior to stress. We revealed components necessary in the formation, stability, and resolution of SGs in ALS-patient iPSC- derived neurons. Excitingly, their depletion suppressed toxicity in animal models (Markmiller et al., Cell 2018)

We have continued to develop means to understand and control the formation and abundance of stress granules. Stress granule formation is frequently accompanied by ubiquitin proteasome system (UPS) impairment and ubiquitylated protein accumulation. In co-corresponding work with Eric Bennett’s lab at UCSD, my postdoc Markmiller tackled the unaddressed relationship between the ubiquitin pathway and SGs. Using pharmacological inhibition of either the ubiquitin or NEDD-activating enzyme (UAE or NAE), we show that UAE inhibition results in rapid loss of global protein ubiquitylation using ubiquitin-specific proteomics. Very interestingly, inhibiting neither UAE nor NAE affected SG formation or disassembly, indicating that active protein ubiquitylation or neddylation is dispensable for SG dynamics. Finally, we show that SGs co-localize primarily with unconjugated ubiquitin rather than polyubiquitylated proteins and suggest that free ubiquitin may alter SG protein interactions (Markmiller et al., Cell Reports 2019).

In 2019, my graduate student Mark Fang published a phenotypic screen using a library of >6,000 compounds to identify pathways that impinge on RNA granule dynamics and discovered a class of small molecules that interrupt the recruitment of proteins into pre-formed RNA granules. Excitingly, several of these compounds extended survival of ALS primary neurons to that of healthy neurons. As part of this study, we also demonstrated that transient, non- lethal cellular stress followed by a recovery period can induce the formation of persistent cytoplasmic TDP-43 aggregates in ALS patient-derived PSC-MNs. These persistent aggregates do not contain canonical SG markers such as G3BP1 and do not frequently occur in unstressed PSC-MNs, demonstrating that transient stress can be used to robustly induce an aging-dependent phenotype in an ALS mutant-specific context (Fang et al., Neuron 2019).

In 2019, my postdoc Markmiller used the transient stress paradigm in combination with a global mRNA localization profiling approach to measure subcellular mRNA distribution landscapes across different cell types and in response to cellular stress in the absence or presence of ALS-associated mutations. We show that the global mRNA localization landscape is shaped by a set of cell type-independent rules based on basic transcript architecture and sequence features and are profoundly disrupted by cellular stress. Remarkably, while stress-induced mRNA localization changes are transitory in wildtype PSC-MNs, HNRNPA2B1-mutant PSC-MNs retain a drastically reshaped mRNA localization profile and display elevated cell death 72h post-stress. Our single, non-toxic stressor also directly induces disease- relevant molecular changes such as splicing-mediated loss of Stathmin-2 (Markmiller et al., Cell Reports 2021).

In an effort to identify novel genetic regulators of stress granules, we developed CRaft-ID, the first pooled CRISPR screening approach using standard imaging readouts that utilizes microRaft arrays, and identified unexpected SG- regulatory proteins. We found that RNA binding proteins that control RNA processing at multiple layers appear to control SG formation and/or abundance, including surprisingly, a nuclear protein snRNP200 involved in splicing (Wheeler et al., Nature Methods 2020). Our ongoing and future work will continue to study some of these proteins such as snRNP200 to understand mechanistically how depletion of these affect cytoplasmic initiation and recovery of SGs. We also continue to unravel dysfunctional RBP-RNA relationships in neurodegeneration.