RNA triggers chronic stress during neuronal aging.

Neurodegenerative diseases are linked with dysregulation of the integrated stress response (ISR), which coordinates cellular homeostasis during and after stress events. Cellular stress can arise from several sources, but there is significant disagreement about which stress might contribute to aging and neurodegeneration. Here, we leverage directed transdifferentiation of human fibroblasts into aged neurons to determine the source of ISR activation.

Neuronal aging causes mislocalization of splicing proteins and unchecked cellular stress.

Aging is one of the most prominent risk factors for neurodegeneration, yet the molecular mechanisms underlying the deterioration of old neurons are mostly unknown. To efficiently study neurodegeneration in the context of aging, we transdifferentiated primary human fibroblasts from aged healthy donors directly into neurons, which retained their aging hallmarks, and we verified key findings in aged human and mouse brain tissue. Here we show that aged neurons are broadly depleted of RNA-binding proteins, especially spliceosome components.

Large-scale map of RNA-binding protein interactomes across the mRNA life cycle.

mRNAs interact with RNA-binding proteins (RBPs) throughout their processing and maturation. While efforts have assigned RBPs to RNA substrates, less exploration has leveraged protein-protein interactions (PPIs) to study proteins in mRNA life-cycle stages. We generated an RNA-aware, RBP-centric PPI map across the mRNA life cycle in human cells by immunopurification-mass spectrometry (IP-MS) of ∼100 endogenous RBPs with and without RNase, augmented by size exclusion chromatography-mass spectrometry (SEC-MS).

High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues.

RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments.

Large-scale map of RNA binding protein interactomes across the mRNA life-cycle.

Messenger RNAs (mRNAs) interact with RNA-binding proteins (RBPs) in diverse ribonucleoprotein complexes (RNPs) during distinct life-cycle stages for their processing and maturation. While substantial attention has focused on understanding RNA regulation by assigning proteins, particularly RBPs, to specific RNA substrates, there has been considerably less exploration leveraging protein-protein interaction (PPI) methodologies to identify and study the role of proteins in mRNA life-cycle stages. To address this gap, we generated an RNA-aware RBP-centric PPI map across the mRNA life-cycle by immunopurification (IP-MS) of ~100 endogenous RBPs across the life-cycle in the presence or absence of RNase, augmented by size exclusion chromatography (SEC-MS).

Regulation of Biomolecular Condensates by Poly(ADP-ribose).

Biomolecular condensates are reversible compartments that form through a process called phase separation. Post-translational modifications like ADP-ribosylation can nucleate the formation of these condensates by accelerating the self-association of proteins. Poly(ADP-ribose) (PAR) chains are remarkably transient modifications with turnover rates on the order of minutes, yet they can be required for the formation of granules in response to oxidative stress, DNA damage, and other stimuli.

A minimal construct of nuclear-import receptor Karyopherin-β2 defines the regions critical for chaperone and disaggregation activity.

Karyopherin-β2 (Kapβ2) is a nuclear-import receptor that recognizes proline-tyrosine nuclear localization signals of diverse cytoplasmic cargo for transport to the nucleus. Kapβ2 cargo includes several disease-linked RNA-binding proteins with prion-like domains, such as FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. These RNA-binding proteins with prion-like domains are linked via pathology and genetics to debilitating degenerative disorders, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy.

Aging RNA granule dynamics in neurodegeneration.

Disordered RNA-binding proteins and repetitive RNA sequences are the main genetic causes of several neurodegenerative diseases, including amyotrophic lateral sclerosis and Huntington's disease. Importantly, these components also seed the formation of cytoplasmic liquid-like granules, like stress granules and P bodies. Emerging evidence demonstrates that healthy granules formed via liquid-liquid phase separation can mature into solid- or gel-like inclusions that persist within the cell.

Poly(ADP-ribose) drives condensation of FUS via a transient interaction.

Poly(ADP-ribose) (PAR) is an RNA-like polymer that regulates an increasing number of biological processes. Dysregulation of PAR is implicated in neurodegenerative diseases characterized by abnormal protein aggregation, including amyotrophic lateral sclerosis (ALS). PAR forms condensates with FUS, an RNA-binding protein linked with ALS, through an unknown mechanism.

Single molecule probing of disordered RNA binding proteins.

Liquid-liquid phase separation of intrinsically disordered proteins is known to underlie diverse pathologies such as neurodegeneration, cancer, and aging. The nucleation step of condensate formation is of critical importance for understanding how healthy and disease-associated condensates differ. Here, we describe four orthogonal single-molecule techniques that enable molecular tracking of the RNA-protein interaction, RNA-induced oligomerization, and kinetics of nucleation.

Single-molecule and ensemble methods to probe RNP nucleation and condensate properties.

Biomolecular condensates often consist of intrinsically disordered protein and RNA molecules, which together promote the formation of membraneless organelles in cells. The nucleation, condensation, and maturation of condensates is a critical yet poorly understood process. Here, we present single-molecule and accompanying ensemble methods to quantify these processes more comprehensively.

Actin filament- and Wiskott-Aldrich syndrome protein-binding sites on fructose-1,6-bisphosphate aldolase are functionally distinct from the active site.

The glycolytic enzyme fructose 1,6-(bis)phosphate aldolase (aldolase) is not only required for efficient utilization of glucose and fructose, but also for cytoskeletal functions like cytokinesis and cell motility. These differing roles are mediated by distinct and discrete binding interactions with aldolase's many binding partners, including actin filaments, Wiskott-Aldrich Syndrome protein (WASP), and Sorting Nexin 9 (SNX9). How these interactions are coordinated on the aldolase homotetramer of 160 kDa is unclear.

ALS/FTLD-Linked Mutations in FUS Glycine Residues Cause Accelerated Gelation and Reduced Interactions with Wild-Type FUS.

The RNA-binding protein fused in sarcoma (FUS) can form pathogenic inclusions in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). Over 70 mutations in Fus are linked to ALS/FTLD. In patients, all Fus mutations are heterozygous, indicating that the mutant drives disease progression despite the presence of wild-type (WT) FUS.

RNA Droplets.

Liquid-liquid phase separation is emerging as the universal mechanism by which membraneless cellular granules form. Despite many previous studies on condensation of intrinsically disordered proteins and low complexity domains, we lack understanding about the role of RNA, which is the essential component of all ribonucleoprotein (RNP) granules. RNA, as an anionic polymer, is inherently an excellent platform for achieving multivalency and can accommodate many RNA binding proteins.

Loss of Dynamic RNA Interaction and Aberrant Phase Separation Induced by Two Distinct Types of ALS/FTD-Linked FUS Mutations.

FUS is a nuclear RNA-binding protein, and its cytoplasmic aggregation is a pathogenic signature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It remains unknown how the FUS-RNA interactions contribute to phase separation and whether its phase behavior is affected by ALS-linked mutations. Here we demonstrate that wild-type FUS binds single-stranded RNA stoichiometrically in a length-dependent manner and that multimers induce highly dynamic interactions with RNA, giving rise to small and fluid condensates.