TDP-43 condensation properties specify its RNA-binding and regulatory repertoire.

Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.

Molecular determinants and signaling effects of PKA RIα phase separation.

Spatiotemporal regulation of intracellular signaling molecules, such as the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), ensures proper cellular function. Liquid-liquid phase separation (LLPS) of the ubiquitous PKA regulatory subunit RIα promotes cAMP compartmentation and signaling specificity. However, the molecular determinants of RIα LLPS remain unclear. Here, we reveal that two separate dimerization interfaces, combined with the cAMP-induced unleashing of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence, drive RIα condensate formation in the cytosol of mammalian cells, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RIα pseudosubstrate region is critically involved in forming a non-canonical R:C complex, which recruits active PKA-C to RIα condensates to maintain low basal PKA activity in the cytosol. Our results suggest that RIα LLPS not only facilitates cAMP compartmentation but also spatially restrains active PKA-C, thus highlighting the functional versatility of biomolecular condensates in driving signaling specificity.

An autoinhibitory switch of the LSD1 disordered region controls enhancer silencing.

Transcriptional coregulators and transcription factors (TFs) contain intrinsically disordered regions (IDRs) that are critical for their association and function in gene regulation. More recently, IDRs have been shown to promote multivalent protein-protein interactions between coregulators and TFs to drive their association into condensates. By contrast, here we demonstrate how the IDR of the corepressor LSD1 excludes TF association, acting as a dynamic conformational switch that tunes repression of active cis-regulatory elements. Hydrogen-deuterium exchange shows that the LSD1 IDR interconverts between transient open and closed conformational states, the latter of which inhibits partitioning of the protein's structured domains with TF condensates. This autoinhibitory switch controls leukemic differentiation by modulating repression of active cis-regulatory elements bound by LSD1 and master hematopoietic TFs. Together, these studies unveil alternative mechanisms by which disordered regions and their dynamic crosstalk with structured regions can shape coregulator-TF interactions to control cis-regulatory landscapes and cell fate.

Blending and separating dynamics of RNA-binding proteins develop architectural splicing networks spreading throughout the nucleus.

The eukaryotic nucleus has a highly organized structure. Although the spatiotemporal arrangement of spliceosomes on nascent RNA drives splicing, the nuclear architecture that directly supports this process remains unclear. Here, we show that RNA-binding proteins (RBPs) assembled on RNA form meshworks in human and mouse cells. Core and accessory RBPs in RNA splicing make two distinct meshworks adjacently but distinctly distributed throughout the nucleus. This is achieved by mutual exclusion dynamics between the charged and uncharged intrinsically disordered regions (IDRs) of RBPs. These two types of meshworks compete for spatial occupancy on pre-mRNA to regulate splicing. Furthermore, the optogenetic enhancement of the RBP meshwork causes aberrant splicing, particularly of genes involved in neurodegeneration. Genetic mutations associated with neurodegenerative diseases are often found in the IDRs of RBPs, and cells harboring these mutations exhibit impaired meshwork formation. Our results uncovered the spatial organization of RBP networks to drive RNA splicing.

Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions.

Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.

An intrinsically disordered region controlling condensation of a circadian clock component and rhythmic transcription in the liver.

Circadian gene transcription is fundamental to metabolic physiology. Here we report that the nuclear receptor REV-ERBα, a repressive component of the molecular clock, forms circadian condensates in the nuclei of mouse liver. These condensates are dictated by an intrinsically disordered region (IDR) located in the protein's hinge region which specifically concentrates nuclear receptor corepressor 1 (NCOR1) at the genome. IDR deletion diminishes the recruitment of NCOR1 and disrupts rhythmic gene transcription in vivo. REV-ERBα condensates are located at high-order transcriptional repressive hubs in the liver genome that are highly correlated with circadian gene repression. Deletion of the IDR disrupts transcriptional repressive hubs and diminishes silencing of target genes by REV-ERBα. This work demonstrates physiological circadian protein condensates containing REV-ERBα whose IDR is required for hub formation and the control of rhythmic gene expression.

"Structure"-function relationships in eukaryotic transcription factors: The role of intrinsically disordered regions in gene regulation.

Many principles of bacterial gene regulation have been foundational to understanding mechanisms of eukaryotic transcription. However, stark structural and functional differences exist between eukaryotic and bacterial transcription factors that complicate inferring properties of the eukaryotic system from that of bacteria. Here, we review those differences, focusing on the impact of intrinsically disordered regions on the thermodynamic and kinetic parameters governing eukaryotic transcription factor interactions-both with other proteins and with chromatin. The prevalence of unstructured domains in eukaryotic transcription factors as well as their known impact on function call for more sophisticated knowledge of what mechanisms they support. Using the evidence available to date, we posit that intrinsically disordered regions are necessary for the complex and integrative functions of eukaryotic transcription factors and that only by understanding their rich biochemistry can we develop a deep molecular understanding of their regulatory mechanisms.

ULK/Atg1: phasing in and out of autophagy.

Autophagy - a highly regulated intracellular degradation process - is pivotal in maintaining cellular homeostasis. Liquid-liquid phase separation (LLPS) is a fundamental mechanism regulating the formation and function of membrane-less compartments. Recent research has unveiled connections between LLPS and autophagy, suggesting that phase separation events may orchestrate the spatiotemporal organization of autophagic machinery and cargo sequestration. The Unc-51-like kinase (ULK)/autophagy-related 1 (Atg1) family of proteins is best known for its regulatory role in initiating autophagy, but there is growing evidence that the functional spectrum of ULK/Atg1 extends beyond autophagy regulation. In this review, we explore the spatial and temporal regulation of the ULK/Atg1 family of kinases, focusing on their recruitment to LLPS-driven compartments, and highlighting their multifaceted functions beyond their traditional role.

How intrinsically disordered proteins order plant gene silencing.

Intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) possess low sequence complexity of amino acids and display non-globular tertiary structures. They can act as scaffolds, form regulatory hubs, or trigger biomolecular condensation to control diverse aspects of biology. Emerging evidence has recently implicated critical roles of IDPs and IDR-contained proteins in nuclear transcription and cytoplasmic post-transcriptional processes, among other molecular functions. We here summarize the concepts and organizing principles of IDPs. We then illustrate recent progress in understanding the roles of key IDPs in machineries that regulate transcriptional and post-transcriptional gene silencing (PTGS) in plants, aiming at highlighting new modes of action of IDPs in controlling biological processes.

The impact of IDR phosphorylation on the RNA binding profiles of proteins.

Due to their capacity to mediate repetitive protein interactions, intrinsically disordered regions (IDRs) are crucial for the formation of various types of protein-RNA complexes. The functions of IDRs are strongly modulated by post-translational modifications (PTMs). Phosphorylation is the most common and well-studied modification of IDRs, which can alter homomeric or heteromeric interactions of proteins and impact their ability to phase separate. Moreover, phosphorylation can influence the RNA-binding properties of proteins, and recent studies demonstrated its selective impact on the global profiles of protein-RNA binding and regulation. These findings highlight the need for further integrative approaches to understand how signalling remodels protein-RNA networks in cells.

TOLLIP acts as a cargo adaptor to promote lysosomal degradation of aberrant ER membrane proteins.

Endoplasmic reticulum (ER) proteostasis is maintained by various catabolic pathways. Lysosomes clear entire ER portions by ER-phagy, while proteasomes selectively clear misfolded or surplus aberrant proteins by ER-associated degradation (ERAD). Recently, lysosomes have also been implicated in the selective clearance of aberrant ER proteins, but the molecular basis remains unclear. Here, we show that the phosphatidylinositol-3-phosphate (PI3P)-binding protein TOLLIP promotes selective lysosomal degradation of aberrant membrane proteins, including an artificial substrate and motoneuron disease-causing mutants of VAPB and Seipin. These cargos are recognized by TOLLIP through its misfolding-sensing intrinsically disordered region (IDR) and ubiquitin-binding CUE domain. In contrast to ER-phagy receptors, which clear both native and aberrant proteins by ER-phagy, TOLLIP selectively clears aberrant cargos by coupling them with the PI3P-dependent lysosomal trafficking without promoting bulk ER turnover. Moreover, TOLLIP depletion augments ER stress after ERAD inhibition, indicating that TOLLIP and ERAD cooperatively safeguard ER proteostasis. Our study identifies TOLLIP as a unique type of cargo-specific adaptor dedicated to the clearance of aberrant ER cargos and provides insights into molecular mechanisms underlying lysosome-mediated quality control of membrane proteins.

BRCA1/BARD1 intrinsically disordered regions facilitate chromatin recruitment and ubiquitylation.

BRCA1/BARD1 is a tumor suppressor E3 ubiquitin (Ub) ligase with roles in DNA damage repair and in transcriptional regulation. BRCA1/BARD1 RING domains interact with nucleosomes to facilitate mono-ubiquitylation of distinct residues on the C-terminal tail of histone H2A. These enzymatic domains constitute a small fraction of the heterodimer, raising the possibility of functional chromatin interactions involving other regions such as the BARD1 C-terminal domains that bind nucleosomes containing the DNA damage signal H2A K15-Ub and H4 K20me0, or portions of the expansive intrinsically disordered regions found in both subunits. Herein, we reveal novel interactions that support robust H2A ubiquitylation activity mediated through a high-affinity, intrinsically disordered DNA-binding region of BARD1. These interactions support BRCA1/BARD1 recruitment to chromatin and sites of DNA damage in cells and contribute to their survival. We also reveal distinct BRCA1/BARD1 complexes that depend on the presence of H2A K15-Ub, including a complex where a single BARD1 subunit spans adjacent nucleosome units. Our findings identify an extensive network of multivalent BARD1-nucleosome interactions that serve as a platform for BRCA1/BARD1-associated functions on chromatin.

Intrinsically Disordered Protein TEX264 Mediates ER-phagy.

Certain proteins and organelles can be selectively degraded by autophagy. Typical substrates and receptors of selective autophagy have LC3-interacting regions (LIRs) that bind to autophagosomal LC3 and GABARAP family proteins. Here, we performed a differential interactome screen using wild-type LC3B and a LIR recognition-deficient mutant and identified TEX264 as a receptor for autophagic degradation of the endoplasmic reticulum (ER-phagy). TEX264 is an ER protein with a single transmembrane domain and a LIR motif. TEX264 interacts with LC3 and GABARAP family proteins more efficiently and is expressed more ubiquitously than previously known ER-phagy receptors. ER-phagy is profoundly blocked by deletion of TEX264 alone and almost completely by additional deletion of FAM134B and CCPG1. A long intrinsically disordered region of TEX264 is required for its ER-phagy receptor function to bridge the gap between the ER and autophagosomal membranes independently of its amino acid sequence. These results suggest that TEX264 is a major ER-phagy receptor.

C9orf72 Poly(PR) Dipeptide Repeats Disturb Biomolecular Phase Separation and Disrupt Nucleolar Function.

Repeat expansion in the C9orf72 gene is the most common cause of the neurodegenerative disorder amyotrophic lateral sclerosis (C9-ALS) and is linked to the unconventional translation of five dipeptide-repeat polypeptides (DPRs). The two enriched in arginine, poly(GR) and poly(PR), infiltrate liquid-like nucleoli, co-localize with the nucleolar protein nucleophosmin (NPM1), and alter the phase separation behavior of NPM1 in vitro. Here, we show that poly(PR) DPRs bind tightly to a long acidic tract within the intrinsically disordered region of NPM1, altering its phase separation with nucleolar partners to the extreme of forming large, soluble complexes that cause droplet dissolution in vitro. In cells, poly(PR) DPRs disperse NPM1 from nucleoli and entrap rRNA in static condensates in a DPR-length-dependent manner. We propose that R-rich DPR toxicity involves disrupting the role of phase separation by NPM1 in organizing ribosomal proteins and RNAs within the nucleolus.

Nascent Pre-rRNA Sorting via Phase Separation Drives the Assembly of Dense Fibrillar Components in the Human Nucleolus.

Fibrillar centers (FCs) and dense fibrillar components (DFCs) are essential morphologically distinct sub-regions of mammalian cell nucleoli for rDNA transcription and pre-rRNA processing. Here, we report that a human nucleolus consists of several dozen FC/DFC units, each containing 2-3 transcriptionally active rDNAs at the FC/DFC border. Pre-rRNA processing factors, such as fibrillarin (FBL), form 18-24 clusters that further assemble into the DFC surrounding the FC. Mechanistically, the 5' end of nascent 47S pre-rRNA binds co-transcriptionally to the RNA-binding domain of FBL. FBL diffuses to the DFC, where local self-association via its glycine- and arginine-rich (GAR) domain forms phase-separated clusters to immobilize FBL-interacting pre-rRNA, thus promoting directional traffic of nascent pre-rRNA while facilitating pre-rRNA processing and DFC formation. These results unveil FC/DFC ultrastructures in nucleoli and suggest a conceptual framework for considering nascent RNA sorting using multivalent interactions of their binding proteins.

The disordered C-terminal tail of fungal LPMOs from phytopathogens mediates protein dimerization and impacts plant penetration.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called CoAA9A, up-regulated during plant infection by Colletotrichum orbiculare, the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates CoAA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.

Ubiquitin-induced RNF168 condensation promotes DNA double-strand break repair.

Rapid accumulation of repair factors at DNA double-strand breaks (DSBs) is essential for DSB repair. Several factors involved in DSB repair have been found undergoing liquid-liquid phase separation (LLPS) at DSB sites to facilitate DNA repair. RNF168, a RING-type E3 ubiquitin ligase, catalyzes H2A.X ubiquitination for recruiting DNA repair factors. Yet, whether RNF168 undergoes LLPS at DSB sites remains unclear. Here, we identified K63-linked polyubiquitin-triggered RNF168 condensation which further promoted RNF168-mediated DSB repair. RNF168 formed liquid-like condensates upon irradiation in the nucleus while purified RNF168 protein also condensed in vitro. An intrinsically disordered region containing amino acids 460-550 was identified as the essential domain for RNF168 condensation. Interestingly, LLPS of RNF168 was significantly enhanced by K63-linked polyubiquitin chains, and LLPS largely enhanced the RNF168-mediated H2A.X ubiquitination, suggesting a positive feedback loop to facilitate RNF168 rapid accumulation and its catalytic activity. Functionally, LLPS deficiency of RNF168 resulted in delayed recruitment of 53BP1 and BRCA1 and subsequent impairment in DSB repair. Taken together, our finding demonstrates the pivotal effect of LLPS in RNF168-mediated DSB repair.

Sequence-independent activity of a predicted long disordered segment of the human papillomavirus type 16 L2 capsid protein during virus entry.

The activity of proteins is thought to be invariably determined by their amino acid sequence or composition, but we show that a long segment of a viral protein can support infection independent of its sequence or composition. During virus entry, the papillomavirus L2 capsid protein protrudes through the endosome membrane into the cytoplasm to bind cellular factors such as retromer required for intracellular virus trafficking. Here, we show that an ~110 amino acid segment of L2 is predicted to be disordered and that large deletions in this segment abolish infectivity of HPV16 pseudoviruses by inhibiting cytoplasmic protrusion of L2, association with retromer, and proper virus trafficking. The activity of these mutants can be restored by insertion of protein segments with diverse sequences, compositions, and chemical properties, including scrambled amino acid sequences, a tandem array of a short sequence, and the intrinsically disordered region of an unrelated cellular protein. The infectivity of mutants with small in-frame deletions in this segment directly correlates with the size of the segment. These results indicate that the length of the disordered segment, not its sequence or composition, determines its activity during HPV16 pseudovirus infection. We propose that a minimal length of L2 is required for it to protrude far enough into the cytoplasm to bind cytoplasmic trafficking factors, but the sequence of this segment is largely irrelevant. Thus, protein segments can carry out complex biological functions such as Human papillomavirus pseudovirus infection in a sequence-independent manner. This finding has important implications for protein function and evolution.

A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling.

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 posttranslational regulation. Here, we characterize a phosphorylation site at Ser325 situated C terminal to the catalytic domain of PTPN22 and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by glycogen synthase kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9-mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p, and Moira domain binding proteins, and next-generation sequencing revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein's properties by deuterium exchange mass spectrometry and NMR spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22.

Integrase-LEDGF/p75 complex triggers the formation of biomolecular condensates that modulate HIV-1 integration efficiency in vitro.

The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS in vitro and if IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the in vitro IN activities. While the 3'-processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes.