Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in Xenopus oocytes.

Proteins containing both intrinsically disordered regions (IDRs) and RNA binding domains (RBDs) can phase separate in vitro, forming bodies similar to cellular biomolecular condensates. However, how IDR and RBD domains contribute to in vivo recruitment of proteins to biomolecular condensates remains poorly understood. Here, we analyzed the roles of IDRs and RBDs in L-bodies, biomolecular condensates present in Xenopus oocytes.

Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in oocytes.

Proteins containing both intrinsically disordered regions (IDRs) and RNA binding domains (RBDs) can phase separate , forming bodies similar to cellular biomolecular condensates. However, how IDR and RBD domains contribute to recruitment of proteins to biomolecular condensates remains poorly understood. Here, we analyzed the roles of IDRs and RBDs in L-bodies, biomolecular condensates present in oocytes.

Fibril formation and ordering of disordered FUS LC driven by hydrophobic interactions.

Biomolecular condensates, protein-rich and dynamic membrane-less organelles, play critical roles in a range of subcellular processes, including membrane trafficking and transcriptional regulation. However, aberrant phase transitions of intrinsically disordered proteins in biomolecular condensates can lead to the formation of irreversible fibrils and aggregates that are linked to neurodegenerative diseases. Despite the implications, the interactions underlying such transitions remain obscure.

Characterization of ConE, the VirB4 Homolog of the Integrative and Conjugative Element ICE of Bacillus subtilis.

Conjugation is a major form of horizontal gene transfer, contributing to bacterial evolution and the acquisition of new traits. During conjugation, a donor cell transfers DNA to a recipient through a specialized DNA translocation channel classified as a type IV secretion system (T4SS). Here, we focused on the T4SS of ICE, an integrative and conjugative element in Bacillus subtilis.

Interactions between FUS and the C-terminal Domain of Nup62 are Sufficient for their Co-phase Separation into Amorphous Assemblies.

Deficient nucleocytoplasmic transport is emerging as a pathogenic feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), including in ALS caused by mutations in Fused in Sarcoma (FUS). Recently, both wild-type and ALS-linked mutant FUS were shown to directly interact with the phenylalanine-glycine (FG)-rich nucleoporin 62 (Nup62) protein, where FUS WT/ Nup62 interactions were enriched within the nucleus but ALS-linked mutant FUS/ Nup62 interactions were enriched within the cytoplasm of cells. Nup62 is a central channel Nup that has a prominent role in forming the selectivity filter within the nuclear pore complex and in regulating effective nucleocytoplasmic transport.

Incorporating NOE-Derived Distances in Conformer Generation of Cyclic Peptides with Distance Geometry.

Nuclear magnetic resonance (NMR) data from NOESY (nuclear Overhauser enhancement spectroscopy) and ROESY (rotating frame Overhauser enhancement spectroscopy) experiments can easily be combined with distance geometry (DG) based conformer generators by modifying the molecular distance bounds matrix. In this work, we extend the modern DG based conformer generator ETKDG, which has been shown to reproduce experimental crystal structures from small molecules to large macrocycles well, to include NOE-derived interproton distances. In noeETKDG, the experimentally derived interproton distances are incorporated into the distance bounds matrix as loose upper (or lower) bounds to generate large conformer sets.

Reversible Kinetic Trapping of FUS Biomolecular Condensates.

Formation of membrane-less organelles by self-assembly of disordered proteins can be triggered by external stimuli such as pH, salt, or temperature. These organelles, called biomolecular condensates, have traditionally been classified as liquids, gels, or solids with limited subclasses. Here, the authors show that a thermal trigger can lead to formation of at least two distinct liquid condensed phases of the fused in sarcoma low complexity (FUS LC) domain.

Molecular interactions contributing to FUS SYGQ LC-RGG phase separation and co-partitioning with RNA polymerase II heptads.

The RNA-binding protein FUS (Fused in Sarcoma) mediates phase separation in biomolecular condensates and functions in transcription by clustering with RNA polymerase II. Specific contact residues and interaction modes formed by FUS and the C-terminal heptad repeats of RNA polymerase II (CTD) have been suggested but not probed directly. Here we show how RGG domains contribute to phase separation with the FUS N-terminal low-complexity domain (SYGQ LC) and RNA polymerase II CTD.

N-terminal acetylation modestly enhances phase separation and reduces aggregation of the low-complexity domain of RNA-binding protein fused in sarcoma.

The RNA-binding protein fused in sarcoma (FUS) assembles via liquid-liquid phase separation (LLPS) into functional RNA granules and aggregates in amyotrophic lateral sclerosis associated neuronal inclusions. Several studies have demonstrated that posttranslational modification (PTM) can significantly alter FUS phase separation and aggregation, particularly charge-altering phosphorylation of the nearly uncharged N-terminal low complexity domain of FUS (FUS LC). However, the occurrence and impact of N-terminal acetylation on FUS phase separation remains unexplored, even though N-terminal acetylation is the most common PTM in mammals and changes the charge at the N-terminus.

SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs.

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human stress granules. Here, we test the possibility that the multivalent RNA-binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) condenses with RNA via liquid-liquid phase separation (LLPS) and that N protein can be recruited in phase-separated forms of human RNA-binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N-terminal IDR and central-linker IDR, as well as the folded C-terminal oligomerization domain, while the folded N-terminal domain and the C-terminal IDR are not required.

SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins.

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) compacts RNA via protein-RNA liquid-liquid phase separation (LLPS) and that N interactions with host RNA-binding proteins are mediated by phase separation. To this end, we created a construct expressing recombinant N fused to a N-terminal maltose binding protein tag which helps keep the oligomeric N soluble for purification.

The (un)structural biology of biomolecular liquid-liquid phase separation using NMR spectroscopy.

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids is a phenomenon that underlies membraneless compartmentalization of the cell. The underlying molecular interactions that underpin biomolecular LLPS have been of increased interest due to the importance of membraneless organelles in facilitating various biological processes and the disease association of several of the proteins that mediate LLPS. Proteins that are able to undergo LLPS often contain intrinsically disordered regions and remain dynamic in solution.

EGFP insertional mutagenesis reveals multiple FXR2P fibrillar states with differing ribosome association in neurons.

RNA-binding proteins (RBPs) function in higher-order assemblages such as RNA granules to regulate RNA localization and translation. The Fragile X homolog FXR2P is an RBP essential for formation of neuronal Fragile X granules that associate with axonal mRNA and ribosomes in the intact brain. However, the FXR2P domains important for assemblage formation in a cellular system are unknown.

Molecular interactions underlying liquid-liquid phase separation of the FUS low-complexity domain.

The low-complexity domain of the RNA-binding protein FUS (FUS LC) mediates liquid-liquid phase separation (LLPS), but the interactions between the repetitive SYGQ-rich sequence of FUS LC that stabilize the liquid phase are not known in detail. By combining NMR and Raman spectroscopy, mutagenesis, and molecular simulation, we demonstrate that heterogeneous interactions involving all residue types underlie LLPS of human FUS LC. We find no evidence that FUS LC adopts conformations with traditional secondary structure elements in the condensed phase; rather, it maintains conformational heterogeneity.