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The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80–90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein’s central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly. The SARS-CoV-2 nucleocapsid (N) protein binds the viral RNA genome and contains two ordered domains flanked by three intrinsically-disordered regions. Here, the authors show that RNA binding induces liquid-liquid phase separation of N, which is driven by its central intrinsically-disordered region and is modulated by phosphorylation. The SARS-CoV-2 Membrane (M) protein also phase-separates with N, and three-component mixtures of N + M + RNA form mutually exclusive compartments containing N + M or N + RNA.

Enhancers act to regulate cell-type-specific gene expression by facilitating the transcription of target genes. In mammalian cells, active or primed enhancers are commonly marked by monomethylation of histone H3 at lysine 4 (H3K4me1) in a cell-type-specific manner. Whether and how this histone modification regulates enhancer-dependent transcription programs in mammals is unclear. In this study, we conducted SILAC mass spectrometry experiments with mononucleosomes and identified multiple H3K4me1-associated proteins, including many involved in chromatin remodeling. We demonstrate that H3K4me1 augments association of the chromatin-remodeling complex BAF to enhancers in vivo and that, in vitro, H3K4me1-marked nucleosomes are more efficiently remodeled by the BAF complex. Crystal structures of the BAF component BAF45C indicate that monomethylation, but not trimethylation, is accommodated by BAF45C’s H3K4-binding site. Our results suggest that H3K4me1 has an active role at enhancers by facilitating binding of the BAF complex and possibly other chromatin regulators. The authors conduct mass spectrometry experiments identifying H3K4me1-associated proteins, including members of the BAF chromatin-remodeling complex. They show that H3K4me1 augments association of the BAF complex with enhancers in vivo and that H3K4me1-marked nucleosomes are more efficiently remodeled by the BAF complex in vitro.

The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, is the most severe public health event of the twenty-first century. While effective vaccines against SARS-CoV-2 have been developed, there remains an urgent need for diagnostics to quickly and accurately detect infections. Antigen tests, particularly those that detect the abundant SARS-CoV-2 Nucleocapsid protein, are a proven method for detecting active SARS-CoV-2 infections. Here we report high-resolution crystal structures of three llama-derived single-domain antibodies that bind the SARS-CoV-2 Nucleocapsid protein with high affinity. Each antibody recognizes a specific folded domain of the protein, with two antibodies recognizing the N-terminal RNA binding domain and one recognizing the C-terminal dimerization domain. The two antibodies that recognize the RNA binding domain affect both RNA binding affinity and RNA-mediated phase separation of the Nucleocapsid protein. All three antibodies recognize highly conserved surfaces on the Nucleocapsid protein, suggesting that they could be used to develop affordable diagnostic tests to detect all circulating SARS-CoV-2 variants.

In meiotic prophase, chromosomes are organized into compacted loop arrays to promote homolog pairing and recombination. Here, we probe the architecture of the mouse spermatocyte genome in early and late meiotic prophase using chromosome conformation capture (Hi-C). Our data support the established loop array model of meiotic chromosomes, and infer loops averaging 0.8–1.0 megabase pairs (Mb) in early prophase and extending to 1.5–2.0 Mb in late prophase as chromosomes compact and homologs undergo synapsis. Topologically associating domains (TADs) are lost in meiotic prophase, suggesting that assembly of the meiotic chromosome axis alters the activity of chromosome-associated cohesin complexes. While TADs are lost, physically separated A and B compartments are maintained in meiotic prophase. Moreover, meiotic DNA breaks and interhomolog crossovers preferentially form in the gene-dense A compartment, revealing a role for chromatin organization in meiotic recombination. Finally, direct detection of interhomolog contacts genome-wide reveals the structural basis for homolog alignment and juxtaposition by the synaptonemal complex.Comparative Hi-C analysis of synchronized mouse spermatocyte populations reveals dynamic changes in chromosome organization during meiotic prophase that permit homolog pairing while sustaining gene expression.

Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction–modification and CRISPR–Cas systems 1 . In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors 2 – 4 . However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation. The nucleus-like compartment formed in bacteria during infection by jumbo phage 201phi2-1 is composed of the bacteriophage protein chimallin, which can self-assemble into closed compartments in vitro.

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 S ensitive C apture of R NA-protein I nteractions in B iological E nvironments), 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. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts. Retrieving interactions between RNA-binding proteins (RBPs) and RNA in endogenous biological contexts remains challenging. Here, the authors develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), which enables highly specific RNA-binding site identification across diverse biological samples.

During meiotic prophase, the essential events of homolog pairing, synapsis, and recombination are coordinated with meiotic progression to promote fidelity and prevent aneuploidy. The conserved AAA+ ATPase PCH-2 coordinates these events to guarantee crossover assurance and accurate chromosome segregation. How PCH-2 accomplishes this coordination is poorly understood. Here, we provide evidence that PCH-2 decelerates pairing, synapsis and recombination in C . elegans by remodeling meiotic HORMADs. We propose that PCH-2 converts the closed versions of these proteins, which drive these meiotic prophase events, to unbuckled conformations, destabilizing interhomolog interactions and delaying meiotic progression. Further, we find that PCH-2 distributes this regulation among three essential meiotic HORMADs in C . elegans : PCH-2 acts through HTP-3 to regulate pairing and synapsis, HIM-3 to promote crossover assurance, and HTP-1 to control meiotic progression. In addition to identifying a molecular mechanism for how PCH-2 regulates interhomolog interactions, our results provide a possible explanation for the expansion of the meiotic HORMAD family as a conserved evolutionary feature of meiosis. Taken together, our work demonstrates that PCH-2’s remodeling of meiotic HORMADs has functional consequences for the rate and fidelity of homolog pairing, synapsis, recombination and meiotic progression, ensuring accurate meiotic chromosome segregation.

The mitotic checkpoint ensures accurate chromosome segregation through assembly of the mitotic checkpoint complex (MCC), a soluble inhibitor of the anaphase-promoting complex/cyclosome (APC/C) produced by unattached kinetochores. MCC is also assembled during interphase by Mad1/Mad2 bound at nuclear pores, thereby preventing premature mitotic exit prior to kinetochore maturation and checkpoint activation. Using degron tagging to rapidly deplete the AAA+ ATPase TRIP13, we show that its catalytic activity is required to maintain a pool of open-state Mad2 for MCC assembly, thereby supporting mitotic checkpoint activation, but is also required for timely mitotic exit through catalytic disassembly of MCC. Strikingly, combining TRIP13 depletion with elimination of APC15-dependent Cdc20 ubiquitination/degradation results in a complete inability to exit mitosis, even when MCC assembly at unattached kinetochores is prevented. Thus, mitotic exit requires MCC produced either in interphase or mitosis to be disassembled by TRIP13-catalyzed removal of Mad2 or APC15-driven ubiquitination/degradation of its Cdc20 subunit. The mitotic checkpoint complex (MCC) is assembled during both mitosis and interphase. Here, the authors use auxin-inducible degron tags to rapidly degrade TRIP13 and find that mitotic exit requires MCC disassembly by TRIP13-catalyzed removal of Mad2 or APC1-driven ubiquitination of Cdc20.

The AAA+ family ATPase TRIP13 is a key regulator of meiotic recombination and the spindle assembly checkpoint, acting on signaling proteins of the conserved HORMA domain family. Here we present the structure of the Caenorhabditis elegans TRIP13 ortholog PCH-2, revealing a new family of AAA+ ATPase protein remodelers. PCH-2 possesses a substrate-recognition domain related to those of the protein remodelers NSF and p97, while its overall hexameric architecture and likely structural mechanism bear close similarities to the bacterial protein unfoldase ClpX. We find that TRIP13, aided by the adapter protein p31(comet), converts the HORMA-family spindle checkpoint protein MAD2 from a signaling-active ‘closed’ conformer to an inactive ‘open’ conformer. We propose that TRIP13 and p31(comet) collaborate to inactivate the spindle assembly checkpoint through MAD2 conformational conversion and disassembly of mitotic checkpoint complexes. A parallel HORMA protein disassembly activity likely underlies TRIP13's critical regulatory functions in meiotic chromosome structure and recombination. The genetic material inside human and other animal cells is made of DNA and is packaged in structures called chromosomes. Before a cell divides, the entire set of chromosomes is copied so that each chromosome is now made of two identical sister ‘chromatids’. Next, the chromosomes line up on a structure called the spindle, which is made of filaments called microtubules. Cells have a surveillance system known as the spindle assembly checkpoint that halts cell division until every chromosome is correctly aligned on the spindle. Once the chromosomes are in place, the checkpoint is turned off and the spindle pulls the chromatids apart so that each daughter cell receives a complete set of chromosomes. A protein called MAD2 plays an important role in the spindle assembly checkpoint. It can adopt two distinct shapes: in the ‘closed’ shape it is active and halts cell division, but in the ‘open’ shape it is inactive and allows cell division to proceed. Another protein called TRIP13 can help turn off the checkpoint, but it is not clear how this works or whether TRIP13 acts on MAD2 directly. Here, Ye et al. studied these proteins using a technique called X-ray crystallography and several biochemical techniques. The experiments show that TRIP13 belongs to a family of proteins known as ‘AAA-ATPases’, which can unfold proteins to alter their activity. Ye et al. found that TRIP13 binds to an adaptor protein that allows it to bind to the closed form of MAD2. TRIP13 then unfolds a part of the MAD2 protein, converting MAD2 into the open shape. Ye et al. propose that, once all chromosomes are lined up on the spindle, TRIP13 turns off the spindle assembly checkpoint by converting closed MAD2 to open MAD2. Also, when cells are not undergoing cell division, TRIP13 may maintain MAD2 in the open shape to prevent cells from turning on the spindle assembly checkpoint at the wrong time. Further work will be needed to show how TRIP13 recognizes the closed form of MAD2, and whether it can act in a similar way on other proteins in the cell.

The meiotic chromosome axis plays key roles in meiotic chromosome organization and recombination, yet the underlying protein components of this structure are highly diverged. Here, we show that ‘axis core proteins’ from budding yeast (Red1), mammals (SYCP2/SYCP3), and plants (ASY3/ASY4) are evolutionarily related and play equivalent roles in chromosome axis assembly. We first identify ‘closure motifs’ in each complex that recruit meiotic HORMADs, the master regulators of meiotic recombination. We next find that axis core proteins form homotetrameric (Red1) or heterotetrameric (SYCP2:SYCP3 and ASY3:ASY4) coiled-coil assemblies that further oligomerize into micron-length filaments. Thus, the meiotic chromosome axis core in fungi, mammals, and plants shares a common molecular architecture, and likely also plays conserved roles in meiotic chromosome axis assembly and recombination control.