School of Medicine

Background

Methods

Findings

Interpretation

Funding

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

Defects in tRNA biogenesis are associated with multiple neurological disorders, yet our understanding of these diseases has been hampered by an inability to determine tRNA expression in individual cell types within a complex tissue. Here, we developed a mouse model in which RNA polymerase III is conditionally epitope tagged in a Cre-dependent manner, allowing us to accurately profile tRNA expression in any cell type in vivo. We investigated tRNA expression in diverse nervous system cell types, revealing dramatic heterogeneity in the expression of tRNA genes between populations. We found that while maintenance of levels of tRNA isoacceptor families is critical for cellular homeostasis, neurons are differentially vulnerable to insults to distinct tRNA isoacceptor families. Cell-type-specific translatome analysis suggests that the balance between tRNA availability and codon demand may underlie such differential resilience. Our work provides a platform for investigating the complexities of mRNA translation and tRNA biology in the brain.

When proteins evolve new activity, a concomitant decrease in stability is often observed because the mutations that confer new activity can destabilize the native fold. In the conventional model of protein evolution, reduced stability is considered a purely deleterious cost of molecular innovation because unstable proteins are prone to aggregation and are sensitive to environmental stressors. However, recent work has revealed that nonnative, often unstable protein conformations play an important role in mediating evolutionary transitions, raising the question of whether instability can itself potentiate the evolution of new activity. We explored this question in a bacteriophage receptor-binding protein during host-range evolution. We studied the properties of the receptor-binding protein of bacteriophage λ before and after host-range evolution and demonstrated that the evolved protein is relatively unstable and may exist in multiple conformations with unique receptor preferences. Through a combination of structural modeling and in vitro oligomeric state analysis, we found that the instability arises from mutations that interfere with trimer formation. This study raises the intriguing possibility that protein instability might play a previously unrecognized role in mediating host-range expansions in viruses.

Several genetic risk factors for Alzheimers disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimers disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimers disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimers disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aβ induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimers disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimers disease.

The RNA-binding protein PARP13 is a primary factor in the innate antiviral response, which suppresses translation and drives decay of bound viral and host RNA. PARP13 interacts with many proteins encoded by interferon-stimulated genes (ISG) to activate antiviral pathways including co-translational addition of ISG15, or ISGylation. We performed enhanced crosslinking immunoprecipitation (eCLIP) and RNA-seq in human cells to investigate PARP13's role in transcriptome regulation for both basal and antiviral states. We find that the antiviral response shifts PARP13 target localization, but not its binding preferences, and that PARP13 supports the expression of ISGylation-related genes, including PARP13's cofactor, TRIM25. PARP13 associates with TRIM25 via RNA-protein interactions, and we elucidate a transcriptome-wide periodicity of PARP13 binding around TRIM25. Taken together, our study implicates PARP13 in creating and maintaining a cellular environment poised for an antiviral response through limiting PARP13 translation, regulating access to distinct mRNA pools, and elevating ISGylation machinery expression.

The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase and is essential for fertility. In S. cerevisiae, the HORMAD protein Hop1 mediates the enrichment of axis proteins at nucleosome-rich islands through a central chromatin-binding region (CBR). Here, we use cryoelectron microscopy to show that the Hop1 CBR directly recognizes bent nucleosomal DNA through a composite interface in its PHD and winged helix-turn-helix domains. Targeted disruption of the Hop1 CBR-nucleosome interface causes a localized reduction of axis protein binding and meiotic DNA double-strand breaks (DSBs) in axis islands and leads to defects in chromosome synapsis. Synthetic effects with mutants of the Hop1 regulator Pch2 suggest that nucleosome binding delays a conformational switch in Hop1 from a DSB-promoting, Pch2-inaccessible state to a DSB-inactive, Pch2-accessible state to regulate the extent of meiotic DSB formation. Phylogenetic analyses of meiotic HORMADs reveal an ancient origin of the CBR, suggesting that the mechanisms we uncover are broadly conserved.

Individuals with Williams syndrome (WS), a neurodevelopmental disorder caused by hemizygous loss of 26-28 genes at 7q11.23, characteristically portray a hypersocial phenotype. Copy-number variations and mutations in one of these genes, GTF2I, are associated with altered sociality and are proposed to underlie hypersociality in WS. However, the contribution of GTF2I to human neurodevelopment remains poorly understood. Here, human cellular models of neurodevelopment, including neural progenitors, neurons, and three-dimensional cortical organoids, are differentiated from CRISPR-Cas9-edited GTF2I-knockout (GTF2I-KO) pluripotent stem cells to investigate the role of GTF2I in human neurodevelopment. GTF2I-KO progenitors exhibit increased proliferation and cell-cycle alterations. Cortical organoids and neurons demonstrate increased cell death and synaptic dysregulation, including synaptic structural dysfunction and decreased electrophysiological activity on a multielectrode array. Our findings suggest that changes in synaptic circuit integrity may be a prominent mediator of the link between alterations in GTF2I and variation in the phenotypic expression of human sociality.

Self-sufficiency (autonomy) in growth signaling, the earliest recognized hallmark of cancer, is fueled by the tumor cell's ability to "secrete-and-sense" growth factors (GFs); this translates into cell survival and proliferation that is self-sustained by autocrine/paracrine secretion. A Golgi-localized circuitry comprised of two GTPase switches has recently been implicated in the orchestration of growth signaling autonomy. Using breast cancer cells that are either endowed or impaired (by gene editing) in their ability to assemble the circuitry for growth signaling autonomy, here we define the transcriptome, proteome, and phenome of such an autonomous state, and unravel its role during cancer progression. We show that autonomy is associated with enhanced molecular programs for stemness, proliferation, and epithelial-mesenchymal plasticity. Autonomy is both necessary and sufficient for anchorage-independent GF-restricted proliferation and resistance to anticancer drugs and is required for metastatic progression. Transcriptomic and proteomic studies show that autonomy is associated, with a surprising degree of specificity, with self-sustained epidermal growth factor receptor (EGFR)/ErbB signaling. Derivation of a gene expression signature for autonomy revealed that growth signaling autonomy is uniquely induced in circulating tumor cells (CTCs), the harshest phase in the life of tumor cells when it is deprived of biologically available epidermal growth factor (EGF). We also show that autonomy in CTCs tracks therapeutic response and prognosticates outcome. These data support a role for growth signaling autonomy in multiple processes essential for the blood-borne dissemination of human breast cancer.