The splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin.

Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes in vivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.

Crym-positive striatal astrocytes gate perseverative behaviour.

Astrocytes are heterogeneous glial cells of the central nervous system1-3. However, the physiological relevance of astrocyte diversity for neural circuits and behaviour remains unclear. Here we show that a specific population of astrocytes in the central striatum expresses µ-crystallin (encoded by Crym in mice and CRYM in humans) that is associated with several human diseases, including neuropsychiatric disorders4-7. In adult mice, reducing the levels of µ-crystallin in striatal astrocytes through CRISPR-Cas9-mediated knockout of Crym resulted in perseverative behaviours, increased fast synaptic excitation in medium spiny neurons and dysfunctional excitatory-inhibitory synaptic balance. Increased perseveration stemmed from the loss of astrocyte-gated control of neurotransmitter release from presynaptic terminals of orbitofrontal cortex-striatum projections. We found that perseveration could be remedied using presynaptic inhibitory chemogenetics8, and that this treatment also corrected the synaptic deficits. Together, our findings reveal converging molecular, synaptic, circuit and behavioural mechanisms by which a molecularly defined and allocated population of striatal astrocytes gates perseveration phenotypes that accompany neuropsychiatric disorders9-12. Our data show that Crym-positive striatal astrocytes have key biological functions within the central nervous system, and uncover astrocyte-neuron interaction mechanisms that could be targeted in treatments for perseveration.

In vivo identification of astrocyte and neuron subproteomes by proximity-dependent biotinylation.

The central nervous system (CNS) comprises diverse and morphologically complex cells. To understand the molecular basis of their physiology, it is crucial to assess proteins expressed within intact cells. Commonly used methods utilize cell dissociation and sorting to isolate specific cell types such as neurons and astrocytes, the major CNS cells. Proteins purified from isolated cells are identified by mass spectrometry-based proteomics. However, dissociation and cell-sorting methods lead to near total loss of cellular morphology, thereby losing proteins from key relevant subcompartments such as processes, end feet, dendrites and axons. Here we provide a systematic protocol for cell- and subcompartment-specific labeling and identification of proteins found within intact astrocytes and neurons in vivo. This protocol utilizes the proximity-dependent biotinylation system BioID2, selectively expressed in either astrocytes or neurons, to label proximal proteins in a cell-specific manner. BioID2 is targeted genetically to assess the subproteomes of subcellular compartments such as the plasma membrane and sites of cell-cell contacts. We describe in detail the expression methods (variable timing), stereotaxic surgeries for expression (1-2 d and then 3 weeks), in vivo protein labeling (7 d), protein isolation (2-3 d), protein identification methods (2-3 d) and data analysis (1 week). The protocol can be applied to any area of the CNS in mouse models of physiological processes and for disease-related research.

ACD15, ACD21, and SLN regulate the accumulation and mobility of MBD6 to silence genes and transposable elements.

DNA methylation mediates silencing of transposable elements and genes in part via recruitment of the Arabidopsis MBD5/6 complex, which contains the methyl-CpG binding domain (MBD) proteins MBD5 and MBD6, and the J-domain containing protein SILENZIO (SLN). Here, we characterize two additional complex members: α-crystalline domain (ACD) containing proteins ACD15 and ACD21. We show that they are necessary for gene silencing, bridge SLN to the complex, and promote higher-order multimerization of MBD5/6 complexes within heterochromatin. These complexes are also highly dynamic, with the mobility of MBD5/6 complexes regulated by the activity of SLN. Using a dCas9 system, we demonstrate that tethering the ACDs to an ectopic site outside of heterochromatin can drive a massive accumulation of MBD5/6 complexes into large nuclear bodies. These results demonstrate that ACD15 and ACD21 are critical components of the gene-silencing MBD5/6 complex and act to drive the formation of higher-order, dynamic assemblies at CG methylation (meCG) sites.

The apoptotic splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin.

Understanding the mechanisms of pre-mRNA splicing and spliceosome assembly is limited by technical challenges to examining spliceosomes in vivo. Here we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of lysed nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA, bound with intronic branch sites prior to the first catalytic step of splicing. Sequencing these pre-mRNA fragments allowed the transcriptome-wide mapping of branch sites with high sensitivity. In addition to known U2 snRNP proteins, these complexes contained the proteins RBM5 and RBM10. RBM5 and RBM10 are alternative splicing regulators that control exons affecting apoptosis and cell proliferation in cancer, but were not previously shown to associate with the U2 snRNP or to play roles in branch site selection. We delineate a common segment of RBM5 and RBM10, separate from their known functional domains, that is required for their interaction with the U2 snRNP. We identify a large set of splicing events regulated by RBM5 and RBM10 and find that they predominantly act as splicing silencers. Disruption of their U2 interaction renders the proteins inactive for repression of many alternative exons. We further find that these proteins assemble on branch sites of nearly all exons across the transcriptome, including those whose splicing is not altered by them. We propose a model where RBM5 and RBM10 act as components of the U2 snRNP complex. From within this complex, they sense structural features of branchpoint recognition to either allow progression to functional spliceosome or rejection of the complex to inhibit splicing.

ACD15, ACD21 and SLN regulate accumulation and mobility of MBD6 to silence genes and transposable elements.

DNA methylation mediates silencing of transposable elements and genes in part via recruitment of the Arabidopsis MBD5/6 complex, which contains the methyl-CpG-binding domain (MBD) proteins MBD5 and MBD6, and the J-domain containing protein SILENZIO (SLN). Here we characterize two additional complex members: α-crystalline domain containing proteins ACD15 and ACD21. We show that they are necessary for gene silencing, bridge SLN to the complex, and promote higher order multimerization of MBD5/6 complexes within heterochromatin. These complexes are also highly dynamic, with the mobility of complex components regulated by the activity of SLN. Using a dCas9 system, we demonstrate that tethering the ACDs to an ectopic site outside of heterochromatin can drive massive accumulation of MBD5/6 complexes into large nuclear bodies. These results demonstrate that ACD15 and ACD21 are critical components of gene silencing complexes that act to drive the formation of higher order, dynamic assemblies.

Chikungunya virus glycoproteins transform macrophages into productive viral dissemination vessels.

Despite their role as innate sentinels, macrophages are cellular reservoirs for chikungunya virus (CHIKV), a highly pathogenic arthropod-borne alphavirus that has caused unprecedented epidemics worldwide. Here, we took interdisciplinary approaches to elucidate the CHIKV determinants that subvert macrophages into virion dissemination vessels. Through comparative infection using chimeric alphaviruses and evolutionary selection analyses, we discovered for the first time that CHIKV glycoproteins E2 and E1 coordinate efficient virion production in macrophages with the domains involved under positive selection. We performed proteomics on CHIKV-infected macrophages to identify cellular proteins interacting with the precursor and/or mature forms of viral glycoproteins. We uncovered two E1-binding proteins, signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 (eIF3k), with novel inhibitory activities against CHIKV production. These results highlight how CHIKV E2 and E1 have been evolutionarily selected for viral dissemination likely through counteracting host restriction factors, making them attractive targets for therapeutic intervention.

The MOM1 complex recruits the RdDM machinery via MORC6 to establish de novo DNA methylation.

MORPHEUS' MOLECULE1 (MOM1) is an Arabidopsis factor previously shown to mediate transcriptional silencing independent of major DNA methylation changes. Here we find that MOM1 localizes with sites of RNA-directed DNA methylation (RdDM). Tethering MOM1 with an artificial zinc finger to an unmethylated FWA promoter leads to establishment of DNA methylation and FWA silencing. This process is blocked by mutations in components of the Pol V arm of the RdDM machinery, as well as by mutation of MICRORCHIDIA 6 (MORC6). We find that at some endogenous RdDM sites, MOM1 is required to maintain DNA methylation and a closed chromatin state. In addition, efficient silencing of newly introduced FWA transgenes is impaired in the mom1 mutant. In addition to RdDM sites, we identify a group of MOM1 peaks at active chromatin near genes that colocalized with MORC6. These findings demonstrate a multifaceted role of MOM1 in genome regulation.

Direct tests of cytochrome c and c1 functions in the electron transport chain of malaria parasites.

The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome (cyt) functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs (c and c-2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c-2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c1 for inducible knockdown. Translational repression of cyt c and cyt c1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c-2 knockdown or knockout had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c-2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c-2 has an unusually open active site in which heme is stably coordinated by only a single axial amino acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution.

MORC proteins regulate transcription factor binding by mediating chromatin compaction in active chromatin regions.

BACKGROUND: The microrchidia (MORC) proteins are a family of evolutionarily conserved GHKL-type ATPases involved in chromatin compaction and gene silencing. Arabidopsis MORC proteins act in the RNA-directed DNA methylation (RdDM) pathway, where they act as molecular tethers to ensure the efficient establishment of RdDM and de novo gene silencing. However, MORC proteins also have RdDM-independent functions although their underlying mechanisms are unknown. RESULTS: In this study, we examine MORC binding regions where RdDM does not occur in order to shed light on the RdDM-independent functions of MORC proteins. We find that MORC proteins compact chromatin and reduce DNA accessibility to transcription factors, thereby repressing gene expression. We also find that MORC-mediated repression of gene expression is particularly important under conditions of stress. MORC-regulated transcription factors can in some cases regulate their own transcription, resulting in feedback loops. CONCLUSIONS: Our findings provide insights into the molecular mechanisms of MORC-mediated chromatin compaction and transcription regulation.

Astrocyte-neuron subproteomes and obsessive-compulsive disorder mechanisms.

Astrocytes and neurons extensively interact in the brain. Identifying astrocyte and neuron proteomes is essential for elucidating the protein networks that dictate their respective contributions to physiology and disease. Here we used cell-specific and subcompartment-specific proximity-dependent biotinylation1 to study the proteomes of striatal astrocytes and neurons in vivo. We evaluated cytosolic and plasma membrane compartments for astrocytes and neurons to discover how these cells differ at the protein level in their signalling machinery. We also assessed subcellular compartments of astrocytes, including end feet and fine processes, to reveal their subproteomes and the molecular basis of essential astrocyte signalling and homeostatic functions. Notably, SAPAP3 (encoded by Dlgap3), which is associated with obsessive-compulsive disorder (OCD) and repetitive behaviours2-8, was detected at high levels in striatal astrocytes and was enriched within specific astrocyte subcompartments where it regulated actin cytoskeleton organization. Furthermore, genetic rescue experiments combined with behavioural analyses and molecular assessments in a mouse model of OCD4 lacking SAPAP3 revealed distinct contributions of astrocytic and neuronal SAPAP3 to repetitive and anxiety-related OCD-like phenotypes. Our data define how astrocytes and neurons differ at the protein level and in their major signalling pathways. Moreover, they reveal how astrocyte subproteomes vary between physiological subcompartments and how both astrocyte and neuronal SAPAP3 mechanisms contribute to OCD phenotypes in mice. Our data indicate that therapeutic strategies that target both astrocytes and neurons may be useful to explore in OCD and potentially other brain disorders.

A gene silencing screen uncovers diverse tools for targeted gene repression in Arabidopsis.

DNA methylation has been utilized for target gene silencing in plants. However, it is not well understood whether other silencing pathways can be also used to manipulate gene expression. Here we performed a gain-of-function screen for proteins that could silence a target gene when fused to an artificial zinc finger. We uncovered many proteins that suppressed gene expression through DNA methylation, histone H3K27me3 deposition, H3K4me3 demethylation, histone deacetylation, inhibition of RNA polymerase II transcription elongation or Ser-5 dephosphorylation. These proteins also silenced many other genes with different efficacies, and a machine learning model could accurately predict the efficacy of each silencer on the basis of various chromatin features of the target loci. Furthermore, some proteins were also able to target gene silencing when used in a dCas9-SunTag system. These results provide a more comprehensive understanding of epigenetic regulatory pathways in plants and provide an armament of tools for targeted gene manipulation.

Transcriptional drifts associated with environmental changes in endothelial cells.

Environmental cues, such as physical forces and heterotypic cell interactions play a critical role in cell function, yet their collective contributions to transcriptional changes are unclear. Focusing on human endothelial cells, we performed broad individual sample analysis to identify transcriptional drifts associated with environmental changes that were independent of genetic background. Global gene expression profiling by RNA sequencing and protein expression by liquid chromatography-mass spectrometry directed proteomics distinguished endothelial cells in vivo from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes. Inclusion of heterotypic interactions by co-culture of endothelial cells with smooth muscle cells normalized approximately 9% of the original in vivo signature. We also identified novel flow dependent genes, as well as genes that necessitate heterotypic cell interactions to mimic the in vivo transcriptome. Our findings highlight specific genes and pathways that rely on contextual information for adequate expression from those that are agnostic of such environmental cues.

Arabidopsis TRB proteins function in H3K4me3 demethylation by recruiting JMJ14.

Arabidopsis telomeric repeat binding factors (TRBs) can bind telomeric DNA sequences to protect telomeres from degradation. TRBs can also recruit Polycomb Repressive Complex 2 (PRC2) to deposit tri-methylation of H3 lysine 27 (H3K27me3) over certain target loci. Here, we demonstrate that TRBs also associate and colocalize with JUMONJI14 (JMJ14) and trigger H3K4me3 demethylation at some loci. The trb1/2/3 triple mutant and the jmj14-1 mutant show an increased level of H3K4me3 over TRB and JMJ14 binding sites, resulting in up-regulation of their target genes. Furthermore, tethering TRBs to the promoter region of genes with an artificial zinc finger (TRB-ZF) successfully triggers target gene silencing, as well as H3K27me3 deposition, and H3K4me3 removal. Interestingly, JMJ14 is predominantly recruited to ZF off-target sites with low levels of H3K4me3, which is accompanied with TRB-ZFs triggered H3K4me3 removal at these loci. These results suggest that TRB proteins coordinate PRC2 and JMJ14 activities to repress target genes via H3K27me3 deposition and H3K4me3 removal.

Proteomic Analysis of Ferrochelatase Interactome in Erythroid and Non-Erythroid Cells.

Heme is an essential cofactor for multiple cellular processes in most organisms. In developing erythroid cells, the demand for heme synthesis is high, but is significantly lower in non-erythroid cells. While the biosynthesis of heme in metazoans is well understood, the tissue-specific regulation of the pathway is less explored. To better understand this, we analyzed the mitochondrial heme metabolon in erythroid and non-erythroid cell lines from the perspective of ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Affinity purification of FLAG-tagged-FECH, together with mass spectrometric analysis, was carried out to identify putative protein partners in human and murine cell lines. Proteins involved in the heme biosynthetic process and mitochondrial organization were identified as the core components of the FECH interactome. Interestingly, in non-erythroid cell lines, the FECH interactome is highly enriched with proteins associated with the tricarboxylic acid (TCA) cycle. Overall, our study shows that the mitochondrial heme metabolon in erythroid and non-erythroid cells has similarities and differences, and suggests new roles for the mitochondrial heme metabolon and heme in regulating metabolic flux and key cellular processes.

Direct Tests of Cytochrome Function in the Electron Transport Chain of Malaria Parasites.

The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs ( c and c -2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c -2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c 1 for inducible knockdown. Translational repression of cyt c and cyt c 1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c -2 knockdown or knock-out had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c -2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c -2 has an unusually open active site in which heme is stably coordinated by only a single axial amino-acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution. SIGNIFICANCE STATEMENT: Mitochondria are critical organelles in eukaryotic cells that drive oxidative metabolism. The mitochondrion of Plasmodium malaria parasites is a major drug target that has many differences from human cells and remains poorly studied. One key difference from humans is that malaria parasites express two cytochrome c proteins that differ significantly from each other and play untested and uncertain roles in the mitochondrial electron transport chain (ETC). Our study revealed that one cyt c is essential for ETC function and parasite viability while the second, more divergent protein has unusual structural and biochemical properties and is not required for growth of blood-stage parasites. This work elucidates key biochemical properties and evolutionary differences in the mitochondrial ETC of malaria parasites.

Neuronal and astrocytic contributions to Huntington's disease dissected with zinc finger protein transcriptional repressors.

Huntington's disease (HD) is caused by expanded CAG repeats in the huntingtin gene (HTT) resulting in expression of mutant HTT proteins (mHTT) with extended polyglutamine tracts, including in striatal neurons and astrocytes. It is unknown whether pathophysiology in vivo can be attenuated by lowering mHTT in either cell type throughout the brain, and the relative contributions of neurons and astrocytes to HD remain undefined. We use zinc finger protein (ZFP) transcriptional repressors to cell-selectively lower mHTT in vivo. Astrocytes display loss of essential functions such as cholesterol metabolism that are partly driven by greater neuronal dysfunctions, which encompass neuromodulation, synaptic, and intracellular signaling pathways. Using transcriptomics, proteomics, electrophysiology, and behavior, we dissect neuronal and astrocytic contributions to HD pathophysiology. Remarkably, brain-wide delivery of neuronal ZFPs results in strong mHTT lowering, rescue of HD-associated behavioral and molecular phenotypes, and significant extension of lifespan, findings that support translational development.

The MOM1 complex recruits the RdDM machinery via MORC6 to establish de novo DNA methylation.

MOM1 is an Arabidopsis factor previously shown to mediate transcriptional silencing independent of major DNA methylation changes. Here we found that MOM1 localizes with sites of RNA-directed DNA methylation (RdDM). Tethering MOM1 with artificial zinc finger to unmethylated FWA promoter led to establishment of DNA methylation and FWA silencing. This process was blocked by mutations in components of the Pol V arm of the RdDM machinery, as well as by mutation of MORC6 . We found that at some endogenous RdDM sites, MOM1 is required to maintain DNA methylation and a closed chromatin state. In addition, efficient silencing of newly introduced FWA transgenes was impaired by mutation of MOM1 or mutation of genes encoding the MOM1 interacting PIAL1/2 proteins. In addition to RdDM sites, we identified a group of MOM1 peaks at active chromatin near genes that colocalized with MORC6. These findings demonstrate a multifaceted role of MOM1 in genome regulation.

CLSTN3ß enforces adipocyte multilocularity to facilitate lipid utilization.

Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3ß) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3ß is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3ß have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3ß is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3ß associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3ß-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.

The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers.

The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1's Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1's helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions via its BRCT and ARD motifs, binds SLF2 analogously to SIMC1 and forms a separate Nse5/6-like complex. Thus, two Nse5/6-like complexes with distinct recruitment domains control human SMC5/6 localization.