Human nuclei contain three RNA polymerases (I, II and III) that transcribe different groups of genes; the active forms of all three are difficult to isolate because they are bound to the ...substructure. Here we describe a purification approach for isolating active RNA polymerase complexes from mammalian cells. After isolation, we analyzed their protein content by mass spectrometry. Each complex represents part of the core of a transcription factory. For example, the RNA polymerase II complex contains subunits unique to RNA polymerase II plus various transcription factors but shares a number of ribonucleoproteins with the other polymerase complexes; it is also rich in polymerase II transcripts. We also describe a native chromosome conformation capture method to confirm that the complexes remain attached to the same pairs of DNA templates found in vivo.
Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA‐binding protein (RBP) that controls translation of select mRNAs. We discovered ...that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP‐bound mRNAs. We show ER stress‐induced activation of Inositol requiring enzyme‐1 (IRE1), an ER‐resident stress‐sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress‐induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.
Synopsis
Targeting IRE1 function and substrate(s) provides a novel therapeutic approach to atherosclerosis. We found a key role for IRE1‐mediated FMRP phosphorylation that suppresses the expression of cholesterol transporters and efferocytosis receptors in macrophages and promotes atherosclerosis progression.
FMRP is a novel IRE1 kinase substrate.
Lipid‐induced, IRE1‐mediated FMRP phosphorylation leads to post‐transcriptional suppression of cholesterol transporters and efferocytosis regulators.
Ablation of IRE1 kinase activity or suppression of FMRP expression enhances efferocytosis and cholesterol transport in vitro and in vivo.
IRE1 kinase inhibition by a small molecule inhibitor or genetic deletion of FMRP in macrophages alleviates atherosclerotic plaque formation.
Targeting IRE1 function and substrate(s) provides a novel therapeutic approach to atherosclerosis. We found a key role for IRE1‐mediated FMRP phosphorylation that suppresses the expression of cholesterol transporters and efferocytosis receptors in macrophages and promotes atherosclerosis progression.
The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) - the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) - ...a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.
In bacteria, protein synthesis can be coupled to transcription, but in eukaryotes it is believed to occur solely in the cytoplasm. Using pulses as short as 5 s, we find that three ...analogues--L-azidohomoalanine, puromycin (detected after attaching fluors using 'click' chemistry or immuno-labeling), and amino acids tagged with 'heavy' 15N and 13C (detected using secondary ion mass spectrometry)--are incorporated into the nucleus and cytoplasm in a process sensitive to translational inhibitors. The nuclear incorporation represents a significant fraction of the total, and labels in both compartments have half-lives of less than a minute; results are consistent with most newly-made peptides being destroyed soon after they are made. As nascent RNA bearing a premature termination codon (detected by fluorescence in situ hybridization) is also eliminated by a mechanism sensitive to a translational inhibitor, the nuclear turnover of peptides is probably a by-product of proof-reading the RNA for stop codons (a process known as nonsense-mediated decay). We speculate that the apparently-wasteful turnover of this previously-hidden ('dark-matter') world of peptide is involved in regulating protein production.
Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain ...complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development.
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•Immunoinformatics reveal precursors for V2-apex bnAbs PCT64 and PG9 in HIV-negative donors•Immunogen design produces ApexGT trimers with affinity for inferred precursors•Cryo-EM structures of trimers bound to precursors or bnAbs assist immunogen design•Membrane-anchored ApexGT trimers retain antigenicity on DNA- or mRNA-transfected cells
Broadly neutralizing antibodies (bnAbs) to the HIV envelope V2-apex are important leads for vaccine design but pose major challenges for precursor priming due to long heavy-chain complementarity-determining region 3 (HCDR3). We prioritize two V2-apex bnAbs, PCT64 and PG9, design and characterize ApexGT trimers that bind bnAb precursors, and develop membrane-bound trimers for mRNA delivery as candidate priming immunogens.
Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA ...polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired‐end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete ‘NFκB factories’. Some factories further specialize in transcribing responsive genes encoding micro‐RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories.
TNFα alpha treatment induces congregation of both coding and non‐coding NFκB target genes in discrete transcription factories, leading to coordinated gene expression.
Viruses can evade the host immune system by displaying numerous glycans on their surface “spike-proteins” that cover immune epitopes. We have developed an ultrasensitive “single-pot” method to assess ...glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for the analysis of site-specific glycosylation of any glycoprotein.
Emerin and lamin B receptor (LBR) are abundant transmembrane proteins of the nuclear envelope that are concentrated at the inner nuclear membrane (INM). Although both proteins interact with chromatin ...and nuclear lamins, they have distinctive biochemical and functional properties. Here, we have deployed proximity labeling using the engineered biotin ligase TurboID (TbID) and quantitative proteomics to compare the neighborhoods of emerin and LBR in cultured mouse embryonic fibroblasts. Our analysis revealed 232 high confidence proximity partners that interact selectively with emerin and/or LBR, 49 of which are shared by both. These included previously characterized NE-concentrated proteins, as well as a host of additional proteins not previously linked to emerin or LBR functions. Many of these are TM proteins of the ER, including two E3 ubiquitin ligases. Supporting these results, we found that 11/12 representative proximity relationships identified by TbID also were detected at the NE with the proximity ligation assay. Overall, this work presents methodology that may be used for large-scale mapping of the landscape of the INM and reveals a group of new proteins with potential functional connections to emerin and LBR.
Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells ...(MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.
Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically ...dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.
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