Broadly Neutralizing Antibodies to HIV and Their Role in Vaccine Design.

HIV employs multiple means to evade the humoral immune response, particularly the elicitation of and recognition by broadly neutralizing antibodies (bnAbs). Such antibodies can act antivirally against a wide spectrum of viruses by targeting relatively conserved regions on the surface HIV envelope trimer spike. Elicitation of and recognition by bnAbs are hindered by the arrangement of spikes on virions and the relatively difficult access to bnAb epitopes on spikes, including the proximity of variable regions and a high density of glycans. Yet, in a small proportion of HIV-infected individuals, potent bnAb responses do develop, and isolation of the corresponding monoclonal antibodies has been facilitated by identification of favorable donors with potent bnAb sera and by development of improved methods for human antibody generation. Molecular studies of recombinant Env trimers, alone and in interaction with bnAbs, are providing new insights that are fueling the development and testing of promising immunogens aimed at the elicitation of bnAbs.

Structural Mechanism of Transport of Mitochondrial Carriers.

Members of the mitochondrial carrier family [solute carrier family 25 (SLC25)] transport nucleotides, amino acids, carboxylic acids, fatty acids, inorganic ions, and vitamins across the mitochondrial inner membrane. They are important for many cellular processes, such as oxidative phosphorylation of lipids and sugars, amino acid metabolism, macromolecular synthesis, ion homeostasis, cellular regulation, and differentiation. Here, we describe the functional elements of the transport mechanism of mitochondrial carriers, consisting of one central substrate-binding site and two gates with salt-bridge networks on either side of the carrier. Binding of the substrate during import causes three gate elements to rotate inward, forming the cytoplasmic network and closing access to the substrate-binding site from the intermembrane space. Simultaneously, three core elements rock outward, disrupting the matrix network and opening the substrate-binding site to the matrix side of the membrane. During export, substrate binding triggers conformational changes involving the same elements but operating in reverse.

Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope.

Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design.

A mouse-specific retrotransposon drives a conserved Cdk2ap1 isoform essential for development.

Retrotransposons mediate gene regulation in important developmental and pathological processes. Here, we characterized the transient retrotransposon induction during preimplantation development of eight mammals. Induced retrotransposons exhibit similar preimplantation profiles across species, conferring gene regulatory activities, particularly through long terminal repeat (LTR) retrotransposon promoters. A mouse-specific MT2B2 retrotransposon promoter generates an N-terminally truncated Cdk2ap1ΔN that peaks in preimplantation embryos and promotes proliferation. In contrast, the canonical Cdk2ap1 peaks in mid-gestation and represses cell proliferation. This MT2B2 promoter, whose deletion abolishes Cdk2ap1ΔN production, reduces cell proliferation and impairs embryo implantation, is developmentally essential. Intriguingly, Cdk2ap1ΔN is evolutionarily conserved in sequence and function yet is driven by different promoters across mammals. The distinct preimplantation Cdk2ap1ΔN expression in each mammalian species correlates with the duration of its preimplantation development. Hence, species-specific transposon promoters can yield evolutionarily conserved, alternative protein isoforms, bestowing them with new functions and species-specific expression to govern essential biological divergence.

A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression.

CRISPR-Cas systems provide prokaryotes with acquired immunity against viruses and plasmids, but how these systems are regulated to prevent autoimmunity is poorly understood. Here, we show that in the S. pyogenes CRISPR-Cas system, a long-form transactivating CRISPR RNA (tracr-L) folds into a natural single guide that directs Cas9 to transcriptionally repress its own promoter (Pcas). Further, we demonstrate that Pcas serves as a critical regulatory node. De-repression causes a dramatic 3,000-fold increase in immunization rates against viruses; however, heightened immunity comes at the cost of increased autoimmune toxicity. Using bioinformatic analyses, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II-A CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.

Splice site m6A methylation prevents binding of U2AF35 to inhibit RNA splicing.

The N6-methyladenosine (m6A) RNA modification is used widely to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (the ortholog of mouse METTL16) deposits an m6A mark on the 3' splice site (AG) of the S-adenosylmethionine (SAM) synthetase pre-mRNA, which inhibits its proper splicing and protein production. The mechanism is triggered by a rich diet and acts as an m6A-mediated switch to stop SAM production and regulate its homeostasis. Although the mammalian SAM synthetase pre-mRNA is not regulated via this mechanism, we show that splicing inhibition by 3' splice site m6A is conserved in mammals. The modification functions by physically preventing the essential splicing factor U2AF35 from recognizing the 3' splice site. We propose that use of splice-site m6A is an ancient mechanism for splicing regulation.

Structural insights into the human D1 and D2 dopamine receptor signaling complexes.

The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-Gs and D2R-Gi signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.

How Is Precursor Messenger RNA Spliced by the Spliceosome?

Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the spliceosome. Together with biochemical and genetic investigations of the past four decades, structural studies of the intact spliceosome at atomic resolution since 2015 have led to mechanistic delineation of RNA splicing with remarkable insights. The spliceosome is proven to be a protein-orchestrated metalloribozyme. Conserved elements of small nuclear RNA (snRNA) constitute the splicing active site with two catalytic metal ions and recognize three conserved intron elements through duplex formation, which are delivered into the splicing active site for branching and exon ligation. The protein components of the spliceosome stabilize the conformation of the snRNA, drive spliceosome remodeling, orchestrate the movement of the RNA elements, and facilitate the splicing reaction. The overall organization of the spliceosome and the configuration of the splicing active site are strictly conserved between human and yeast.

Intergenerationally Maintained Histone H4 Lysine 16 Acetylation Is Instructive for Future Gene Activation.

Before zygotic genome activation (ZGA), the quiescent genome undergoes reprogramming to transition into the transcriptionally active state. However, the mechanisms underlying euchromatin establishment during early embryogenesis remain poorly understood. Here, we show that histone H4 lysine 16 acetylation (H4K16ac) is maintained from oocytes to fertilized embryos in Drosophila and mammals. H4K16ac forms large domains that control nucleosome accessibility of promoters prior to ZGA in flies. Maternal depletion of MOF acetyltransferase leading to H4K16ac loss causes aberrant RNA Pol II recruitment, compromises the 3D organization of the active genomic compartments during ZGA, and causes downregulation of post-zygotically expressed genes. Germline depletion of histone deacetylases revealed that other acetyl marks cannot compensate for H4K16ac loss in the oocyte. Moreover, zygotic re-expression of MOF was neither able to restore embryonic viability nor onset of X chromosome dosage compensation. Thus, maternal H4K16ac provides an instructive function to the offspring, priming future gene activation.

Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.

Oligopeptide Signaling through TbGPR89 Drives Trypanosome Quorum Sensing.

Trypanosome parasites control their virulence and spread by using quorum sensing (QS) to generate transmissible "stumpy forms" in their host bloodstream. However, the QS signal "stumpy induction factor" (SIF) and its reception mechanism are unknown. Although trypanosomes lack G protein-coupled receptor signaling, we have identified a surface GPR89-family protein that regulates stumpy formation. TbGPR89 is expressed on bloodstream "slender form" trypanosomes, which receive the SIF signal, and when ectopically expressed, TbGPR89 drives stumpy formation in a SIF-pathway-dependent process. Structural modeling of TbGPR89 predicts unexpected similarity to oligopeptide transporters (POT), and when expressed in bacteria, TbGPR89 transports oligopeptides. Conversely, expression of an E. coli POT in trypanosomes drives parasite differentiation, and oligopeptides promote stumpy formation in vitro. Furthermore, the expression of secreted trypanosome oligopeptidases generates a paracrine signal that accelerates stumpy formation in vivo. Peptidase-generated oligopeptide QS signals being received through TbGPR89 provides a mechanism for both trypanosome SIF production and reception.

The Molecular Basis of G Protein-Coupled Receptor Activation.

G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into α and ßγ subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.

Protein AMPylation by an Evolutionarily Conserved Pseudokinase.

Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities.

Ultraconserved Enhancers Are Required for Normal Development.

Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting.

Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system.

In multicellular organisms, duplicated genes can diverge through tissue-specific gene expression patterns, as exemplified by highly regulated expression of RUNX transcription factor paralogs with apparent functional redundancy. Here we asked what cell-type-specific biologies might be supported by the selective expression of RUNX paralogs during Langerhans cell and inducible regulatory T cell differentiation. We uncovered functional nonequivalence between RUNX paralogs. Selective expression of native paralogs allowed integration of transcription factor activity with extrinsic signals, while non-native paralogs enforced differentiation even in the absence of exogenous inducers. DNA binding affinity was controlled by divergent amino acids within the otherwise highly conserved RUNT domain and evolutionary reconstruction suggested convergence of RUNT domain residues toward submaximal strength. Hence, the selective expression of gene duplicates in specialized cell types can synergize with the acquisition of functional differences to enable appropriate gene expression, lineage choice and differentiation in the mammalian immune system.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

The functions and unique features of long intergenic non-coding RNA.

Long intergenic non-coding RNA (lincRNA) genes have diverse features that distinguish them from mRNA-encoding genes and exercise functions such as remodelling chromatin and genome architecture, RNA stabilization and transcription regulation, including enhancer-associated activity. Some genes currently annotated as encoding lincRNAs include small open reading frames (smORFs) and encode functional peptides and thus may be more properly classified as coding RNAs. lincRNAs may broadly serve to fine-tune the expression of neighbouring genes with remarkable tissue specificity through a diversity of mechanisms, highlighting our rapidly evolving understanding of the non-coding genome.

Identification of constrained sequence elements across 239 primate genomes.

Noncoding DNA is central to our understanding of human gene regulation and complex diseases1,2, and measuring the evolutionary sequence constraint can establish the functional relevance of putative regulatory elements in the human genome3-9. Identifying the genomic elements that have become constrained specifically in primates has been hampered by the faster evolution of noncoding DNA compared to protein-coding DNA10, the relatively short timescales separating primate species11, and the previously limited availability of whole-genome sequences12. Here we construct a whole-genome alignment of 239 species, representing nearly half of all extant species in the primate order. Using this resource, we identified human regulatory elements that are under selective constraint across primates and other mammals at a 5% false discovery rate. We detected 111,318 DNase I hypersensitivity sites and 267,410 transcription factor binding sites that are constrained specifically in primates but not across other placental mammals and validate their cis-regulatory effects on gene expression. These regulatory elements are enriched for human genetic variants that affect gene expression and complex traits and diseases. Our results highlight the important role of recent evolution in regulatory sequence elements differentiating primates, including humans, from other placental mammals.

Identification of plant transcriptional activation domains.

Gene expression in Arabidopsis is regulated by more than 1,900 transcription factors (TFs), which have been identified genome-wide by the presence of well-conserved DNA-binding domains. Activator TFs contain activation domains (ADs) that recruit coactivator complexes; however, for nearly all Arabidopsis TFs, we lack knowledge about the presence, location and transcriptional strength of their ADs1. To address this gap, here we use a yeast library approach to experimentally identify Arabidopsis ADs on a proteome-wide scale, and find that more than half of the Arabidopsis TFs contain an AD. We annotate 1,553 ADs, the vast majority of which are, to our knowledge, previously unknown. Using the dataset generated, we develop a neural network to accurately predict ADs and to identify sequence features that are necessary to recruit coactivator complexes. We uncover six distinct combinations of sequence features that result in activation activity, providing a framework to interrogate the subfunctionalization of ADs. Furthermore, we identify ADs in the ancient AUXIN RESPONSE FACTOR family of TFs, revealing that AD positioning is conserved in distinct clades. Our findings provide a deep resource for understanding transcriptional activation, a framework for examining function in intrinsically disordered regions and a predictive model of ADs.

Release of a ubiquitin brake activates OsCERK1-triggered immunity in rice.

Plant pattern-recognition receptors perceive microorganism-associated molecular patterns to activate immune signalling1,2. Activation of the pattern-recognition receptor kinase CERK1 is essential for immunity, but tight inhibition of receptor kinases in the absence of pathogen is crucial to prevent autoimmunity3,4. Here we find that the U-box ubiquitin E3 ligase OsCIE1 acts as a molecular brake to inhibit OsCERK1 in rice. During homeostasis, OsCIE1 ubiquitinates OsCERK1, reducing its kinase activity. In the presence of the microorganism-associated molecular pattern chitin, active OsCERK1 phosphorylates OsCIE1 and blocks its E3 ligase activity, thus releasing the brake and promoting immunity. Phosphorylation of a serine within the U-box of OsCIE1 prevents its interaction with E2 ubiquitin-conjugating enzymes and serves as a phosphorylation switch. This phosphorylation site is conserved in E3 ligases from plants to animals. Our work identifies a ligand-released brake that enables dynamic immune regulation.