High-throughput sequencing of single neuron projections reveals spatial organization in the olfactory cortex.

In most sensory modalities, neuronal connectivity reflects behaviorally relevant stimulus features, such as spatial location, orientation, and sound frequency. By contrast, the prevailing view in the olfactory cortex, based on the reconstruction of dozens of neurons, is that connectivity is random. Here, we used high-throughput sequencing-based neuroanatomical techniques to analyze the projections of 5,309 mouse olfactory bulb and 30,433 piriform cortex output neurons at single-cell resolution. Surprisingly, statistical analysis of this much larger dataset revealed that the olfactory cortex connectivity is spatially structured. Single olfactory bulb neurons targeting a particular location along the anterior-posterior axis of piriform cortex also project to matched, functionally distinct, extra-piriform targets. Moreover, single neurons from the targeted piriform locus also project to the same matched extra-piriform targets, forming triadic circuit motifs. Thus, as in other sensory modalities, olfactory information is routed at early stages of processing to functionally diverse targets in a coordinated manner.

Molecular topography of an entire nervous system.

We have produced gene expression profiles of all 302 neurons of the C. elegans nervous system that match the single-cell resolution of its anatomy and wiring diagram. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families. For example, each neuron class expresses distinct codes of ∼23 neuropeptide genes and ∼36 neuropeptide receptors, delineating a complex and expansive "wireless" signaling network. To demonstrate the utility of this comprehensive gene expression catalog, we used computational approaches to (1) identify cis-regulatory elements for neuron-specific gene expression and (2) reveal adhesion proteins with potential roles in process placement and synaptic specificity. Our expression data are available at https://cengen.org and can be interrogated at the web application CengenApp. We expect that this neuron-specific directory of gene expression will spur investigations of underlying mechanisms that define anatomy, connectivity, and function throughout the C. elegans nervous system.

Atlas of Subcellular RNA Localization Revealed by APEX-Seq.

We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

Machine learning predictions of MHC-II specificities reveal alternative binding mode of class II epitopes.

CD4+ T cells orchestrate the adaptive immune response against pathogens and cancer by recognizing epitopes presented on class II major histocompatibility complex (MHC-II) molecules. The high polymorphism of MHC-II genes represents an important hurdle toward accurate prediction and identification of CD4+ T cell epitopes. Here we collected and curated a dataset of 627,013 unique MHC-II ligands identified by mass spectrometry. This enabled us to precisely determine the binding motifs of 88 MHC-II alleles across humans, mice, cattle, and chickens. Analysis of these binding specificities combined with X-ray crystallography refined our understanding of the molecular determinants of MHC-II motifs and revealed a widespread reverse-binding mode in HLA-DP ligands. We then developed a machine-learning framework to accurately predict binding specificities and ligands of any MHC-II allele. This tool improves and expands predictions of CD4+ T cell epitopes and enables us to discover viral and bacterial epitopes following the aforementioned reverse-binding mode.

Structural Basis of Egg Coat-Sperm Recognition at Fertilization.

Recognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1-3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion.

Basis of gene-specific transcription regulation by the Integrator complex.

The Integrator complex attenuates gene expression via the premature termination of RNA polymerase II (RNAP2) at promoter-proximal pausing sites. It is required for stimulus response, cell differentiation, and neurodevelopment, but how gene-specific and adaptive regulation by Integrator is achieved remains unclear. Here, we identify two sites on human Integrator subunits 13/14 that serve as binding hubs for sequence-specific transcription factors (TFs) and other transcription effector complexes. When Integrator is attached to paused RNAP2, these hubs are positioned upstream of the transcription bubble, consistent with simultaneous TF-promoter tethering. The TFs co-localize with Integrator genome-wide, increase Integrator abundance on target genes, and co-regulate responsive transcriptional programs. For instance, sensory cilia formation induced by glucose starvation depends on Integrator-TF contacts. Our data suggest TF-mediated promoter recruitment of Integrator as a widespread mechanism for targeted transcription regulation.

Evolutionary origins and interactomes of human, young microproteins and small peptides translated from short open reading frames.

All species continuously evolve short open reading frames (sORFs) that can be templated for protein synthesis and may provide raw materials for evolutionary adaptation. We analyzed the evolutionary origins of 7,264 recently cataloged human sORFs and found that most were evolutionarily young and had emerged de novo. We additionally identified 221 previously missed sORFs potentially translated into peptides of up to 15 amino acids-all of which are smaller than the smallest human microprotein annotated to date. To investigate the bioactivity of sORF-encoded small peptides and young microproteins, we subjected 266 candidates to a mass-spectrometry-based interactome screen with motif resolution. Based on these interactomes and additional cellular assays, we can associate several candidates with mRNA splicing, translational regulation, and endocytosis. Our work provides insights into the evolutionary origins and interaction potential of young and small proteins, thereby helping to elucidate this underexplored territory of the human proteome.

"Stripe" transcription factors provide accessibility to co-binding partners in mammalian genomes.

Regulatory elements activate promoters by recruiting transcription factors (TFs) to specific motifs. Notably, TF-DNA interactions often depend on cooperativity with colocalized partners, suggesting an underlying cis-regulatory syntax. To explore TF cooperativity in mammals, we analyze ∼500 mouse and human primary cells by combining an atlas of TF motifs, footprints, ChIP-seq, transcriptomes, and accessibility. We uncover two TF groups that colocalize with most expressed factors, forming stripes in hierarchical clustering maps. The first group includes lineage-determining factors that occupy DNA elements broadly, consistent with their key role in tissue-specific transcription. The second one, dubbed universal stripe factors (USFs), comprises ∼30 SP, KLF, EGR, and ZBTB family members that recognize overlapping GC-rich sequences in all tissues analyzed. Knockouts and single-molecule tracking reveal that USFs impart accessibility to colocalized partners and increase their residence time. Mammalian cells have thus evolved a TF superfamily with overlapping DNA binding that facilitate chromatin accessibility.

Multiple, short protein binding motifs in ORC1 and CDC6 control the initiation of DNA replication.

The initiation of DNA replication involves cell cycle-dependent assembly and disassembly of protein complexes, including the origin recognition complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short linear protein motifs (SLiMs) within intrinsically disordered regions (IDRs) in ORC1 and CDC6 mediate cyclin-CDK-dependent and independent protein-protein interactions, conditional on the cell cycle phase. A domain within the ORC1 IDR is required for interaction between the ORC1 and CDC6 AAA+ domains in G1, whereas the same domain prevents CDC6-ORC1 interaction during mitosis. Then, during late G1, this domain facilitates ORC1 destruction by a SKP2-cyclin A-CDK2-dependent mechanism. During G1, the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interactions. The CDC6 IDR regulates self-interaction by ORC1, thereby controlling ORC1 protein levels. Protein phosphatase 1 binds directly to a SLiM in the ORC1 IDR, causing ORC1 de-phosphorylation upon mitotic exit, increasing ORC1 protein, and promoting pre-RC assembly.

RNA fine-tunes estrogen receptor-alpha binding on low-affinity DNA motifs for transcriptional regulation.

Transcription factors (TFs) regulate gene expression by binding with varying strengths to DNA via their DNA-binding domain. Additionally, some TFs also interact with RNA, which modulates transcription factor binding to chromatin. However, whether RNA-mediated TF binding results in differential transcriptional outcomes remains unknown. In this study, we demonstrate that estrogen receptor α (ERα), a ligand-activated TF, interacts with RNA in a ligand-dependent manner. Defects in RNA binding lead to genome-wide loss of ERα recruitment, particularly at weaker ERα-motifs. Furthermore, ERα mobility in the nucleus increases in the absence of its RNA-binding capacity. Unexpectedly, this increased mobility coincides with robust polymerase loading and transcription of ERα-regulated genes that harbor low-strength motifs. However, highly stable binding of ERα on chromatin negatively impacts ligand-dependent transcription. Collectively, our results suggest that RNA interactions spatially confine ERα on low-affinity sites to fine-tune gene transcription.

Small RNAs, Large Networks: Posttranscriptional Regulons in Gram-Negative Bacteria.

Small regulatory RNA (sRNAs) are key mediators of posttranscriptional gene control in bacteria. Assisted by RNA-binding proteins, a single sRNA often modulates the expression of dozens of genes, and thus sRNAs frequently adopt central roles in regulatory networks. Posttranscriptional regulation by sRNAs comes with several unique features that cannot be achieved by transcriptional regulators. However, for optimal network performance, transcriptional and posttranscriptional control mechanisms typically go hand-in-hand. This view is reflected by the ever-growing class of mixed network motifs involving sRNAs and transcription factors, which are ubiquitous in biology and whose regulatory properties we are beginning to understand. In addition, sRNA activity can be antagonized by base-pairing with sponge RNAs, adding yet another layer of complexity to these networks. In this article, we summarize the regulatory concepts underlying sRNA-mediated gene control in bacteria and discuss how sRNAs shape the output of a network, focusing on several key examples.

Substrate and phosphorylation site selection by phosphoprotein phosphatases.

Dynamic protein phosphorylation and dephosphorylation are essential regulatory mechanisms that ensure proper cellular signaling and biological functions. Deregulation of either reaction has been implicated in several human diseases. Here, we focus on the mechanisms that govern the specificity of the dephosphorylation reaction. Most cellular serine/threonine dephosphorylation is catalyzed by 13 highly conserved phosphoprotein phosphatase (PPP) catalytic subunits, which form hundreds of holoenzymes by binding to regulatory and scaffolding subunits. PPP holoenzymes recognize phosphorylation site consensus motifs and interact with short linear motifs (SLiMs) or structural elements distal to the phosphorylation site. We review recent advances in understanding the mechanisms of PPP site-specific dephosphorylation preference and substrate recruitment and highlight examples of their interplay in the regulation of cell division.

SLiM-binding pockets: an attractive target for broad-spectrum antivirals.

Short linear motif (SLiM)-mediated interactions offer a unique strategy for viral intervention due to their compact interfaces, ease of convergent evolution, and key functional roles. Consequently, many viruses extensively mimic host SLiMs to hijack or deregulate cellular pathways and the same motif-binding pocket is often targeted by numerous unrelated viruses. A toolkit of therapeutics targeting commonly mimicked SLiMs could provide prophylactic and therapeutic broad-spectrum antivirals and vastly improve our ability to treat ongoing and future viral outbreaks. In this opinion article, we discuss the therapeutic relevance of SLiMs, advocating their suitability as targets for broad-spectrum antiviral inhibitors.

i-Motif DNA: identification, formation, and cellular functions.

An i-motif (iM) is a four-stranded (quadruplex) DNA structure that folds from cytosine (C)-rich sequences. iMs can fold under many different conditions in vitro, which paves the way for their formation in living cells. iMs are thought to play key roles in various DNA transactions, notably in the regulation of genome stability, gene transcription, mRNA translation, DNA replication, telomere and centromere functions, and human diseases. We summarize the different techniques used to assess the folding of iMs in vitro and provide an overview of the internal and external factors that affect their formation and stability in vivo. We describe the possible biological relevance of iMs and propose directions towards their use as target in biology.

Systematic identification and characterization of repressive domains in Drosophila transcription factors.

All multicellular life relies on differential gene expression, determined by regulatory DNA elements and DNA-binding transcription factors that mediate activation and repression via cofactor recruitment. While activators have been extensively characterized, repressors are less well studied: the identities and properties of their repressive domains (RDs) are typically unknown and the specific co-repressors (CoRs) they recruit have not been determined. Here, we develop a high-throughput, next-generation sequencing-based screening method, repressive-domain (RD)-seq, to systematically identify RDs in complex DNA-fragment libraries. Screening more than 200,000 fragments covering the coding sequences of all transcription-related proteins in Drosophila melanogaster, we identify 195 RDs in known repressors and in proteins not previously associated with repression. Many RDs contain recurrent short peptide motifs, which are conserved between fly and human and are required for RD function, as demonstrated by motif mutagenesis. Moreover, we show that RDs that contain one of five distinct repressive motifs interact with and depend on different CoRs, such as Groucho, CtBP, Sin3A, or Smrter. These findings advance our understanding of repressors, their sequences, and the functional impact of sequence-altering mutations and should provide a valuable resource for further studies.

Regulatory features aid interpretation of 3'UTR variants.

Our ability to determine the clinical impact of variants in 3' untranslated regions (UTRs) of genes remains poor. We provide a thorough analysis of 3' UTR variants from several datasets. Variants in putative regulatory elements, including RNA-binding protein motifs, eCLIP peaks, and microRNA sites, are up to 16 times more likely than variants not in these elements to have gene expression and phenotype associations. Variants in regulatory motifs result in allele-specific protein binding in cell lines and allele-specific gene expression differences in population studies. In addition, variants in shared regions of alternatively polyadenylated isoforms and those proximal to polyA sites are more likely to affect gene expression and phenotype. Finally, pathogenic 3' UTR variants in ClinVar are up to 20 times more likely than benign variants to fall in a regulatory site. We incorporated these findings into RegVar, a software tool that interprets regulatory elements and annotations for any 3' UTR variant and predicts whether the variant is likely to affect gene expression or phenotype. This tool will help prioritize variants for experimental studies and identify pathogenic variants in individuals.

The C-Terminal Domain of RNA Polymerase II Is a Multivalent Targeting Sequence that Supports Drosophila Development with Only Consensus Heptads.

The C-terminal domain (CTD) of RNA polymerase II (Pol II) is composed of repeats of the consensus YSPTSPS and is an essential binding scaffold for transcription-associated factors. Metazoan CTDs have well-conserved lengths and sequence compositions arising from the evolution of divergent motifs, features thought to be essential for development. On the contrary, we show that a truncated CTD composed solely of YSPTSPS repeats supports Drosophila viability but that a CTD with enough YSPTSPS repeats to match the length of the wild-type Drosophila CTD is defective. Furthermore, a fluorescently tagged CTD lacking the rest of Pol II dynamically enters transcription compartments, indicating that the CTD functions as a signal sequence. However, CTDs with too many YSPTSPS repeats are more prone to localize to static nuclear foci separate from the chromosomes. We propose that the sequence complexity of the CTD offsets aberrant behavior caused by excessive repetitive sequences without compromising its targeting function.

Promoter Distortion and Opening in the RNA Polymerase II Cleft.

Transcription initiation requires opening of promoter DNA in the RNA polymerase II (Pol II) pre-initiation complex (PIC), but it remains unclear how this is achieved. Here we report the cryo-electron microscopic (cryo-EM) structure of a yeast PIC that contains underwound, distorted promoter DNA in the closed Pol II cleft. The DNA duplex axis is offset at the upstream edge of the initially melted DNA region (IMR) where DNA opening begins. Unstable IMRs are found in a subset of yeast promoters that we show can still initiate transcription after depletion of the transcription factor (TF) IIH (TFIIH) translocase Ssl2 (XPB in human) from the nucleus in vivo. PIC-induced DNA distortions may thus prime the IMR for melting and may explain how unstable IMRs that are predicted in promoters of Pol I and Pol III can open spontaneously. These results suggest that DNA distortion in the polymerase cleft is a general mechanism that contributes to promoter opening.

Harnessing the power of artificial intelligence to advance cell therapy.

Cell therapies are powerful technologies in which human cells are reprogrammed for therapeutic applications such as killing cancer cells or replacing defective cells. The technologies underlying cell therapies are increasing in effectiveness and complexity, making rational engineering of cell therapies more difficult. Creating the next generation of cell therapies will require improved experimental approaches and predictive models. Artificial intelligence (AI) and machine learning (ML) methods have revolutionized several fields in biology including genome annotation, protein structure prediction, and enzyme design. In this review, we discuss the potential of combining experimental library screens and AI to build predictive models for the development of modular cell therapy technologies. Advances in DNA synthesis and high-throughput screening techniques enable the construction and screening of libraries of modular cell therapy constructs. AI and ML models trained on this screening data can accelerate the development of cell therapies by generating predictive models, design rules, and improved designs.

The spindle checkpoint proteins BUB1 and BUBR1: (SLiM)ming down to the basics.

Benzimidazole 1 (BUB1) and budding uninhibited by benzimidazole 1-related 1 (BUBR1) are multidomain paralogs with key roles in chromosome alignment during mitosis and the spindle assembly checkpoint (SAC), an evolutionarily conserved signaling pathway that monitors errors in chromosome segregation during cell division in eukaryotes. Although BUB1 and BUBR1 share a similar domain organization and short linear interaction motifs (SLiMs), they control distinct aspects of chromosome congression and the SAC. Here we discuss the roles of BUB1 and BUBR1 SLiMs in mitosis and complement this with additional insights gleamed from studying their evolution. We show that BUB1 and BUBR1 SLiMs form highly specific interactions that are carefully orchestrated in space and time and contend that they define BUB1 and BUBR1 as organizing hubs that drive SAC signaling and ensure genome stability.