The genomic and cellular basis of biosynthetic innovation in rove beetles.

How evolution at the cellular level potentiates macroevolutionary change is central to understanding biological diversification. The >66,000 rove beetle species (Staphylinidae) form the largest metazoan family. Combining genomic and cell type transcriptomic insights spanning the largest clade, Aleocharinae, we retrace evolution of two cell types comprising a defensive gland-a putative catalyst behind staphylinid megadiversity. We identify molecular evolutionary steps leading to benzoquinone production by one cell type via a mechanism convergent with plant toxin release systems, and synthesis by the second cell type of a solvent that weaponizes the total secretion. This cooperative system has been conserved since the Early Cretaceous as Aleocharinae radiated into tens of thousands of lineages. Reprogramming each cell type yielded biochemical novelties enabling ecological specialization-most dramatically in symbionts that infiltrate social insect colonies via host-manipulating secretions. Our findings uncover cell type evolutionary processes underlying the origin and evolvability of a beetle chemical innovation.

RNA promotes the formation of spatial compartments in the nucleus.

RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.

SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.

Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus.

Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.

Viscoelasticity and advective flow of RNA underlies nucleolar form and function.

The nucleolus is the largest biomolecular condensate and facilitates transcription, processing, and assembly of ribosomal RNA (rRNA). Although nucleolar function is thought to require multiphase liquid-like properties, nucleolar fluidity and its connection to the highly coordinated transport and biogenesis of ribosomal subunits are poorly understood. Here, we use quantitative imaging, mathematical modeling, and pulse-chase nucleotide labeling to examine nucleolar material properties and rRNA dynamics. The mobility of rRNA is several orders of magnitude slower than that of nucleolar proteins, with rRNA steadily moving away from the transcriptional sites in a slow (∼1 Å/s), radially directed fashion. This constrained but directional mobility, together with polymer physics-based calculations, suggests that nascent rRNA forms an entangled gel, whose constant production drives outward flow. We propose a model in which progressive maturation of nascent rRNA reduces its initial entanglement, fluidizing the nucleolar periphery to facilitate the release of assembled pre-ribosomal particles.

Genome organization around nuclear speckles drives mRNA splicing efficiency.

The nucleus is highly organized, such that factors involved in the transcription and processing of distinct classes of RNA are confined within specific nuclear bodies1,2. One example is the nuclear speckle, which is defined by high concentrations of protein and noncoding RNA regulators of pre-mRNA splicing3. What functional role, if any, speckles might play in the process of mRNA splicing is unclear4,5. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs and higher co-transcriptional splicing levels than genes that are located farther from nuclear speckles. Gene organization around nuclear speckles is dynamic between cell types, and changes in speckle proximity lead to differences in splicing efficiency. Finally, directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Together, our results integrate the long-standing observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a crucial role for dynamic three-dimensional spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.

High-Resolution Mapping of Multiway Enhancer-Promoter Interactions Regulating Pathogen Detection.

Eukaryotic gene expression regulation involves thousands of distal regulatory elements. Understanding the quantitative contribution of individual enhancers to gene expression is critical for assessing the role of disease-associated genetic risk variants. Yet, we lack the ability to accurately link genes with their distal regulatory elements. To address this, we used 3D enhancer-promoter (E-P) associations identified using split-pool recognition of interactions by tag extension (SPRITE) to build a predictive model of gene expression. Our model dramatically outperforms models using genomic proximity and can be used to determine the quantitative impact of enhancer loss on gene expression in different genetic backgrounds. We show that genes that form stable E-P hubs have less cell-to-cell variability in gene expression. Finally, we identified transcription factors that regulate stimulation-dependent E-P interactions. Together, our results provide a framework for understanding quantitative contributions of E-P interactions and associated genetic variants to gene expression.

Integrative genome modeling platform reveals essentiality of rare contact events in 3D genome organizations.

A multitude of sequencing-based and microscopy technologies provide the means to unravel the relationship between the three-dimensional organization of genomes and key regulatory processes of genome function. Here, we develop a multimodal data integration approach to produce populations of single-cell genome structures that are highly predictive for nuclear locations of genes and nuclear bodies, local chromatin compaction and spatial segregation of functionally related chromatin. We demonstrate that multimodal data integration can compensate for systematic errors in some of the data and can greatly increase accuracy and coverage of genome structure models. We also show that alternative combinations of different orthogonal data sources can converge to models with similar predictive power. Moreover, our study reveals the key contributions of low-frequency ('rare') interchromosomal contacts to accurately predicting the global nuclear architecture, including the positioning of genes and chromosomes. Overall, our results highlight the benefits of multimodal data integration for genome structure analysis, available through the Integrative Genome Modeling software package.

3D genome organization around nuclear speckles drives mRNA splicing efficiency.

The nucleus is highly organized such that factors involved in transcription and processing of distinct classes of RNA are organized within specific nuclear bodies. One such nuclear body is the nuclear speckle, which is defined by high concentrations of protein and non-coding RNA regulators of pre-mRNA splicing. What functional role, if any, speckles might play in the process of mRNA splicing remains unknown. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs, and higher co-transcriptional splicing levels relative to genes that are located farther from nuclear speckles. We show that directed recruitment of a pre-mRNA to nuclear speckles is sufficient to drive increased mRNA splicing levels. Finally, we show that gene organization around nuclear speckles is highly dynamic with differential localization between cell types corresponding to differences in Pol II occupancy. Together, our results integrate the longstanding observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a critical role for dynamic 3D spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.

The genomic and cellular basis of biosynthetic innovation in rove beetles.

How evolution at the cellular level potentiates change at the macroevolutionary level is a major question in evolutionary biology. With >66,000 described species, rove beetles (Staphylinidae) comprise the largest metazoan family. Their exceptional radiation has been coupled to pervasive biosynthetic innovation whereby numerous lineages bear defensive glands with diverse chemistries. Here, we combine comparative genomic and single-cell transcriptomic data from across the largest rove beetle clade, Aleocharinae. We retrace the functional evolution of two novel secretory cell types that together comprise the tergal gland-a putative catalyst behind Aleocharinae's megadiversity. We identify key genomic contingencies that were critical to the assembly of each cell type and their organ-level partnership in manufacturing the beetle's defensive secretion. This process hinged on evolving a mechanism for regulated production of noxious benzoquinones that appears convergent with plant toxin release systems, and synthesis of an effective benzoquinone solvent that weaponized the total secretion. We show that this cooperative biosynthetic system arose at the Jurassic-Cretaceous boundary, and that following its establishment, both cell types underwent ∼150 million years of stasis, their chemistry and core molecular architecture maintained almost clade-wide as Aleocharinae radiated globally into tens of thousands of lineages. Despite this deep conservation, we show that the two cell types have acted as substrates for the emergence of adaptive, biochemical novelties-most dramatically in symbiotic lineages that have infiltrated social insect colonies and produce host behavior-manipulating secretions. Our findings uncover genomic and cell type evolutionary processes underlying the origin, functional conservation and evolvability of a chemical innovation in beetles.

Essential Roles for RNA in Shaping Nuclear Organization.

It has long been proposed that nuclear RNAs might play an important role in organizing the structure of the nucleus. Initial experiments performed more than 30 years ago found that global disruption of RNA led to visible rearrangements of nuclear organization. Yet, this idea remained controversial for many years, in large part because it was unclear what specific RNAs might be involved, and which specific nuclear structures might be dependent on RNA. Over the past few years, the contributions of RNA to organizing nuclear structures have become clearer with the discovery that many nuclear bodies are enriched for specific noncoding RNAs (ncRNAs); in specific cases, ncRNAs have been shown to be essential for establishment and maintenance of these nuclear structures. More recently, many different ncRNAs have been shown to play critical roles in initiating the three-dimensional (3D) spatial organization of DNA, RNA, and protein molecules in the nucleus. These examples, combined with global imaging and genomic experiments, have begun to paint a picture of a broader role for RNA in nuclear organization and to uncover a unifying mechanism that may explain why RNA is a uniquely suited molecule for this role. In this review, we provide an overview of the history of RNA and nuclear structure and discuss key examples of RNA-mediated bodies, the global roles of ncRNAs in shaping nuclear structure, and emerging insights into mechanisms of RNA-mediated nuclear organization.

Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription.

Mammalian genomes are organized into three-dimensional DNA structures called A/B compartments that are associated with transcriptional activity/inactivity. However, whether these structures are simply correlated with gene expression or are permissive/impermissible to transcription has remained largely unknown because we lack methods to measure DNA organization and transcription simultaneously. Recently, we developed RNA & DNA (RD)-SPRITE, which enables genome-wide measurements of the spatial organization of RNA and DNA. Here we show that RD-SPRITE measures genomic structure surrounding nascent pre-mRNAs and maps their spatial contacts. We find that transcription occurs within B compartments-with multiple active genes simultaneously colocalizing within the same B compartment-and at genes proximal to nucleoli. These results suggest that localization near or within nuclear structures thought to be inactive does not preclude transcription and that active transcription can occur throughout the nucleus. In general, we anticipate RD-SPRITE will be a powerful tool for exploring relationships between genome structure and transcription.

SPRITE: a genome-wide method for mapping higher-order 3D interactions in the nucleus using combinatorial split-and-pool barcoding.

A fundamental question in gene regulation is how cell-type-specific gene expression is influenced by the packaging of DNA within the nucleus of each cell. We recently developed Split-Pool Recognition of Interactions by Tag Extension (SPRITE), which enables mapping of higher-order interactions within the nucleus. SPRITE works by cross-linking interacting DNA, RNA and protein molecules and then mapping DNA-DNA spatial arrangements through an iterative split-and-pool barcoding method. All DNA molecules within a cross-linked complex are barcoded by repeatedly splitting complexes across a 96-well plate, ligating molecules with a unique tag sequence, and pooling all complexes into a single well before repeating the tagging. Because all molecules in a cross-linked complex are covalently attached, they will sort together throughout each round of split-and-pool and will obtain the same series of SPRITE tags, which we refer to as a barcode. The DNA fragments and their associated barcodes are sequenced, and all reads sharing identical barcodes are matched to reconstruct interactions. SPRITE accurately maps pairwise DNA interactions within the nucleus and measures higher-order spatial contacts occurring among up to thousands of simultaneously interacting molecules. Here, we provide a detailed protocol for the experimental steps of SPRITE, including a video ( https://youtu.be/6SdWkBxQGlg ). Furthermore, we provide an automated computational pipeline available on GitHub that allows experimenters to seamlessly generate SPRITE interaction matrices starting with raw fastq files. The protocol takes ~5 d from cell cross-linking to high-throughput sequencing for the experimental steps and 1 d for data processing.

Single-cell measurement of higher-order 3D genome organization with scSPRITE.

Although three-dimensional (3D) genome organization is central to many aspects of nuclear function, it has been difficult to measure at the single-cell level. To address this, we developed 'single-cell split-pool recognition of interactions by tag extension' (scSPRITE). scSPRITE uses split-and-pool barcoding to tag DNA fragments in the same nucleus and their 3D spatial arrangement. Because scSPRITE measures multiway DNA contacts, it generates higher-resolution maps within an individual cell than can be achieved by proximity ligation. We applied scSPRITE to thousands of mouse embryonic stem cells and detected known genome structures, including chromosome territories, active and inactive compartments, and topologically associating domains (TADs) as well as long-range inter-chromosomal structures organized around various nuclear bodies. We observe that these structures exhibit different levels of heterogeneity across the population, with TADs representing dynamic units of genome organization across cells. We expect that scSPRITE will be a critical tool for studying genome structure within heterogeneous populations.

Noncoding RNAs: biology and applications-a Keystone Symposia report.

The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium "Noncoding RNAs: Biology and Applications" brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications.