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.

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.

Mammalian genomes encode thousands of long non-coding RNAs (lncRNAs) that play important roles in diverse biological processes. As a class, lncRNAs are generally enriched in the nucleus and specifically within the chromatin-associated fraction. Consistent with their localization, many lncRNAs have been implicated in the regulation of gene expression and in shaping three-dimensional nuclear organization. Here, we discuss the evidence that many nuclear-retained lncRNAs can interact with various chromatin regulatory proteins and recruit them to specific sites on DNA to regulate gene expression. Furthermore, we discuss the role of specific lncRNAs in shaping nuclear organization and their emerging mechanisms. Based on these examples, we propose a model that explains how lncRNAs may shape aspects of nuclear organization to regulate gene expression.

No abstract

The nucleus is a highly organized arrangement of RNA, DNA, and protein molecules that are compartmentalized within three-dimensional (3D) structures involved in shared functional and regulatory processes. Although RNA has long been proposed to play a global role in organizing nuclear structure, exploring the role of RNA in shaping nuclear structure has remained a challenge because no existing methods can simultaneously measure RNA-RNA, RNA-DNA, and DNA-DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the location of all RNAs relative to DNA and other RNAs. Using this approach, we identify many RNAs that are localized near their transcriptional loci (RNA-DNA) together with other diffusible ncRNAs (RNA-RNA) within higher-order DNA structures (DNA-DNA). These RNA-chromatin compartments span three major classes of nuclear functions: RNA processing (including ribosome biogenesis, mRNA splicing, snRNA biogenesis, and histone mRNA processing), heterochromatin assembly, and gene regulation. More generally, we identify hundreds of ncRNAs that form stable nuclear compartments in spatial proximity to their transcriptional loci. We find that dozens of nuclear compartments require RNA to guide protein regulators into these 3D structures, and focusing on several ncRNAs, we show that these ncRNAs specifically regulate heterochromatin assembly and the expression of genes contained within these compartments. Together, our results demonstrate a unique mechanism by which RNA acts to shape nuclear structure by forming high concentration territories immediately upon transcription, binding to diffusible regulators, and guiding them into spatial compartments to regulate a wide range of essential nuclear functions.

In eukaryotes, the nucleus is organized into a three dimensional structure consisting of both local interactions such as those between enhancers and promoters, and long-range higher-order structures such as nuclear bodies. This organization is central to many aspects of nuclear function, including DNA replication, transcription, and cell cycle progression. Nuclear structure intrinsically occurs within single cells; however, measuring such a broad spectrum of 3D DNA interactions on a genome-wide scale and at the single cell level has been a great challenge. To address this, we developed single-cell split-pool recognition of interactions by tag extension (scSPRITE), a new method that enables measurements of genome-wide maps of 3D DNA structure in thousands of individual nuclei. scSPRITE maximizes the number of DNA contacts detected per cell enabling high-resolution genome structure maps within each cells and is easy-touse and cost-effective. scSPRITE accurately detects chromosome territories, active and inactive compartments, topologically associating domains (TADs), and higher-order structures within .

SUMMARY In cells, specific regulators often compete for limited amounts of a core enzymatic resource. It is typically assumed that competition leads to partitioning of core enzyme molecules among regulators at constant levels. Alternatively, however, different regulatory species could time share, or take turns utilizing, the core resource. Using quantitative time-lapse microscopy, we analyzed sigma factor activity dynamics, and their competition for RNA polymerase, in individual Bacillus subtilis cells under energy stress. Multiple alternative sigma factors were activated in ~1-hr pulses in stochastic and repetitive fashion. Pairwise analysis revealed that two sigma factors rarely pulse simultaneously and that some pairs are anti-correlated, indicating that RNAP utilization alternates among different sigma factors. Mathematical modeling revealed how stochastic time-sharing dynamics can emerge from pulse-generating sigma factor regulatory circuits actively competing for RNAP. Time sharing provides a mechanism for cells to dynamically control the distribution of cell states within a population. Since core molecular components are limiting in many other systems, time sharing may represent a general mode of regulation.

c Bacteria use a chemical communication process called quorum sensing to monitor cell density and to alter behavior in response to fluctuations in population numbers. Previous studies with Vibrio harveyi have shown that LuxR, the master quorum-sensing regulator, activates and represses >600 genes. These include six genes that encode homologs of the Escherichia coli Bet and ProU systems for synthesis and transport, respectively, of glycine betaine, an osmoprotectant used during osmotic stress. Here we show that LuxR activates expression of the glycine betaine operon betIBA-proXWV, which enhances growth recovery under osmotic stress conditions. BetI, an autorepressor of the V. harveyi betIBA-proXWV operon, activates the expression of genes encoding regulatory small RNAs that control quorum-sensing transitions. Connecting quorum-sensing and glycine betaine pathways presumably enables V. harveyi to tune its execution of collective behaviors to its tolerance to stress.