Nucleolar RNA polymerase II drives ribosome biogenesis.

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.

Integration of DNA damage responses with dynamic spatial genome organization.

The maintenance of genome stability and cellular homeostasis depends on the temporal and spatial coordination of successive events constituting the classical DNA damage response (DDR). Recent findings suggest close integration and coordination of DDR signaling with specific cellular processes. The mechanisms underlying such coordination remain unclear. We review emerging crosstalk between DNA repair factors, chromatin remodeling, replication, transcription, spatial genome organization, cytoskeletal forces, and liquid-liquid phase separation (LLPS) in mediating DNA repair. We present an overarching DNA repair framework within which these dynamic processes intersect in nuclear space over time. Collectively, this interplay ensures the efficient assembly of DNA repair proteins onto shifting genome structures to preserve genome stability and cell survival.

Excessive transcription-replication conflicts are a vulnerability of BRCA1-mutant cancers.

BRCA1 mutations are associated with increased breast and ovarian cancer risk. BRCA1-mutant tumors are high-grade, recurrent, and often become resistant to standard therapies. Herein, we performed a targeted CRISPR-Cas9 screen and identified MEPCE, a methylphosphate capping enzyme, as a synthetic lethal interactor of BRCA1. Mechanistically, we demonstrate that depletion of MEPCE in a BRCA1-deficient setting led to dysregulated RNA polymerase II (RNAPII) promoter-proximal pausing, R-loop accumulation, and replication stress, contributing to transcription-replication collisions. These collisions compromise genomic integrity resulting in loss of viability of BRCA1-deficient cells. We also extend these findings to another RNAPII-regulating factor, PAF1. This study identifies a new class of synthetic lethal partners of BRCA1 that exploit the RNAPII pausing regulation and highlight the untapped potential of transcription-replication collision-inducing factors as unique potential therapeutic targets for treating cancers associated with BRCA1 mutations.

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells.

Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.

Phase Separation as a Melting Pot for DNA Repeats.

Genome expression and stability are dependent on biological processes that control repetitive DNA sequences and nuclear compartmentalization. The phase separation of macromolecules has recently emerged as a major player in the control of biological pathways. Here, we summarize recent studies that collectively reveal intersections between phase separation, repetitive DNA elements, and nuclear compartments. These intersections modulate fundamental processes, including gene expression, DNA repair, and cellular lifespan, in the context of health and diseases such as cancer and neurodegeneration.

The nuclear envelope in genome organization, expression and stability.

Non-random positioning of chromosomal domains relative to each other and to nuclear landmarks is a common feature of eukaryotic genomes. In particular, the distribution of DNA loci relative to the nuclear periphery has been linked to both transcriptional activation and repression. Nuclear pores and other integral membrane protein complexes are key players in the dynamic organization of the genome in the nucleus, and recent advances in our understanding of the molecular networks that organize genomes at the nuclear periphery point to a further role for non-random locus positioning in DNA repair, recombination and stability.

Catch the live show: Visualizing damaged DNA in vivo.

The health of an organism is intimately linked to its ability to repair damaged DNA. Importantly, DNA repair processes are highly dynamic. This highlights the necessity of characterizing DNA repair in live cells. Advanced genome editing and imaging approaches allow us to visualize damaged DNA and its associated factors in real time. Here, we summarize both established and recent methods that are used to induce DNA damage and visualize damaged DNA and its repair in live cells.

Intersection of calorie restriction and magnesium in the suppression of genome-destabilizing RNA-DNA hybrids.

Dietary calorie restriction is a broadly acting intervention that extends the lifespan of various organisms from yeast to mammals. On another front, magnesium (Mg2+) is an essential biological metal critical to fundamental cellular processes and is commonly used as both a dietary supplement and treatment for some clinical conditions. If connections exist between calorie restriction and Mg2+ is unknown. Here, we show that Mg2+, acting alone or in response to dietary calorie restriction, allows eukaryotic cells to combat genome-destabilizing and lifespan-shortening accumulations of RNA-DNA hybrids, or R-loops. In an R-loop accumulation model of Pbp1-deficient Saccharomyces cerevisiae, magnesium ions guided by cell membrane Mg2+ transporters Alr1/2 act via Mg2+-sensitive R-loop suppressors Rnh1/201 and Pif1 to restore R-loop suppression, ribosomal DNA stability and cellular lifespan. Similarly, human cells deficient in ATXN2, the human ortholog of Pbp1, exhibit nuclear R-loop accumulations repressible by Mg2+ in a process that is dependent on the TRPM7 Mg2+ transporter and the RNaseH1 R-loop suppressor. Thus, we identify Mg2+ as a biochemical signal of beneficial calorie restriction, reveal an R-loop suppressing function for human ATXN2 and propose that practical magnesium supplementation regimens can be used to combat R-loop accumulation linked to the dysfunction of disease-linked human genes.

Role for perinuclear chromosome tethering in maintenance of genome stability.

Repetitive DNA sequences, which constitute half the genome in some organisms, often undergo homologous recombination. This can instigate genomic instability resulting from a gain or loss of DNA. Assembly of DNA into silent chromatin is generally thought to serve as a mechanism ensuring repeat stability by limiting access to the recombination machinery. Consistent with this notion is the observation, in the budding yeast Saccharomyces cerevisiae, that stability of the highly repetitive ribosomal DNA (rDNA) sequences requires a Sir2-containing chromatin silencing complex that also inhibits transcription from foreign promoters and transposons inserted within the repeats by a process called rDNA silencing. Here we describe a protein network that stabilizes rDNA repeats of budding yeast by means of interactions between rDNA-associated silencing proteins and two proteins of the inner nuclear membrane (INM). Deletion of either the INM or silencing proteins reduces perinuclear rDNA positioning, disrupts the nucleolus-nucleoplasm boundary, induces the formation of recombination foci, and destabilizes the repeats. In addition, artificial targeting of rDNA repeats to the INM suppresses the instability observed in cells lacking an rDNA-associated silencing protein that is typically required for peripheral tethering of the repeats. Moreover, in contrast to Sir2 and its associated nucleolar factors, the INM proteins are not required for rDNA silencing, indicating that Sir2-dependent silencing is not sufficient to inhibit recombination within the rDNA locus. These findings demonstrate a role for INM proteins in the perinuclear localization of chromosomes and show that tethering to the nuclear periphery is required for the stability of rDNA repeats. The INM proteins studied here are conserved and have been implicated in chromosome organization in metazoans. Our results therefore reveal an ancient mechanism in which interactions between INM proteins and chromosomal proteins ensure genome stability.

DNA double-strand break-capturing nuclear envelope tubules drive DNA repair.

Current models suggest that DNA double-strand breaks (DSBs) can move to the nuclear periphery for repair. It is unclear to what extent human DSBs display such repositioning. Here we show that the human nuclear envelope localizes to DSBs in a manner depending on DNA damage response (DDR) kinases and cytoplasmic microtubules acetylated by α-tubulin acetyltransferase-1 (ATAT1). These factors collaborate with the linker of nucleoskeleton and cytoskeleton complex (LINC), nuclear pore complex (NPC) protein NUP153, nuclear lamina and kinesins KIF5B and KIF13B to generate DSB-capturing nuclear envelope tubules (dsbNETs). dsbNETs are partly supported by nuclear actin filaments and the circadian factor PER1 and reversed by kinesin KIFC3. Although dsbNETs promote repair and survival, they are also co-opted during poly(ADP-ribose) polymerase (PARP) inhibition to restrain BRCA1-deficient breast cancer cells and are hyper-induced in cells expressing the aging-linked lamin A mutant progerin. In summary, our results advance understanding of nuclear structure-function relationships, uncover a nuclear-cytoplasmic DDR and identify dsbNETs as critical factors in genome organization and stability.

SARS-CoV-2 targets ribosomal RNA biogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hinders host gene expression, curbing defenses and licensing viral protein synthesis and virulence. During SARS-CoV-2 infection, the virulence factor non-structural protein 1 (Nsp1) targets the mRNA entry channel of mature cytoplasmic ribosomes, limiting translation. We show that Nsp1 also restrains translation by targeting nucleolar ribosome biogenesis. SARS-CoV-2 infection disrupts 18S and 28S ribosomal RNA (rRNA) processing. Expression of Nsp1 recapitulates the processing defects. Nsp1 abrogates rRNA production without altering the expression of critical processing factors or nucleolar organization. Instead, Nsp1 localizes to the nucleolus, interacting with precursor-rRNA and hindering its maturation separately from the viral protein's role in restricting mature ribosomes. Thus, SARS-CoV-2 Nsp1 limits translation by targeting ribosome biogenesis and mature ribosomes. These findings revise our understanding of how SARS-CoV-2 Nsp1 controls human protein synthesis, suggesting that efforts to counter Nsp1's effect on translation should consider the protein's impact from ribosome manufacturing to mature ribosomes.

Protocol to use RNaseH1-based CRISPR to modulate locus-associated R-loops.

Modulating R-loop triplex nucleic acid structures reveals their roles across the genome. However, common approaches cannot ascribe functions to R-loops in a locus-associated manner. This protocol presents the use of a locus-associated R-loop-modulating system (dubbed LasR), which employs an inducible RNaseH1-EGFP-dCas9 chimaera. We detail the in silico design of sgRNAs and their transfection with the chimaera, and outline steps confirming RNaseH1-EGFP-dCas9 expression, localization, locus-targeted association, and R-loop modulation in cis or trans using immunoblotting, microscopy, and chromatin and DNA-RNA immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Abraham et al. (2020).

A small molecule antagonist of SMN disrupts the interaction between SMN and RNAP II.

Survival of motor neuron (SMN) functions in diverse biological pathways via recognition of symmetric dimethylarginine (Rme2s) on proteins by its Tudor domain, and deficiency of SMN leads to spinal muscular atrophy. Here we report a potent and selective antagonist with a 4-iminopyridine scaffold targeting the Tudor domain of SMN. Our structural and mutagenesis studies indicate that both the aromatic ring and imino groups of compound 1 contribute to its selective binding to SMN. Various on-target engagement assays support that compound 1 specifically recognizes SMN in a cellular context and prevents the interaction of SMN with the R1810me2s of RNA polymerase II subunit POLR2A, resulting in transcription termination and R-loop accumulation mimicking SMN depletion. Thus, in addition to the antisense, RNAi and CRISPR/Cas9 techniques, potent SMN antagonists could be used as an efficient tool to understand the biological functions of SMN.

HIF activation by pH-dependent nucleolar sequestration of VHL.

Hypoxia and acidosis occur in a wide variety of physiological and pathological settings that include muscle stress, tumour development and ischaemic disorders. A central element in the adaptive response to cellular hypoxia is HIF (hypoxia-inducible factor), a transcription factor that activates an array of genes implicated in oxygen homeostasis, tumour vascularization and ischaemic preconditioning. HIF is activated by hypoxia, but undergoes degradation by the VHL (von Hippel-Lindau) tumour suppressor protein in the presence of oxygen. Here, we demonstrate that hypoxia induction or normoxic acidosis can neutralize the function of VHL by triggering its nucleolar sequestration, a regulatory mechanism of protein function that is observed rarely. VHL is confined to nucleoli until neutral pH conditions are reinstated. Nucleolar sequestration of VHL enables HIF to evade destruction in the presence of oxygen and activate its target genes. Our findings suggest that an increase in hydrogen ions elicits a transient and reversible loss of VHL function by promoting its nucleolar sequestration.

Interphase microtubules in nuclear organization and genome maintenance.

Microtubules are major cytoskeletal components mediating fundamental cellular processes, including cell division. Recent evidence suggests that microtubules also regulate the nucleus during the cell cycle's interphase stage. Deciphering such roles of microtubules should uncover direct crosstalk between the nucleus and cytoplasm, impacting genome function and organismal health. Here, we review emerging roles for microtubules in interphase genome regulation. We explore how microtubules exert cytoplasmic forces on the nucleus or transport molecular cargo, including DNA, into or within the nucleus. We also describe how microtubules perform these functions by establishing transient or stable connections with nuclear envelope elements. Lastly, we discuss how the regulation of the nucleus by microtubules impacts genome organization and repair. Together, the literature indicates that interphase microtubules are critical regulators of nuclear structure and genome stability.

RNF168 regulates R-loop resolution and genomic stability in BRCA1/2-deficient tumors.

Germline mutations in BRCA1 and BRCA2 (BRCA1/2) genes considerably increase breast and ovarian cancer risk. Given that tumors with these mutations have elevated genomic instability, they exhibit relative vulnerability to certain chemotherapies and targeted treatments based on poly (ADP-ribose) polymerase (PARP) inhibition. However, the molecular mechanisms that influence cancer risk and therapeutic benefit or resistance remain only partially understood. BRCA1 and BRCA2 have also been implicated in the suppression of R-loops, triple-stranded nucleic acid structures composed of a DNA:RNA hybrid and a displaced ssDNA strand. Here, we report that loss of RNF168, an E3 ubiquitin ligase and DNA double-strand break (DSB) responder, remarkably protected Brca1-mutant mice against mammary tumorigenesis. We demonstrate that RNF168 deficiency resulted in accumulation of R-loops in BRCA1/2-mutant breast and ovarian cancer cells, leading to DSBs, senescence, and subsequent cell death. Using interactome assays, we identified RNF168 interaction with DHX9, a helicase involved in the resolution and removal of R-loops. Mechanistically, RNF168 directly ubiquitylated DHX9 to facilitate its recruitment to R-loop-prone genomic loci. Consequently, loss of RNF168 impaired DHX9 recruitment to R-loops, thereby abrogating its ability to resolve R-loops. The data presented in this study highlight a dependence of BRCA1/2-defective tumors on factors that suppress R-loops and reveal a fundamental RNF168-mediated molecular mechanism that governs cancer development and vulnerability.

Regulation of ubiquitin ligase dynamics by the nucleolus.

Cellular pathways relay information through dynamic protein interactions. We have assessed the kinetic properties of the murine double minute protein (MDM2) and von Hippel-Lindau (VHL) ubiquitin ligases in living cells under physiological conditions that alter the stability of their respective p53 and hypoxia-inducible factor substrates. Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded. The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability. After signal termination, the nucleolus is able to rapidly release these proteins from static detention, thereby restoring their high mobility profiles. A protein surface region of VHL's beta-sheet domain was identified as a discrete [H+]-responsive nucleolar detention signal that targets the VHL/Cullin-2 ubiquitin ligase complex to nucleoli in response to physiological fluctuations in environmental pH. Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.

Identification of a common subnuclear localization signal.

Proteins share peptidic sequences, such as a nuclear localization signal (NLS), which guide them to particular membrane-bound compartments. Similarities have also been observed within different classes of signals that target proteins to membrane-less subnuclear compartments. Common localization signals affect spatial and temporal subcellular organization and are thought to allow the coordinated response of different molecular networks to a given signaling cue. Here we identify a higher-order and predictive code, {[RR(I/L)X(3)r]((n, n > or = 1))+[L(phi/N)(V/L)]((n,n>1))}, that establishes high-affinity interactions between a group of proteins and the nucleolus in response to a specific signal. This position-independent code is referred to as a nucleolar detention signal regulated by H(+) (NoDS(H+)) and the class of proteins includes the cIAP2 apoptotic regulator, VHL ubiquitylation factor, HSC70 heat shock protein and RNF8 transcription regulator. By identifying a common subnuclear targeting consensus sequence, our work reveals rules governing the dynamics of subnuclear organization and ascribes new modes of regulation to several proteins with diverse steady-state distributions and dynamic properties.

Biomolecular condensates as arbiters of biochemical reactions inside the nucleus.

Liquid-liquid phase separation (LLPS) has emerged as a central player in the assembly of membraneless compartments termed biomolecular condensates. These compartments are dynamic structures that can condense or dissolve under specific conditions to regulate molecular functions. Such properties allow biomolecular condensates to rapidly respond to changing endogenous or environmental conditions. Here, we review emerging roles for LLPS within the nuclear space, with a specific emphasis on genome organization, expression and repair. Our review highlights the emerging notion that biomolecular condensates regulate the sequential engagement of molecules in multistep biological processes.

Mobility and Repair of Damaged DNA: Random or Directed?

The increased mobility of damaged DNA within the nucleus can promote genome stability and cell survival. New cell biology approaches have indicated that damaged DNA mobility exhibits random and directed movements during DNA repair. Here, we review recent studies that collectively reveal that cooperation between different molecular mechanisms, which underlie increases in the random and directional motion of damaged DNA, can promote genome repair. We also review the latest approaches that can be used to distinguish between random and directed motions of damaged DNA or other biological molecules. Detailed understanding of the mechanisms behind the increased motion of damaged DNA within the nucleus will reveal more of the secrets of genome organization and stability while potentially pointing to novel research and therapeutic tools.