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).

RNA-cDNA hybrids mediate transposition via different mechanisms.

Retrotransposons can represent half of eukaryotic genomes. Retrotransposon dysregulation destabilizes genomes and has been linked to various human diseases. Emerging regulators of retromobility include RNA-DNA hybrid-containing structures known as R-loops. Accumulation of these structures at the transposons of yeast 1 (Ty1) elements has been shown to increase Ty1 retromobility through an unknown mechanism. Here, via a targeted genetic screen, we identified the rnh1Δ rad27Δ yeast mutant, which lacked both the Ty1 inhibitor Rad27 and the RNA-DNA hybrid suppressor Rnh1. The mutant exhibited elevated levels of Ty1 cDNA-associated RNA-DNA hybrids that promoted Ty1 mobility. Moreover, in this rnh1Δ rad27Δ mutant, but not in the double RNase H mutant rnh1Δ rnh201Δ, RNA-DNA hybrids preferentially existed as duplex nucleic acid structures and increased Ty1 mobility in a Rad52-dependent manner. The data indicate that in cells lacking RNA-DNA hybrid and Ty1 repressors, elevated levels of RNA-cDNA hybrids, which are associated with duplex nucleic acid structures, boost Ty1 mobility via a Rad52-dependent mechanism. In contrast, in cells lacking RNA-DNA hybrid repressors alone, elevated levels of RNA-cDNA hybrids, which are associated with triplex nucleic acid structures, boost Ty1 mobility via a Rad52-independent process. We propose that duplex and triplex RNA-DNA hybrids promote transposon mobility via Rad52-dependent or -independent mechanisms.

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.

Conserved Pbp1/Ataxin-2 regulates retrotransposon activity and connects polyglutamine expansion-driven protein aggregation to lifespan-controlling rDNA repeats.

Ribosomal DNA (rDNA) repeat instability and protein aggregation are thought to be two major and independent drivers of cellular aging. Pbp1, the yeast ortholog of human ATXN2, maintains rDNA repeat stability and lifespan via suppression of RNA-DNA hybrids. ATXN2 polyglutamine expansion drives neurodegeneration causing spinocerebellar ataxia type 2 and promoting amyotrophic lateral sclerosis. Here, molecular characterization of Pbp1 revealed that its knockout or subjection to disease-modeling polyQ expansion represses Ty1 (Transposons of Yeast) retrotransposons by respectively promoting Trf4-depedendent RNA turnover and Ty1 Gag protein aggregation. This aggregation, but not its impact on retrotransposition, compromises rDNA repeat stability and shortens lifespan by hyper-activating Trf4-dependent turnover of intergenic ncRNA within the repeats. We uncover a function for the conserved Pbp1/ATXN2 proteins in the promotion of retrotransposition, create and describe powerful yeast genetic models of ATXN2-linked neurodegenerative diseases, and connect the major aging mechanisms of rDNA instability and protein aggregation.

Nuclear microtubule filaments mediate non-linear directional motion of chromatin and promote DNA repair.

Damaged DNA shows increased mobility, which can promote interactions with repair-conducive nuclear pore complexes (NPCs). This apparently random mobility is paradoxically abrogated upon disruption of microtubules or kinesins, factors that typically cooperate to mediate the directional movement of macromolecules. Here, we resolve this paradox by uncovering DNA damage-inducible intranuclear microtubule filaments (DIMs) that mobilize damaged DNA and promote repair. Upon DNA damage, relief of centromeric constraint induces DIMs that cooperate with the Rad9 DNA damage response mediator and Kar3 kinesin motor to capture DNA lesions, which then linearly move along dynamic DIMs. Decreasing and hyper-inducing DIMs respectively abrogates and hyper-activates repair. Accounting for DIM dynamics across cell populations by measuring directional changes of damaged DNA reveals that it exhibits increased non-linear directional behavior in nuclear space. Abrogation of DIM-dependent processes or repair-promoting factors decreases directional behavior. Thus, inducible and dynamic nuclear microtubule filaments directionally mobilize damaged DNA and promote repair.

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.

Perinuclear tethers license telomeric DSBs for a broad kinesin- and NPC-dependent DNA repair process.

DNA double-strand breaks (DSBs) are often targeted to nuclear pore complexes (NPCs) for repair. How targeting is achieved and the DNA repair pathways involved in this process remain unclear. Here, we show that the kinesin-14 motor protein complex (Cik1-Kar3) cooperates with chromatin remodellers to mediate interactions between subtelomeric DSBs and the Nup84 nuclear pore complex to ensure cell survival via break-induced replication (BIR), an error-prone DNA repair process. Insertion of a DNA zip code near the subtelomeric DSB site artificially targets it to NPCs hyperactivating this repair mechanism. Kinesin-14 and Nup84 mediate BIR-dependent repair at non-telomeric DSBs whereas perinuclear telomere tethers are only required for telomeric BIR. Furthermore, kinesin-14 plays a critical role in telomerase-independent telomere maintenance. Thus, we uncover roles for kinesin and NPCs in DNA repair by BIR and reveal that perinuclear telomere anchors license subtelomeric DSBs for this error-prone DNA repair mechanism.

Roles for Pbp1 and caloric restriction in genome and lifespan maintenance via suppression of RNA-DNA hybrids.

Intergenic transcription within repetitive loci such as the ribosomal DNA (rDNA) repeats of yeast commonly triggers aberrant recombination. Major mechanisms suppressing aberrant rDNA recombination rely on chromatin silencing or RNAPII repression at intergenic spacers within the repeats. We find ancient processes operating at rDNA intergenic spacers and other loci to maintain genome stability via repression of RNA-DNA hybrids. The yeast Ataxin-2 protein Pbp1 binds noncoding RNA, suppresses RNA-DNA hybrids, and prevents aberrant rDNA recombination. Repression of RNA-DNA hybrids in Pbp1-deficient cells through RNaseH overexpression, deletion of the G4DNA-stabilizing Stm1, or caloric restriction operating via RNaseH/Pif1 restores rDNA stability. Pbp1 also limits hybrids at non-rDNA G4DNA loci including telomeres. Moreover, cells lacking Pbp1 have a short replicative lifespan that is extended upon hybrid suppression. Thus, we find roles for Pbp1 in genome maintenance and reveal that caloric restriction counteracts Pbp1 deficiencies by engaging RNaseH and Pif1.

Enforcement of a lifespan-sustaining distribution of Sir2 between telomeres, mating-type loci, and rDNA repeats by Rif1.

Telomere dysfunction is linked with genome instability and premature aging. Roles for sirtuin proteins at telomeres are thought to promote lifespan in yeast and mammals. However, replicative lifespan of the budding yeast Saccharomyces cerevisiae shortens upon deletion of Rif1, a protein that limits the recruitment of the sirtuin histone deacetylase Sir2 to telomeres. Here we show that Rif1 maintains replicative lifespan by ultimately stabilizing another age-related chromosomal domain harboring the ribosomal DNA (rDNA) repeats. Deletion of Rif1 increases Sir2 localization to telomeres and the silent mating-type loci, while releasing a pool of the histone deacetylase from the intergenic spacer 1 (IGS1) of rDNA. This is accompanied by a disruption of IGS1 silent chromatin assembly and increases in aberrant recombination within rDNA repeats. Lifespan defects linked with Rif1 deletion are abolished if rDNA repeats are forcibly stabilized via deletion of the replication fork-blocking protein Fob1. In addition, Sir2 overexpression prevents Rif1 deletion from disrupting Sir2 at IGS1 and shortening lifespan. Moreover, subjecting cells lacking Rif1 to caloric restriction increases IGS1 histone deacetylation and lifespan, while uncovering novel genetic interactions between RIF1 and SIR2. Our data indicate that Rif1 maintains lifespan-sustaining levels of Sir2 at rDNA by preventing excessive recruitment of the histone deacetylase to telomeric and silent mating-type loci. As sirtuin histone deacetylases, such as Sir2 or mammalian SIRT6, each operate at multiple age-related loci, we propose that factors limiting the localization of sirtuins to certain age-related loci can promote lifespan-sustaining roles of these sirtuins elsewhere in the genome.

Target identification by chromatographic co-elution: monitoring of drug-protein interactions without immobilization or chemical derivatization.

Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely monitoring the interaction of drugs with cellular proteins under nearly physiological conditions in vitro based on simple liquid chromatographic separations of cell-free lysates. Correlative proteomic analysis of drug-bound protein fractions by shotgun sequencing is then performed to identify candidate target(s). The method is highly reproducible, does not require immobilization or derivatization of drug or protein, and is applicable to diverse natural products and synthetic compounds. The capability of TICC to detect known drug-protein target physical interactions (K(d) range: micromolar to nanomolar) is demonstrated both qualitatively and quantitatively. We subsequently used TICC to uncover the sterol biosynthetic enzyme Erg6p as a novel putative anti-fungal target. Furthermore, TICC identified Asc1 and Dak1, a core 40 S ribosomal protein that represses gene expression, and dihydroxyacetone kinase involved in stress adaptation, respectively, as novel yeast targets of a dopamine receptor agonist.

Perinuclear cohibin complexes maintain replicative life span via roles at distinct silent chromatin domains.

Heterochromatin, or silent chromatin, preferentially resides at the nuclear envelope. Telomeres and rDNA repeats are the two major perinuclear silent chromatin domains of Saccharomyces cerevisiae. The Cohibin protein complex maintains rDNA repeat stability in part through silent chromatin assembly and perinuclear rDNA anchoring. We report here a role for Cohibin at telomeres and show that functions of the complex at chromosome ends and rDNA maintain replicative life span. Cohibin binds LEM/SUN domain-containing nuclear envelope proteins and telomere-associated factors. Disruption of Cohibin or the envelope proteins abrogates telomere localization and silent chromatin assembly within subtelomeres. Loss of Cohibin limits Sir2 histone deacetylase localization to chromosome ends, disrupts subtelomeric DNA stability, and shortens life span even when rDNA repeats are stabilized. Restoring telomeric Sir2 concentration abolishes chromatin and life span defects linked to the loss of telomeric Cohibin. Our work uncovers roles for Cohibin complexes and reveals relationships between nuclear compartmentalization, chromosome stability, and aging.

Recent advances and method development for drug target identification.

Although it is commonly recognized that most drugs cause inhibition or activation of function by physically binding to one or more gene products, the direct interactions of bioactive small molecules with specific gene products, or targets, is often not well characterized. From a therapeutic perspective, it is nevertheless essential to know a drug's binding partner(s) to understand the mechanism of action and anticipate possible side effects to avoid costly clinical failures. This knowledge is increasingly important as the prevalence of polypharmacy expands to include drugs that engage multiple targets. This review provides a succinct overview of several recent approaches that employ genetics, proteomics, expression profiling or bioinformatics procedures for the systematic characterization of the targets of bioactive compounds. The continuous improvement and advancement of existing technologies is critically discussed and we offer a perspective on the future of innovative emerging new generation technologies.