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).

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.

Non-Coding RNA Molecules Connect Calorie Restriction and Lifespan.

Calorie restriction (CR) is a broadly effective environmental intervention that extends life by operating through numerous biological processes. Here, we discuss how non-coding RNA (ncRNA) molecules act as mediators and targets of lifespan-extending CR. We also highlight how these RNA molecules connect CR to its effects on genome stability, cell metabolism, programmed cell death, senescence, cancer, and neurodegeneration. We anticipate that an advanced understanding of the connections between CR and non-coding RNA will provide unique insights into aging mechanisms while pointing to novel approaches aimed at modulating aging and age-related diseases.

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.

Roles for miR-375 in Neuroendocrine Differentiation and Tumor Suppression via Notch Pathway Suppression in Merkel Cell Carcinoma.

Dysfunction of key miRNA pathways regulating basic cellular processes is a common driver of many cancers. However, the biological roles and/or clinical applications of such pathways in Merkel cell carcinoma (MCC), a rare but lethal cutaneous neuroendocrine (NE) malignancy, have yet to be determined. Previous work has established that miR-375 is highly expressed in MCC tumors, but its biological role in MCC remains unknown. Herein, we show that elevated miR-375 expression is a specific feature of well-differentiated MCC cell lines that express NE markers. In contrast, miR-375 is strikingly down-regulated in highly aggressive, undifferentiated MCC cell lines. Enforced miR-375 expression in these cells induced NE differentiation, and opposed cancer cell viability, migration, invasion, and survival, pointing to tumor-suppressive roles for miR-375. Mechanistically, miR-375-driven phenotypes were caused by the direct post-transcriptional repression of multiple Notch pathway proteins (Notch2 and RBPJ) linked to cancer and regulation of cell fate. Thus, we detail a novel molecular axis linking tumor-suppressive miR-375 and Notch with NE differentiation and cancer cell behavior in MCC. Our findings identify miR-375 as a putative regulator of NE differentiation, provide insight into the cell of origin of MCC, and suggest that miR-375 silencing may promote aggressive cancer cell behavior through Notch disinhibition.

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.

Non-coding RNA in neural function, disease, and aging.

Declining brain and neurobiological function is arguably one of the most common features of human aging. The study of conserved aging processes as well as the characterization of various neurodegenerative diseases using different genetic models such as yeast, fly, mouse, and human systems is uncovering links to non-coding RNAs. These links implicate a variety of RNA-regulatory processes, including microRNA function, paraspeckle formation, RNA-DNA hybrid regulation, nucleolar RNAs and toxic RNA clearance, amongst others. Here we highlight these connections and reveal over-arching themes or questions related to recently appreciated roles of non-coding RNA in neural function and dysfunction across lifespan.