Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli 1,2. 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. Various proteins self-organize via liquid-liquid phase separation (LLPS) into nucleolar subdomains, which are needed for highly stereotyped ribosome assembly 1,2. At fibrillar centres in the heart of mammalian nucleoli, the major rRNA molecules needed to assemble ribosomes are generated by Pol-I-dependent transcription of rRNA genes within ribosomal DNA (rDNA) repeats 1,3. Within rDNA, rRNA genes are separated by large intergenic spacers (IGSs) (Extended Data Fig. 1a). At nucleolar rRNA genes, Pol I synthesizes precursor rRNAs (pre-rRNAs) that are processed into mature 28S, 18S and 5.8S rRNA molecules as they migrate to the granular component at the nucleolar periphery. Outside nucleoli, Pol III synthesizes 5S rRNA molecules that are targeted to nucleoli for processing. Mature rRNAs are packaged into 40S and 60S ribosomal subunits for export to the cytoplasm. Traditionally, the nucleolar Pol I and nucleoplasmic Pol III are viewed as the sole mammalian RNA polymerases that directly mediate housekeeping ribosome biogenesis. Interestingly, in the budding yeast Saccharomyces cerevisiae, Pol II is physically enriched at rDNA IGSs, but this phenomenon is deleterious because it drives ageing without affecting rRNA expression 3-5. It is unclear whether nucleolar Pol II exists in higher organisms or directly promotes ribosome biogenesis in any species. Active Pol II at rDNA IGSs To investigate whether Pol II exists within human nucleoli, we first used immunofluorescence coupled to super-resolution microscopy. Within nucleoli, which we...

Genetic loci are non-randomly arranged in the nucleus of the cell. This order, which is important to overall genome expression and stability, is maintained by a growing number of factors including the nuclear envelope, various genetic elements and dedicated protein complexes. Here, we review evidence supporting roles for non-coding RNAs (ncRNAs) in the regulation of spatial genome organization and its impact on gene expression and cell survival. Specifically, we discuss how ncRNAs from single-copy and repetitive DNA loci contribute to spatial genome organization by impacting perinuclear chromosome tethering, major nuclear compartments, chromatin looping, and various chromosomal structures. Overall, our analysis of the literature highlights central functions for ncRNAs and their transcription in the modulation of spatial genome organization with connections to human health and disease.

R-loops exert varied beneficial or detrimental effects. To assess the function of an R-loop at a specific genetic locus, we had developed an inducible RNaseH1-EGFP-dCas9 (RED) protein chimaera as part of a locus-associated R-loop-modulating system (LasR). LasR is compatible with R-loop modulation in trans, which targets RED to one locus to repress R-loops at another spatially proximal site. Here we use the LasR system for R-loop modulation in cis, which consists of targeting RED directly to an R-loop. The combination of LasR in cis and trans will be essential to ascribe functions to specific R-loops within varied molecular contexts and study designs.

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Highlights The matrix protein collagen VIII influences conduit artery compliance. Collagen VIII negatively regulates elastin. Collagen VIII KO increases elastin synthesis and content in carotid arteries. Collagen VIII and elastin exert opposing regulation of smooth muscle cell phenotypes.