Nucleolar RNA polymerase II drives ribosome biogenesis

Proteins are universally manufactured by ribosomes, which are assembled within major nuclear compartments called nucleoli(1,2). Current paradigms suggest that only RNA polymerase I and III (Pol I and Pol III) directly mediate the expression of ribosomal RNA (rRNA) components of ribosomes. Here, we show that RNA polymerase II (Pol II) inside of human nucleoli operates directly near genes encoding rRNA to drive its expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates an R-loop shield 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. The disruptive sincRNAs are unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma, or locus-associated R-loop repression via an RNaseH1-EGFP-dCas9 system (denoted ‘red laser’). We reveal a nucleolar Pol II-dependent mechanism driving ribosome biogenesis, identify disease-associated disruption of nucleoli by non-coding RNAs, and establish locus-targeted R-loop modulation. Our findings revise the current schema 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 in health and disease.