Nucleolar RNA polymerase II drives ribosome biogenesis

Abstract

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...

DNA folds threaten genetic stability and can be leveraged for chemotherapy

“…342,348 In a recent study, R-loops generated by RNA polymerase II localised to non-coding regions in ribosomal DNA and had positive regulatory effects on ribosomal RNA expression by RNA Pol1 in the nucleoli. 351 Aberrant R-loop formation threatens genomic stability, notably inducing replication and translational stress as they form roadblocks to polymerase processivity that promote DNA breaks and activate both ATM and ATR pathways. However, the relationship between DNA damage and R-loop formation is intricate as R-loops trigger DNA damage and conversely DNA damage favours R-loop formation.…”

Biomolecular condensates as arbiters of biochemical reactions inside the nucleus

“…LLPS allows for the enrichment of specific factors while excluding others from the condensates, thereby creating a unique environment that either favours or restricts certain biochemical reactions 5 . LLPS drives the formation of multiple membraneless compartments, including but not limited to P granules, the nucleolus, facultative heterochromatin, transcriptional complexes and DNA repair centres 6 – 14 . These phase-separated compartments are dynamic, and the processes mediating their formation and dissolution are tightly regulated.…”

Nucleolar TFIIE plays a role in ribosomal biogenesis and performance

“…So far, a nucleolar localization and function of general transcription factors of RNA polymerase II was reported for TFIIH (Hoogstraten, Nigg et al, 2002, Iben et al, 2002). A seminal publication indicates that nucleolar RNA polymerase II transcription is essential for proper rRNA biogenesis and processing (Abraham, Khosraviani et al, 2020). The authors showed that RNA polymerase II transcribes intergenic spacers (IGSs) of the rDNA to maintain nucleolar organization and hence rRNA synthesis and maturation.…”