The NuRD chromatin remodeling complex regulates tRNA genes through direct interactions with RNA polymerase III [Nuclei run-on (nascent RNA) profiling]

RNA polymerase III (Pol III) synthesizes noncoding RNAs essential for translation, RNA processing, and transcription regulation. Its activity aligns with cellular growth and nutrient availability, and its dysregulation is linked to diseases like breast cancer and leukodystrophy. Despite its importance, Pol III regulators remain largely unidentified due to a lack of exploration of Pol III-centered protein-protein interactions (PPIs). To address this need, we conducted high throughput proteomic experiments and identified over 300 novel Pol III interactors, including Pol II-related transcription factors, nuclear import factors, and splicing factors. Most notably, our proteomic survey uncovers complex-complex interactions between Pol III and the NuRD (Nucleosome Remodeling and Deacetylase) complex. NuRD complex is a known Pol II co-repressor, but its recruitment to and effect on Pol III remains uncharacterized. Using innovative large-scale NuRD binding pattern meta-study, NuRD ChIP-seq combined with Pol III inhibition, and Pol III activity profiling in mammalian cell lines, we revealed that the NuRD is recruited by Pol III itself and fine-tunes Pol III transcription activity. We found that NuRD exhibits a binding preference for highly active Pol III genes, and inhibition of Pol III transcription reduced the enrichment of NuRD subunits. Functionally, inhibiting NuRD deacetylase activity releases its inhibition on Pol III transcription and increases its dynamic range. In addition, our ATAC-seq analysis suggests NuRD is potentially involved in establishing nucleosome positioning through its ATP-dependent remodeler activity. Beyond identifying NuRD as a novel Pol III regulator, our survey of Pol III interactions unexpectedly identifies physical interactions between Pol III and mRNA splicing factors. We demonstrate that Pol III depletion leads to rewiring of splicing efficiencies for specific mRNA subpopulations, suggesting a previously unrecognized interplay between Pol III and RNA processing factors. Together, these findings advance our understanding of Pol III interactome, uncovering new layers of transcriptional control and unexpected functional consequences of Pol III interactions in human cells.