Ribosomal RNA modification in nucleoli and disease

ABSTRACT Nucleoli produce 1-10 million ribosomes per cell. They are membraneless organelles in the nu- cleus formed by liquid-liquid phase separation (LLPS). Dysregulation of ribosome synthesis causes many inherited and somatic diseases. How phase separation contributes to ribosome biogenesis is un-

known. We focus on the dense fibrillar component (DFC) of the nucleolus. Nascent pre-ribosomal RNAs (rRNAs) extend into the DFC as they are modified, folded, and processed with the aid of hun- dreds of biogenesis factors and modifying small nucleolar ribonucleoproteins (snoRNPs). How these processes coalesce to produce properly processed and folded rRNA that is fully modified at some 200

nucleotides is a mystery. To elucidate this complex but efficient process, we need to understand how the DFC is formed, how it enables cotranscriptional RNA modification, and how it is impacted by inher- ited disease. Having identified the snoRNP chaperone Nopp140 and the pseudouridine synthase NAP57 (aka dyskerin and Cbf5) while studying snoRNPs for over 30-years, puts us in a favorable posi-

tion to successfully address these questions in the following three aims. One, determine how the dense fibrillar component of the nucleolus forms, two, identify the mechanism of cotranscriptional RNA modifi- cation, and three, define the impact of dyskeratosis congenita. We show in preliminary data that

Nopp140 concentrates snoRNPs through interaction with their intrinsically disordered domains. We identify a novel disordered C-terminal domain in an RNA polymerase I subunit PAF49 that also binds Nopp140. Based on these data we hypothesize that snoRNPs concentrate at pol I via Nopp140 to co- transcriptionally modify rRNA. This process is supported by LLPS, which forms the DFC. Additionally,

missense mutations in NAP57 and other RNP components impact both processes, LLPS and rRNA modification. In this application, we will test these hypotheses. Understanding biogenesis and function of the DFC, the link between snoRNP abundance and rRNA modification, and the impact of inherited disease provides a basis for therapeutic intervention.