Principles of Co-Transcriptional Ribosome Assembly Nucleation and Quality Control

Author

• Rafal Piwowarczyk

Date of Award

2026

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Sebastian Klinge

Abstract

The ribosome is a universally conserved macromolecular machine responsible for protein synthesis across all domains of life. It is composed of two subunits, the small ribosomal subunit (SSU) and the large ribosomal subunit (LSU), which come together during translation to form the functional ribosome. In Saccharomyces cerevisiae, the mature SSU consists of the 18𝑆 ribosomal RNA (rRNA) and 33 ribosomal proteins, while the LSU contains the 5𝑆, 5.8𝑆, and 25𝑆 rRNAs alongside 46 different ribosomal proteins. Crucially, the key functional centers of the ribosome are composed of rRNA and are distributed throughout both subunits. This mechanistic dependence on the rRNA structure emphasizes the importance of its correct folding during biogenesis. Ribosome assembly is a highly regulated, hierarchical process that begins with the transcription of the precursor rRNA (pre-rRNA) in the nucleolus before transitioning through the nucleoplasm and cytoplasm, where mature ribosomes enter the translation pool. The process requires the coordinated action of over 200 assembly factors that cleave, modify, and remodel the pre-rRNA transcript, guiding its folding. While the last decade has seen significant advances in our understanding of the post- transcriptional maturation of the LSU, the molecular transitions governing its co-transcriptional biogenesis remain poorly defined. The research presented in my doctoral work elucidates the earliest stages of co-transcriptional LSU assembly, beginning with the stabilization of the 5′ end of the pre-rRNA transcript. Through a combination of biochemical and structural biology methods, I isolated and structurally characterized a series of novel co-transcriptional LSU assembly intermediates that reveal the stepwise maturation of the pre-ribosomal particle. Characterization of the Pwp1 RNP, the smallest known LSU assembly intermediate, reveals the function of Pwp1 as a structural scaffold that autonomously nucleates the assembly of the 5′ end of the LSU pre-rRNA (Chapter 2). This particle subsequently undergoes modular expansion through the installation of the Internal Transcribed Spacer 2 (ITS2) and the Nop12-mediated docking of premature 5.8𝑆 rRNA (Chapter 3). The following intermediate, the Noc1–Noc2 RNP, characterized by Zahra A. Sanghai, captures the independent folding of another major rRNA module (Domain II) (Chapter 4). The molecular logic underpinning these transitions is further examined through rRNA engineering and biochemical experiments described in Chapter 5, leading to a revised model of co-transcriptional LSU assembly (Chapter 6). Together, these findings offer unprecedented insight into the molecular logic of early co- transcriptional LSU assembly, describing a series of molecular checkpoints used to interrogate the correct folding of pre-rRNA modules. The intermediates and regulatory principles characterized in this work establish a foundation for future structural and functional studies of the early co- transcriptional LSU assembly pathway.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

License and Reuse Information

This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

Recommended Citation

Piwowarczyk, Rafal, "Principles of Co-Transcriptional Ribosome Assembly Nucleation and Quality Control" (2026). Student Theses and Dissertations. 862.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/862