Molecular Mechanisms underlying the assembly and biogenesis of RNA polymerases I and III

• Applicant: Professor Dr. Christoph Engel
• Subject Area: Biochemistry ; Structural Biology ; Cell Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 567301236

The synthesis of RNA from a DNA template is a critical step in gene expression and involves three distinct RNA polymerases (Pols) in eukaryotic cells, which transcribe different classes of RNAs. Recent research has revealed much detail about Pol structure, function, and regulation. However, key aspects of how Pols are assembled into functional enzymes from their up to 17 distinct protein subunits, remain unclear. Here, we present a comprehensive strategy that weaves together two teams and follows both top-down and bottom-up approaches that aim to uncover the fundamental mechanisms underlying the assembly of Pol I and III. We will uncover the hierarchy of Pol I / III subunit assembly in yeast, explore the role of the conserved assembly factor Rbs1 and show how co-translational subunit association is achieved. The current understanding of Pol I and III assembly pathways is built on limited literature and preliminary data. We were already able to show that early assembly steps involve the four subunits shared between Pol I and III, followed by the integration of the second-largest subunit of either polymerase assisted by the protein Rbs1. Rbs1 also stabilizes early subunit mRNA levels and interacts with mRNA metabolism proteins, suggesting a link between Pol assembly and mRNA decay machinery. We envisage Rbs1 as an assembly factor that bridges translated proteins and translating mRNAs through a network of protein-protein and RNA-protein interactions, which we aim to elucidate with the results of the proposed work programme. Specifically, this proposal focuses on two objectives: First, we will validate the hypothesis that Pol I and III subunit assembly occurs co-translationally, facilitated by Rbs1. We will use various molecular approaches and in-cell analysis of mutated Rbs1 to study how these interactions guide the initial formation of Pol complexes. Alongside, we will also assess the composition of early Pol-subassemblies. Second, we will determine the sequence of Pol I/III subunit assembly, identifying the involved chaperones and assembly checkpoints. Cryo-EM reconstructions of Pol I/III assembly intermediates will be determined using already implemented genetic tools that allow the deletion of otherwise essential enzyme subunits. Alongside this top-down approach, early assembly intermediates will be recombinantly obtained in the presence of Rbs1 for their structural and functional characterization. By achieving these aims, we will enhance the understanding of Pol biogenesis mechanisms and lay the groundwork for investigating the cellular consequences of Pol I or III assembly defects. While our focus is on establishing a solid mechanistic framework, future research will explore the biological effects and potential treatments for human diseases related to impaired Pol I or III assembly, such as Treacher-Collins syndrome and Hypomyelinating Leukodystrophy.

• DFG Programme: Research Grants
• International Connection: Poland
• Partner Organisation: Narodowe Centrum Nauki (NCN)
• Cooperation Partner: Professor Tomasz Turowski, Ph.D.