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Abstract

In eukaryotes, the task of RNA synthesis is divided between three RNA polymerases (RNAPs). RNA polymerase I (Pol I) produces ribosomal RNA (rRNA), and Pol III specialises in making short, non-transcribed RNAs, which together constitute the majority of the RNA pool. Dysregulation of Pol I and Pol III function leads to complex diseases such as cancer or developmental disorders like Treacher Collins Syndrome or Hypomyelinating Leukodystrophy. Traditionally, RNAPs have been studied in Saccharomyces cerevisiae. Yet, to gain insights into the complex human disorders and allow targeted drug design, understanding the structure and function of human RNAPs is crucial. Here, I present insights into the structure and function of the human Pol I and explore the role of human Pol I and Pol III in the aetiology of developmental disorders. Firstly, I employed the CRISPR-Cas9 genome editing technique to endogenously tag a subunit shared exclusively between Pol I and III (the RPAC1 subunit). Using introduced affinity tags, I purified native complexes to high homogeneity. Additionally, recombinant expression systems for producing human Pol I and its co-factors have been established. Cryo-electron microscopy (cryo-EM) allowed me to determine the structure of human Pol I in various functional states such as an elongating complex, bound to initiation factor RRN3 and bound to an open DNA scaffold, at 2.7 to 3.3 Å resolution. In the elongating Pol I structure, I could observe a double-stranded RNA in the RNA exit tunnel, which may support Pol I processivity. Analysis of the stalk sub-complex revealed that in humans, it constitutes only one subunit allowing the stalk to bend, for example, upon binding the initiation factor RRN3. Different modes of the human Pol I inactivation could also be explored. Finally, I could use the high-resolution structure of the human Pol I, and the recently solved structure of the human Pol III, to map disease-associated mutations. While some of those mutations are located in the subunits shared between Pol I and III, they might affect the two complexes differently. To further study the role of those mutations, I used the CRISPR-Cas9 system to introduce selected mutations into the endogenous subunits. Using inserted tags, I could study the effects of mutations on the localisation and assembly of the human Pol I and Pol III complexes. Further studies will help in understanding the molecular phenotypes underlying complex developmental disorders.