--- OA_place: publisher _id: '19431' abstract: - lang: eng text: "Gene expression is crucial for cell differentiation, development and survival of\r\norganisms. It consists of several steps, starting with transcription that is mediated by\r\nRNA polymerases. These are protein machineries transcribing and producing different\r\ntypes of RNAs. Although, the individual steps of transcription by RNA polymerase II\r\n(Pol II) as well as the structure of Pol II has been extensively studied, surprisingly,\r\nthere is still little known about its regulation and assembly in cytoplasm. Among the\r\nproteins that are important in biogenesis of Pol II are RNA polymerase II associating\r\nproteins (RPAP) and small GPN-loop GTPases (GPN). Both of these protein groups\r\nwere shown to take essential part in assembly of Pol II.\r\nThe aim of this project was to deepen our knowledge in regulation of Pol II in\r\nthe cytoplasm as well as the proteins involved in this process. Techniques of structural\r\nbiology, biochemistry and cell biology were employed to study and characterize cytoplasmic Pol II and its interacting partners.\r\nThis study shows for the first time the structure of cytoplasmic Pol II at high\r\nresolution. The structure also reveals proteins interacting with Pol II in cytoplasm,\r\nnamely GDOWN1, RPAP2. Comparing the structure of cytoplasmic Pol II with transcribing Pol II revealed striking difference in clamp region that is not in closed state.\r\nFurthermore, GDOWN1 and RPAP2 make steric clashes with various transcription\r\nfactors bound to Pol II during different stages of transcription. Even though GPN1 and\r\nGPN3 proteins were not resolved in the cytoplasmic Pol II structure, they are part of\r\nthe complex and their interaction with Pol II was confirmed in vitro. RPAP2 stabilizes\r\nthese proteins on Pol II and several experiments suggest that they interact with the\r\nclamp region. In addition, GDOWN1, RPAP2 and GPNs might keep clamp in open or\r\npartially open state. Based on these results I propose a novel model of regulation of\r\nPol II in cytoplasm. GDOWN1, RPAP2, GPN1 and GPN3 bind to Pol II in cytoplasm\r\nand doing so they can prevent pre-mature binding of DNA or RNA and different transcription factors to Pol II in cytoplasm or before engaging in transcription nucleus.\r\nThis research contributes to the current knowledge of molecular mechanisms\r\nof Pol II regulation in cytoplasm." acknowledged_ssus: - _id: LifeSc - _id: EM-Fac - _id: ScienComp acknowledgement: 'I would also like to acknowledge the ISTA Facilities: Lab Support Facility, Protein Services and Electron Microscopy Facility (EMF) and Scientific Computing. EMF for their support during data collections and troubleshooting, especially Valentin. Scientific Computing for solving quickly any issues related with cluster.' alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Annamaria full_name: Hlavata, Annamaria id: 36062FEC-F248-11E8-B48F-1D18A9856A87 last_name: Hlavata citation: ama: Hlavata A. Regulation of Cytoplasmic RNA Polymerase II. 2025. doi:10.15479/10.15479/AT-ISTA-19431 apa: Hlavata, A. (2025). Regulation of Cytoplasmic RNA Polymerase II. Institute of Science and Technology Austria. https://doi.org/10.15479/10.15479/AT-ISTA-19431 chicago: Hlavata, Annamaria. “Regulation of Cytoplasmic RNA Polymerase II.” Institute of Science and Technology Austria, 2025. https://doi.org/10.15479/10.15479/AT-ISTA-19431. ieee: A. Hlavata, “Regulation of Cytoplasmic RNA Polymerase II,” Institute of Science and Technology Austria, 2025. ista: Hlavata A. 2025. Regulation of Cytoplasmic RNA Polymerase II. Institute of Science and Technology Austria. mla: Hlavata, Annamaria. Regulation of Cytoplasmic RNA Polymerase II. Institute of Science and Technology Austria, 2025, doi:10.15479/10.15479/AT-ISTA-19431. short: A. Hlavata, Regulation of Cytoplasmic RNA Polymerase II, Institute of Science and Technology Austria, 2025. corr_author: '1' date_created: 2025-03-20T12:52:47Z date_published: 2025-03-20T00:00:00Z date_updated: 2026-04-07T11:46:32Z day: '20' ddc: - '572' degree_awarded: PhD department: - _id: GradSch - _id: CaBe doi: 10.15479/10.15479/AT-ISTA-19431 file: - access_level: closed checksum: b7ddf424ffe95f8c767c53c8bb62d4f3 content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: ahlavata date_created: 2025-03-24T12:48:36Z date_updated: 2026-03-20T23:30:04Z embargo_to: open_access file_id: '19448' file_name: PhD_Thesis_Hlavata_final_submission.docx file_size: 23506747 relation: source_file - access_level: open_access checksum: 6c5a59c9bac467c3d0b3ffb8ea6d9fd4 content_type: application/pdf creator: ahlavata date_created: 2025-03-24T12:51:10Z date_updated: 2026-03-20T23:30:04Z embargo: 2026-03-20 file_id: '19449' file_name: PhD_Thesis_Hlavata_final_submission_update.pdf file_size: 9478591 relation: main_file file_date_updated: 2026-03-20T23:30:04Z has_accepted_license: '1' language: - iso: eng month: '03' oa: 1 oa_version: Published Version page: '83' publication_identifier: eissn: - 2663-337X isbn: - 978-3-99078-055-8 publication_status: published publisher: Institute of Science and Technology Austria status: public supervisor: - first_name: Carrie A full_name: Bernecky, Carrie A id: 2CB9DFE2-F248-11E8-B48F-1D18A9856A87 last_name: Bernecky orcid: 0000-0003-0893-7036 title: Regulation of Cytoplasmic RNA Polymerase II type: dissertation user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd year: '2025' ... --- OA_place: publisher _id: '18477' abstract: - lang: eng text: "ADAR1 is broadly expressed across various tissues and is vital in regulating pathways\r\nassociated with innate immune responses. ADAR1 marks double-stranded RNA as \"self\"\r\nthrough its A-to-I editing activity, effectively repressing autoimmunity and maintaining\r\nimmune tolerance. This editing process has been detected at millions of sites across the\r\nhuman genome. However, the mechanism underlying ADAR1's substrate selectivity\r\nproperties remains largely unclear, with much of the current knowledge derived from\r\ncomparisons to its more extensively studied homolog, ADAR2. By studying ADAR1 in complex\r\nwith its RNA substrates and applying a combination of biochemical techniques and structural\r\nstudies using CryoEM, we aim to gain a more comprehensive understanding of the substrate\r\nselectivity characteristics of ADAR1.\r\nIn this thesis, the purification protocol for ADAR1 was successfully optimized, resulting in the\r\nfirst report in the literature to achieve high protein purity and activity. This advancement\r\nenabled the investigation of complex formation between ADAR1 and various RNA substrates,\r\nleading to the identification of optimal conditions for preparing the cryoEM sample. However,\r\ndespite comprehensive optimization of the cryo-EM conditions, the resulting data lacked the\r\ndesired quality, highlighting the need for similar rigorous optimization of the RNA substrates\r\nto facilitate structural studies of the ADAR1-RNA complex. The study was complemented by\r\nAlphaFold predictions, which provided some insights into this mechanism.\r\nMoreover, during this project I established a collaboration with a research group focused on\r\nstudying ADAR homologs. Notably ADAR homologs were identified in bivalve species, and it\r\nwas further demonstrated that ADAR and its A-to-I editing activity are upregulated in Pacific\r\noysters during infections with Ostreid herpesvirus-1—a highly infectious virus that leads to\r\nsignificant losses in oyster populations globally. I successfully purified oyster ADAR and\r\nprepared in vitro edited RNA for nanopore sequencing—a direct sequencing technology\r\ncapable of detecting modified nucleotides without the need for reverse transcription. The\r\ncollaborators initiated optimization of this nanopore-based approach. However, current\r\ntechnological limitations still constrain the reliable detection of modified nucleotides.\r\nThe project also examined the impact of RNA editing on RNA binding and filament formation\r\nby MDA5, a key cytosolic dsRNA sensor that triggers an interferon response. A primary target\r\nof ADAR1's editing activity is RNA derived from repetitive elements present in the genome,\r\nparticularly Alu elements forming double-stranded RNA. When unedited, these RNA\r\nsequences are recognized by MDA5. However, the mechanisms by which MDA5 interacts with\r\nAlu RNAs, as well as the role of A-to-I editing in influencing this binding, are still not well\r\nunderstood.\r\nThe interaction between MDA5 and Alu elements, was successfully established. This was\r\nachieved through the testing of different RNA variants and the evaluation of filament\r\nformation using binding techniques and electron microscopy imaging. This groundwork has\r\nset the conditions for further evaluation using CryoEM. Furthermore, the effects of A-to-I\r\nediting on the binding properties of MDA5 with Alu RNA were investigated. Given the recent\r\nresearch that has provided new insights into MDA5's interaction with dsRNA, it is essential to\r\nrevise the experimental setup to integrate these findings before moving forward with the\r\nCryoEM sample analysis." acknowledged_ssus: - _id: EM-Fac - _id: LifeSc alternative_title: - ISTA Thesis article_processing_charge: No author: - first_name: Beata M full_name: Kaczmarek, Beata M id: 36FA4AFA-F248-11E8-B48F-1D18A9856A87 last_name: Kaczmarek citation: ama: Kaczmarek BM. Biochemical and structural insights into ADAR1 RNA editing. 2024. doi:10.15479/at:ista:18477 apa: Kaczmarek, B. M. (2024). Biochemical and structural insights into ADAR1 RNA editing. Institute of Science and Technology Austria. https://doi.org/10.15479/at:ista:18477 chicago: Kaczmarek, Beata M. “Biochemical and Structural Insights into ADAR1 RNA Editing.” Institute of Science and Technology Austria, 2024. https://doi.org/10.15479/at:ista:18477. ieee: B. M. Kaczmarek, “Biochemical and structural insights into ADAR1 RNA editing,” Institute of Science and Technology Austria, 2024. ista: Kaczmarek BM. 2024. Biochemical and structural insights into ADAR1 RNA editing. Institute of Science and Technology Austria. mla: Kaczmarek, Beata M. Biochemical and Structural Insights into ADAR1 RNA Editing. Institute of Science and Technology Austria, 2024, doi:10.15479/at:ista:18477. short: B.M. Kaczmarek, Biochemical and Structural Insights into ADAR1 RNA Editing, Institute of Science and Technology Austria, 2024. corr_author: '1' date_created: 2024-10-27T07:35:13Z date_published: 2024-10-29T00:00:00Z date_updated: 2026-04-07T13:23:59Z day: '29' ddc: - '572' degree_awarded: PhD department: - _id: GradSch - _id: CaBe doi: 10.15479/at:ista:18477 file: - access_level: closed checksum: 2053294ea4d770c495e4cc501e2a218b content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: bkaczmar date_created: 2024-10-29T11:56:36Z date_updated: 2025-10-29T23:30:02Z embargo_to: open_access file_id: '18485' file_name: 20241029_PhD_thesis_BKaczmarek.docx file_size: 23136626 relation: source_file - access_level: open_access checksum: 8ce857a4cd44b776791eaf180ac9dbb3 content_type: application/pdf creator: bkaczmar date_created: 2024-10-29T11:56:44Z date_updated: 2025-10-29T23:30:02Z embargo: 2025-10-29 file_id: '18486' file_name: 20241029_PhD_thesis_BKaczmarek.pdf file_size: 11707360 relation: main_file file_date_updated: 2025-10-29T23:30:02Z has_accepted_license: '1' language: - iso: eng month: '10' oa: 1 oa_version: Published Version page: '124' publication_identifier: isbn: - 978-3-99078-045-9 issn: - 2663-337X publication_status: published publisher: Institute of Science and Technology Austria status: public supervisor: - first_name: Carrie A full_name: Bernecky, Carrie A id: 2CB9DFE2-F248-11E8-B48F-1D18A9856A87 last_name: Bernecky orcid: 0000-0003-0893-7036 title: Biochemical and structural insights into ADAR1 RNA editing tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: dissertation user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd year: '2024' ...