[{"doi":"10.15479/10.15479/AT-ISTA-19431","citation":{"short":"A. Hlavata, Regulation of Cytoplasmic RNA Polymerase II, Institute of Science and Technology Austria, 2025.","ama":"Hlavata A. Regulation of Cytoplasmic RNA Polymerase II. 2025. doi:<a href=\"https://doi.org/10.15479/10.15479/AT-ISTA-19431\">10.15479/10.15479/AT-ISTA-19431</a>","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.","chicago":"Hlavata, Annamaria. “Regulation of Cytoplasmic RNA Polymerase II.” Institute of Science and Technology Austria, 2025. <a href=\"https://doi.org/10.15479/10.15479/AT-ISTA-19431\">https://doi.org/10.15479/10.15479/AT-ISTA-19431</a>.","apa":"Hlavata, A. (2025). <i>Regulation of Cytoplasmic RNA Polymerase II</i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/10.15479/AT-ISTA-19431\">https://doi.org/10.15479/10.15479/AT-ISTA-19431</a>","mla":"Hlavata, Annamaria. <i>Regulation of Cytoplasmic RNA Polymerase II</i>. Institute of Science and Technology Austria, 2025, doi:<a href=\"https://doi.org/10.15479/10.15479/AT-ISTA-19431\">10.15479/10.15479/AT-ISTA-19431</a>."},"abstract":[{"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.","lang":"eng"}],"publication_status":"published","corr_author":"1","type":"dissertation","publisher":"Institute of Science and Technology Austria","supervisor":[{"id":"2CB9DFE2-F248-11E8-B48F-1D18A9856A87","last_name":"Bernecky","first_name":"Carrie A","full_name":"Bernecky, Carrie A","orcid":"0000-0003-0893-7036"}],"has_accepted_license":"1","acknowledged_ssus":[{"_id":"LifeSc"},{"_id":"EM-Fac"},{"_id":"ScienComp"}],"year":"2025","ddc":["572"],"language":[{"iso":"eng"}],"article_processing_charge":"No","month":"03","file_date_updated":"2026-03-20T23:30:04Z","page":"83","date_published":"2025-03-20T00:00:00Z","publication_identifier":{"isbn":["978-3-99078-055-8"],"eissn":["2663-337X"]},"oa":1,"date_created":"2025-03-20T12:52:47Z","oa_version":"Published Version","_id":"19431","file":[{"file_id":"19448","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","date_updated":"2026-03-20T23:30:04Z","access_level":"closed","relation":"source_file","file_size":23506747,"checksum":"b7ddf424ffe95f8c767c53c8bb62d4f3","date_created":"2025-03-24T12:48:36Z","creator":"ahlavata","embargo_to":"open_access","file_name":"PhD_Thesis_Hlavata_final_submission.docx"},{"file_size":9478591,"date_created":"2025-03-24T12:51:10Z","checksum":"6c5a59c9bac467c3d0b3ffb8ea6d9fd4","relation":"main_file","file_name":"PhD_Thesis_Hlavata_final_submission_update.pdf","embargo":"2026-03-20","creator":"ahlavata","file_id":"19449","access_level":"open_access","content_type":"application/pdf","date_updated":"2026-03-20T23:30:04Z"}],"user_id":"ba8df636-2132-11f1-aed0-ed93e2281fdd","status":"public","date_updated":"2026-04-07T11:46:32Z","author":[{"first_name":"Annamaria","last_name":"Hlavata","full_name":"Hlavata, Annamaria","id":"36062FEC-F248-11E8-B48F-1D18A9856A87"}],"title":"Regulation of Cytoplasmic RNA Polymerase II","alternative_title":["ISTA Thesis"],"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.","degree_awarded":"PhD","OA_place":"publisher","department":[{"_id":"GradSch"},{"_id":"CaBe"}],"day":"20"},{"article_processing_charge":"No","license":"https://creativecommons.org/licenses/by/4.0/","month":"10","file_date_updated":"2025-10-29T23:30:02Z","page":"124","acknowledged_ssus":[{"_id":"EM-Fac"},{"_id":"LifeSc"}],"year":"2024","ddc":["572"],"language":[{"iso":"eng"}],"publication_status":"published","publisher":"Institute of Science and Technology Austria","corr_author":"1","type":"dissertation","supervisor":[{"id":"2CB9DFE2-F248-11E8-B48F-1D18A9856A87","full_name":"Bernecky, Carrie A","orcid":"0000-0003-0893-7036","first_name":"Carrie A","last_name":"Bernecky"}],"has_accepted_license":"1","doi":"10.15479/at:ista:18477","citation":{"apa":"Kaczmarek, B. M. (2024). <i>Biochemical and structural insights into ADAR1 RNA editing</i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/at:ista:18477\">https://doi.org/10.15479/at:ista:18477</a>","mla":"Kaczmarek, Beata M. <i>Biochemical and Structural Insights into ADAR1 RNA Editing</i>. Institute of Science and Technology Austria, 2024, doi:<a href=\"https://doi.org/10.15479/at:ista:18477\">10.15479/at:ista:18477</a>.","short":"B.M. Kaczmarek, Biochemical and Structural Insights into ADAR1 RNA Editing, Institute of Science and Technology Austria, 2024.","ama":"Kaczmarek BM. Biochemical and structural insights into ADAR1 RNA editing. 2024. doi:<a href=\"https://doi.org/10.15479/at:ista:18477\">10.15479/at:ista:18477</a>","ieee":"B. M. Kaczmarek, “Biochemical and structural insights into ADAR1 RNA editing,” Institute of Science and Technology Austria, 2024.","chicago":"Kaczmarek, Beata M. “Biochemical and Structural Insights into ADAR1 RNA Editing.” Institute of Science and Technology Austria, 2024. <a href=\"https://doi.org/10.15479/at:ista:18477\">https://doi.org/10.15479/at:ista:18477</a>.","ista":"Kaczmarek BM. 2024. Biochemical and structural insights into ADAR1 RNA editing. Institute of Science and Technology Austria."},"abstract":[{"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.","lang":"eng"}],"author":[{"full_name":"Kaczmarek, Beata M","last_name":"Kaczmarek","first_name":"Beata M","id":"36FA4AFA-F248-11E8-B48F-1D18A9856A87"}],"title":"Biochemical and structural insights into ADAR1 RNA editing","alternative_title":["ISTA Thesis"],"degree_awarded":"PhD","OA_place":"publisher","department":[{"_id":"GradSch"},{"_id":"CaBe"}],"day":"29","user_id":"ba8df636-2132-11f1-aed0-ed93e2281fdd","status":"public","date_updated":"2026-04-07T13:23:59Z","tmp":{"short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)"},"date_created":"2024-10-27T07:35:13Z","oa_version":"Published Version","_id":"18477","file":[{"file_name":"20241029_PhD_thesis_BKaczmarek.docx","creator":"bkaczmar","embargo_to":"open_access","date_created":"2024-10-29T11:56:36Z","checksum":"2053294ea4d770c495e4cc501e2a218b","file_size":23136626,"relation":"source_file","access_level":"closed","date_updated":"2025-10-29T23:30:02Z","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","file_id":"18485"},{"file_id":"18486","access_level":"open_access","content_type":"application/pdf","date_updated":"2025-10-29T23:30:02Z","file_size":11707360,"checksum":"8ce857a4cd44b776791eaf180ac9dbb3","date_created":"2024-10-29T11:56:44Z","relation":"main_file","embargo":"2025-10-29","file_name":"20241029_PhD_thesis_BKaczmarek.pdf","creator":"bkaczmar"}],"date_published":"2024-10-29T00:00:00Z","publication_identifier":{"isbn":["978-3-99078-045-9"],"issn":["2663-337X"]},"oa":1}]