[{"doi":"10.1038/s41594-024-01448-7","acknowledgement":"We thank the members of the Bernecky laboratory for helpful discussions and A. Hlavata for providing Pol II for use in the fluorescence anisotropy binding assay. We thank V.-V. Hodirnau for SerialEM data collection and support with EPU data collection. We thank D. Slade (Max Perutz Laboratories and Medical University of Vienna, Vienna, Austria) for the wild-type TFIIF expression plasmid. We thank N. Thompson and R. Burgess (McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI, USA) for the 8WG16 hybridoma cell line. We thank C. Plaschka and M. Loose for critical reading of the manuscript. This work was supported by Austrian Science Fund (FWF) grant no. P34185 (DOI 10.55776/P34185) (C.B.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. This research was further supported by the Scientific Service Units of ISTA through resources provided by the Laboratory Support Facility, Electron Microscopy Facility, Scientific Computing and the Preclinical Facility.","article_type":"original","oa":1,"author":[{"id":"4AC7D980-F248-11E8-B48F-1D18A9856A87","last_name":"Tluckova","first_name":"Katarina","full_name":"Tluckova, Katarina"},{"last_name":"Kaczmarek","id":"36FA4AFA-F248-11E8-B48F-1D18A9856A87","first_name":"Beata M","full_name":"Kaczmarek, Beata M"},{"first_name":"Anita P","full_name":"Testa Salmazo, Anita P","id":"41F1F098-F248-11E8-B48F-1D18A9856A87","last_name":"Testa Salmazo"},{"id":"2CB9DFE2-F248-11E8-B48F-1D18A9856A87","last_name":"Bernecky","first_name":"Carrie A","full_name":"Bernecky, Carrie A","orcid":"0000-0003-0893-7036"}],"date_published":"2025-04-01T00:00:00Z","day":"01","abstract":[{"text":"Transcription by RNA polymerase II (Pol II) can be repressed by noncoding RNA, including the human RNA Alu. However, the mechanism by which endogenous RNAs repress transcription remains unclear. Here we present cryogenic-electron microscopy structures of Pol II bound to Alu RNA, which reveal that Alu RNA mimics how DNA and RNA bind to Pol II during transcription elongation. Further, we show how distinct domains of the general transcription factor TFIIF control repressive activity. Together, we reveal how a noncoding RNA can regulate mammalian gene expression.","lang":"eng"}],"publisher":"Springer Nature","file":[{"date_created":"2025-04-16T08:17:27Z","date_updated":"2025-04-16T08:17:27Z","file_size":9306639,"content_type":"application/pdf","creator":"dernst","file_name":"2025_NatureStrucMolBiol_Tluckova.pdf","success":1,"access_level":"open_access","checksum":"2919b30b271f395888e880076a680d73","relation":"main_file","file_id":"19573"}],"article_processing_charge":"Yes (in subscription journal)","citation":{"ieee":"K. Tluckova, B. M. Kaczmarek, A. P. Testa Salmazo, and C. Bernecky, “Mechanism of mammalian transcriptional repression by noncoding RNA,” <i>Nature Structural &#38; Molecular Biology</i>, vol. 32. Springer Nature, pp. 607–612, 2025.","ista":"Tluckova K, Kaczmarek BM, Testa Salmazo AP, Bernecky C. 2025. Mechanism of mammalian transcriptional repression by noncoding RNA. Nature Structural &#38; Molecular Biology. 32, 607–612.","chicago":"Tluckova, Katarina, Beata M Kaczmarek, Anita P Testa Salmazo, and Carrie Bernecky. “Mechanism of Mammalian Transcriptional Repression by Noncoding RNA.” <i>Nature Structural &#38; Molecular Biology</i>. Springer Nature, 2025. <a href=\"https://doi.org/10.1038/s41594-024-01448-7\">https://doi.org/10.1038/s41594-024-01448-7</a>.","apa":"Tluckova, K., Kaczmarek, B. M., Testa Salmazo, A. P., &#38; Bernecky, C. (2025). Mechanism of mammalian transcriptional repression by noncoding RNA. <i>Nature Structural &#38; Molecular Biology</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41594-024-01448-7\">https://doi.org/10.1038/s41594-024-01448-7</a>","short":"K. Tluckova, B.M. Kaczmarek, A.P. Testa Salmazo, C. Bernecky, Nature Structural &#38; Molecular Biology 32 (2025) 607–612.","mla":"Tluckova, Katarina, et al. “Mechanism of Mammalian Transcriptional Repression by Noncoding RNA.” <i>Nature Structural &#38; Molecular Biology</i>, vol. 32, Springer Nature, 2025, pp. 607–12, doi:<a href=\"https://doi.org/10.1038/s41594-024-01448-7\">10.1038/s41594-024-01448-7</a>.","ama":"Tluckova K, Kaczmarek BM, Testa Salmazo AP, Bernecky C. Mechanism of mammalian transcriptional repression by noncoding RNA. <i>Nature Structural &#38; Molecular Biology</i>. 2025;32:607-612. doi:<a href=\"https://doi.org/10.1038/s41594-024-01448-7\">10.1038/s41594-024-01448-7</a>"},"ddc":["570"],"file_date_updated":"2025-04-16T08:17:27Z","publication_identifier":{"eissn":["1545-9985"],"issn":["1545-9993"]},"external_id":{"pmid":["39762629"],"isi":["001390268000001"]},"intvolume":"        32","year":"2025","date_created":"2025-01-08T11:20:20Z","has_accepted_license":"1","title":"Mechanism of mammalian transcriptional repression by noncoding RNA","pmid":1,"corr_author":"1","scopus_import":"1","APC_amount":"12348 EUR","department":[{"_id":"CaBe"}],"OA_place":"publisher","type":"journal_article","language":[{"iso":"eng"}],"project":[{"grant_number":"P34185","name":"Regulation of mammalian transcription by noncoding RNA","_id":"c08a6700-5a5b-11eb-8a69-82a722b2bc30"}],"publication":"Nature Structural & Molecular Biology","related_material":{"record":[{"status":"public","id":"14644","relation":"earlier_version"}]},"quality_controlled":"1","acknowledged_ssus":[{"_id":"LifeSc"},{"_id":"EM-Fac"},{"_id":"ScienComp"},{"_id":"PreCl"}],"license":"https://creativecommons.org/licenses/by/4.0/","page":"607-612","_id":"18778","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","oa_version":"Published Version","publication_status":"published","volume":32,"month":"04","isi":1,"OA_type":"hybrid","tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode"},"status":"public","date_updated":"2025-11-20T10:28:36Z"},{"language":[{"iso":"eng"}],"publication":"Nature Reviews Molecular Cell Biology","scopus_import":"1","corr_author":"1","department":[{"_id":"CaBe"}],"type":"journal_article","isi":1,"volume":26,"publication_status":"published","month":"06","OA_type":"closed access","date_updated":"2025-09-30T11:20:36Z","status":"public","article_number":"415","quality_controlled":"1","oa_version":"None","_id":"19465","user_id":"317138e5-6ab7-11ef-aa6d-ffef3953e345","author":[{"id":"2CB9DFE2-F248-11E8-B48F-1D18A9856A87","last_name":"Bernecky","first_name":"Carrie A","full_name":"Bernecky, Carrie A","orcid":"0000-0003-0893-7036"}],"date_published":"2025-06-01T00:00:00Z","day":"01","doi":"10.1038/s41580-025-00844-1","article_type":"letter_note","external_id":{"pmid":["40155512"],"isi":["001455740100001"]},"publication_identifier":{"eissn":["1471-0080"],"issn":["1471-0072"]},"date_created":"2025-03-31T10:07:22Z","year":"2025","intvolume":"        26","pmid":1,"title":"Understanding the machinery that reads the genome","article_processing_charge":"No","publisher":"Springer Nature","citation":{"mla":"Bernecky, Carrie. “Understanding the Machinery That Reads the Genome.” <i>Nature Reviews Molecular Cell Biology</i>, vol. 26, 415, Springer Nature, 2025, doi:<a href=\"https://doi.org/10.1038/s41580-025-00844-1\">10.1038/s41580-025-00844-1</a>.","ama":"Bernecky C. Understanding the machinery that reads the genome. <i>Nature Reviews Molecular Cell Biology</i>. 2025;26. doi:<a href=\"https://doi.org/10.1038/s41580-025-00844-1\">10.1038/s41580-025-00844-1</a>","short":"C. Bernecky, Nature Reviews Molecular Cell Biology 26 (2025).","chicago":"Bernecky, Carrie. “Understanding the Machinery That Reads the Genome.” <i>Nature Reviews Molecular Cell Biology</i>. Springer Nature, 2025. <a href=\"https://doi.org/10.1038/s41580-025-00844-1\">https://doi.org/10.1038/s41580-025-00844-1</a>.","apa":"Bernecky, C. (2025). Understanding the machinery that reads the genome. <i>Nature Reviews Molecular Cell Biology</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41580-025-00844-1\">https://doi.org/10.1038/s41580-025-00844-1</a>","ista":"Bernecky C. 2025. Understanding the machinery that reads the genome. Nature Reviews Molecular Cell Biology. 26, 415.","ieee":"C. Bernecky, “Understanding the machinery that reads the genome,” <i>Nature Reviews Molecular Cell Biology</i>, vol. 26. Springer Nature, 2025."}},{"PlanS_conform":"1","article_number":"930","quality_controlled":"1","_id":"20077","user_id":"317138e5-6ab7-11ef-aa6d-ffef3953e345","oa_version":"Published Version","isi":1,"publication_status":"published","month":"07","volume":14,"OA_type":"gold","tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode"},"date_updated":"2025-09-30T14:10:07Z","status":"public","department":[{"_id":"CaBe"}],"type":"journal_article","OA_place":"publisher","language":[{"iso":"eng"}],"publication":"Biology","file":[{"date_updated":"2025-07-31T09:11:09Z","file_size":1885781,"content_type":"application/pdf","file_name":"2025_Biology_Rosani.pdf","creator":"dernst","success":1,"date_created":"2025-07-31T09:11:09Z","file_id":"20097","checksum":"f5e059e66803fa54249c1db029aef0f6","access_level":"open_access","relation":"main_file"}],"article_processing_charge":"Yes","publisher":"MDPI","DOAJ_listed":"1","citation":{"mla":"Rosani, Umberto, et al. “Ancestral Origin and Functional Expression of a Hyaluronic Acid Pathway Complement in Mussels.” <i>Biology</i>, vol. 14, no. 8, 930, MDPI, 2025, doi:<a href=\"https://doi.org/10.3390/biology14080930\">10.3390/biology14080930</a>.","ama":"Rosani U, Altan N, Venier P, Bortoletto E, Volpi N, Bernecky C. Ancestral origin and functional expression of a hyaluronic acid pathway complement in mussels. <i>Biology</i>. 2025;14(8). doi:<a href=\"https://doi.org/10.3390/biology14080930\">10.3390/biology14080930</a>","short":"U. Rosani, N. Altan, P. Venier, E. Bortoletto, N. Volpi, C. Bernecky, Biology 14 (2025).","chicago":"Rosani, Umberto, Nehir Altan, Paola Venier, Enrico Bortoletto, Nicola Volpi, and Carrie Bernecky. “Ancestral Origin and Functional Expression of a Hyaluronic Acid Pathway Complement in Mussels.” <i>Biology</i>. MDPI, 2025. <a href=\"https://doi.org/10.3390/biology14080930\">https://doi.org/10.3390/biology14080930</a>.","apa":"Rosani, U., Altan, N., Venier, P., Bortoletto, E., Volpi, N., &#38; Bernecky, C. (2025). Ancestral origin and functional expression of a hyaluronic acid pathway complement in mussels. <i>Biology</i>. MDPI. <a href=\"https://doi.org/10.3390/biology14080930\">https://doi.org/10.3390/biology14080930</a>","ieee":"U. Rosani, N. Altan, P. Venier, E. Bortoletto, N. Volpi, and C. Bernecky, “Ancestral origin and functional expression of a hyaluronic acid pathway complement in mussels,” <i>Biology</i>, vol. 14, no. 8. MDPI, 2025.","ista":"Rosani U, Altan N, Venier P, Bortoletto E, Volpi N, Bernecky C. 2025. Ancestral origin and functional expression of a hyaluronic acid pathway complement in mussels. Biology. 14(8), 930."},"file_date_updated":"2025-07-31T09:11:09Z","ddc":["570"],"external_id":{"isi":["001557922100001"]},"publication_identifier":{"issn":["2079-7737"]},"date_created":"2025-07-25T08:28:26Z","year":"2025","intvolume":"        14","has_accepted_license":"1","title":"Ancestral origin and functional expression of a hyaluronic acid pathway complement in mussels","doi":"10.3390/biology14080930","acknowledgement":"This research was funded by the Italian Ministry of University and Research (MIUR), grant ID: P2022JEEMT (Developing a tool for the study of haplotype diversity in Mytilus galloprovincialis (HAMIGA)).","issue":"8","article_type":"original","author":[{"full_name":"Rosani, Umberto","first_name":"Umberto","last_name":"Rosani"},{"last_name":"Altan","first_name":"Nehir","full_name":"Altan, Nehir"},{"full_name":"Venier, Paola","first_name":"Paola","last_name":"Venier"},{"full_name":"Bortoletto, Enrico","first_name":"Enrico","last_name":"Bortoletto"},{"first_name":"Nicola","full_name":"Volpi, Nicola","last_name":"Volpi"},{"first_name":"Carrie A","full_name":"Bernecky, Carrie A","orcid":"0000-0003-0893-7036","id":"2CB9DFE2-F248-11E8-B48F-1D18A9856A87","last_name":"Bernecky"}],"oa":1,"date_published":"2025-07-24T00:00:00Z","day":"24","abstract":[{"text":"Hyaluronic acid (HA) is a key extracellular matrix component of vertebrates, where it mediates cell adhesion, immune regulation, and tissue remodeling through its interaction with specific receptors. Although HA has been detected in a few invertebrate species, the lack of fundamental components of the molecular HA pathway poses relevant objections about its functional role in these species. Mining genomic and transcriptomic data, we considered the conservation of the gene locus encoding for the extracellular link protein (XLINK) in marine mussels as well as its expression patterns. Structural and phylogenetic analyses were undertaken to evaluate possible similarities with vertebrate orthologs and to infer the origin of this gene in invertebrates. Biochemical analysis was used to quantify HA in tissues of Mytilus galloprovincialis. As a result, we confirm that the mussel can produce HA (up to 1.02 ng/mg in mantle) and that its genome encodes two XLINK gene loci. These loci are conserved in Mytilidae species and show a complex evolutionary path. Mussel XLINK genes appeared to be expressed during developmental stages in three mussel species, ranking in the top 100 expressed genes in M. trossulus at 17 h post-fertilization. In conclusion, the presence of HA and an active gene with the potential to bind HA suggests that mussels have the potential to synthesize and use HA and are among the few invertebrates encoding this gene.","lang":"eng"}]},{"corr_author":"1","doi":"10.64898/2025.12.10.692585","acknowledgement":"We thank A. Salmazo for assistance with Pol II purification. We thank staff at the VBCF Proteomics facility for immunoprecipitation-mass spectrometry analysis, and J.A. Stopp for assistance with IP-MS data visualization. This research was further supported by the Scientific Service Units (SSUs) of IST Austria through resources provided by the Lab Support Facility (LSF), Electron Microscopy (EMF), Scientific Computing (SciComp), and the Preclinical Facility (PCF).","department":[{"_id":"CaBe"}],"type":"preprint","oa":1,"author":[{"first_name":"Annamaria","full_name":"Hlavata, Annamaria","last_name":"Hlavata","id":"36062FEC-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Neuditschko","first_name":"Benjamin","full_name":"Neuditschko, Benjamin"},{"first_name":"Ulla","full_name":"Schellhaas, Ulla","last_name":"Schellhaas"},{"last_name":"Plaschka","first_name":"Clemens","full_name":"Plaschka, Clemens"},{"last_name":"Herzog","first_name":"Franz","full_name":"Herzog, Franz"},{"full_name":"Bernecky, Carrie A","first_name":"Carrie A","orcid":"0000-0003-0893-7036","id":"2CB9DFE2-F248-11E8-B48F-1D18A9856A87","last_name":"Bernecky"}],"language":[{"iso":"eng"}],"date_published":"2025-12-10T00:00:00Z","day":"10","abstract":[{"text":"RNA polymerase II (Pol II) must be assembled in the cytoplasm before it enters the nucleus, where it transcribes protein-coding genes. Although transcription by Pol II is intensively studied, how this central multi-subunit enzyme is made and the role of dedicated factors remains unclear. Here, we report the integrative structural analysis of a native human Pol II from the cytoplasm captured near the end of biogenesis. The complex contained Gdown1 and three biogenesis factors – RPAP2 and the critical small GTPases GPN1 and GPN3. Cryo-EM analysis of the complex revealed how Gdown1 and RPAP2 associate with Pol II and prevent the premature association of transcription factors. Further biochemical and cryo-EM analysis revealed how RPAP2 recruits GPN1–GPN3 to the complex, and how the assembly of the RPAP2–GPN1–GPN3 complex is controlled by GTP hydrolysis. The combined results uncover a network of interactions that chaperone cytoplasmic Pol II to prevent aberrant interactions, reveal a GTP-controlled switch during the final stages of Pol II biogenesis, and suggest a general mechanism for the action of GPN-loop GTPase family of enzymes.","lang":"eng"}],"publisher":"bioRxiv","article_processing_charge":"No","citation":{"apa":"Hlavata, A., Neuditschko, B., Schellhaas, U., Plaschka, C., Herzog, F., &#38; Bernecky, C. (2025). Structure of cytoplasmic RNA polymerase II. bioRxiv. <a href=\"https://doi.org/10.64898/2025.12.10.692585\">https://doi.org/10.64898/2025.12.10.692585</a>","chicago":"Hlavata, Annamaria, Benjamin Neuditschko, Ulla Schellhaas, Clemens Plaschka, Franz Herzog, and Carrie Bernecky. “Structure of Cytoplasmic RNA Polymerase II.” bioRxiv, 2025. <a href=\"https://doi.org/10.64898/2025.12.10.692585\">https://doi.org/10.64898/2025.12.10.692585</a>.","ieee":"A. Hlavata, B. Neuditschko, U. Schellhaas, C. Plaschka, F. Herzog, and C. Bernecky, “Structure of cytoplasmic RNA polymerase II.” bioRxiv, 2025.","ista":"Hlavata A, Neuditschko B, Schellhaas U, Plaschka C, Herzog F, Bernecky C. 2025. Structure of cytoplasmic RNA polymerase II. <a href=\"https://doi.org/10.64898/2025.12.10.692585\">10.64898/2025.12.10.692585</a>.","mla":"Hlavata, Annamaria, et al. <i>Structure of Cytoplasmic RNA Polymerase II</i>. bioRxiv, 2025, doi:<a href=\"https://doi.org/10.64898/2025.12.10.692585\">10.64898/2025.12.10.692585</a>.","ama":"Hlavata A, Neuditschko B, Schellhaas U, Plaschka C, Herzog F, Bernecky C. Structure of cytoplasmic RNA polymerase II. 2025. doi:<a href=\"https://doi.org/10.64898/2025.12.10.692585\">10.64898/2025.12.10.692585</a>","short":"A. Hlavata, B. Neuditschko, U. Schellhaas, C. Plaschka, F. Herzog, C. Bernecky, (2025)."},"acknowledged_ssus":[{"_id":"LifeSc"},{"_id":"EM-Fac"},{"_id":"ScienComp"},{"_id":"PreCl"}],"_id":"20804","oa_version":"None","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","publication_status":"published","month":"12","year":"2025","date_created":"2025-12-11T13:33:27Z","main_file_link":[{"open_access":"1","url":"https://doi.org/10.64898/2025.12.10.692585"}],"title":"Structure of cytoplasmic RNA polymerase II","date_updated":"2025-12-15T09:48:22Z","status":"public"},{"file_date_updated":"2026-03-20T23:30:04Z","ddc":["572"],"citation":{"ista":"Hlavata A. 2025. Regulation of Cytoplasmic RNA Polymerase II. Institute of Science and Technology Austria.","ieee":"A. Hlavata, “Regulation of Cytoplasmic RNA Polymerase II,” Institute of Science and Technology Austria, 2025.","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>","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>.","short":"A. Hlavata, Regulation of Cytoplasmic RNA Polymerase II, Institute of Science and Technology Austria, 2025.","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>.","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>"},"publisher":"Institute of Science and Technology Austria","article_processing_charge":"No","file":[{"embargo_to":"open_access","date_created":"2025-03-24T12:48:36Z","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","creator":"ahlavata","file_name":"PhD_Thesis_Hlavata_final_submission.docx","date_updated":"2026-03-20T23:30:04Z","file_size":23506747,"checksum":"b7ddf424ffe95f8c767c53c8bb62d4f3","access_level":"closed","relation":"source_file","file_id":"19448"},{"content_type":"application/pdf","creator":"ahlavata","file_name":"PhD_Thesis_Hlavata_final_submission_update.pdf","date_updated":"2026-03-20T23:30:04Z","file_size":9478591,"embargo":"2026-03-20","date_created":"2025-03-24T12:51:10Z","file_id":"19449","access_level":"open_access","checksum":"6c5a59c9bac467c3d0b3ffb8ea6d9fd4","relation":"main_file"}],"title":"Regulation of Cytoplasmic RNA Polymerase II","has_accepted_license":"1","date_created":"2025-03-20T12:52:47Z","year":"2025","publication_identifier":{"eissn":["2663-337X"],"isbn":["978-3-99078-055-8"]},"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.","doi":"10.15479/10.15479/AT-ISTA-19431","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"}],"day":"20","date_published":"2025-03-20T00:00:00Z","author":[{"id":"36062FEC-F248-11E8-B48F-1D18A9856A87","last_name":"Hlavata","full_name":"Hlavata, Annamaria","first_name":"Annamaria"}],"oa":1,"_id":"19431","page":"83","user_id":"ba8df636-2132-11f1-aed0-ed93e2281fdd","oa_version":"Published Version","acknowledged_ssus":[{"_id":"LifeSc"},{"_id":"EM-Fac"},{"_id":"ScienComp"}],"date_updated":"2026-04-07T11:46:32Z","status":"public","degree_awarded":"PhD","publication_status":"published","month":"03","type":"dissertation","OA_place":"publisher","department":[{"_id":"GradSch"},{"_id":"CaBe"}],"alternative_title":["ISTA Thesis"],"corr_author":"1","supervisor":[{"full_name":"Bernecky, Carrie A","first_name":"Carrie A","orcid":"0000-0003-0893-7036","id":"2CB9DFE2-F248-11E8-B48F-1D18A9856A87","last_name":"Bernecky"}],"language":[{"iso":"eng"}]},{"language":[{"iso":"eng"}],"publication":"Journal of Cell Science","project":[{"grant_number":"665385","call_identifier":"H2020","_id":"2564DBCA-B435-11E9-9278-68D0E5697425","name":"International IST Doctoral Program"},{"_id":"bd76d395-d553-11ed-ba76-f678c14f9033","name":"Peptide receptors for auxin canalization in Arabidopsis","grant_number":"I06123"}],"ec_funded":1,"corr_author":"1","scopus_import":"1","type":"journal_article","OA_place":"publisher","department":[{"_id":"MaLo"},{"_id":"JiFr"},{"_id":"CaBe"}],"OA_type":"hybrid","publication_status":"published","volume":137,"month":"04","isi":1,"date_updated":"2025-09-04T13:49:45Z","status":"public","tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode"},"quality_controlled":"1","article_number":"jcs.261720","related_material":{"record":[{"status":"public","id":"14591","relation":"earlier_version"}]},"_id":"15330","user_id":"317138e5-6ab7-11ef-aa6d-ffef3953e345","oa_version":"Published Version","acknowledged_ssus":[{"_id":"EM-Fac"},{"_id":"LifeSc"},{"_id":"Bio"}],"date_published":"2024-04-01T00:00:00Z","oa":1,"author":[{"id":"390C1120-F248-11E8-B48F-1D18A9856A87","last_name":"Gnyliukh","full_name":"Gnyliukh, Nataliia","first_name":"Nataliia","orcid":"0000-0002-2198-0509"},{"last_name":"Johnson","id":"46A62C3A-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-2739-8843","full_name":"Johnson, Alexander J","first_name":"Alexander J"},{"full_name":"Nagel, MK","first_name":"MK","last_name":"Nagel"},{"id":"2DB5D88C-D7B3-11E9-B8FD-7907E6697425","last_name":"Monzer","first_name":"Aline","full_name":"Monzer, Aline"},{"last_name":"Babic","id":"db566d23-f6e0-11ea-865d-e6f270e968e7","first_name":"David","full_name":"Babic, David"},{"last_name":"Hlavata","id":"36062FEC-F248-11E8-B48F-1D18A9856A87","first_name":"Annamaria","full_name":"Hlavata, Annamaria"},{"last_name":"Alotaibi","first_name":"SS","full_name":"Alotaibi, SS"},{"last_name":"Isono","first_name":"E","full_name":"Isono, E"},{"id":"462D4284-F248-11E8-B48F-1D18A9856A87","last_name":"Loose","first_name":"Martin","full_name":"Loose, Martin","orcid":"0000-0001-7309-9724"},{"last_name":"Friml","id":"4159519E-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-8302-7596","first_name":"Jiří","full_name":"Friml, Jiří"}],"abstract":[{"text":"Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development by controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scission machinery in plants, but the precise roles of these proteins in this process are not fully understood. Here, we characterised the roles of Plant Dynamin-Related Proteins 2 (DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to Dynamins’ recruiters, like Endophilin and Amphiphysin, in the CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the sh3p123 triple-mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggests that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME.","lang":"eng"}],"day":"01","doi":"10.1242/jcs.261720","article_type":"original","issue":"8","acknowledgement":"Nataliia Gnyliukh was partially funded by the European Union’s Horizon 2020 research and\r\ninnovation program (2018-2020) under the Marie Sklodowska-Curie Grant (agreement no.\r\n665385). Taif University Researchers Supporting Project: TURSP-HC2022/02. and Austrian\r\nScience Fund (FWF): I 6123-B.We thank Prof. Eileen Lafer and Liping Wang for their suggestions regarding the optimisation of protein expression and purification. We thank Prof. Sebastian Y. Bednarek for the useful comments and constructive criticism of the project. We thank Maciek Adamowski for providing genetic material. This research was supported by the Scientific Service Units (SSU) of IST-Austria through resources provided by the Electron microscopy (EMF), Lab Support Facility (LSF) (particularly Dorota Jaworska) and the Bioimaging Facility (BIF).","intvolume":"       137","year":"2024","date_created":"2024-04-19T09:54:59Z","publication_identifier":{"eissn":["1477-9137"],"issn":["0021-9533"]},"external_id":{"isi":["001266917100005"],"pmid":["38506228"]},"pmid":1,"title":"Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana","has_accepted_license":"1","citation":{"chicago":"Gnyliukh, Nataliia, Alexander J Johnson, MK Nagel, Aline Monzer, David Babic, Annamaria Hlavata, SS Alotaibi, E Isono, Martin Loose, and Jiří Friml. “Role of Dynamin-Related Proteins 2 and SH3P2 in Clathrin-Mediated Endocytosis in Arabidopsis Thaliana.” <i>Journal of Cell Science</i>. The Company of Biologists, 2024. <a href=\"https://doi.org/10.1242/jcs.261720\">https://doi.org/10.1242/jcs.261720</a>.","apa":"Gnyliukh, N., Johnson, A. J., Nagel, M., Monzer, A., Babic, D., Hlavata, A., … Friml, J. (2024). Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana. <i>Journal of Cell Science</i>. The Company of Biologists. <a href=\"https://doi.org/10.1242/jcs.261720\">https://doi.org/10.1242/jcs.261720</a>","ieee":"N. Gnyliukh <i>et al.</i>, “Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana,” <i>Journal of Cell Science</i>, vol. 137, no. 8. The Company of Biologists, 2024.","ista":"Gnyliukh N, Johnson AJ, Nagel M, Monzer A, Babic D, Hlavata A, Alotaibi S, Isono E, Loose M, Friml J. 2024. Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana. Journal of Cell Science. 137(8), jcs. 261720.","mla":"Gnyliukh, Nataliia, et al. “Role of Dynamin-Related Proteins 2 and SH3P2 in Clathrin-Mediated Endocytosis in Arabidopsis Thaliana.” <i>Journal of Cell Science</i>, vol. 137, no. 8, jcs. 261720, The Company of Biologists, 2024, doi:<a href=\"https://doi.org/10.1242/jcs.261720\">10.1242/jcs.261720</a>.","ama":"Gnyliukh N, Johnson AJ, Nagel M, et al. Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana. <i>Journal of Cell Science</i>. 2024;137(8). doi:<a href=\"https://doi.org/10.1242/jcs.261720\">10.1242/jcs.261720</a>","short":"N. Gnyliukh, A.J. Johnson, M. Nagel, A. Monzer, D. Babic, A. Hlavata, S. Alotaibi, E. Isono, M. Loose, J. Friml, Journal of Cell Science 137 (2024)."},"file":[{"date_created":"2025-01-09T08:41:16Z","creator":"dernst","success":1,"file_name":"2024_JourCellScience_Gnyliukh.pdf","content_type":"application/pdf","date_updated":"2025-01-09T08:41:16Z","file_size":25845948,"relation":"main_file","access_level":"open_access","checksum":"6dc023f0cc7052ad3cf0a42589d2e30f","file_id":"18792"}],"publisher":"The Company of Biologists","article_processing_charge":"Yes (via OA deal)","ddc":["570"],"file_date_updated":"2025-01-09T08:41:16Z"},{"date_updated":"2025-09-08T14:42:50Z","status":"public","tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode"},"OA_type":"hybrid","volume":84,"month":"12","publication_status":"published","isi":1,"oa_version":"Published Version","_id":"18553","page":"4740-4757.e12","user_id":"317138e5-6ab7-11ef-aa6d-ffef3953e345","acknowledged_ssus":[{"_id":"LifeSc"},{"_id":"PreCl"}],"quality_controlled":"1","publication":"Molecular Cell","language":[{"iso":"eng"}],"OA_place":"publisher","type":"journal_article","department":[{"_id":"CaBe"}],"scopus_import":"1","title":"STK19 drives transcription-coupled repair by stimulating repair complex stability, RNA Pol II ubiquitylation, and TFIIH recruitment","pmid":1,"has_accepted_license":"1","intvolume":"        84","date_created":"2024-11-15T12:12:54Z","year":"2024","publication_identifier":{"issn":["1097-2765"]},"external_id":{"isi":["001395711300001"],"pmid":["39547223"]},"ddc":["570"],"file_date_updated":"2025-01-13T11:17:35Z","citation":{"short":"A.R. Ramadhin, S.-H. Lee, D. Zhou, A.P. Testa Salmazo, C. Gonzalo-Hansen, M. van Sluis, C.M.A. Blom, R.C. Janssens, A. Raams, D. Dekkers, K. Bezstarosti, D. Slade, W. Vermeulen, A. Pines, J.A.A. Demmers, C. Bernecky, T.K. Sixma, J.A. Marteijn, Molecular Cell 84 (2024) 4740–4757.e12.","ama":"Ramadhin AR, Lee S-H, Zhou D, et al. STK19 drives transcription-coupled repair by stimulating repair complex stability, RNA Pol II ubiquitylation, and TFIIH recruitment. <i>Molecular Cell</i>. 2024;84(24):4740-4757.e12. doi:<a href=\"https://doi.org/10.1016/j.molcel.2024.10.030\">10.1016/j.molcel.2024.10.030</a>","mla":"Ramadhin, Anisha R., et al. “STK19 Drives Transcription-Coupled Repair by Stimulating Repair Complex Stability, RNA Pol II Ubiquitylation, and TFIIH Recruitment.” <i>Molecular Cell</i>, vol. 84, no. 24, Elsevier, 2024, p. 4740–4757.e12, doi:<a href=\"https://doi.org/10.1016/j.molcel.2024.10.030\">10.1016/j.molcel.2024.10.030</a>.","ista":"Ramadhin AR, Lee S-H, Zhou D, Testa Salmazo AP, Gonzalo-Hansen C, van Sluis M, Blom CMA, Janssens RC, Raams A, Dekkers D, Bezstarosti K, Slade D, Vermeulen W, Pines A, Demmers JAA, Bernecky C, Sixma TK, Marteijn JA. 2024. STK19 drives transcription-coupled repair by stimulating repair complex stability, RNA Pol II ubiquitylation, and TFIIH recruitment. Molecular Cell. 84(24), 4740–4757.e12.","ieee":"A. R. Ramadhin <i>et al.</i>, “STK19 drives transcription-coupled repair by stimulating repair complex stability, RNA Pol II ubiquitylation, and TFIIH recruitment,” <i>Molecular Cell</i>, vol. 84, no. 24. Elsevier, p. 4740–4757.e12, 2024.","chicago":"Ramadhin, Anisha R., Shun-Hsiao Lee, Di Zhou, Anita P Testa Salmazo, Camila Gonzalo-Hansen, Marjolein van Sluis, Cindy M.A. Blom, et al. “STK19 Drives Transcription-Coupled Repair by Stimulating Repair Complex Stability, RNA Pol II Ubiquitylation, and TFIIH Recruitment.” <i>Molecular Cell</i>. Elsevier, 2024. <a href=\"https://doi.org/10.1016/j.molcel.2024.10.030\">https://doi.org/10.1016/j.molcel.2024.10.030</a>.","apa":"Ramadhin, A. R., Lee, S.-H., Zhou, D., Testa Salmazo, A. P., Gonzalo-Hansen, C., van Sluis, M., … Marteijn, J. A. (2024). STK19 drives transcription-coupled repair by stimulating repair complex stability, RNA Pol II ubiquitylation, and TFIIH recruitment. <i>Molecular Cell</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.molcel.2024.10.030\">https://doi.org/10.1016/j.molcel.2024.10.030</a>"},"file":[{"content_type":"application/pdf","file_name":"2024_MolecularCell_Ramadhin.pdf","creator":"dernst","success":1,"date_updated":"2025-01-13T11:17:35Z","file_size":25071994,"date_created":"2025-01-13T11:17:35Z","file_id":"18844","checksum":"e051e2766b2d424983778f742cb7c5ed","access_level":"open_access","relation":"main_file"}],"publisher":"Elsevier","article_processing_charge":"No","abstract":[{"lang":"eng","text":"Transcription-coupled nucleotide excision repair (TC-NER) efficiently eliminates DNA damage that impedes gene transcription by RNA polymerase II (RNA Pol II). TC-NER is initiated by the recognition of lesion-stalled RNA Pol II by CSB, which recruits the CRL4CSA ubiquitin ligase and UVSSA. RNA Pol II ubiquitylation at RPB1-K1268 by CRL4CSA serves as a critical TC-NER checkpoint, governing RNA Pol II stability and initiating DNA damage excision by TFIIH recruitment. However, the precise regulatory mechanisms of CRL4CSA activity and TFIIH recruitment remain elusive. Here, we reveal human serine/threonine-protein kinase 19 (STK19) as a TC-NER factor, which is essential for correct DNA damage removal and subsequent transcription restart. Cryogenic electron microscopy (cryo-EM) studies demonstrate that STK19 is an integral part of the RNA Pol II-TC-NER complex, bridging CSA, UVSSA, RNA Pol II, and downstream DNA. STK19 stimulates TC-NER complex stability and CRL4CSA activity, resulting in efficient RNA Pol II ubiquitylation and correct UVSSA and TFIIH binding. These findings underscore the crucial role of STK19 as a core TC-NER component."}],"day":"19","date_published":"2024-12-19T00:00:00Z","oa":1,"author":[{"last_name":"Ramadhin","full_name":"Ramadhin, Anisha R.","first_name":"Anisha R."},{"full_name":"Lee, Shun-Hsiao","first_name":"Shun-Hsiao","last_name":"Lee"},{"full_name":"Zhou, Di","first_name":"Di","last_name":"Zhou"},{"last_name":"Testa Salmazo","id":"41F1F098-F248-11E8-B48F-1D18A9856A87","full_name":"Testa Salmazo, Anita P","first_name":"Anita P"},{"last_name":"Gonzalo-Hansen","full_name":"Gonzalo-Hansen, Camila","first_name":"Camila"},{"last_name":"van Sluis","full_name":"van Sluis, Marjolein","first_name":"Marjolein"},{"first_name":"Cindy M.A.","full_name":"Blom, Cindy M.A.","last_name":"Blom"},{"first_name":"Roel C.","full_name":"Janssens, Roel C.","last_name":"Janssens"},{"last_name":"Raams","full_name":"Raams, Anja","first_name":"Anja"},{"last_name":"Dekkers","first_name":"Dick","full_name":"Dekkers, Dick"},{"first_name":"Karel","full_name":"Bezstarosti, Karel","last_name":"Bezstarosti"},{"full_name":"Slade, Dea","first_name":"Dea","last_name":"Slade"},{"first_name":"Wim","full_name":"Vermeulen, Wim","last_name":"Vermeulen"},{"full_name":"Pines, Alex","first_name":"Alex","last_name":"Pines"},{"last_name":"Demmers","first_name":"Jeroen A.A.","full_name":"Demmers, Jeroen A.A."},{"full_name":"Bernecky, Carrie A","first_name":"Carrie A","orcid":"0000-0003-0893-7036","id":"2CB9DFE2-F248-11E8-B48F-1D18A9856A87","last_name":"Bernecky"},{"last_name":"Sixma","full_name":"Sixma, Titia K.","first_name":"Titia K."},{"full_name":"Marteijn, Jurgen A.","first_name":"Jurgen A.","last_name":"Marteijn"}],"article_type":"original","issue":"24","acknowledgement":"We thank N. Thompson and R. Burgess for the 8WG16 hybridoma cell line. This research was further supported by the Scientific Service Units (SSU) of IST Austria through resources provided by the Lab Support Facility (LSF) and the Preclinical Facility (PCF). This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society. Research at the Netherlands Cancer Institute is supported by institutional grants of the Dutch Cancer Society and the Dutch Ministry of Health, Welfare and Sport. This study was supported by a VICI (VI.C.182.025) and a TOP Grant (714.017.003) of the Netherlands Organization for Scientific Research.","doi":"10.1016/j.molcel.2024.10.030"},{"type":"journal_article","department":[{"_id":"CaBe"}],"scopus_import":"1","publication":"Journal of Medicinal Chemistry","language":[{"iso":"eng"}],"page":"8609-8629","_id":"18945","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","oa_version":"None","quality_controlled":"1","date_updated":"2025-01-29T09:19:15Z","status":"public","OA_type":"closed access","volume":67,"publication_status":"published","month":"05","article_type":"original","acknowledgement":"R.M.C. and K.B.M. are grateful for support by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) (grants 2013/50724–5 and 2014/50897–0), Embrapii (Empresa Brasileira de Pesquisa e Inovação Industrial), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) (grant 465651/2014–3) and Aché Laboratórios Farmacêuticos. R.M.C. and O.G. are also grateful for support by the Structural Genomics Consortium, a registered charity (1097737) that receives funds from AbbVie, Bayer AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genentech, Genome Canada through the Ontario Genomics Institute (OGI-196), EU/EFPIA/OICR/McGill/KTH/Diamond, Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN Grant 875510), Janssen, Merck KGaA, Merck & Co., Pfizer, Takeda, and Wellcome. B.L. and M.H. are grateful for support from the Swedish Research Council, Swedish Cancer Society, Karolinska Institutet and The Mark Foundation for Cancer Research. R.A.M.S. (2016/25320–6 and 2018/23322–7), A.S.S. (2019/14275–8), S.N.S.V (2018/09475–5), V.M.A. (2022/00743–2) and M.R.C. (2021/04853–4) were recipients of fellowships from the Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP. C.V.R. (88887.146077/2017–00), J.E.T. (88887.373547/2019–00) and P.Z.R (88887.136432/2017–00) were the recipient of fellowships from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES.\r\nWe thank all members of CQMED-UNICAMP for their help and support. We thank the staff of the Life Sciences Core Facility (LaCTAD) at UNICAMP for the Genomics and Mass Spectrometry analysis. We thank the NMR facility at UNICAMP Chemistry Institute for its assistance. We thank the staff at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6M detector on the 24-ID-C beamline is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source; a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. We thank Diamond Light Source for access to beamline I24. The authors thank Tammy Havener (SGC-UNC), Abid Hussain Sayyid (KI), and Yiqiu Yang (KI) for valuable discussions and technical support.","issue":"11","doi":"10.1021/acs.jmedchem.3c02250","abstract":[{"lang":"eng","text":"Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division."}],"day":"23","date_published":"2024-05-23T00:00:00Z","author":[{"full_name":"de Souza Gama, Fernando H.","first_name":"Fernando H.","last_name":"de Souza Gama"},{"last_name":"Dutra","full_name":"Dutra, Luiz A.","first_name":"Luiz A."},{"last_name":"Hawgood","full_name":"Hawgood, Michael","first_name":"Michael"},{"first_name":"Caio Vinícius","full_name":"dos Reis, Caio Vinícius","last_name":"dos Reis"},{"last_name":"Serafim","full_name":"Serafim, Ricardo A. M.","first_name":"Ricardo A. M."},{"last_name":"Ferreira","full_name":"Ferreira, Marcos A.","first_name":"Marcos A."},{"first_name":"Bruno V. M.","full_name":"Teodoro, Bruno V. M.","last_name":"Teodoro"},{"first_name":"Jéssica Emi","full_name":"Takarada, Jéssica Emi","last_name":"Takarada"},{"full_name":"Santiago, André S.","first_name":"André S.","last_name":"Santiago"},{"full_name":"Balourdas, Dimitrios-Ilias","first_name":"Dimitrios-Ilias","last_name":"Balourdas"},{"last_name":"Nilsson","first_name":"Ann-Sofie","full_name":"Nilsson, Ann-Sofie"},{"last_name":"Urien","first_name":"Bruno","full_name":"Urien, Bruno"},{"last_name":"Almeida","full_name":"Almeida, Vitor M.","first_name":"Vitor M."},{"last_name":"Gileadi","full_name":"Gileadi, Carina","first_name":"Carina"},{"last_name":"Ramos","full_name":"Ramos, Priscila Z.","first_name":"Priscila Z."},{"full_name":"Testa Salmazo, Anita P","first_name":"Anita P","id":"41F1F098-F248-11E8-B48F-1D18A9856A87","last_name":"Testa Salmazo"},{"last_name":"Vasconcelos","full_name":"Vasconcelos, Stanley N. S.","first_name":"Stanley N. S."},{"first_name":"Micael R.","full_name":"Cunha, Micael R.","last_name":"Cunha"},{"last_name":"Mueller","full_name":"Mueller, Susanne","first_name":"Susanne"},{"first_name":"Stefan","full_name":"Knapp, Stefan","last_name":"Knapp"},{"last_name":"Massirer","first_name":"Katlin B.","full_name":"Massirer, Katlin B."},{"first_name":"Jonathan M.","full_name":"Elkins, Jonathan M.","last_name":"Elkins"},{"last_name":"Gileadi","full_name":"Gileadi, Opher","first_name":"Opher"},{"full_name":"Mascarello, Alessandra","first_name":"Alessandra","last_name":"Mascarello"},{"last_name":"Lemmens","full_name":"Lemmens, Bennie B. L. G.","first_name":"Bennie B. L. G."},{"full_name":"Guimarães, Cristiano R. W.","first_name":"Cristiano R. W.","last_name":"Guimarães"},{"last_name":"Azevedo","first_name":"Hatylas","full_name":"Azevedo, Hatylas"},{"last_name":"Couñago","full_name":"Couñago, Rafael M.","first_name":"Rafael M."}],"citation":{"short":"F.H. de Souza Gama, L.A. Dutra, M. Hawgood, C.V. dos Reis, R.A.M. Serafim, M.A. Ferreira, B.V.M. Teodoro, J.E. Takarada, A.S. Santiago, D.-I. Balourdas, A.-S. Nilsson, B. Urien, V.M. Almeida, C. Gileadi, P.Z. Ramos, A.P. Testa Salmazo, S.N.S. Vasconcelos, M.R. Cunha, S. Mueller, S. Knapp, K.B. Massirer, J.M. Elkins, O. Gileadi, A. Mascarello, B.B.L.G. Lemmens, C.R.W. Guimarães, H. Azevedo, R.M. Couñago, Journal of Medicinal Chemistry 67 (2024) 8609–8629.","ama":"de Souza Gama FH, Dutra LA, Hawgood M, et al. Novel dihydropteridinone derivatives as potent inhibitors of the understudied human kinases vaccinia-related kinase 1 and casein kinase 1δ/ε. <i>Journal of Medicinal Chemistry</i>. 2024;67(11):8609-8629. doi:<a href=\"https://doi.org/10.1021/acs.jmedchem.3c02250\">10.1021/acs.jmedchem.3c02250</a>","mla":"de Souza Gama, Fernando H., et al. “Novel Dihydropteridinone Derivatives as Potent Inhibitors of the Understudied Human Kinases Vaccinia-Related Kinase 1 and Casein Kinase 1δ/ε.” <i>Journal of Medicinal Chemistry</i>, vol. 67, no. 11, American Chemical Society, 2024, pp. 8609–29, doi:<a href=\"https://doi.org/10.1021/acs.jmedchem.3c02250\">10.1021/acs.jmedchem.3c02250</a>.","ieee":"F. H. de Souza Gama <i>et al.</i>, “Novel dihydropteridinone derivatives as potent inhibitors of the understudied human kinases vaccinia-related kinase 1 and casein kinase 1δ/ε,” <i>Journal of Medicinal Chemistry</i>, vol. 67, no. 11. American Chemical Society, pp. 8609–8629, 2024.","ista":"de Souza Gama FH, Dutra LA, Hawgood M, dos Reis CV, Serafim RAM, Ferreira MA, Teodoro BVM, Takarada JE, Santiago AS, Balourdas D-I, Nilsson A-S, Urien B, Almeida VM, Gileadi C, Ramos PZ, Testa Salmazo AP, Vasconcelos SNS, Cunha MR, Mueller S, Knapp S, Massirer KB, Elkins JM, Gileadi O, Mascarello A, Lemmens BBLG, Guimarães CRW, Azevedo H, Couñago RM. 2024. Novel dihydropteridinone derivatives as potent inhibitors of the understudied human kinases vaccinia-related kinase 1 and casein kinase 1δ/ε. Journal of Medicinal Chemistry. 67(11), 8609–8629.","apa":"de Souza Gama, F. H., Dutra, L. A., Hawgood, M., dos Reis, C. V., Serafim, R. A. M., Ferreira, M. A., … Couñago, R. M. (2024). Novel dihydropteridinone derivatives as potent inhibitors of the understudied human kinases vaccinia-related kinase 1 and casein kinase 1δ/ε. <i>Journal of Medicinal Chemistry</i>. American Chemical Society. <a href=\"https://doi.org/10.1021/acs.jmedchem.3c02250\">https://doi.org/10.1021/acs.jmedchem.3c02250</a>","chicago":"Souza Gama, Fernando H. de, Luiz A. Dutra, Michael Hawgood, Caio Vinícius dos Reis, Ricardo A. M. Serafim, Marcos A. Ferreira, Bruno V. M. Teodoro, et al. “Novel Dihydropteridinone Derivatives as Potent Inhibitors of the Understudied Human Kinases Vaccinia-Related Kinase 1 and Casein Kinase 1δ/ε.” <i>Journal of Medicinal Chemistry</i>. American Chemical Society, 2024. <a href=\"https://doi.org/10.1021/acs.jmedchem.3c02250\">https://doi.org/10.1021/acs.jmedchem.3c02250</a>."},"article_processing_charge":"No","publisher":"American Chemical Society","pmid":1,"title":"Novel dihydropteridinone derivatives as potent inhibitors of the understudied human kinases vaccinia-related kinase 1 and casein kinase 1δ/ε","year":"2024","date_created":"2025-01-29T09:14:19Z","intvolume":"        67","external_id":{"pmid":["38780468"]},"publication_identifier":{"eissn":["1520-4804"],"issn":["0022-2623"]}},{"oa_version":"Published Version","_id":"18477","user_id":"ba8df636-2132-11f1-aed0-ed93e2281fdd","page":"124","acknowledged_ssus":[{"_id":"EM-Fac"},{"_id":"LifeSc"}],"status":"public","date_updated":"2026-04-07T13:23:59Z","tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode"},"degree_awarded":"PhD","month":"10","publication_status":"published","type":"dissertation","OA_place":"publisher","department":[{"_id":"GradSch"},{"_id":"CaBe"}],"alternative_title":["ISTA Thesis"],"supervisor":[{"id":"2CB9DFE2-F248-11E8-B48F-1D18A9856A87","last_name":"Bernecky","first_name":"Carrie A","full_name":"Bernecky, Carrie A","orcid":"0000-0003-0893-7036"}],"corr_author":"1","language":[{"iso":"eng"}],"ddc":["572"],"file_date_updated":"2025-10-29T23:30:02Z","citation":{"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>.","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>","short":"B.M. Kaczmarek, Biochemical and Structural Insights into ADAR1 RNA Editing, Institute of Science and Technology Austria, 2024.","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>","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.","ieee":"B. M. Kaczmarek, “Biochemical and structural insights into ADAR1 RNA editing,” Institute of Science and Technology Austria, 2024."},"publisher":"Institute of Science and Technology Austria","article_processing_charge":"No","file":[{"date_updated":"2025-10-29T23:30:02Z","file_size":23136626,"content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","file_name":"20241029_PhD_thesis_BKaczmarek.docx","creator":"bkaczmar","date_created":"2024-10-29T11:56:36Z","embargo_to":"open_access","file_id":"18485","checksum":"2053294ea4d770c495e4cc501e2a218b","access_level":"closed","relation":"source_file"},{"date_updated":"2025-10-29T23:30:02Z","file_size":11707360,"embargo":"2025-10-29","content_type":"application/pdf","file_name":"20241029_PhD_thesis_BKaczmarek.pdf","creator":"bkaczmar","date_created":"2024-10-29T11:56:44Z","file_id":"18486","access_level":"open_access","checksum":"8ce857a4cd44b776791eaf180ac9dbb3","relation":"main_file"}],"title":"Biochemical and structural insights into ADAR1 RNA editing","has_accepted_license":"1","date_created":"2024-10-27T07:35:13Z","year":"2024","publication_identifier":{"isbn":["978-3-99078-045-9"],"issn":["2663-337X"]},"doi":"10.15479/at:ista:18477","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"}],"day":"29","date_published":"2024-10-29T00:00:00Z","oa":1,"author":[{"full_name":"Kaczmarek, Beata M","first_name":"Beata M","last_name":"Kaczmarek","id":"36FA4AFA-F248-11E8-B48F-1D18A9856A87"}]},{"ec_funded":1,"doi":"10.1101/2023.10.09.561523","corr_author":"1","type":"preprint","OA_place":"repository","department":[{"_id":"JiFr"},{"_id":"MaLo"},{"_id":"CaBe"}],"date_published":"2023-10-10T00:00:00Z","author":[{"first_name":"Nataliia","full_name":"Gnyliukh, Nataliia","orcid":"0000-0002-2198-0509","id":"390C1120-F248-11E8-B48F-1D18A9856A87","last_name":"Gnyliukh"},{"last_name":"Johnson","id":"46A62C3A-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0002-2739-8843","full_name":"Johnson, Alexander J","first_name":"Alexander J"},{"full_name":"Nagel, Marie-Kristin","first_name":"Marie-Kristin","last_name":"Nagel"},{"id":"2DB5D88C-D7B3-11E9-B8FD-7907E6697425","last_name":"Monzer","full_name":"Monzer, Aline","first_name":"Aline"},{"first_name":"Annamaria","full_name":"Hlavata, Annamaria","id":"36062FEC-F248-11E8-B48F-1D18A9856A87","last_name":"Hlavata"},{"full_name":"Isono, Erika","first_name":"Erika","last_name":"Isono"},{"last_name":"Loose","id":"462D4284-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-7309-9724","full_name":"Loose, Martin","first_name":"Martin"},{"first_name":"Jiří","full_name":"Friml, Jiří","orcid":"0000-0002-8302-7596","id":"4159519E-F248-11E8-B48F-1D18A9856A87","last_name":"Friml"}],"language":[{"iso":"eng"}],"oa":1,"publication":"bioRxiv","abstract":[{"lang":"eng","text":"Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development by controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scissin machinery in plants, but the precise roles of these proteins in this process is not fully understood. Here, we characterised the roles of Plant Dynamin-Related Proteins 2 (DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to Dynamins’ recruiters, like Endophilin and Amphiphysin, in the CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the Dsh3p1,2,3 triple-mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggests that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME. One Sentence Summary In contrast to predictions based on mammalian systems, plant Dynamin-related proteins 2 are recruited to the site of Clathrin-mediated endocytosis independently of BAR-SH3 proteins."}],"project":[{"grant_number":"665385","call_identifier":"H2020","name":"International IST Doctoral Program","_id":"2564DBCA-B435-11E9-9278-68D0E5697425"}],"day":"10","citation":{"short":"N. Gnyliukh, A.J. Johnson, M.-K. Nagel, A. Monzer, A. Hlavata, E. Isono, M. Loose, J. Friml, BioRxiv (n.d.).","ama":"Gnyliukh N, Johnson AJ, Nagel M-K, et al. Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in plants. <i>bioRxiv</i>. doi:<a href=\"https://doi.org/10.1101/2023.10.09.561523\">10.1101/2023.10.09.561523</a>","mla":"Gnyliukh, Nataliia, et al. “Role of Dynamin-Related Proteins 2 and SH3P2 in Clathrin-Mediated Endocytosis in Plants.” <i>BioRxiv</i>, doi:<a href=\"https://doi.org/10.1101/2023.10.09.561523\">10.1101/2023.10.09.561523</a>.","ieee":"N. Gnyliukh <i>et al.</i>, “Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in plants,” <i>bioRxiv</i>. .","ista":"Gnyliukh N, Johnson AJ, Nagel M-K, Monzer A, Hlavata A, Isono E, Loose M, Friml J. Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in plants. bioRxiv, <a href=\"https://doi.org/10.1101/2023.10.09.561523\">10.1101/2023.10.09.561523</a>.","chicago":"Gnyliukh, Nataliia, Alexander J Johnson, Marie-Kristin Nagel, Aline Monzer, Annamaria Hlavata, Erika Isono, Martin Loose, and Jiří Friml. “Role of Dynamin-Related Proteins 2 and SH3P2 in Clathrin-Mediated Endocytosis in Plants.” <i>BioRxiv</i>, n.d. <a href=\"https://doi.org/10.1101/2023.10.09.561523\">https://doi.org/10.1101/2023.10.09.561523</a>.","apa":"Gnyliukh, N., Johnson, A. J., Nagel, M.-K., Monzer, A., Hlavata, A., Isono, E., … Friml, J. (n.d.). Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in plants. <i>bioRxiv</i>. <a href=\"https://doi.org/10.1101/2023.10.09.561523\">https://doi.org/10.1101/2023.10.09.561523</a>"},"related_material":{"record":[{"relation":"later_version","status":"public","id":"15330"},{"status":"public","id":"14510","relation":"dissertation_contains"}]},"article_processing_charge":"No","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","_id":"14591","oa_version":"Preprint","acknowledged_ssus":[{"_id":"EM-Fac"},{"_id":"LifeSc"},{"_id":"Bio"}],"date_created":"2023-11-22T10:17:49Z","year":"2023","publication_status":"draft","month":"10","status":"public","date_updated":"2026-07-03T22:34:14Z","title":"Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in plants","main_file_link":[{"url":"https://doi.org/10.1101/2023.10.09.561523","open_access":"1"}]},{"scopus_import":"1","department":[{"_id":"CaBe"}],"type":"journal_article","language":[{"iso":"eng"}],"publication":"Life Science Alliance","keyword":["Health","Toxicology and Mutagenesis","Plant Science","Biochemistry","Genetics and Molecular Biology (miscellaneous)","Ecology"],"article_number":"e202201568","quality_controlled":"1","_id":"12051","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","oa_version":"Published Version","isi":1,"volume":5,"month":"09","publication_status":"published","tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode"},"status":"public","date_updated":"2024-10-21T06:01:48Z","doi":"10.26508/lsa.202201568","acknowledgement":"The authors especially thank Philip Gunkel for his contribution. We thank all\r\npast and present members of the Engel lab, Achim Griesenbeck, Colyn Crane-\r\nRobinson, Christophe Lotz, Marlene Vayssieres, Klaus Grasser, Herbert Tschochner, and Philipp Milkereit for help and discussion; Gerhard Lehmann and Nobert Eichner for IT support; Joost Zomerdijk for UBF-constructs, Volker Cordes for the Hela P2 cell line; Remco Sprangers for shared cell culture; Dina Grohmann and the Archaea Center for fermentation; and Thomas\r\nDresselhaus for access to fluorescence microscopes. This work was in part supported by the Emmy-Noether Programm (DFG grant no. EN 1204/1-1 to C Engel) of the German Research Council and Collaborative Research Center 960 (TP-A8 to C Engel).","issue":"11","article_type":"original","author":[{"first_name":"Julia L","full_name":"Daiß, Julia L","last_name":"Daiß"},{"last_name":"Pilsl","first_name":"Michael","full_name":"Pilsl, Michael"},{"last_name":"Straub","first_name":"Kristina","full_name":"Straub, Kristina"},{"first_name":"Andrea","full_name":"Bleckmann, Andrea","last_name":"Bleckmann"},{"last_name":"Höcherl","first_name":"Mona","full_name":"Höcherl, Mona"},{"last_name":"Heiss","first_name":"Florian B","full_name":"Heiss, Florian B"},{"first_name":"Guillermo","full_name":"Abascal-Palacios, Guillermo","last_name":"Abascal-Palacios"},{"first_name":"Ewan P","full_name":"Ramsay, Ewan P","last_name":"Ramsay"},{"first_name":"Katarina","full_name":"Tluckova, Katarina","last_name":"Tluckova","id":"4AC7D980-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Mars, Jean-Clement","first_name":"Jean-Clement","last_name":"Mars"},{"full_name":"Fürtges, Torben","first_name":"Torben","last_name":"Fürtges"},{"last_name":"Bruckmann","first_name":"Astrid","full_name":"Bruckmann, Astrid"},{"full_name":"Rudack, Till","first_name":"Till","last_name":"Rudack"},{"full_name":"Bernecky, Carrie A","first_name":"Carrie A","orcid":"0000-0003-0893-7036","id":"2CB9DFE2-F248-11E8-B48F-1D18A9856A87","last_name":"Bernecky"},{"last_name":"Lamour","first_name":"Valérie","full_name":"Lamour, Valérie"},{"first_name":"Konstantin","full_name":"Panov, Konstantin","last_name":"Panov"},{"full_name":"Vannini, Alessandro","first_name":"Alessandro","last_name":"Vannini"},{"last_name":"Moss","first_name":"Tom","full_name":"Moss, Tom"},{"full_name":"Engel, Christoph","first_name":"Christoph","last_name":"Engel"}],"oa":1,"date_published":"2022-09-01T00:00:00Z","day":"01","abstract":[{"text":"Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This “dock II” domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor–binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain–containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.","lang":"eng"}],"article_processing_charge":"No","publisher":"Life Science Alliance","file":[{"content_type":"application/pdf","file_name":"2022_LifeScienceAlliance_Daiss.pdf","creator":"dernst","success":1,"file_size":3183129,"date_updated":"2022-09-08T06:41:14Z","date_created":"2022-09-08T06:41:14Z","file_id":"12062","checksum":"4201d876a3e5e8b65e319d03300014ad","access_level":"open_access","relation":"main_file"}],"citation":{"short":"J.L. Daiß, M. Pilsl, K. Straub, A. Bleckmann, M. Höcherl, F.B. Heiss, G. Abascal-Palacios, E.P. Ramsay, K. Tluckova, J.-C. Mars, T. Fürtges, A. Bruckmann, T. Rudack, C. Bernecky, V. Lamour, K. Panov, A. Vannini, T. Moss, C. Engel, Life Science Alliance 5 (2022).","ama":"Daiß JL, Pilsl M, Straub K, et al. The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans. <i>Life Science Alliance</i>. 2022;5(11). doi:<a href=\"https://doi.org/10.26508/lsa.202201568\">10.26508/lsa.202201568</a>","mla":"Daiß, Julia L., et al. “The Human RNA Polymerase I Structure Reveals an HMG-like Docking Domain Specific to Metazoans.” <i>Life Science Alliance</i>, vol. 5, no. 11, e202201568, Life Science Alliance, 2022, doi:<a href=\"https://doi.org/10.26508/lsa.202201568\">10.26508/lsa.202201568</a>.","ieee":"J. L. Daiß <i>et al.</i>, “The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans,” <i>Life Science Alliance</i>, vol. 5, no. 11. Life Science Alliance, 2022.","ista":"Daiß JL, Pilsl M, Straub K, Bleckmann A, Höcherl M, Heiss FB, Abascal-Palacios G, Ramsay EP, Tluckova K, Mars J-C, Fürtges T, Bruckmann A, Rudack T, Bernecky C, Lamour V, Panov K, Vannini A, Moss T, Engel C. 2022. The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans. Life Science Alliance. 5(11), e202201568.","chicago":"Daiß, Julia L, Michael Pilsl, Kristina Straub, Andrea Bleckmann, Mona Höcherl, Florian B Heiss, Guillermo Abascal-Palacios, et al. “The Human RNA Polymerase I Structure Reveals an HMG-like Docking Domain Specific to Metazoans.” <i>Life Science Alliance</i>. Life Science Alliance, 2022. <a href=\"https://doi.org/10.26508/lsa.202201568\">https://doi.org/10.26508/lsa.202201568</a>.","apa":"Daiß, J. L., Pilsl, M., Straub, K., Bleckmann, A., Höcherl, M., Heiss, F. B., … Engel, C. (2022). The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans. <i>Life Science Alliance</i>. Life Science Alliance. <a href=\"https://doi.org/10.26508/lsa.202201568\">https://doi.org/10.26508/lsa.202201568</a>"},"ddc":["570"],"file_date_updated":"2022-09-08T06:41:14Z","external_id":{"isi":["000972702600001"]},"publication_identifier":{"issn":["2575-1077"]},"date_created":"2022-09-06T18:45:23Z","year":"2022","intvolume":"         5","has_accepted_license":"1","title":"The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans"},{"abstract":[{"lang":"eng","text":"MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer’s DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer⋅miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways."}],"day":"03","date_published":"2022-11-03T00:00:00Z","oa":1,"author":[{"last_name":"Zapletal","full_name":"Zapletal, David","first_name":"David"},{"last_name":"Taborska","full_name":"Taborska, Eliska","first_name":"Eliska"},{"last_name":"Pasulka","full_name":"Pasulka, Josef","first_name":"Josef"},{"full_name":"Malik, Radek","first_name":"Radek","last_name":"Malik"},{"first_name":"Karel","full_name":"Kubicek, Karel","last_name":"Kubicek"},{"last_name":"Zanova","full_name":"Zanova, Martina","first_name":"Martina"},{"full_name":"Much, Christian","first_name":"Christian","last_name":"Much"},{"first_name":"Marek","full_name":"Sebesta, Marek","last_name":"Sebesta"},{"last_name":"Buccheri","full_name":"Buccheri, Valeria","first_name":"Valeria"},{"full_name":"Horvat, Filip","first_name":"Filip","last_name":"Horvat"},{"full_name":"Jenickova, Irena","first_name":"Irena","last_name":"Jenickova"},{"last_name":"Prochazkova","first_name":"Michaela","full_name":"Prochazkova, Michaela"},{"first_name":"Jan","full_name":"Prochazka, Jan","last_name":"Prochazka"},{"first_name":"Matyas","full_name":"Pinkas, Matyas","last_name":"Pinkas"},{"full_name":"Novacek, Jiri","first_name":"Jiri","last_name":"Novacek"},{"first_name":"Diego F.","full_name":"Joseph, Diego F.","last_name":"Joseph"},{"last_name":"Sedlacek","first_name":"Radislav","full_name":"Sedlacek, Radislav"},{"first_name":"Carrie A","full_name":"Bernecky, Carrie A","orcid":"0000-0003-0893-7036","id":"2CB9DFE2-F248-11E8-B48F-1D18A9856A87","last_name":"Bernecky"},{"first_name":"Dónal","full_name":"O’Carroll, Dónal","last_name":"O’Carroll"},{"last_name":"Stefl","full_name":"Stefl, Richard","first_name":"Richard"},{"full_name":"Svoboda, Petr","first_name":"Petr","last_name":"Svoboda"}],"article_type":"original","issue":"21","acknowledgement":"We thank Kristian Vlahovicek (University of Zagreb) for support of bioinformatics analyses and Vladimir Benes (EMBL Sequencing Facility) and Genomics and Bioinformatics Core Facility at the Institute of Molecular Genetics for help with RNA sequencing. The main funding was provided by the Czech Science Foundation (EXPRO grant 20-03950X to P.S. and 22-19896S to R. Stefl). Early stages of the work were supported by European Research Council grants under the European Union’s Horizon 2020 Research and Innovation Programme (grants 647403 to P.S. and 649030 to R. Stefl). V.B., D.F.J., and F.H. were in part supported by PhD student fellowships from the Charles University; this work will be in part fulfilling requirements for a PhD degree as “school work.” Funding of D.Z. included the OP RDE project “Internal Grant Agency of Masaryk University” no. CZ.02.2.69/0.0/0.0/19_073/0016943. The Ministry of Education, Youth, and Sports of the Czech Republic (MEYS CR) provided institutional support for CEITEC 2020 project LQ1601. For technical support, we acknowledge EMBL Monterotondo’s genome engineering and transgenic core facilities, the Czech Centre for Phenogenomics at the Institute of Molecular Genetics (supported by RVO 68378050 from the Czech Academy of Sciences and LM2018126 and CZ.02.1.01/0.0/0.0/18_046/0015861 CCP Infrastructure Upgrade II from MEYS CR), the Cryo-EM and Proteomics Core Facilities (CEITEC, Masaryk University) supported by the CIISB research infrastructure (LM2018127 from MEYS CR), and support from the Scientific Service Units of ISTA through resources from the Electron Microscopy Facility. Computational resources included e-Infrastruktura CZ (LM2018140) and ELIXIR-CZ (LM2018131) projects by MEYS CR and the Croatian National Centres of Research Excellence in Personalized Healthcare (#KK.01.1.1.01.0010) and Data Science and Advanced Cooperative Systems (#KK.01.1.1.01.0009) projects funded by the European Structural and Investment Funds grants.","doi":"10.1016/j.molcel.2022.10.010","title":"Structural and functional basis of mammalian microRNA biogenesis by Dicer","pmid":1,"has_accepted_license":"1","intvolume":"        82","date_created":"2023-01-12T12:05:36Z","year":"2022","publication_identifier":{"issn":["1097-2765"]},"external_id":{"pmid":["36332606"],"isi":["000898565300011"]},"file_date_updated":"2023-01-24T09:29:02Z","ddc":["570"],"citation":{"short":"D. Zapletal, E. Taborska, J. Pasulka, R. Malik, K. Kubicek, M. Zanova, C. Much, M. Sebesta, V. Buccheri, F. Horvat, I. Jenickova, M. Prochazkova, J. Prochazka, M. Pinkas, J. Novacek, D.F. Joseph, R. Sedlacek, C. Bernecky, D. O’Carroll, R. Stefl, P. Svoboda, Molecular Cell 82 (2022) 4064–4079.e13.","ama":"Zapletal D, Taborska E, Pasulka J, et al. Structural and functional basis of mammalian microRNA biogenesis by Dicer. <i>Molecular Cell</i>. 2022;82(21):4064-4079.e13. doi:<a href=\"https://doi.org/10.1016/j.molcel.2022.10.010\">10.1016/j.molcel.2022.10.010</a>","mla":"Zapletal, David, et al. “Structural and Functional Basis of Mammalian MicroRNA Biogenesis by Dicer.” <i>Molecular Cell</i>, vol. 82, no. 21, Elsevier, 2022, p. 4064–4079.e13, doi:<a href=\"https://doi.org/10.1016/j.molcel.2022.10.010\">10.1016/j.molcel.2022.10.010</a>.","ieee":"D. Zapletal <i>et al.</i>, “Structural and functional basis of mammalian microRNA biogenesis by Dicer,” <i>Molecular Cell</i>, vol. 82, no. 21. Elsevier, p. 4064–4079.e13, 2022.","ista":"Zapletal D, Taborska E, Pasulka J, Malik R, Kubicek K, Zanova M, Much C, Sebesta M, Buccheri V, Horvat F, Jenickova I, Prochazkova M, Prochazka J, Pinkas M, Novacek J, Joseph DF, Sedlacek R, Bernecky C, O’Carroll D, Stefl R, Svoboda P. 2022. Structural and functional basis of mammalian microRNA biogenesis by Dicer. Molecular Cell. 82(21), 4064–4079.e13.","chicago":"Zapletal, David, Eliska Taborska, Josef Pasulka, Radek Malik, Karel Kubicek, Martina Zanova, Christian Much, et al. “Structural and Functional Basis of Mammalian MicroRNA Biogenesis by Dicer.” <i>Molecular Cell</i>. Elsevier, 2022. <a href=\"https://doi.org/10.1016/j.molcel.2022.10.010\">https://doi.org/10.1016/j.molcel.2022.10.010</a>.","apa":"Zapletal, D., Taborska, E., Pasulka, J., Malik, R., Kubicek, K., Zanova, M., … Svoboda, P. (2022). Structural and functional basis of mammalian microRNA biogenesis by Dicer. <i>Molecular Cell</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.molcel.2022.10.010\">https://doi.org/10.1016/j.molcel.2022.10.010</a>"},"file":[{"success":1,"file_name":"2022_MolecularCell_Zapletal.pdf","creator":"dernst","content_type":"application/pdf","file_size":7368534,"date_updated":"2023-01-24T09:29:02Z","date_created":"2023-01-24T09:29:02Z","file_id":"12354","relation":"main_file","checksum":"999e443b54e4fdaa2542ca5a97619731","access_level":"open_access"}],"article_processing_charge":"No","publisher":"Elsevier","publication":"Molecular Cell","keyword":["Cell Biology","Molecular Biology"],"language":[{"iso":"eng"}],"type":"journal_article","department":[{"_id":"CaBe"}],"scopus_import":"1","date_updated":"2023-08-04T08:57:17Z","status":"public","tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode"},"publication_status":"published","volume":82,"month":"11","isi":1,"_id":"12143","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","page":"4064-4079.e13","oa_version":"Published Version","acknowledged_ssus":[{"_id":"EM-Fac"}],"quality_controlled":"1"},{"tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode"},"date_updated":"2024-10-21T06:02:05Z","status":"public","volume":12,"publication_status":"published","month":"10","isi":1,"_id":"10163","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","oa_version":"Published Version","related_material":{"link":[{"url":"https://www.biorxiv.org/content/10.1101/2020.02.11.943159","relation":"earlier_version","description":"Preprint "}]},"quality_controlled":"1","article_number":"6078","keyword":["general physics and astronomy","general biochemistry","genetics and molecular biology","general chemistry"],"publication":"Nature Communications","language":[{"iso":"eng"}],"department":[{"_id":"CaBe"}],"type":"journal_article","scopus_import":"1","has_accepted_license":"1","title":"PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC","publication_identifier":{"eissn":["2041-1723"]},"external_id":{"isi":["000709050300001"]},"intvolume":"        12","year":"2021","date_created":"2021-10-20T14:40:32Z","file_date_updated":"2021-10-21T13:51:49Z","ddc":["610"],"file":[{"date_created":"2021-10-21T13:51:49Z","success":1,"file_name":"2021_NatComm_Appel.pdf","creator":"cchlebak","content_type":"application/pdf","file_size":5111706,"date_updated":"2021-10-21T13:51:49Z","relation":"main_file","access_level":"open_access","checksum":"d99fcd51aebde19c21314e3de0148007","file_id":"10169"}],"article_processing_charge":"No","publisher":"Springer Nature","citation":{"ieee":"L.-M. Appel <i>et al.</i>, “PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC,” <i>Nature Communications</i>, vol. 12, no. 1. Springer Nature, 2021.","ista":"Appel L-M, Franke V, Bruno M, Grishkovskaya I, Kasiliauskaite A, Kaufmann T, Schoeberl UE, Puchinger MG, Kostrhon S, Ebenwaldner C, Sebesta M, Beltzung E, Mechtler K, Lin G, Vlasova A, Leeb M, Pavri R, Stark A, Akalin A, Stefl R, Bernecky C, Djinovic-Carugo K, Slade D. 2021. PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC. Nature Communications. 12(1), 6078.","chicago":"Appel, Lisa-Marie, Vedran Franke, Melania Bruno, Irina Grishkovskaya, Aiste Kasiliauskaite, Tanja Kaufmann, Ursula E. Schoeberl, et al. “PHF3 Regulates Neuronal Gene Expression through the Pol II CTD Reader Domain SPOC.” <i>Nature Communications</i>. Springer Nature, 2021. <a href=\"https://doi.org/10.1038/s41467-021-26360-2\">https://doi.org/10.1038/s41467-021-26360-2</a>.","apa":"Appel, L.-M., Franke, V., Bruno, M., Grishkovskaya, I., Kasiliauskaite, A., Kaufmann, T., … Slade, D. (2021). PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC. <i>Nature Communications</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41467-021-26360-2\">https://doi.org/10.1038/s41467-021-26360-2</a>","short":"L.-M. Appel, V. Franke, M. Bruno, I. Grishkovskaya, A. Kasiliauskaite, T. Kaufmann, U.E. Schoeberl, M.G. Puchinger, S. Kostrhon, C. Ebenwaldner, M. Sebesta, E. Beltzung, K. Mechtler, G. Lin, A. Vlasova, M. Leeb, R. Pavri, A. Stark, A. Akalin, R. Stefl, C. Bernecky, K. Djinovic-Carugo, D. Slade, Nature Communications 12 (2021).","ama":"Appel L-M, Franke V, Bruno M, et al. PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC. <i>Nature Communications</i>. 2021;12(1). doi:<a href=\"https://doi.org/10.1038/s41467-021-26360-2\">10.1038/s41467-021-26360-2</a>","mla":"Appel, Lisa-Marie, et al. “PHF3 Regulates Neuronal Gene Expression through the Pol II CTD Reader Domain SPOC.” <i>Nature Communications</i>, vol. 12, no. 1, 6078, Springer Nature, 2021, doi:<a href=\"https://doi.org/10.1038/s41467-021-26360-2\">10.1038/s41467-021-26360-2</a>."},"day":"19","abstract":[{"text":"The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.","lang":"eng"}],"oa":1,"author":[{"first_name":"Lisa-Marie","full_name":"Appel, Lisa-Marie","last_name":"Appel"},{"last_name":"Franke","first_name":"Vedran","full_name":"Franke, Vedran"},{"last_name":"Bruno","first_name":"Melania","full_name":"Bruno, Melania"},{"full_name":"Grishkovskaya, Irina","first_name":"Irina","last_name":"Grishkovskaya"},{"first_name":"Aiste","full_name":"Kasiliauskaite, Aiste","last_name":"Kasiliauskaite"},{"last_name":"Kaufmann","first_name":"Tanja","full_name":"Kaufmann, Tanja"},{"full_name":"Schoeberl, Ursula E.","first_name":"Ursula E.","last_name":"Schoeberl"},{"last_name":"Puchinger","first_name":"Martin G.","full_name":"Puchinger, Martin G."},{"full_name":"Kostrhon, Sebastian","first_name":"Sebastian","last_name":"Kostrhon"},{"full_name":"Ebenwaldner, Carmen","first_name":"Carmen","last_name":"Ebenwaldner"},{"last_name":"Sebesta","full_name":"Sebesta, Marek","first_name":"Marek"},{"full_name":"Beltzung, Etienne","first_name":"Etienne","last_name":"Beltzung"},{"last_name":"Mechtler","first_name":"Karl","full_name":"Mechtler, Karl"},{"last_name":"Lin","full_name":"Lin, Gen","first_name":"Gen"},{"last_name":"Vlasova","full_name":"Vlasova, Anna","first_name":"Anna"},{"last_name":"Leeb","first_name":"Martin","full_name":"Leeb, Martin"},{"last_name":"Pavri","full_name":"Pavri, Rushad","first_name":"Rushad"},{"last_name":"Stark","first_name":"Alexander","full_name":"Stark, Alexander"},{"last_name":"Akalin","first_name":"Altuna","full_name":"Akalin, Altuna"},{"last_name":"Stefl","first_name":"Richard","full_name":"Stefl, Richard"},{"id":"2CB9DFE2-F248-11E8-B48F-1D18A9856A87","last_name":"Bernecky","full_name":"Bernecky, Carrie A","first_name":"Carrie A","orcid":"0000-0003-0893-7036"},{"first_name":"Kristina","full_name":"Djinovic-Carugo, Kristina","last_name":"Djinovic-Carugo"},{"first_name":"Dea","full_name":"Slade, Dea","last_name":"Slade"}],"date_published":"2021-10-19T00:00:00Z","issue":"1","acknowledgement":"D.S. thanks Claudine Kraft, Renée Schroeder, Verena Jantsch, Franz Klein and Peter Schlögelhofer for support. We thank Anita Testa Salmazo for help with purifying Pol II; Matthias Geyer and Robert Düster for sharing DYRK1A kinase; Felix Hartmann and Clemens Plaschka for help with mass photometry; Goran Kokic for design of the arrest assay sequences; Petra van der Lelij for help with generating mESC KO; Maximilian Freilinger for help with the purification of mEGFP-CTD; Stefan Ameres, Nina Fasching and Brian Reichholf for advice on SLAM-seq and for sharing reagents; Laura Gallego Valle for advice regarding LLPS assays; Krzysztof Chylinski for advice regarding CRISPR/Cas9 methodology; VBCF Protein Technologies facility for purifying PHF3 and providing gRNAs and Cas9; VBCF NGS facility for sequencing; Monoclonal antibody facility at the Helmholtz center for Pol II antibodies; Friedrich Propst and Elzbieta Kowalska for advice and for sharing materials; Egon Ogris for sharing materials; Martin Eilers for recommending a ChIP-grade TFIIS antibody; Susanne Opravil, Otto Hudecz, Markus Hartl and Natascha Hartl for mass spectrometry analysis; staff of the X-ray beamlines at the ESRF in Grenoble for their excellent support; Christa Bücker, Anton Meinhart, Clemens Plaschka and members of the Slade lab for critical comments on the manuscript; Life Science Editors for editing assistance. M.B. and D.S. acknowledge support by the FWF-funded DK ‘Chromosome Dynamics’. T.K. is a recipient of the DOC fellowship from the Austrian Academy of Sciences. U.S. is supported by the L’Oreal for Women in Science Austria Fellowship and the Austrian Science Fund (FWF T 795-B30). M.L is supported by the Vienna Science and Technology Fund (WWTF, VRG14-006). R.S. is supported by the Czech Science Foundation (15-17670 S and 21-24460 S), Ministry of Education, Youths and Sports of the Czech Republic (CEITEC 2020 project (LQ1601)), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant agreement no. 649030); this publication reflects only the author’s view and the Research Executive Agency is not responsible for any use that may be made of the information it contains. M.S. is supported by the Czech Science Foundation (GJ20-21581Y). K.D.C. research is supported by the Austrian Science Fund (FWF) Projects I525 and I1593, P22276, P19060, and W1221, Federal Ministry of Economy, Family and Youth through the initiative ‘Laura Bassi Centres of Expertise’, funding from the Centre of Optimized Structural Studies No. 253275, the Wellcome Trust Collaborative Award (201543/Z/16), COST action BM1405 Non-globular proteins - from sequence to structure, function and application in molecular physiopathology (NGP-NET), the Vienna Science and Technology Fund (WWTF LS17-008), and by the University of Vienna. This project was funded by the MFPL start-up grant, the Vienna Science and Technology Fund (WWTF LS14-001), and the Austrian Science Fund (P31546-B28 and W1258 “DK: Integrative Structural Biology”) to D.S.","article_type":"original","doi":"10.1038/s41467-021-26360-2"},{"issue":"36","acknowledgement":"We thank the staff of the macromolecular crystallography (MX) and SAXS beamlines at the European Synchrotron Radiation facility, Diamond, and Swiss Light Source for excellent support, and the Life Sciences Facility of the Institute of Science and Technology Austria for usage of the rheometer. We thank Life Sciences editors for editing assistance. EM data were\r\nrecorded at the EM Facility of the Vienna BioCenter Core Facilities (Austria). Confocal microscopy was carried out at the Advanced Instrument Research Facility, Jawaharlal Nehru University. K.D.-C.’s research was supported by the Initial Training Network MUZIC (ITN-MUZIC) (N°238423), Austrian Science Fund (FWF) Projects I525, I1593, P22276, P19060, and W1221, Laura Bassi Centre of Optimized Structural Studies (N°253275), a Wellcome Trust Collaborative Award (201543/Z/16/Z), COST Action BM1405, Vienna Science and Technology Fund (WWTF) Chemical Biology Project LS17-008, and Christian Doppler Laboratory for High-Content Structural Biology and Biotechnology. K.Z., J.L.A., C.S., E.A.G., and A.S. were supported by the University of Vienna, J.K. by a Wellcome Trust Collaborative Award and by the Centre of Optimized Structural Studies, M.P. by FWF Project I1593, E.d.A.R. ITN-MUZIC, and FWF Projects I525 and I1593, and T.C.M. and L.C. by FWF Project I 2408-B22. E.A.G. acknowledges the PhD program Structure and Interaction of Biological Macromolecules. M.B. acknowledges the University Grant Commission, India, for a senior research fellowship. A.B. acknowledges a JC Bose Fellowship from the Science Engineering Research Council. ","article_type":"original","doi":"10.1073/pnas.1917269117","day":"08","abstract":[{"lang":"eng","text":"The actin cytoskeleton, a dynamic network of actin filaments and associated F-actin–binding proteins, is fundamentally important in eukaryotes. α-Actinins are major F-actin bundlers that are inhibited by Ca2+ in nonmuscle cells. Here we report the mechanism of Ca2+-mediated regulation of Entamoeba histolytica α-actinin-2 (EhActn2) with features expected for the common ancestor of Entamoeba and higher eukaryotic α-actinins. Crystal structures of Ca2+-free and Ca2+-bound EhActn2 reveal a calmodulin-like domain (CaMD) uniquely inserted within the rod domain. Integrative studies reveal an exceptionally high affinity of the EhActn2 CaMD for Ca2+, binding of which can only be regulated in the presence of physiological concentrations of Mg2+. Ca2+ binding triggers an increase in protein multidomain rigidity, reducing conformational flexibility of F-actin–binding domains via interdomain cross-talk and consequently inhibiting F-actin bundling. In vivo studies uncover that EhActn2 plays an important role in phagocytic cup formation and might constitute a new drug target for amoebic dysentery."}],"oa":1,"author":[{"last_name":"Pinotsis","full_name":"Pinotsis, Nikos","first_name":"Nikos"},{"first_name":"Karolina","full_name":"Zielinska, Karolina","last_name":"Zielinska"},{"last_name":"Babuta","first_name":"Mrigya","full_name":"Babuta, Mrigya"},{"first_name":"Joan L.","full_name":"Arolas, Joan L.","last_name":"Arolas"},{"last_name":"Kostan","full_name":"Kostan, Julius","first_name":"Julius"},{"last_name":"Khan","full_name":"Khan, Muhammad Bashir","first_name":"Muhammad Bashir"},{"first_name":"Claudia","full_name":"Schreiner, Claudia","last_name":"Schreiner"},{"id":"41F1F098-F248-11E8-B48F-1D18A9856A87","last_name":"Testa Salmazo","first_name":"Anita P","full_name":"Testa Salmazo, Anita P"},{"last_name":"Ciccarelli","first_name":"Luciano","full_name":"Ciccarelli, Luciano"},{"last_name":"Puchinger","first_name":"Martin","full_name":"Puchinger, Martin"},{"full_name":"Gkougkoulia, Eirini A.","first_name":"Eirini A.","last_name":"Gkougkoulia"},{"last_name":"Ribeiro","first_name":"Euripedes de Almeida","full_name":"Ribeiro, Euripedes de Almeida"},{"first_name":"Thomas C.","full_name":"Marlovits, Thomas C.","last_name":"Marlovits"},{"last_name":"Bhattacharya","first_name":"Alok","full_name":"Bhattacharya, Alok"},{"last_name":"Djinovic-Carugo","first_name":"Kristina","full_name":"Djinovic-Carugo, Kristina"}],"date_published":"2020-09-08T00:00:00Z","ddc":["570"],"article_processing_charge":"No","publisher":"National Academy of Sciences","citation":{"ista":"Pinotsis N, Zielinska K, Babuta M, Arolas JL, Kostan J, Khan MB, Schreiner C, Testa Salmazo AP, Ciccarelli L, Puchinger M, Gkougkoulia EA, Ribeiro E de A, Marlovits TC, Bhattacharya A, Djinovic-Carugo K. 2020. Calcium modulates the domain flexibility and function of an α-actinin similar to the ancestral α-actinin. Proceedings of the National Academy of Sciences of the United States of America. 117(36), 22101–22112.","ieee":"N. Pinotsis <i>et al.</i>, “Calcium modulates the domain flexibility and function of an α-actinin similar to the ancestral α-actinin,” <i>Proceedings of the National Academy of Sciences of the United States of America</i>, vol. 117, no. 36. National Academy of Sciences, pp. 22101–22112, 2020.","apa":"Pinotsis, N., Zielinska, K., Babuta, M., Arolas, J. L., Kostan, J., Khan, M. B., … Djinovic-Carugo, K. (2020). Calcium modulates the domain flexibility and function of an α-actinin similar to the ancestral α-actinin. <i>Proceedings of the National Academy of Sciences of the United States of America</i>. National Academy of Sciences. <a href=\"https://doi.org/10.1073/pnas.1917269117\">https://doi.org/10.1073/pnas.1917269117</a>","chicago":"Pinotsis, Nikos, Karolina Zielinska, Mrigya Babuta, Joan L. Arolas, Julius Kostan, Muhammad Bashir Khan, Claudia Schreiner, et al. “Calcium Modulates the Domain Flexibility and Function of an α-Actinin Similar to the Ancestral α-Actinin.” <i>Proceedings of the National Academy of Sciences of the United States of America</i>. National Academy of Sciences, 2020. <a href=\"https://doi.org/10.1073/pnas.1917269117\">https://doi.org/10.1073/pnas.1917269117</a>.","short":"N. Pinotsis, K. Zielinska, M. Babuta, J.L. Arolas, J. Kostan, M.B. Khan, C. Schreiner, A.P. Testa Salmazo, L. Ciccarelli, M. Puchinger, E.A. Gkougkoulia, E. de A. Ribeiro, T.C. Marlovits, A. Bhattacharya, K. Djinovic-Carugo, Proceedings of the National Academy of Sciences of the United States of America 117 (2020) 22101–22112.","ama":"Pinotsis N, Zielinska K, Babuta M, et al. Calcium modulates the domain flexibility and function of an α-actinin similar to the ancestral α-actinin. <i>Proceedings of the National Academy of Sciences of the United States of America</i>. 2020;117(36):22101-22112. doi:<a href=\"https://doi.org/10.1073/pnas.1917269117\">10.1073/pnas.1917269117</a>","mla":"Pinotsis, Nikos, et al. “Calcium Modulates the Domain Flexibility and Function of an α-Actinin Similar to the Ancestral α-Actinin.” <i>Proceedings of the National Academy of Sciences of the United States of America</i>, vol. 117, no. 36, National Academy of Sciences, 2020, pp. 22101–12, doi:<a href=\"https://doi.org/10.1073/pnas.1917269117\">10.1073/pnas.1917269117</a>."},"pmid":1,"title":"Calcium modulates the domain flexibility and function of an α-actinin similar to the ancestral α-actinin","publication_identifier":{"eissn":["1091-6490"],"issn":["0027-8424"]},"external_id":{"pmid":["32848067"]},"intvolume":"       117","year":"2020","date_created":"2024-03-04T10:03:52Z","department":[{"_id":"CaBe"}],"type":"journal_article","publication":"Proceedings of the National Academy of Sciences of the United States of America","language":[{"iso":"eng"}],"acknowledged_ssus":[{"_id":"LifeSc"}],"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","_id":"15061","oa_version":"Published Version","page":"22101-22112","quality_controlled":"1","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1073/pnas.191726911"}],"status":"public","date_updated":"2026-06-18T17:45:21Z","month":"09","volume":117,"publication_status":"published"},{"pmid":1,"title":"Stochastic activation and bistability in a Rab GTPase regulatory network","intvolume":"       117","year":"2020","date_created":"2020-03-12T05:32:26Z","publication_identifier":{"eissn":["1091-6490"],"issn":["0027-8424"]},"external_id":{"pmid":["32161136"],"isi":["000521821800040"]},"citation":{"chicago":"Bezeljak, Urban, Hrushikesh Loya, Beata M Kaczmarek, Timothy E. Saunders, and Martin Loose. “Stochastic Activation and Bistability in a Rab GTPase Regulatory Network.” <i>Proceedings of the National Academy of Sciences of the United States of America</i>. National Academy of Sciences, 2020. <a href=\"https://doi.org/10.1073/pnas.1921027117\">https://doi.org/10.1073/pnas.1921027117</a>.","apa":"Bezeljak, U., Loya, H., Kaczmarek, B. M., Saunders, T. E., &#38; Loose, M. (2020). Stochastic activation and bistability in a Rab GTPase regulatory network. <i>Proceedings of the National Academy of Sciences of the United States of America</i>. National Academy of Sciences. <a href=\"https://doi.org/10.1073/pnas.1921027117\">https://doi.org/10.1073/pnas.1921027117</a>","ista":"Bezeljak U, Loya H, Kaczmarek BM, Saunders TE, Loose M. 2020. Stochastic activation and bistability in a Rab GTPase regulatory network. Proceedings of the National Academy of Sciences of the United States of America. 117(12), 6504–6549.","ieee":"U. Bezeljak, H. Loya, B. M. Kaczmarek, T. E. Saunders, and M. Loose, “Stochastic activation and bistability in a Rab GTPase regulatory network,” <i>Proceedings of the National Academy of Sciences of the United States of America</i>, vol. 117, no. 12. National Academy of Sciences, pp. 6504–6549, 2020.","ama":"Bezeljak U, Loya H, Kaczmarek BM, Saunders TE, Loose M. Stochastic activation and bistability in a Rab GTPase regulatory network. <i>Proceedings of the National Academy of Sciences of the United States of America</i>. 2020;117(12):6504-6549. doi:<a href=\"https://doi.org/10.1073/pnas.1921027117\">10.1073/pnas.1921027117</a>","mla":"Bezeljak, Urban, et al. “Stochastic Activation and Bistability in a Rab GTPase Regulatory Network.” <i>Proceedings of the National Academy of Sciences of the United States of America</i>, vol. 117, no. 12, National Academy of Sciences, 2020, pp. 6504–49, doi:<a href=\"https://doi.org/10.1073/pnas.1921027117\">10.1073/pnas.1921027117</a>.","short":"U. Bezeljak, H. Loya, B.M. Kaczmarek, T.E. Saunders, M. Loose, Proceedings of the National Academy of Sciences of the United States of America 117 (2020) 6504–6549."},"publisher":"National Academy of Sciences","article_processing_charge":"No","abstract":[{"text":"The eukaryotic endomembrane system is controlled by small GTPases of the Rab family, which are activated at defined times and locations in a switch-like manner. While this switch is well understood for an individual protein, how regulatory networks produce intracellular activity patterns is currently not known. Here, we combine in vitro reconstitution experiments with computational modeling to study a minimal Rab5 activation network. We find that the molecular interactions in this system give rise to a positive feedback and bistable collective switching of Rab5. Furthermore, we find that switching near the critical point is intrinsically stochastic and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, our findings reveal how biochemical signaling networks control vesicle trafficking pathways and how their nonequilibrium properties define the spatiotemporal organization of the cell.","lang":"eng"}],"day":"24","date_published":"2020-03-24T00:00:00Z","oa":1,"author":[{"full_name":"Bezeljak, Urban","first_name":"Urban","orcid":"0000-0003-1365-5631","id":"2A58201A-F248-11E8-B48F-1D18A9856A87","last_name":"Bezeljak"},{"full_name":"Loya, Hrushikesh","first_name":"Hrushikesh","last_name":"Loya"},{"last_name":"Kaczmarek","id":"36FA4AFA-F248-11E8-B48F-1D18A9856A87","full_name":"Kaczmarek, Beata M","first_name":"Beata M"},{"full_name":"Saunders, Timothy E.","first_name":"Timothy E.","last_name":"Saunders"},{"orcid":"0000-0001-7309-9724","full_name":"Loose, Martin","first_name":"Martin","last_name":"Loose","id":"462D4284-F248-11E8-B48F-1D18A9856A87"}],"article_type":"original","issue":"12","doi":"10.1073/pnas.1921027117","date_updated":"2026-04-08T07:24:55Z","status":"public","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1101/776567"}],"month":"03","volume":117,"publication_status":"published","isi":1,"page":"6504-6549","_id":"7580","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","oa_version":"Preprint","acknowledged_ssus":[{"_id":"Bio"},{"_id":"LifeSc"}],"quality_controlled":"1","related_material":{"link":[{"relation":"press_release","description":"News on IST Homepage","url":"https://ist.ac.at/en/news/proteins-as-molecular-switches/"}],"record":[{"relation":"dissertation_contains","id":"8341","status":"public"}]},"publication":"Proceedings of the National Academy of Sciences of the United States of America","project":[{"name":"Reconstitution of cell polarity and axis determination in a cell-free system","_id":"2599F062-B435-11E9-9278-68D0E5697425","grant_number":"RGY0083/2016"}],"language":[{"iso":"eng"}],"type":"journal_article","department":[{"_id":"MaLo"},{"_id":"CaBe"}],"scopus_import":"1"},{"publication_identifier":{"eissn":["2045-2322"]},"external_id":{"pmid":["32042057"],"isi":["000560694800012"]},"intvolume":"        10","year":"2020","date_created":"2020-02-16T23:00:49Z","has_accepted_license":"1","title":"Nuclear translocation of glutaminase GLS2 in human cancer cells associates with proliferation arrest and differentiation","pmid":1,"file":[{"file_name":"2020_ScientificReport_Lopez.pdf","creator":"dernst","content_type":"application/pdf","date_updated":"2020-07-14T12:47:59Z","file_size":4703751,"date_created":"2020-02-18T07:43:21Z","file_id":"7495","relation":"main_file","checksum":"c780bd87476a9c9e12668ff66de3dc96","access_level":"open_access"}],"article_processing_charge":"No","publisher":"Springer Nature","citation":{"short":"A.R. López De La Oliva, J.A. Campos-Sandoval, M.C. Gómez-García, C. Cardona, M. Martín-Rufián, F.J. Sialana, L. Castilla, N. Bae, C. Lobo, A. Peñalver, M. García-Frutos, D. Carro, V. Enrique, J.C. Paz, R.G. Mirmira, A. Gutiérrez, F.J. Alonso, J.A. Segura, J.M. Matés, G. Lubec, J. Márquez, Scientific Reports 10 (2020).","mla":"López De La Oliva, Amada R., et al. “Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation.” <i>Scientific Reports</i>, vol. 10, no. 1, 2259, Springer Nature, 2020, doi:<a href=\"https://doi.org/10.1038/s41598-020-58264-4\">10.1038/s41598-020-58264-4</a>.","ama":"López De La Oliva AR, Campos-Sandoval JA, Gómez-García MC, et al. Nuclear translocation of glutaminase GLS2 in human cancer cells associates with proliferation arrest and differentiation. <i>Scientific reports</i>. 2020;10(1). doi:<a href=\"https://doi.org/10.1038/s41598-020-58264-4\">10.1038/s41598-020-58264-4</a>","ieee":"A. R. López De La Oliva <i>et al.</i>, “Nuclear translocation of glutaminase GLS2 in human cancer cells associates with proliferation arrest and differentiation,” <i>Scientific reports</i>, vol. 10, no. 1. Springer Nature, 2020.","ista":"López De La Oliva AR, Campos-Sandoval JA, Gómez-García MC, Cardona C, Martín-Rufián M, Sialana FJ, Castilla L, Bae N, Lobo C, Peñalver A, García-Frutos M, Carro D, Enrique V, Paz JC, Mirmira RG, Gutiérrez A, Alonso FJ, Segura JA, Matés JM, Lubec G, Márquez J. 2020. Nuclear translocation of glutaminase GLS2 in human cancer cells associates with proliferation arrest and differentiation. Scientific reports. 10(1), 2259.","chicago":"López De La Oliva, Amada R., José A. Campos-Sandoval, María C. Gómez-García, Carolina Cardona, Mercedes Martín-Rufián, Fernando J. Sialana, Laura Castilla, et al. “Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation.” <i>Scientific Reports</i>. Springer Nature, 2020. <a href=\"https://doi.org/10.1038/s41598-020-58264-4\">https://doi.org/10.1038/s41598-020-58264-4</a>.","apa":"López De La Oliva, A. R., Campos-Sandoval, J. A., Gómez-García, M. C., Cardona, C., Martín-Rufián, M., Sialana, F. J., … Márquez, J. (2020). Nuclear translocation of glutaminase GLS2 in human cancer cells associates with proliferation arrest and differentiation. <i>Scientific Reports</i>. Springer Nature. <a href=\"https://doi.org/10.1038/s41598-020-58264-4\">https://doi.org/10.1038/s41598-020-58264-4</a>"},"file_date_updated":"2020-07-14T12:47:59Z","ddc":["570"],"oa":1,"author":[{"full_name":"López De La Oliva, Amada R.","first_name":"Amada R.","last_name":"López De La Oliva"},{"full_name":"Campos-Sandoval, José A.","first_name":"José A.","last_name":"Campos-Sandoval"},{"full_name":"Gómez-García, María C.","first_name":"María C.","last_name":"Gómez-García"},{"last_name":"Cardona","first_name":"Carolina","full_name":"Cardona, Carolina"},{"full_name":"Martín-Rufián, Mercedes","first_name":"Mercedes","last_name":"Martín-Rufián"},{"last_name":"Sialana","first_name":"Fernando J.","full_name":"Sialana, Fernando J."},{"full_name":"Castilla, Laura","first_name":"Laura","last_name":"Castilla"},{"full_name":"Bae, Narkhyun","first_name":"Narkhyun","last_name":"Bae","id":"3A5F7CD8-F248-11E8-B48F-1D18A9856A87"},{"full_name":"Lobo, Carolina","first_name":"Carolina","last_name":"Lobo"},{"last_name":"Peñalver","full_name":"Peñalver, Ana","first_name":"Ana"},{"last_name":"García-Frutos","first_name":"Marina","full_name":"García-Frutos, Marina"},{"full_name":"Carro, David","first_name":"David","last_name":"Carro"},{"full_name":"Enrique, Victoria","first_name":"Victoria","last_name":"Enrique"},{"first_name":"José C.","full_name":"Paz, José C.","last_name":"Paz"},{"full_name":"Mirmira, Raghavendra G.","first_name":"Raghavendra G.","last_name":"Mirmira"},{"full_name":"Gutiérrez, Antonia","first_name":"Antonia","last_name":"Gutiérrez"},{"last_name":"Alonso","first_name":"Francisco J.","full_name":"Alonso, Francisco J."},{"last_name":"Segura","first_name":"Juan A.","full_name":"Segura, Juan A."},{"full_name":"Matés, José M.","first_name":"José M.","last_name":"Matés"},{"last_name":"Lubec","first_name":"Gert","full_name":"Lubec, Gert"},{"last_name":"Márquez","full_name":"Márquez, Javier","first_name":"Javier"}],"date_published":"2020-02-10T00:00:00Z","day":"10","abstract":[{"lang":"eng","text":"Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase."}],"doi":"10.1038/s41598-020-58264-4","issue":"1","article_type":"original","publication_status":"published","volume":10,"month":"02","isi":1,"tmp":{"name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode"},"date_updated":"2026-04-02T11:51:06Z","status":"public","related_material":{"link":[{"url":"https://doi.org/10.1038/s41598-020-80651-0","relation":"erratum"}]},"quality_controlled":"1","article_number":"2259","user_id":"ba8df636-2132-11f1-aed0-ed93e2281fdd","_id":"7487","oa_version":"Published Version","language":[{"iso":"eng"}],"publication":"Scientific reports","scopus_import":"1","department":[{"_id":"CaBe"}],"type":"journal_article"}]