@unpublished{20804,
  abstract     = {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.},
  author       = {Hlavata, Annamaria and Neuditschko, Benjamin and Schellhaas, Ulla and Plaschka, Clemens and Herzog, Franz and Bernecky, Carrie A},
  publisher    = {bioRxiv},
  title        = {{Structure of cytoplasmic RNA polymerase II}},
  doi          = {10.64898/2025.12.10.692585},
  year         = {2025},
}

@phdthesis{19431,
  abstract     = {Gene expression is crucial for cell differentiation, development and survival of
organisms. It consists of several steps, starting with transcription that is mediated by
RNA polymerases. These are protein machineries transcribing and producing different
types of RNAs. Although, the individual steps of transcription by RNA polymerase II
(Pol II) as well as the structure of Pol II has been extensively studied, surprisingly,
there is still little known about its regulation and assembly in cytoplasm. Among the
proteins that are important in biogenesis of Pol II are RNA polymerase II associating
proteins (RPAP) and small GPN-loop GTPases (GPN). Both of these protein groups
were shown to take essential part in assembly of Pol II.
The aim of this project was to deepen our knowledge in regulation of Pol II in
the cytoplasm as well as the proteins involved in this process. Techniques of structural
biology, biochemistry and cell biology were employed to study and characterize cytoplasmic Pol II and its interacting partners.
This study shows for the first time the structure of cytoplasmic Pol II at high
resolution. The structure also reveals proteins interacting with Pol II in cytoplasm,
namely 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.
Furthermore, GDOWN1 and RPAP2 make steric clashes with various transcription
factors bound to Pol II during different stages of transcription. Even though GPN1 and
GPN3 proteins were not resolved in the cytoplasmic Pol II structure, they are part of
the complex and their interaction with Pol II was confirmed in vitro. RPAP2 stabilizes
these proteins on Pol II and several experiments suggest that they interact with the
clamp region. In addition, GDOWN1, RPAP2 and GPNs might keep clamp in open or
partially open state. Based on these results I propose a novel model of regulation of
Pol II in cytoplasm. GDOWN1, RPAP2, GPN1 and GPN3 bind to Pol II in cytoplasm
and 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.
This research contributes to the current knowledge of molecular mechanisms
of Pol II regulation in cytoplasm.},
  author       = {Hlavata, Annamaria},
  isbn         = {978-3-99078-055-8},
  issn         = {2663-337X},
  pages        = {83},
  publisher    = {Institute of Science and Technology Austria},
  title        = {{Regulation of Cytoplasmic RNA Polymerase II}},
  doi          = {10.15479/10.15479/AT-ISTA-19431},
  year         = {2025},
}

@article{15330,
  abstract     = {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.},
  author       = {Gnyliukh, Nataliia and Johnson, Alexander J and Nagel, MK and Monzer, Aline and Babic, David and Hlavata, Annamaria and Alotaibi, SS and Isono, E and Loose, Martin and Friml, Jiří},
  issn         = {1477-9137},
  journal      = {Journal of Cell Science},
  number       = {8},
  publisher    = {The Company of Biologists},
  title        = {{Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana}},
  doi          = {10.1242/jcs.261720},
  volume       = {137},
  year         = {2024},
}

@unpublished{14591,
  abstract     = {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.},
  author       = {Gnyliukh, Nataliia and Johnson, Alexander J and Nagel, Marie-Kristin and Monzer, Aline and Hlavata, Annamaria and Isono, Erika and Loose, Martin and Friml, Jiří},
  booktitle    = {bioRxiv},
  title        = {{Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in plants}},
  doi          = {10.1101/2023.10.09.561523},
  year         = {2023},
}