---
_id: '20804'
abstract:
- lang: eng
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.
acknowledged_ssus:
- _id: LifeSc
- _id: EM-Fac
- _id: ScienComp
- _id: PreCl
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).
article_processing_charge: No
author:
- first_name: Annamaria
full_name: Hlavata, Annamaria
id: 36062FEC-F248-11E8-B48F-1D18A9856A87
last_name: Hlavata
- first_name: Benjamin
full_name: Neuditschko, Benjamin
last_name: Neuditschko
- first_name: Ulla
full_name: Schellhaas, Ulla
last_name: Schellhaas
- first_name: Clemens
full_name: Plaschka, Clemens
last_name: Plaschka
- first_name: Franz
full_name: Herzog, Franz
last_name: Herzog
- first_name: Carrie A
full_name: Bernecky, Carrie A
id: 2CB9DFE2-F248-11E8-B48F-1D18A9856A87
last_name: Bernecky
orcid: 0000-0003-0893-7036
citation:
ama: Hlavata A, Neuditschko B, Schellhaas U, Plaschka C, Herzog F, Bernecky C. Structure
of cytoplasmic RNA polymerase II. 2025. doi:10.64898/2025.12.10.692585
apa: Hlavata, A., Neuditschko, B., Schellhaas, U., Plaschka, C., Herzog, F., &
Bernecky, C. (2025). Structure of cytoplasmic RNA polymerase II. bioRxiv. https://doi.org/10.64898/2025.12.10.692585
chicago: Hlavata, Annamaria, Benjamin Neuditschko, Ulla Schellhaas, Clemens Plaschka,
Franz Herzog, and Carrie Bernecky. “Structure of Cytoplasmic RNA Polymerase II.”
bioRxiv, 2025. https://doi.org/10.64898/2025.12.10.692585.
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. 10.64898/2025.12.10.692585.
mla: Hlavata, Annamaria, et al. Structure of Cytoplasmic RNA Polymerase II.
bioRxiv, 2025, doi:10.64898/2025.12.10.692585.
short: A. Hlavata, B. Neuditschko, U. Schellhaas, C. Plaschka, F. Herzog, C. Bernecky,
(2025).
corr_author: '1'
date_created: 2025-12-11T13:33:27Z
date_published: 2025-12-10T00:00:00Z
date_updated: 2025-12-15T09:48:22Z
day: '10'
department:
- _id: CaBe
doi: 10.64898/2025.12.10.692585
language:
- iso: eng
main_file_link:
- open_access: '1'
url: https://doi.org/10.64898/2025.12.10.692585
month: '12'
oa: 1
oa_version: None
publication_status: published
publisher: bioRxiv
status: public
title: Structure of cytoplasmic RNA polymerase II
type: preprint
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
year: '2025'
...
---
OA_place: publisher
_id: '19431'
abstract:
- lang: eng
text: "Gene expression is crucial for cell differentiation, development and survival
of\r\norganisms. It consists of several steps, starting with transcription that
is mediated by\r\nRNA polymerases. These are protein machineries transcribing
and producing different\r\ntypes of RNAs. Although, the individual steps of transcription
by RNA polymerase II\r\n(Pol II) as well as the structure of Pol II has been extensively
studied, surprisingly,\r\nthere is still little known about its regulation and
assembly in cytoplasm. Among the\r\nproteins that are important in biogenesis
of Pol II are RNA polymerase II associating\r\nproteins (RPAP) and small GPN-loop
GTPases (GPN). Both of these protein groups\r\nwere shown to take essential part
in assembly of Pol II.\r\nThe aim of this project was to deepen our knowledge
in regulation of Pol II in\r\nthe cytoplasm as well as the proteins involved in
this process. Techniques of structural\r\nbiology, biochemistry and cell biology
were employed to study and characterize cytoplasmic Pol II and its interacting
partners.\r\nThis study shows for the first time the structure of cytoplasmic
Pol II at high\r\nresolution. The structure also reveals proteins interacting
with Pol II in cytoplasm,\r\nnamely GDOWN1, RPAP2. Comparing the structure of
cytoplasmic Pol II with transcribing Pol II revealed striking difference in clamp
region that is not in closed state.\r\nFurthermore, GDOWN1 and RPAP2 make steric
clashes with various transcription\r\nfactors bound to Pol II during different
stages of transcription. Even though GPN1 and\r\nGPN3 proteins were not resolved
in the cytoplasmic Pol II structure, they are part of\r\nthe complex and their
interaction with Pol II was confirmed in vitro. RPAP2 stabilizes\r\nthese proteins
on Pol II and several experiments suggest that they interact with the\r\nclamp
region. In addition, GDOWN1, RPAP2 and GPNs might keep clamp in open or\r\npartially
open state. Based on these results I propose a novel model of regulation of\r\nPol
II in cytoplasm. GDOWN1, RPAP2, GPN1 and GPN3 bind to Pol II in cytoplasm\r\nand
doing so they can prevent pre-mature binding of DNA or RNA and different transcription
factors to Pol II in cytoplasm or before engaging in transcription nucleus.\r\nThis
research contributes to the current knowledge of molecular mechanisms\r\nof Pol
II regulation in cytoplasm."
acknowledged_ssus:
- _id: LifeSc
- _id: EM-Fac
- _id: ScienComp
acknowledgement: 'I would also like to acknowledge the ISTA Facilities: Lab Support
Facility, Protein Services and Electron Microscopy Facility (EMF) and Scientific
Computing. EMF for their support during data collections and troubleshooting, especially
Valentin. Scientific Computing for solving quickly any issues related with cluster.'
alternative_title:
- ISTA Thesis
article_processing_charge: No
author:
- first_name: Annamaria
full_name: Hlavata, Annamaria
id: 36062FEC-F248-11E8-B48F-1D18A9856A87
last_name: Hlavata
citation:
ama: Hlavata A. Regulation of Cytoplasmic RNA Polymerase II. 2025. doi:10.15479/10.15479/AT-ISTA-19431
apa: Hlavata, A. (2025). Regulation of Cytoplasmic RNA Polymerase II. Institute
of Science and Technology Austria. https://doi.org/10.15479/10.15479/AT-ISTA-19431
chicago: Hlavata, Annamaria. “Regulation of Cytoplasmic RNA Polymerase II.” Institute
of Science and Technology Austria, 2025. https://doi.org/10.15479/10.15479/AT-ISTA-19431.
ieee: A. Hlavata, “Regulation of Cytoplasmic RNA Polymerase II,” Institute of Science
and Technology Austria, 2025.
ista: Hlavata A. 2025. Regulation of Cytoplasmic RNA Polymerase II. Institute of
Science and Technology Austria.
mla: Hlavata, Annamaria. Regulation of Cytoplasmic RNA Polymerase II. Institute
of Science and Technology Austria, 2025, doi:10.15479/10.15479/AT-ISTA-19431.
short: A. Hlavata, Regulation of Cytoplasmic RNA Polymerase II, Institute of Science
and Technology Austria, 2025.
corr_author: '1'
date_created: 2025-03-20T12:52:47Z
date_published: 2025-03-20T00:00:00Z
date_updated: 2026-04-07T11:46:32Z
day: '20'
ddc:
- '572'
degree_awarded: PhD
department:
- _id: GradSch
- _id: CaBe
doi: 10.15479/10.15479/AT-ISTA-19431
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language:
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oa_version: Published Version
page: '83'
publication_identifier:
eissn:
- 2663-337X
isbn:
- 978-3-99078-055-8
publication_status: published
publisher: Institute of Science and Technology Austria
status: public
supervisor:
- first_name: Carrie A
full_name: Bernecky, Carrie A
id: 2CB9DFE2-F248-11E8-B48F-1D18A9856A87
last_name: Bernecky
orcid: 0000-0003-0893-7036
title: Regulation of Cytoplasmic RNA Polymerase II
type: dissertation
user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd
year: '2025'
...
---
OA_place: publisher
OA_type: hybrid
_id: '15330'
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 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.
acknowledged_ssus:
- _id: EM-Fac
- _id: LifeSc
- _id: Bio
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)."
article_number: jcs.261720
article_processing_charge: Yes (via OA deal)
article_type: original
author:
- first_name: Nataliia
full_name: Gnyliukh, Nataliia
id: 390C1120-F248-11E8-B48F-1D18A9856A87
last_name: Gnyliukh
orcid: 0000-0002-2198-0509
- first_name: Alexander J
full_name: Johnson, Alexander J
id: 46A62C3A-F248-11E8-B48F-1D18A9856A87
last_name: Johnson
orcid: 0000-0002-2739-8843
- first_name: MK
full_name: Nagel, MK
last_name: Nagel
- first_name: Aline
full_name: Monzer, Aline
id: 2DB5D88C-D7B3-11E9-B8FD-7907E6697425
last_name: Monzer
- first_name: David
full_name: Babic, David
id: db566d23-f6e0-11ea-865d-e6f270e968e7
last_name: Babic
- first_name: Annamaria
full_name: Hlavata, Annamaria
id: 36062FEC-F248-11E8-B48F-1D18A9856A87
last_name: Hlavata
- first_name: SS
full_name: Alotaibi, SS
last_name: Alotaibi
- first_name: E
full_name: Isono, E
last_name: Isono
- first_name: Martin
full_name: Loose, Martin
id: 462D4284-F248-11E8-B48F-1D18A9856A87
last_name: Loose
orcid: 0000-0001-7309-9724
- first_name: Jiří
full_name: Friml, Jiří
id: 4159519E-F248-11E8-B48F-1D18A9856A87
last_name: Friml
orcid: 0000-0002-8302-7596
citation:
ama: Gnyliukh N, Johnson AJ, Nagel M, et al. Role of dynamin-related proteins 2
and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana. Journal
of Cell Science. 2024;137(8). doi:10.1242/jcs.261720
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. Journal of Cell Science. The Company
of Biologists. https://doi.org/10.1242/jcs.261720
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.” Journal of Cell Science. The Company of Biologists,
2024. https://doi.org/10.1242/jcs.261720.
ieee: N. Gnyliukh et al., “Role of dynamin-related proteins 2 and SH3P2 in
clathrin-mediated endocytosis in Arabidopsis thaliana,” Journal of Cell Science,
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.” Journal of Cell Science,
vol. 137, no. 8, jcs. 261720, The Company of Biologists, 2024, doi:10.1242/jcs.261720.
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).
corr_author: '1'
date_created: 2024-04-19T09:54:59Z
date_published: 2024-04-01T00:00:00Z
date_updated: 2025-09-04T13:49:45Z
day: '01'
ddc:
- '570'
department:
- _id: MaLo
- _id: JiFr
- _id: CaBe
doi: 10.1242/jcs.261720
ec_funded: 1
external_id:
isi:
- '001266917100005'
pmid:
- '38506228'
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checksum: 6dc023f0cc7052ad3cf0a42589d2e30f
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creator: dernst
date_created: 2025-01-09T08:41:16Z
date_updated: 2025-01-09T08:41:16Z
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file_name: 2024_JourCellScience_Gnyliukh.pdf
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language:
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license: https://creativecommons.org/licenses/by/4.0/
month: '04'
oa: 1
oa_version: Published Version
pmid: 1
project:
- _id: 2564DBCA-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '665385'
name: International IST Doctoral Program
- _id: bd76d395-d553-11ed-ba76-f678c14f9033
grant_number: I06123
name: Peptide receptors for auxin canalization in Arabidopsis
publication: Journal of Cell Science
publication_identifier:
eissn:
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issn:
- 0021-9533
publication_status: published
publisher: The Company of Biologists
quality_controlled: '1'
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relation: earlier_version
status: public
scopus_import: '1'
status: public
title: Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis
in Arabidopsis thaliana
tmp:
image: /images/cc_by.png
legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
short: CC BY (4.0)
type: journal_article
user_id: 317138e5-6ab7-11ef-aa6d-ffef3953e345
volume: 137
year: '2024'
...
---
OA_place: repository
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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.
acknowledged_ssus:
- _id: EM-Fac
- _id: LifeSc
- _id: Bio
article_processing_charge: No
author:
- first_name: Nataliia
full_name: Gnyliukh, Nataliia
id: 390C1120-F248-11E8-B48F-1D18A9856A87
last_name: Gnyliukh
orcid: 0000-0002-2198-0509
- first_name: Alexander J
full_name: Johnson, Alexander J
id: 46A62C3A-F248-11E8-B48F-1D18A9856A87
last_name: Johnson
orcid: 0000-0002-2739-8843
- first_name: Marie-Kristin
full_name: Nagel, Marie-Kristin
last_name: Nagel
- first_name: Aline
full_name: Monzer, Aline
id: 2DB5D88C-D7B3-11E9-B8FD-7907E6697425
last_name: Monzer
- first_name: Annamaria
full_name: Hlavata, Annamaria
id: 36062FEC-F248-11E8-B48F-1D18A9856A87
last_name: Hlavata
- first_name: Erika
full_name: Isono, Erika
last_name: Isono
- first_name: Martin
full_name: Loose, Martin
id: 462D4284-F248-11E8-B48F-1D18A9856A87
last_name: Loose
orcid: 0000-0001-7309-9724
- first_name: Jiří
full_name: Friml, Jiří
id: 4159519E-F248-11E8-B48F-1D18A9856A87
last_name: Friml
orcid: 0000-0002-8302-7596
citation:
ama: Gnyliukh N, Johnson AJ, Nagel M-K, et al. Role of dynamin-related proteins
2 and SH3P2 in clathrin-mediated endocytosis in plants. bioRxiv. doi:10.1101/2023.10.09.561523
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. bioRxiv. https://doi.org/10.1101/2023.10.09.561523
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.” BioRxiv,
n.d. https://doi.org/10.1101/2023.10.09.561523.
ieee: N. Gnyliukh et al., “Role of dynamin-related proteins 2 and SH3P2 in
clathrin-mediated endocytosis in plants,” bioRxiv. .
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, 10.1101/2023.10.09.561523.
mla: Gnyliukh, Nataliia, et al. “Role of Dynamin-Related Proteins 2 and SH3P2 in
Clathrin-Mediated Endocytosis in Plants.” BioRxiv, doi:10.1101/2023.10.09.561523.
short: N. Gnyliukh, A.J. Johnson, M.-K. Nagel, A. Monzer, A. Hlavata, E. Isono,
M. Loose, J. Friml, BioRxiv (n.d.).
corr_author: '1'
date_created: 2023-11-22T10:17:49Z
date_published: 2023-10-10T00:00:00Z
date_updated: 2026-06-18T22:30:30Z
day: '10'
department:
- _id: JiFr
- _id: MaLo
- _id: CaBe
doi: 10.1101/2023.10.09.561523
ec_funded: 1
language:
- iso: eng
main_file_link:
- open_access: '1'
url: https://doi.org/10.1101/2023.10.09.561523
month: '10'
oa: 1
oa_version: Preprint
project:
- _id: 2564DBCA-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '665385'
name: International IST Doctoral Program
publication: bioRxiv
publication_status: draft
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relation: dissertation_contains
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title: Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis
in plants
type: preprint
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
year: '2023'
...