---
APC_amount: 12348 EUR
OA_place: publisher
OA_type: hybrid
_id: '18778'
abstract:
- lang: eng
  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.
acknowledged_ssus:
- _id: LifeSc
- _id: EM-Fac
- _id: ScienComp
- _id: PreCl
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_processing_charge: Yes (in subscription journal)
article_type: original
author:
- first_name: Katarina
  full_name: Tluckova, Katarina
  id: 4AC7D980-F248-11E8-B48F-1D18A9856A87
  last_name: Tluckova
- first_name: Beata M
  full_name: Kaczmarek, Beata M
  id: 36FA4AFA-F248-11E8-B48F-1D18A9856A87
  last_name: Kaczmarek
- first_name: Anita P
  full_name: Testa Salmazo, Anita P
  id: 41F1F098-F248-11E8-B48F-1D18A9856A87
  last_name: Testa Salmazo
- 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: 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>
  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>
  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>.
  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.
  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>.
  short: K. Tluckova, B.M. Kaczmarek, A.P. Testa Salmazo, C. Bernecky, Nature Structural
    &#38; Molecular Biology 32 (2025) 607–612.
corr_author: '1'
date_created: 2025-01-08T11:20:20Z
date_published: 2025-04-01T00:00:00Z
date_updated: 2025-11-20T10:28:36Z
day: '01'
ddc:
- '570'
department:
- _id: CaBe
doi: 10.1038/s41594-024-01448-7
external_id:
  isi:
  - '001390268000001'
  pmid:
  - '39762629'
file:
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  file_name: 2025_NatureStrucMolBiol_Tluckova.pdf
  file_size: 9306639
  relation: main_file
  success: 1
file_date_updated: 2025-04-16T08:17:27Z
has_accepted_license: '1'
intvolume: '        32'
isi: 1
language:
- iso: eng
month: '04'
oa: 1
oa_version: Published Version
page: 607-612
pmid: 1
project:
- _id: c08a6700-5a5b-11eb-8a69-82a722b2bc30
  grant_number: P34185
  name: Regulation of mammalian transcription by noncoding RNA
publication: Nature Structural & Molecular Biology
publication_identifier:
  eissn:
  - 1545-9985
  issn:
  - 1545-9993
publication_status: published
publisher: Springer Nature
quality_controlled: '1'
related_material:
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  - id: '14644'
    relation: earlier_version
    status: public
scopus_import: '1'
status: public
title: Mechanism of mammalian transcriptional repression by noncoding RNA
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: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 32
year: '2025'
...
---
OA_place: publisher
_id: '18477'
abstract:
- lang: eng
  text: "ADAR1 is broadly expressed across various tissues and is vital in regulating
    pathways\r\nassociated with innate immune responses. ADAR1 marks double-stranded
    RNA as \"self\"\r\nthrough its A-to-I editing activity, effectively repressing
    autoimmunity and maintaining\r\nimmune tolerance. This editing process has been
    detected at millions of sites across the\r\nhuman genome. However, the mechanism
    underlying ADAR1's substrate selectivity\r\nproperties remains largely unclear,
    with much of the current knowledge derived from\r\ncomparisons to its more extensively
    studied homolog, ADAR2. By studying ADAR1 in complex\r\nwith its RNA substrates
    and applying a combination of biochemical techniques and structural\r\nstudies
    using CryoEM, we aim to gain a more comprehensive understanding of the substrate\r\nselectivity
    characteristics of ADAR1.\r\nIn this thesis, the purification protocol for ADAR1
    was successfully optimized, resulting in the\r\nfirst report in the literature
    to achieve high protein purity and activity. This advancement\r\nenabled the investigation
    of complex formation between ADAR1 and various RNA substrates,\r\nleading to the
    identification of optimal conditions for preparing the cryoEM sample. However,\r\ndespite
    comprehensive optimization of the cryo-EM conditions, the resulting data lacked
    the\r\ndesired quality, highlighting the need for similar rigorous optimization
    of the RNA substrates\r\nto facilitate structural studies of the ADAR1-RNA complex.
    The study was complemented by\r\nAlphaFold predictions, which provided some insights
    into this mechanism.\r\nMoreover, during this project I established a collaboration
    with a research group focused on\r\nstudying ADAR homologs. Notably ADAR homologs
    were identified in bivalve species, and it\r\nwas further demonstrated that ADAR
    and its A-to-I editing activity are upregulated in Pacific\r\noysters during infections
    with Ostreid herpesvirus-1—a highly infectious virus that leads to\r\nsignificant
    losses in oyster populations globally. I successfully purified oyster ADAR and\r\nprepared
    in vitro edited RNA for nanopore sequencing—a direct sequencing technology\r\ncapable
    of detecting modified nucleotides without the need for reverse transcription.
    The\r\ncollaborators initiated optimization of this nanopore-based approach. However,
    current\r\ntechnological limitations still constrain the reliable detection of
    modified nucleotides.\r\nThe project also examined the impact of RNA editing on
    RNA binding and filament formation\r\nby MDA5, a key cytosolic dsRNA sensor that
    triggers an interferon response. A primary target\r\nof ADAR1's editing activity
    is RNA derived from repetitive elements present in the genome,\r\nparticularly
    Alu elements forming double-stranded RNA. When unedited, these RNA\r\nsequences
    are recognized by MDA5. However, the mechanisms by which MDA5 interacts with\r\nAlu
    RNAs, as well as the role of A-to-I editing in influencing this binding, are still
    not well\r\nunderstood.\r\nThe interaction between MDA5 and Alu elements, was
    successfully established. This was\r\nachieved through the testing of different
    RNA variants and the evaluation of filament\r\nformation using binding techniques
    and electron microscopy imaging. This groundwork has\r\nset the conditions for
    further evaluation using CryoEM. Furthermore, the effects of A-to-I\r\nediting
    on the binding properties of MDA5 with Alu RNA were investigated. Given the recent\r\nresearch
    that has provided new insights into MDA5's interaction with dsRNA, it is essential
    to\r\nrevise the experimental setup to integrate these findings before moving
    forward with the\r\nCryoEM sample analysis."
acknowledged_ssus:
- _id: EM-Fac
- _id: LifeSc
alternative_title:
- ISTA Thesis
article_processing_charge: No
author:
- first_name: Beata M
  full_name: Kaczmarek, Beata M
  id: 36FA4AFA-F248-11E8-B48F-1D18A9856A87
  last_name: Kaczmarek
citation:
  ama: Kaczmarek BM. Biochemical and structural insights into ADAR1 RNA editing. 2024.
    doi:<a href="https://doi.org/10.15479/at:ista:18477">10.15479/at:ista:18477</a>
  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>.
  ieee: B. M. Kaczmarek, “Biochemical and structural insights into ADAR1 RNA editing,”
    Institute of Science and Technology Austria, 2024.
  ista: Kaczmarek BM. 2024. Biochemical and structural insights into ADAR1 RNA editing.
    Institute of Science and Technology Austria.
  mla: Kaczmarek, Beata M. <i>Biochemical and Structural Insights into ADAR1 RNA Editing</i>.
    Institute of Science and Technology Austria, 2024, doi:<a href="https://doi.org/10.15479/at:ista:18477">10.15479/at:ista:18477</a>.
  short: B.M. Kaczmarek, Biochemical and Structural Insights into ADAR1 RNA Editing,
    Institute of Science and Technology Austria, 2024.
corr_author: '1'
date_created: 2024-10-27T07:35:13Z
date_published: 2024-10-29T00:00:00Z
date_updated: 2026-04-07T13:23:59Z
day: '29'
ddc:
- '572'
degree_awarded: PhD
department:
- _id: GradSch
- _id: CaBe
doi: 10.15479/at:ista:18477
file:
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  checksum: 2053294ea4d770c495e4cc501e2a218b
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  creator: bkaczmar
  date_created: 2024-10-29T11:56:36Z
  date_updated: 2025-10-29T23:30:02Z
  embargo_to: open_access
  file_id: '18485'
  file_name: 20241029_PhD_thesis_BKaczmarek.docx
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  date_created: 2024-10-29T11:56:44Z
  date_updated: 2025-10-29T23:30:02Z
  embargo: 2025-10-29
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  file_name: 20241029_PhD_thesis_BKaczmarek.pdf
  file_size: 11707360
  relation: main_file
file_date_updated: 2025-10-29T23:30:02Z
has_accepted_license: '1'
language:
- iso: eng
month: '10'
oa: 1
oa_version: Published Version
page: '124'
publication_identifier:
  isbn:
  - 978-3-99078-045-9
  issn:
  - 2663-337X
publication_status: published
publisher: Institute of Science and Technology Austria
status: public
supervisor:
- first_name: Carrie A
  full_name: Bernecky, Carrie A
  id: 2CB9DFE2-F248-11E8-B48F-1D18A9856A87
  last_name: Bernecky
  orcid: 0000-0003-0893-7036
title: Biochemical and structural insights into ADAR1 RNA editing
tmp:
  image: /images/cc_by.png
  legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
  name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
  short: CC BY (4.0)
type: dissertation
user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd
year: '2024'
...
---
_id: '7580'
abstract:
- lang: eng
  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.
acknowledged_ssus:
- _id: Bio
- _id: LifeSc
article_processing_charge: No
article_type: original
author:
- first_name: Urban
  full_name: Bezeljak, Urban
  id: 2A58201A-F248-11E8-B48F-1D18A9856A87
  last_name: Bezeljak
  orcid: 0000-0003-1365-5631
- first_name: Hrushikesh
  full_name: Loya, Hrushikesh
  last_name: Loya
- first_name: Beata M
  full_name: Kaczmarek, Beata M
  id: 36FA4AFA-F248-11E8-B48F-1D18A9856A87
  last_name: Kaczmarek
- first_name: Timothy E.
  full_name: Saunders, Timothy E.
  last_name: Saunders
- first_name: Martin
  full_name: Loose, Martin
  id: 462D4284-F248-11E8-B48F-1D18A9856A87
  last_name: Loose
  orcid: 0000-0001-7309-9724
citation:
  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>
  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>
  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>.
  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.
  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.
  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.
date_created: 2020-03-12T05:32:26Z
date_published: 2020-03-24T00:00:00Z
date_updated: 2026-04-08T07:24:55Z
day: '24'
department:
- _id: MaLo
- _id: CaBe
doi: 10.1073/pnas.1921027117
external_id:
  isi:
  - '000521821800040'
  pmid:
  - '32161136'
intvolume: '       117'
isi: 1
issue: '12'
language:
- iso: eng
main_file_link:
- open_access: '1'
  url: https://doi.org/10.1101/776567
month: '03'
oa: 1
oa_version: Preprint
page: 6504-6549
pmid: 1
project:
- _id: 2599F062-B435-11E9-9278-68D0E5697425
  grant_number: RGY0083/2016
  name: Reconstitution of cell polarity and axis determination in a cell-free system
publication: Proceedings of the National Academy of Sciences of the United States
  of America
publication_identifier:
  eissn:
  - 1091-6490
  issn:
  - 0027-8424
publication_status: published
publisher: National Academy of Sciences
quality_controlled: '1'
related_material:
  link:
  - description: News on IST Homepage
    relation: press_release
    url: https://ist.ac.at/en/news/proteins-as-molecular-switches/
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  - id: '8341'
    relation: dissertation_contains
    status: public
scopus_import: '1'
status: public
title: Stochastic activation and bistability in a Rab GTPase regulatory network
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 117
year: '2020'
...