---
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
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has_accepted_license: '1'
language:
- iso: eng
license: https://creativecommons.org/licenses/by/4.0/
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:
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  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'
...