@phdthesis{22745,
  author       = {Michalik, David},
  issn         = {2663-337X},
  pages        = {182},
  publisher    = {Institute of Science and Technology Austria},
  title        = {{Mechanistic insights into MDA5 selectivity and regulation}},
  doi          = {10.15479/AT-ISTA-22745},
  year         = {2026},
}

@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},
}

@phdthesis{18477,
  abstract     = {ADAR1 is broadly expressed across various tissues and is vital in regulating pathways
associated with innate immune responses. ADAR1 marks double-stranded RNA as "self"
through its A-to-I editing activity, effectively repressing autoimmunity and maintaining
immune tolerance. This editing process has been detected at millions of sites across the
human genome. However, the mechanism underlying ADAR1's substrate selectivity
properties remains largely unclear, with much of the current knowledge derived from
comparisons to its more extensively studied homolog, ADAR2. By studying ADAR1 in complex
with its RNA substrates and applying a combination of biochemical techniques and structural
studies using CryoEM, we aim to gain a more comprehensive understanding of the substrate
selectivity characteristics of ADAR1.
In this thesis, the purification protocol for ADAR1 was successfully optimized, resulting in the
first report in the literature to achieve high protein purity and activity. This advancement
enabled the investigation of complex formation between ADAR1 and various RNA substrates,
leading to the identification of optimal conditions for preparing the cryoEM sample. However,
despite comprehensive optimization of the cryo-EM conditions, the resulting data lacked the
desired quality, highlighting the need for similar rigorous optimization of the RNA substrates
to facilitate structural studies of the ADAR1-RNA complex. The study was complemented by
AlphaFold predictions, which provided some insights into this mechanism.
Moreover, during this project I established a collaboration with a research group focused on
studying ADAR homologs. Notably ADAR homologs were identified in bivalve species, and it
was further demonstrated that ADAR and its A-to-I editing activity are upregulated in Pacific
oysters during infections with Ostreid herpesvirus-1—a highly infectious virus that leads to
significant losses in oyster populations globally. I successfully purified oyster ADAR and
prepared in vitro edited RNA for nanopore sequencing—a direct sequencing technology
capable of detecting modified nucleotides without the need for reverse transcription. The
collaborators initiated optimization of this nanopore-based approach. However, current
technological limitations still constrain the reliable detection of modified nucleotides.
The project also examined the impact of RNA editing on RNA binding and filament formation
by MDA5, a key cytosolic dsRNA sensor that triggers an interferon response. A primary target
of ADAR1's editing activity is RNA derived from repetitive elements present in the genome,
particularly Alu elements forming double-stranded RNA. When unedited, these RNA
sequences are recognized by MDA5. However, the mechanisms by which MDA5 interacts with
Alu RNAs, as well as the role of A-to-I editing in influencing this binding, are still not well
understood.
The interaction between MDA5 and Alu elements, was successfully established. This was
achieved through the testing of different RNA variants and the evaluation of filament
formation using binding techniques and electron microscopy imaging. This groundwork has
set the conditions for further evaluation using CryoEM. Furthermore, the effects of A-to-I
editing on the binding properties of MDA5 with Alu RNA were investigated. Given the recent
research that has provided new insights into MDA5's interaction with dsRNA, it is essential to
revise the experimental setup to integrate these findings before moving forward with the
CryoEM sample analysis.},
  author       = {Kaczmarek, Beata M},
  isbn         = {978-3-99078-045-9},
  issn         = {2663-337X},
  pages        = {124},
  publisher    = {Institute of Science and Technology Austria},
  title        = {{Biochemical and structural insights into ADAR1 RNA editing}},
  doi          = {10.15479/at:ista:18477},
  year         = {2024},
}