@article{18778,
  abstract     = {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.},
  author       = {Tluckova, Katarina and Kaczmarek, Beata M and Testa Salmazo, Anita P and Bernecky, Carrie A},
  issn         = {1545-9985},
  journal      = {Nature Structural & Molecular Biology},
  pages        = {607--612},
  publisher    = {Springer Nature},
  title        = {{Mechanism of mammalian transcriptional repression by noncoding RNA}},
  doi          = {10.1038/s41594-024-01448-7},
  volume       = {32},
  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},
}

@article{7580,
  abstract     = {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.},
  author       = {Bezeljak, Urban and Loya, Hrushikesh and Kaczmarek, Beata M and Saunders, Timothy E. and Loose, Martin},
  issn         = {1091-6490},
  journal      = {Proceedings of the National Academy of Sciences of the United States of America},
  number       = {12},
  pages        = {6504--6549},
  publisher    = {National Academy of Sciences},
  title        = {{Stochastic activation and bistability in a Rab GTPase regulatory network}},
  doi          = {10.1073/pnas.1921027117},
  volume       = {117},
  year         = {2020},
}