Year: 2025
Type: dissertation
Abstract: In mammalian cells, many RNAs and RNA-binding proteins form dynamic biomolecular condensates, such as stress granules, processing bodies, and nuclear speckles. Although these condensates are highly concentrated with messenger RNA (mRNAs), their exact role in condensate formation remains unclear. To assess the contributions of mRNA to stress granule formation, I used microinjection to directly deliver reporter mRNAs into the cytoplasm of human cells. I showed that microinjected mRNAs robustly trigger and accumulate in both stress granules and processing bodies. Stress granule assembly required Protein Kinase R (PKR), which is typically stimulated by double-stranded RNAs (dsRNAs) present during viral infection. I corroborated that the in vitro transcription of reporter mRNAs by T7 RNA Polymerase introduced undesired PKR-stimulating ligands. Incorporating uridine analogs during in vitro transcription, or treating the bulk reaction with RNaseIII post-transcription, which cleaves and degrades dsRNAs, greatly reduced PKR activation and stress granule formation. However, microinjecting excess amounts of RNaseIII-treated mRNAs still induced stress granules independently of PKR, supporting current models of mRNAs acting as scaffolding platforms for stress granule assembly. In parallel, this work provides insight on whether uridine analogs in mRNAs mitigate PKR signaling in cells by blocking RNA-PKR interactions or by minimizing the production of PKR-stimulating ligands by T7 RNA Polymerase, as documented by other groups. Since mRNA-based therapeutics often use T7 RNA Polymerase with uridine analogs that may impede with translation, our results implore alternative ways to circumvent PKR activation by therapeutic mRNAs in cells. In addition to cytoplasmic condensates, we also identified and characterized a novel type of nuclear condensate, which we call YTH-bodies, that concentrate mRNAs with N6-methyl-adenosine (m6A) modifications, physically associate with nuclear speckles, and may play a role in retaining and degrading misprocessed mRNAs. Overall, our analyses help to elucidate the role of RNA in the formation of stress granules, provide mechanistic insight behind how uridine analog incorporation reduces PKR activation and stress granule assembly, and delineate the interplay between mRNA metabolism, the m6A modification and nuclear condensates.
Source: TSpace (University of Toronto)
Author: Ihn, Sean (Sung Jin)
Language: English
Topic: RNA Research and Splicing
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7139637468