<p>A-to-I mRNA editing is an important co- or post-transcriptional event that can recode the heredity information through adenosine deaminase. It is mediated by <u>a</u>denosine <u>d</u>e<u>a</u>minases <u>a</u>cting on <u>R</u>NA (ADAR) in animals. Due to the lack of ADAR orthologs, yeast and filamentous fungi are assumed to have no A-to-I mRNA editing. However, genome-wide A-to-I mRNA editing was discovered in the plant pathogenic fungus <i>Fusarium graminearum</i> that occurs specifically during sexual reproduction in 2016. In a previous study all the predicted adenosine/cytosine deaminase genes except <i>FgTAD2 </i>and <i>FgTAD3</i>, the orthologs of yeast <i>TAD2 </i>and <i>TAD3</i>, were found to be dispensable for A-to-I mRNA editing in <i>F. graminearum</i>. <i>TAD2</i> and <i>TAD3</i> encode ADATs (<u>a</u>denosine <u>d</u>e<u>a</u>minase acting on <u>t</u>RNA) that mediate A-to-I editing at A34 of tRNA. In this study, <i>FgTAD2</i> was found to have two isoforms based on RNA-seq analysis. Whereas the longer isoform was predominant during vegetative growth, the expression of the short one was significantly increase and likely acts as the major isoform during sexual stage. Because deletion of <i>FgTAD2</i> appeared to be lethal, the RIP (<u>r</u>epeat-<u>i</u>nduced <u>p</u>oint mutation) approach was used to generate point mutations. A total of 16 RIP mutations were identified in <i>FgTAD2</i>after sequencing analysis with 8 ascospore progeny that were normal in vegetative growth but defective in sexual reproduction. Two of them were verified by introducing specific point mutations into the endogenous <i>FgTAD2</i> allele in the wild-type strain. In addition to genetic approaches, we developed an <i>in vitro</i> assay to detect the deaminase activity of FgTad2. The FgTad2 protein complex purified from perithecia formed by transformants of <i>F. graminearum</i> expressing FgTad2-6xHis by immunoprecipitation was found to catalyze A-to-I editing in a mRNA substrate. Like yeast Tad3, FgTad3 has the E to V mutation in its catalytic core that likely abolishes its ADAT activity but it forms heterodimers with FgTad2 based on co-immunoprecipitation assays. Because <i>FgTAD2</i> and <i>FgTAD3</i> were constitutively expressed and the FgTad2/FgTad3 protein complex purified from vegetative hyphae had no A-to-I RNA editing activity in <i>in vitro</i> assays, it is likely that stage-specific co-factors present in perithecia interact with FgTad2/FgTad3 ADATs (lack of RNA binding domains) and enable the editing of mRNA. Affinity purification and mass spectrometry were conducted with the FgTad2-S-tag and FgTad3-S-tag transformants. Among the putative FgTad2- and FgTad3-interacting proteins, Gad1 was confirmed to interact with FgTad2 specifically during sexual reproduction. Surprisingly, both the number of editing sites and editing levels were increased in the <i>gad1</i> mutant, indicating that Gad1 affects A-to-I mRNA editing in a negative way. Overall, genetic studies and <i>in vitro</i> assays showed that FgTad2, possibly together with FgTad3, is responsible for A-to-I mRNA editing. Proteins co-immunoprecipitated with FgTad2 likely contains co-factors interacting with FgTad2/FgTad3 for stage-specific A-to-I mRNA by these two ADATs in <i>F. graminearum</i>. </p>

Mobile group II introns insert site-specifically into DNA target sites through a mechanism ("retrohoming") that involves reverse splicing of the intron RNA into the DNA and its subsequent reverse transcription by an intron-encoded protein (IEP) that is associated with the RNA in a ribonucleoprotein (RNP) complex. Characterization of this RNP complex and its retrohoming activities have enabled the development of programmable mobile group II intron gene targeting vectors routinely used in prokaryotic organisms. Building upon recent research by our lab to develop gene targeting in Xenopus laevis and Drosophila melanogaster using the group II intron Ll.LtrB from Lactococcus lactis, I describe work to extend this system to mammalian cells. I demonstrate that group II intron RNPs can be delivered to mammalian cells efficiently and produced in vivo via a CMV/T7 hybrid expression system. Using a robust single-strand annealing assay to detect homologous recombination induced by double-strand breaks (DSBs), I found that group II intron-mediated DSBs are efficiently repaired by mammalian cells. Despite varied approaches, I failed to detect endogenous group II intron-mediated gene targeting in human and mouse cells in culture. Gene expression microarray analysis and in vivo imaging of RNP molecules indicated that group II intron RNPs are sequestered away from the genome and induce host innate immune responses. I also investigated how the C-terminal DNA-binding domain of the Ll.LtrB IEP contributes to DNA target site recognition. Building upon previous mass spectrophotometric analysis of site-specific UV-crosslinking, I used genetic and biochemical analyses to identify potential protein contacts for key target site residues T-23 and T+5. Genetic selection of mutants in a region contacting T+5 led to identification of LtrA variants with increased retrohoming efficiency. My results provide evidence that the DNA-binding domain of a group II intron reverse transcriptase functions in DNA target site recognition and suggest new methods for changing its DNA target specificity and targeting efficiency. / text

This thesis characterizes RNA footprints of several RNA-binding proteins (RBPs) thatare involved in U-insertion/deletion, A-to-I, and C-to-U RNA editing in Trypanosoma brucei. Relying on iCLIP data and biochemical methods it shows that two paralogs proteins from the MRB1 complex regulate distinct editing fates of the mitochondrial transcripts. Further, this thesis provides evidence where the combinatorial interplay of RBPs might fine-tune the levels of edited mRNA. Finally, the presented thesis adds to the growing evidence of the importance of RBPs in post-transcriptional gene regulation.

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Motivation: SARS-CoV-2 is the virus responsible for the COVID-19 pandemic that currently impacts our world. SARS-CoV-2 is an enveloped, positive sense single stranded RNA virus and like other RNA viruses is known to form RNA secondary structure in its genome. In related viruses the secondary structures are responsible for fulfilling roles including proper expression of viral gene products and possibly regulation of viral genome replication. I hypothesize that SARS-CoV-2 may be capable of forming additional secondary structures beyond what is already known and that those secondary structures are identifiable on the basis of sequence conservation with related RNA viruses. Results: By repurposing and expanding an existing computational pipeline de- signed for the detection of structural RNAs in vertebrates, I identified 40 regions of the SARS-CoV-2 genome highly likely to form secondary structure. Partial re- identification of known secondary structures in the SARS-CoV-2 genome was achieved. To further explore the role these structures may fill, the 9 most conservatively pre- dicted structures were analyzed in wild viral samples collected from three Canadian provinces, and distinct patterns of mutation were observed. The 40 regions identi- fied by my modified pipeline were compared against three contemporary works and the differences between findings were quantified. Lastly, Variants of Concern for SARS-CoV-2 were analyzed for prevalent but poorly reported mutations that may influence RNA secondary structure. Code developed for this work is available at https://github.com/aziesel/MSc. / Graduate / 2023-04-06

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Major fraction of eukaryotic genomes is composed of transposons. Mobilization of these transposons leads to mutations and genomic instability. In animals, these selfish genetic elements are regulated by a class of small RNAs called PIWI interacting RNAs (piRNAs). Thus host piRNA pathway acts as a defense against pathogenic transposons. Many piRNA pathway genes are rapidly evolving indicating that they are involved in a host-pathogen arms race. In my thesis, I investigated the nature of this arms race by checking functional consequences of the sequence diversity in piRNA pathway genes. In order to study the functional consequences of the divergence in piRNA pathway genes, we swapped piRNA pathway genes between two sibling Drosophila species, Drosophila melanogaster and Drosophila simulans. We focused on RDC complex, composed of Rhino, Deadlock and Cutoff, which specifies piRNA clusters and regulates transcription from clusters. None of the D. simulans RDC complex proteins function in D. melanogaster. Rhino and Deadlock interact and colocalize in D. simulans and D. melanogaster, but D. simulans Rhino does not bind D. melanogaster Deadlock, due to substitutions in the rapidly evolving Shadow domain. Cutoff from D. simulans stably binds and traps D. melanogaster Deadlock. Adaptive evolution has thus generated cross-species incompatibilities in the piRNA pathway which may contribute in reproductive isolation.

The Piwi interacting RNA pathway (piRNA) transcriptionally and post-transcriptionally silences transposons in the germline to maintain host genome integrity and faithful transmission of the genetic materials. In Drosophilaovaries, maternally loaded piRNAs kick-start piRNA biogenesis and convert precursor transcripts into piRNAs to replenish the piRNA pool during oogenesis. piRNA clusters are the genomic source of piRNA precursors, which are determined by the HP1 homolog Rhino and accessary factors. Rhino specifically binds to piRNA cluster chromatin. I was intrigued by how Rhino localizes to piRNA clusters to specify piRNA precursors. TREX is a conserved mRNA biogenesis complex composed of UAP56 and the THO complex. Identification of UAP56 as a cluster transcript-processing factor established the link between piRNA biogenesis and the general mRNA processing machinery. In my thesis, I investigated the functions of UAP56 and THO in piRNA cluster transcript processing. I characterized an RNP specific to cluster transcripts, defined by binding with both factors, which is distinct from RNP of bulk mRNA transcripts, and found that assembly of these RNPs depends on Rhino. These findings imply that piRNA precursors are specified co-transcriptionally. Additionally, I found that TREX mutants lead to a loss of Rhino binding specificity. I propose that Rhino and TREX co-transcriptionally scan for cluster and transposon sequences to establish loci that produce piRNA precursors. Surprisingly, I also discovered a piRNA-independent function for TREX in transposon silencing. I showed that TREX mutants lead to transcriptionally activation of a number of transposon families without affecting their piRNA biogenesis and piRNA mediated repressive histone modifications. I propose that TREX could mediate a conserved transposon silencing mechanism.