Cell surface RNA (csRNA) is a recent discovery in the field of RNA biology and has been implicated in playing important roles in many biological processes due to its extracellular properties. To understand the biogenesis, regulation, and function of csRNA, it is critical to develop methods to detect, isolate, and confidently characterize membrane-bound csRNA. Previously, csRNA has been profiled using methods based on cell membrane isolation that are expensive, laborious, and with unsatisfactory specificity and sensitivity . In this study, we use metabolic labeling and chemical cross-linking techniques to specifically label csRNA with biotin handles. We intended to use this technique for separating biotin-labeled csRNA from total RNA samples for characterization purposes. The primary materials that were used to label such csRNAs are 4-Thiouridine (4sU), an unnatural nucleotide analogue, and S-(2-aminoethyl)-ester-methanesulfonothioic-acid-biotin (MTSEA-biotin), a crosslinker designed specifically to label 4sU. By deploying these tools to cell lines such as HEK293T and HeLa, csRNA is detectable by Enhanced Chemiluminescent detection via Dot Blot. Furthermore, to separate biotin-labeled csRNA from total RNA, streptavidin-coated magnetic bead separation procedures could be used as a promising method for purifying csRNA from total RNA, for RNAseq characterization. This study highlights the processes of establishing the csRNA detection protocol and describes the current status and issues with developing the streptavidin-coated magnetic beads separation method. / Master of Science in Life Sciences / The 'central dogma' is a term that describes the process of DNA (a template-like molecule that holds all genetic coding within cells) transcribing into mRNA (a messenger molecule that transports this message to the ribosome) which is then translated into proteins (large, complex molecular machinery that is responsible for many biochemical functions within the body). However, RNA has been found to have a much wider range of functions than just being an intermediate messenger between DNA and proteins. Recently, short snippets of single nucleotide RNA strands have been discovered to be present on the outer cell membrane of certain mammalian cell types. The function of cell surface RNA (csRNA) is largely undiscovered, however, csRNA are likely involved in cell-cell interactions similar to outer membrane proteins, lipids, and carbohydrates. Currently, methods involved in detecting and characterizing csRNA are laborious, time extensive, and with unsatisfactory specificity and sensitivity. This study aims to develop novel methods to detect csRNA on different cell types in an undemanding and trustworthy manner to speed up research timelines while maintaining high confidence in results. Our design is to use metabolic labeling and click-chemistry to 'label' the csRNA. In this study, we describe early signs of detecting csRNA and how this was achieved. Additionally, the current status for separating and profiling csRNA sequences is discussed.

Since the initial description of cancer stem cells (CSCs) as a self-renewing subpopulation of malignant cells with tumor-initiating capacity, a growing body of evidence has supported the existence of CSCs in virtually every tumor type. Our previous work in colorectal cancer has identified the transcription factor CDX1 as a key regulator of colorectal CSC differentiation. CDX1 expression is frequently lost in colorectal cancer, resulting in more aggressive, poorly differentiated tumors with higher proportions of CSCs. Many miRNAs have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in colorectal cancer, remain poorly understood. We began by identifying miRNAs downstream of CDX1 by using high-throughput small-RNA sequencing to profile miRNA expression in two pairs of colorectal cancer cell lines with stable CDX1 overexpression or knockdown. Validation of candidates identified by RNAseq in a larger cell line panel revealed miR-215 to be most significantly correlated with CDX1 expression. ChIP-qPCR and promoter reporter assays confirmed that CDX1 directly transactivates miR-215 transcription. MiR-215 is depleted in FACS-enriched CSCs compared to unsorted samples. Overexpression of miR-215 in poorly-differentiated, highly clonogenic cell lines causes growth arrest and a dramatic decrease in colony formation. miR-215 knockdown using a miRNA sponge causes an increase in clonogenicity and impairs differentiation in CDX1-high cell lines. Indeed, the effects of CDX1 expression on both gene expression and colony morphology can be attenuated by miR-215 inhibition, indicating that miR-215 is a functional mediator of CDX1. Microarray studies following miR-215 overexpression indicate that miR-215 induces terminal differentiation-associated growth arrest, due in part to direct silencing of BMI1 expression and de-repression of BMI1 target genes including CDKN1A. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in colorectal cancer. We further characterize another miRNA-transcription factor axis in colorectal cancer, and we identify the novel miR-3189-3p as a potent effector of cell death with potential therapeutic implications.

RNA modifications and processing events are important modulators of global gene expression. Genomic mutations in the RNA methylase NSun2 and the alternative splicing factor Srsf2 are linked to neurological disorders and cancer in humans, respectively. NSun2 methylates cytosine-5 in most tRNAs and, to a lesser extent, other ncRNAs and mRNAs. Srsf2 is a critical component of the spliceosome and interacts with abundant ncRNAs that are methylated by NSun2. However, how precisely these processes effect homeostasis is largely unexplored. Therefore, the main aims of my PhD were (1) to dissect the molecular mechanisms of NSun2-mediated RNA methylation pathways that regulate cell survival under normal conditions and in response to oxidative stress, and (2) to investigate the importance of Srsf2 in stem cells using skin as a model system. In the context of RNA modifications, firstly I described how NSun2-expressing cells enrich for transcripts related to enhanced cell survival. Subsequently, by metabolically profiling wildtype and patient-derived dermal fibroblasts carrying loss-of-function mutations in the NSUN2 gene, I showed that the absence of NSun2 is synonymous to an energy-saving, low-translating and stressed cellular state. I further confirmed that lack of NSun2 was sufficient to instigate a cellular stress response, by monitoring BIRC5, a member of the inhibitor of apoptosis family. To further answer whether lack of NSun2 enhanced the susceptibility of patient cells to external stress stimuli, I next exposed them to oxidative stress and measured transcriptional and translational changes. I discovered that NSun2 is required to adapt global protein synthesis to the stress response, while NSun2-depleted cells failed to do so. This was concurrent with NSun2-depleted cells enriching for transcripts related to mRNA degradation and negative regulators of protein translation in response to stress. Generally, since loss of NSun2-driven methylation in tRNAs triggers their cleavage into small ncRNA fragments by angiogenin, I asked how angiogenin or tRNA-derived ncRNAs affect translation levels. In the presence of NSun2, angiogenin alone did not reduce global protein synthesis, yet tRNA fragmentation was required to modulate translation levels. Finally, to uncover how the lack of NSun2 influenced tRNA cleavage and methylation patterns in response to stress, I exposed wildtype and patient cells to sodium arsenite and measured the abundance of tRNA-derived fragments and occurrence of methylation events. With this I discovered unique tRNA fragmentation patterns and global RNA methylation profiles for wildtype and NSun2-depleted cells, that can account for the underlying molecular and phenotypical differences in response to stress. In the context of alternative splicing, and since the cellular functions of Srsf2 are largely unknown, I explored its role in cellular survival and differentiation. By conditionally deleting SRSF2 in two different stem cell populations of the mouse epidermis, I observed significant thickening of the epidermis, altered expression of cell proliferation and stem cell differentiation markers, and distorted hair follicle structures. Moreover, I demonstrated that lack of Srsf2 promotes skin regeneration following injury, thus strongly indicating that Srsf2 is required for normal skin development and regeneration after injury. In summary, my research suggests that NSun2-mediated RNA methylation pathways orchestrate transcriptional and translational programmes in response to external stress stimuli, and my studies are the first to show that the alternative splicing factor Srsf2 is required for stem cell differentiation in skin.

<p>Gene expression is a complex process by which genetic information flow from genes to proteins. Factors regulating gene expression are diverse ranging from sequence elements on DNA, to various types of RNA, to proteins. These factors are categorized into two main categories, <em>cis</em>-acting elements and <em>trans</em>-acting elements. <em>PHO84</em> is a budding yeast gene that was previously reported to be regulated by its cognate antisense transcripts both in <em>cis</em> and in <em>trans</em>. The antisense transcripts of <em>PHO84</em> are a group of long non-coding RNAs (lncRNAs). In my project, I performed RNA-seq and TT-seq analysis to investigate the global correlation of sense/antisense pairs, which showed that the model of sense/antisense negative correlation is not always true for <em>PHO84</em> locus as well as others. I conducted a series of gene expression analysis experiments to decipher the mechanisms regulating <em>PHO84</em> gene, which showed that the 3’ untranslated region (3’UTR) of <em>PHO84</em> plays a regulatory role in the sense expression, an activity not linked to the antisense transcript levels. I also performed a genetic screen to identify <em>trans</em>-acting protein factors that promote the 3’UTR-dependent regulation at <em>PHO84</em> locus.</p> <p>Taken together, I provided insights on both <em>cis</em>- and <em>trans</em>-acting elements controlling the expression of the model gene <em>PHO84</em>. Such information can be taken further and be applied to other higher organisms, with possible implication in the identification of key players in human diseases arising from gene expression dysregulation. </p>

The translation of mRNA into functional proteins is essential for all life. In eukaryotes, aberrant RNAs containing sequence features that stall or severely slow down ribosomes are subject to translation-dependent quality control. Targets include mRNAs encoding a strong secondary structure (No-Go Decay; NGD) or stretches of positively-charged amino acids (Peptide-dependent Translation Arrest/Ribosome Quality Control; PDTA/RQC), mRNAs lacking an in-frame stop codon (Non-Stop Decay; NSD), or defective 18S rRNAs (18S Nonfunctional rRNA Decay; 18S NRD). Previous work from our lab showed that the S. cerevisiae NGD factors DOM34 and HBS1, and PDTA/RQC factor ASC1, all participate in the kinetics of 18S NRD. Upon further investigation of 18S NRD, our research revealed the critical role of ribosomal protein S3 (RPS3), thus adding to the emerging evidence that the ribosome senses its own translational status. While aberrant mRNAs mentioned above can occur endogenously, damaging agents, such as oxidative stress or UV irradiation, can negatively affect the chemical integrity of RNA. Such lesions could lead to translation errors and ribosome stalling. However, current tools to monitor the fate of damaged RNA are quite limited and only provide a low-resolution picture. Therefore, we sought to develop a deep-sequencing method to detect damaged RNA, taking advantage of reverse transcriptase's ability to insert a mutation across a damaged site. Using oxidized RNA as a model damaged RNA, our preliminary data showed increased G>T mutations in oxidized RNA. This method provides the foundation for future work aimed at understanding how cells deal with damaged RNA.

Der eukaryotische Zellzyklus ist auf allen Ebenen der Genexpres- sion reguliert. Sowohl breit angelegte genetische Screens als auch funktionale Studien zu den beteiligten Proteinen haben unser Ver- ständnis dieses fundamentalen Prozesses geprägt. In dieser Arbeit behandle ich räumliche Aspekte der post-transkriptionalen Regulation des Zellzyklus, die mit lichtmikroskopischen Einzelzell- und Einzel- molekülmethoden experimentell zugänglich werden. Insbesondere untersuchte ich die subzelluläre Lokalisierung der messenger RNA von CLB2, einem zentralen Regulator der Mitose im eukaryotischen Modellorganismus Saccharomyces cerevisiae (Bierhefe). Frühere Studien zeigten, dass diese RNA sich im Laufe des vegetativen Zellwachstums in der entstehenden Tochterzelle, der Knospe, anreichert. Mithilfe modernster Fluoreszenzmikroskopie charakterisierte ich die Bewe- gung und Verteilung einzelner CLB2 messenger RNA-Moleküle auf Zeitskalen von Millisekunden bis hin zur Generationszeit dieser He- fen. Ich zeigte, dass sich mit Hilfe von Multifokusmikroskopie unter Verwendung optimierter Fluoreszenzmarker und der Entwicklung objektiver Analysemethoden die Bewegung einzelner RNA-Moleküle zwischen Mutterzelle und Knospe nachvollziehen lässt. Dazu präsen- tiere ich eine Methode um die beobachteten Trajektorien der messenger RNA mathematischen Analysen der Systembiologie zugänglich zu machen. Weiterhin gab die Beobachtung der Verteilung einzelner CLB2 messenger RNA Moleküle über den Zellzyklus hinweg mittels einer neuartigen Lichtblattmikroskopie (Lattice Light Sheet Microscopy) Hinweise auf eine bisher unbekannte Dynamik in der Lokalisierung dieser messenger RNA. Die hier entwickelten Methoden ermöglichen eine quantitative Untersuchung räumlicher Aspekte der posttranskrip- tionalen Zellzyklusregulation. / The eukaryotic cell cycle is regulated on all levels of gene expression. Genetic screens and functional studies of the involved proteins have shaped our understanding of this fundamental process. In this thesis I use single cell and single molecule light microscopy methods to investigate spatial aspects of post-transcriptional cell cycle regulation. I investigated the subcellular localization of CLB2 mRNA, a central regulator of mitosis in the eukaryotic model organism Saccharomyces cerevisiae (baker’s yeast). Previous studies have shown that that this messenger RNA is enriched in the emerging daughter cell, the bud, during vegetative growth. Using pre-commercial fluorescence micro- scopes I characterized the dynamics and partitioning of single CLB2 mRNA on time scales from milliseconds to the generation time of this yeast. I demonstrate that using aberration corrected multifocus mi- croscopy, optimized fluorescent markers, and here developed objective analysis methods, the translocation of single mRNA molecules be- tween mother and bud can be observed. In addition, I report a method to make these trajectories available for the mathematical approaches of Systems Biology. Further, the observation of single CLB2 mRNA partitioning throughout the cell cycle with the use of lattice light sheet microscopy suggested a previously unknown localization behavior of the transcript. The methods developed here enable a quantitative analysis of spatial aspects of post-transcriptional cell cycle regulation.