E2F1 is a transcription factor which paradoxically has major influence on both apoptosis and cell cycle progression. One of the most important questions in E2F1 biology therefore is the mechanism underlying regulation of these opposing physiological outcomes. Post-translational modifications (PTM) provide proteins with an additional layer of complexity, potentially altering interactions with partner DNA and protein. The importance of arginine methylation has recently been implicated in modulating the activity of the tumour suppression pathway proteins, p53 and E2F1. Previous studies have established that the methyltransferase, PRMT5, is responsible for the symmetrical dimethylation of E2F1, which inhibits its pro-apoptotic activity. In this thesis, E2F1 was found to be a substrate of PRMT1, which catalysed asymmetrical dimethylation of E2F1 at arginine 109. In addition, a positive correlation was found between the percentage of apoptotic cells and levels of PRMT1. Conversely, an increase in cancer cell colony formation was shown when the site of PRMT1 methylation on E2F1 was changed from arginine to lysine at position 109. These findings suggested a growth inhibition effect by PRMT1 methylation on E2F1. At the transcriptional level, depletion of PRMT1 increased E2F1 binding to the promoter region of Cdc6, a cell cycle regulator, and decreased binding to the promoter region of Apaf1, which has a pro- apoptotic role. Genome-wide ChIP-sequencing technology was undertaken and results further clarified that the depletion of PRMT1 preferably enriched E2F1 binding to promoters of positive regulators of cell proliferation and promoters of the cell cycle. Collectively, the findings of this thesis suggested that the opposing roles E2F1 demonstrated in promoting both cell proliferation and apoptosis was due to different types of arginine methylation which trigger E2F1 binding to different promoters. Lastly, arginine methylation was shown to influence protein-protein interactions. PRMT5 induction resulted in the identification by mass spectrometry of β-catenin as an E2F1 interacting partner. As the Wnt/β-catenin signalling pathway is broadly recognised as having pro- cell proliferation activity, this finding is consistent with previous reports that suggest the oncogenic role PRMT5 methylation has on E2F1.

Breast cancer is the most commonly diagnosed cancer amongst Canadian women. Though numerous treatments are available, in many instances tumours become refractory or recur. Therefore, understanding the biological events that lead to the progression and therapeutic resistance of breast cancer is essential for the development of novel treatment options for this disease. Numerous members of the protein arginine methyltransferase (PRMT) family, which are the enzymes responsible for catalyzing methylation on arginine residues are aberrantly regulated in breast cancer. Hence, understanding the precise contribution of PRMTs to the development and progression of breast cancer is important. This Thesis will present my findings on the alternatively spliced PRMT1 isoform, PRMT1v2, previously identified to be overexpressed in breast cancer cell lines and here shown to promote breast cancer cell survival and invasion. Second, a novel role is ascribed to PRMT6, another PRMT aberrantly expressed in breast cancer. PRMT6 promotes chemoresistance to the drug bortezomib by mediating stress granule formation through down-regulation of eIF4E. Increased stress granule formation in bortezomib-resistant cancer cells promotes cell survival. Third, DDX3, a prototypical PRMT substrate which is overexpressed in breast cancer cell lines and stimulates transformation of mammary epithelial cells is a novel substrate of PRMT1, CARM1, and PRMT6. Lastly, TDRD3, a reader/effector of arginine methylation also overexpressed in breast tumours regulates breast cancer cell proliferation, anchorage-independent growth and cell motility and invasion.

Identification of therapeutically relevant molecules is necessary for the advancement of non-viral reprogramming of human cells for regenerative medicine. We have developed a novel non-viral model system that transforms primary human dermal fibroblasts into cells with induced regeneration competence (iRC). Low oxygen-mediated effects of fibroblast growth factor FGF2 lead to an increased cellular lifespan with a two fold increase in population doublings before senescence, remaining non-tumorigenic when injected into SCID mice while maintaining regeneration competence. This system allows us to study molecules that participate in increased cellular lifespan in a non-tumorigenic system. Analysis of chromatin modification enzymes by hybridization array, RT-PCR, and Western blots revealed upregulation of the arginine methyltransferase PRMT8 in iRC cells, challenging the paradigm that PRMT8 is solely expressed in brain tissue at the plasma membrane. Possibly leading to the erroneous conclusions that PRMT8 is brain specific at the plasma membrane is the fact that PRMT8 has several mRNA variants and protein isoforms. Here, I report expression of a novel PRMT8 variant in human dermal fibroblasts. Essential participation of PRMT8 in cellular proliferation was identified as a novel function for this enzyme through siRNA-mediated knockdown in both non-tumorigenic and tumorigenic cell lines. While other members of the PRMT family have known roles in cell cycle progression, I show for the first time that PRMT8 expression is reduced in both natural senescence and by premature induction of replicative senescence using sub-cytotoxic levels of hydrogen peroxide, implicating a correlation between PRMT8 expression and cell cycle progression. However, PRMT8 overexpression causes no significant change in the number of population doublings or the amount of time spent in culture prior to senescence, and does not alter the expression of key cell cycle regulatory genes. These results suggest that maintenance of PRMT8 expression is critical for cellular proliferation, but overexpression of PRMT8 alone is not sufficient to increase cellular lifespan. I determined that oxygen is the primary mediator of PRMT8 upregulation in the iRC system and therefore investigate histone occupancy of the PRMT8 promoter at hypoxia response elements. Through this analysis, I found bivalent occupancy regardless of culture conditions, indicating that PRMT8 maintains a state of poised readiness for transcriptional accessibility. The mechanism by which PRMT8 participates in cellular proliferation was investigated through binding partner identification. A binding partner of endogenous PRMT8 is identified here for the first time as FGF2 using co-IP and mass spectrometry. As iRC cells demonstrate a unique phenotype that uncouples the mechanisms of increased lifespan from tumorigenesis, I investigated the feasibility of PRMT8 as a cancer biomarker by mining publically available data in light of our own. I showed that PRMT8 is not only expressed in a variety of cancers, but that its expression is amplified. Moreover, PRMT8 expression significantly correlates to patient survival in specific cancers, strengthening the feasibility of this molecule as a biomarker. Aberrant expression of most PRMT family members has been described in various cancers, and specific PRMT variants are currently being used as prognostic markers. As such, I analyzed variant-specific PRMT8 expression in primary cancer cell lines and show that tumorigenic glioblastomas express PRMT8 mRNA variant 2. These data suggest that PRMT8 is a viable candidate for further study as a prognostic cancer biomarker, specifically for brain cancer.

Protein arginine methylation is an important post-translational modification that is vital in regulating various cellular processes such as gene transcription, cell signaling, and RNA processing. Protein arginine methyltransferases (PRMTs) are responsible for performing this important modification. PRMT1 (protein arginine methyltransferase 1) and Hmt1 (hnRNP methyltransferase 1) are the predominant PRMTs in humans and yeast, respectively. Despite growing momentum in this field, relatively little is understood about PRMT regulation. Further work discovering how PRMTs are regulated will greatly advance our understanding of diseases where PRMTs have been implicated, such as heart disease, viral pathogenesis, and cancer. It has been discovered that a human protein called hCaf1 (human Ccr4-associated factor 1) is a regulator of PRMT1 with respect to certain substrates, and also colocalizes with PRMT1. We present data that suggest the yeast homolog of hCaf1, Pop2, may also perform a similar function on Hmt1. We provide data on the expression and purification of a truncation of Pop2 from S. cerevisiae, including the temperature sensitivity of one construct of Pop2 and its susceptibility to precipitation. We also demonstrated concentration-dependent inhibition of Hmt1-catalyzed methylation of histone H4 by Pop2 in vitro. Yeast cell lysates also showed altered patterns of methylation in the presence and absence of Pop2 in vivo. In an effort to understand the mechanism employed by Pop2 to accomplish this regulatory function, pull-downs were performed suggesting that Pop2 directly interacts with histone H4, a substrate of Hmt1. Mutagenic studies with Pop2 suggested a region that may be responsible for this interaction. Given these data, we hypothesized that Pop2 is able to inhibit the methylation of histone H4 via a substrate-sequestering mechanism. Further experimentation will determine the precise interaction surfaces of Pop2 and substrate, and continue to define the details of methylation inhibition by Pop2, including the scope of its influence in the cell.

Proteins of the nuclear envelope play important roles in a variety of cellular processes e.g. transport of proteins between the nucleus and cytoplasm, co-ordination of nuclear and cytoplasmic events, anchoring of chromatin to the nuclear periphery and regulation of transcription. Defects in proteins of the nuclear envelope and the nuclear pore complexes have been related to a number of human diseases. To understand the cellular functions in which nuclear envelope proteins participate it is crucial to map the functions of these proteins. The present study was done in order to characterize the role of three different proteins in functions related to the nuclear envelope in the yeast Saccharomyces cerevisiae. The arginine methyltransferase Rmt2 was demonstrated to associate with proteins of the nuclear pore complexes and to influence nuclear export. In addition, Rmt2 was found to interact with the Lsm4 protein involved in RNA degradation, splicing and ribosome biosynthesis. These results provide support for a role of Rmt2 at the nuclear periphery and potentially in nuclear transport and RNA processing. The integral membrane protein Cwh43 was localized to the inner nuclear membrane and was also found at the nucleolus. A nuclear function for Cwh43 was demonstrated by its ability to bind DNA in vitro. A link to nucleolar functions was demonstrated by genetic analysis. Furthermore, Cwh43 is interacting with signalling pathways perhaps acting as a sensor for signals transmitted from the cytoplasm to the nucleus. The Myr1 protein was found to be membrane-associated and to interact with proteins involved in vesicular traffic. Overexpression of Myr1 affects nuclear morphology and nuclear pore distribution suggesting a function in membrane dynamics. In conclusion, the presented results aid in a deeper understanding of functions related to the nuclear envelope in revealing a novel link between arginine methylation and the nuclear periphery, identifying a novel inner nuclear membrane protein and a new membrane-associated protein.

Spinal muscular atrophy is an autosomal recessive genetic disease that results from the loss and/or degeneration of alpha motor neurons in the lower part of the spinal cord. With ~ 1 in 6000 live births per year being affected, this disease is the second leading cause of infant death and is caused by the loss or decrease of the Survival of Motor Neuron protein (SMN). While a lot is known about the role that SMN plays in the cytoplasmic assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), it remains a crucial question in the field to gain a better understanding of what specific/distinct function(s) SMN might have in motor neurons. We have identified novel interactions between SMN and two RNA-binding proteins (RBPs) known to be components of axonal RNA granules. More specifically, we demonstrated that SMN interacts with HuD and SERBP1 in a direct fashion in foci-like structures along neurites of motor neuron-derived cells. We have also demonstrated that the SMN/HuD interaction is required for the localization of HuD into RNA granules in neurites of motor neuron-derived cells. Furthermore, I have shown that SERBP1 is down-regulated in the absence of normal levels of SMN and, most importantly, that over-expression of SERBP1 can rescue SMA-like neuronal defects using a cell culture model of the disease. These findings may help shed light on the non-canonical molecular pathway(s) involving SMN and RBPs in motor neurons and underscores the possible therapeutic benefits of targeting these RBPs in the treatment of SMA.

Spinal muscular atrophy is an autosomal recessive genetic disease that results from the loss and/or degeneration of alpha motor neurons in the lower part of the spinal cord. With ~ 1 in 6000 live births per year being affected, this disease is the second leading cause of infant death and is caused by the loss or decrease of the Survival of Motor Neuron protein (SMN). While a lot is known about the role that SMN plays in the cytoplasmic assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), it remains a crucial question in the field to gain a better understanding of what specific/distinct function(s) SMN might have in motor neurons. We have identified novel interactions between SMN and two RNA-binding proteins (RBPs) known to be components of axonal RNA granules. More specifically, we demonstrated that SMN interacts with HuD and SERBP1 in a direct fashion in foci-like structures along neurites of motor neuron-derived cells. We have also demonstrated that the SMN/HuD interaction is required for the localization of HuD into RNA granules in neurites of motor neuron-derived cells. Furthermore, I have shown that SERBP1 is down-regulated in the absence of normal levels of SMN and, most importantly, that over-expression of SERBP1 can rescue SMA-like neuronal defects using a cell culture model of the disease. These findings may help shed light on the non-canonical molecular pathway(s) involving SMN and RBPs in motor neurons and underscores the possible therapeutic benefits of targeting these RBPs in the treatment of SMA.

The dysfunction of autophagy pathways has been linked to the development and progression of numerous human diseases, in particular neurological disorders and cancer. Investigating these pathological autophagy mechanisms is essential to gain insights into the underlying disease mechanisms, identify novel biomarkers, and develop targeted therapies. In this thesis, I present three manuscripts that investigate the regulatory mechanisms of autophagy machinery in human diseases. In the first manuscript (Chitiprolu et al., 2018), we investigated the mechanism of p62-mediated selective autophagic clearance of RNA stress granules implicated in Amyotrophic Lateral Sclerosis (ALS). Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. By employing mass spectrometry, high resolution imaging and biochemical assays, we demonstrated that the autophagy receptor p62 associates with C9ORF72 to eliminate stress granules by autophagy. This requires p62 to associate with proteins that are symmetrically methylated on arginines. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients. The second manuscript (Guo, Chitiprolu et al., 2014) describes the mechanism by which autophagy degrades retrotransposon RNA from both long and short interspersed elements, thereby preventing new retrotransposon insertions into the genome. By employing quantitative imaging tools, we demonstrated that retrotransposon RNA localizes to RNA granules that are selectively degraded by the autophagy receptors NDP52 and p62. Mice lacking a copy of Atg6/Beclin1, a gene critical for autophagy, also accumulate both retrotransposon RNA and genomic insertions. This suggests a mechanism for the increased tumorigenesis upon autophagy inhibition and therefore a role for autophagy in tempering evolutionary change. Finally, the third manuscript (Guo, Chitiprolu et al., 2017) examines the intersection of autophagy machinery with exosome release and function in cancer metastasis. By employing dynamic light scattering, Nanosight particle tracking, electron microscopy, super-resolution imaging and Western blotting, we robustly quantified exosome identity and purity in multiple cell lines. We demonstrated that exosome production is strongly reduced in cells lacking Atg5 and Atg16L1, but this is independent of Atg7 and canonical autophagy. The effect of Atg5 on exosome production promotes the migration and in vivo metastasis of orthotopic breast cancer cells. These findings delineate autophagy-independent pathways by which autophagy-related genes can contribute to metastasis. Taken together, data presented in the three manuscripts highlight the molecular mechanisms of autophagy core machinery proteins and selective receptors such as Atg5, p62 and NDP52, in the pathogenesis of cancer and neurodegeneration. In these diseases characterized by mutations in autophagy pathways, the mechanisms we uncover provide insights into their causes and serve as potential therapeutic targets.

Spinal muscular atrophy is an autosomal recessive genetic disease that results from the loss and/or degeneration of alpha motor neurons in the lower part of the spinal cord. With ~ 1 in 6000 live births per year being affected, this disease is the second leading cause of infant death and is caused by the loss or decrease of the Survival of Motor Neuron protein (SMN). While a lot is known about the role that SMN plays in the cytoplasmic assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), it remains a crucial question in the field to gain a better understanding of what specific/distinct function(s) SMN might have in motor neurons. We have identified novel interactions between SMN and two RNA-binding proteins (RBPs) known to be components of axonal RNA granules. More specifically, we demonstrated that SMN interacts with HuD and SERBP1 in a direct fashion in foci-like structures along neurites of motor neuron-derived cells. We have also demonstrated that the SMN/HuD interaction is required for the localization of HuD into RNA granules in neurites of motor neuron-derived cells. Furthermore, I have shown that SERBP1 is down-regulated in the absence of normal levels of SMN and, most importantly, that over-expression of SERBP1 can rescue SMA-like neuronal defects using a cell culture model of the disease. These findings may help shed light on the non-canonical molecular pathway(s) involving SMN and RBPs in motor neurons and underscores the possible therapeutic benefits of targeting these RBPs in the treatment of SMA.

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