<p>Germ cells represent the immortal line: they are guardians of a totipotent genome and are essential for the genetic survival of an individual organism and ultimately a species. An error at any stage in development (specification, migration, colonization, differentiation, adult maintenance) can lead to one of two disastrous outcomes: (1) germ cell death or (2) unchecked growth and proliferation leading to tumorigenesis. The work in this dissertation utilizes a classic mouse model (<italic>Ter</italic>) resulting in both of these phenotypes to further explore the molecular mechanisms important for development of germ cells. </p> <p>A homozygous nonsense mutation (<italic>Ter</italic>) in murine <italic>Dnd1</italic> (<italic>Dnd1<super>Ter/Ter</super></italic>) results in a significant (but not complete) early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129/SvJ background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost, develop into teratomas, or what factors directly control the strain-specific phenotype variation. </p> <p>Work here demonstrates that <italic>Dnd1</italic> expression is restricted to germ cells and that the <italic>Ter</italic> mutant defect is cell autonomous. The early loss of germ cells is due in part to BAX&ndash;mediated apoptosis which also affects the incidence of tumorigenesis on a mixed genetic background. Moreover, tumor formation is-specific to the male developmental pathway and not dependent on sex chromosome composition of the germ cell (XX vs. XY). Despite normal initiation of the male somatic pathway, mutant germ cells fail to differentiate as pro&ndash;spermatogonia and instead prematurely enter meiosis.</p> <p>Results here also reveal that, on a 129/SvJ background, many mutant germ cells fail to commit to the male differentiation pathway, instead maintain expression of the pluripotency markers, NANOG, SOX2, and OCT4, and initiate teratoma formation at the stage when male germ cells normally enter mitotic arrest. RNA immunoprecipitation experiments reveal that mouse DND1 directly binds a group of transcripts that encode negative regulators of the cell cycle, including <italic>p27Kip1</italic>, which is not translated in <italic>Dnd1<super>Ter/Ter</super></italic> germ cells. Additionally, overexpression of DND1 in a teratocarcinoma cell line leads to significant alteration of pathways controlling the G1/S checkpoint and the RB tumor suppressor protein. This strongly suggests that DND1 regulates mitotic arrest in male germ cells through regulation of cell cycle genes, serving as a gatekeeper to prevent the activation of a pluripotent program leading to teratoma formation. Furthermore, strain&ndash;specific morphological and expression level differences possibly account for sensitivity to tumor development.</p> / Dissertation

<p>SR proteins are a family of splicing factors involved in the regulation of both constitutive and alternative splicing of pre-mRNAs. Despite years of studies, several big questions still remain: how the expression levels of SR proteins are regulated; what are the underlying mechanisms responsible for SR proteins-mediated gene regulation; what are the physiological targets of SR proteins in vivo. In my dissertation study, I am focusing on two members of the family, SF2/ASF and SRp20, to study their functional involvement in regulating microRNA/mRNA biogenesis and their own expression. </p><p>Negative feedback regulation is a common mechanism maintaining the steady-state level of SR proteins (i.e. SC35 and SRp20), and several mechanism may be involved. In order to test if miRNAs are also involved in such negative feedbacks, small RNA sequencing was used to identify differentially expressed miRNAs after SF2/ASF overexpression in an inducible stable cell line system. Among the 40 differentially expressed miRNAs, miR-7 is particularly interesting, because it is also predicted to target SF2/ASF, which forms a negative feedback regulation. This is indeed the case as shown by luciferase reporter assay and overexpression/knocking down of miR-7 in vivo. To our knowledge, this is the first identified negative feedback circuit between a SR protein and a miRNA, which may be a general mechanism in regulating SR protein homeostasis.</p><p>To characterize the mechanism underlying SF2/ASF-enhanced miRNA biogenesis, I have employed a series of molecular and biochemical approaches to pinpoint the key molecular interactions in a minigene system, which is consist of miR-7 embedded intron and the flanking exons of its host gene. By manipulating the splicing pattern of such minigene, I have uncovered a splicing-independent function of SF2/ASF in regulating miRNA biogenesis. Directly binding between SF2/ASF protein and pri-miR-7 was demonstrated by Cross-linking and immunoprecipitation assay (CLIP) and RNA affinity purification. The precise binding site was then pinpointed by combining computational prediction and mutagenesis assay. Finally, by using in vitro pri-miRNA processing assay, I showed that SF2/ASF can promote the Drosha cleavage step of pri-miR-7 through directly association with the predicted binding site. So far, this is the first SR protein discovered, which is directly involved in miRNA biogenesis. Moreover, our preliminary data also suggested that SF2/ASF may promote miRNA biogenesis in other steps after Drosha cleavage; and different SR proteins can regulate miRNA biogenesis in a substrate-specific manner. Taken together, SR family of splicing factors may be broadly involved in miRNA biogenesis through direct interactions.</p><p>In order to study the general involvement of SR proteins in RNA biogenesis, one important step stone is to have a better profile of their targets in vivo. To achieve this, I focused on SRp20, another classic SR protein. Photoactivatable-Ribonucleoside-Enhanced Cross-linking and immunoprecipitation assay combined with deep sequencing (PAR-CLIP-seq) was used to identify the binding partners of SRp20 globally, which is subsidized by candidate gene validations. Consistent with the literature, I found that SRp20 primarily targets exonic regions for splicing regulation, and such interactions are likely to be sequence dependent on the CWWCW motif. Surprisingly, I also observed extensive binding between SRp20 and the 3' UTRs of mRNA, which may affect the choice of alternative polyadenylation sites. The underlying mechanisms are being investigated by a variety of molecular methods. </p><p>In summary, I have identified a subset of miRNAs, the expression of which can be regulated by SF2/ASF in a splicing independent manner. This is the first SR protein identified in regulating miRNA biogenesis. One of the upregulated miRNAs, miRNA-7 can form a negative feedback with SF2/ASF by negatively regulating the expression of SF2/ASF on translational level. By using PAR-CLIP method, I have identified the genome-wide binding partners of SRp20 in vivo. When SRp20 binds to the exonic regions, it potentially affects the alternative splicing patterns of nearby introns. Interestingly, the 3' end choices for a subset of genes may be regulated by SRp20 through directly binding, which may be a new mechanism for the regulation of 3' end processing.</p> / Dissertation

Both microRNAs (miRNAs) and RNA-binding proteins (RBPs) regulate post- transcriptional events, but the post-transcriptional contribution to the global mammalian transcriptomes is still not well understood. In this study we study the synergistic interaction between microRNAs that inhibit gene production, and a special RBP, HuR, that positively regulates mRNA stability. We examined their relationship in terms of spatial, conservational and expressional perspective. We show comprehensive mapping of HuR binding sites by combination of its structural and sequential preferences; and cross-platform normalization method within a process of refining miRNA and HuR binding site mapping. Finally, we observed co-evolution of miRNA and HuR binding sites by looking at their proximity and conservation levels. Collectively, our data suggest that mammalian microRNAs and HuR, with seemingly opposing regulatory effects, cooperatively regulate their mutual targets.

Cellular messenger RNAs (mRNAs) exist almost exclusively in the context of ribonucleoprotein complexes (RNPs), which are largely responsible for the coordinated regulation of mRNA fate, and in particular, the post-transcriptional regulation of mRNA stability and translation. RNA- binding proteins, antisense RNAs, and microRNAs represent three major classes of post- transcriptional regulatory factors that interact with target mRNAs. Significantly, these interactions are dynamically regulated under both basal and stress conditions, such as hypoxia. Given the prominent contributions of post-transcriptional regulation to overall gene expression, a more comprehensive understanding of the underlying mechanisms is required. In this thesis, we present exciting new evidence for the functional importance of post- transcriptional gene regulation, especially in the vascular endothelium. Firstly, we show that the formation of hnRNP E1-containing RNPs contributes significantly to the remarkable basal stability of endothelial nitric oxide synthase (eNOS) mRNAs in endothelial cells by protecting them from inhibitory post-transcriptional forces. However, hypoxia impairs such RNP formation through hnRNP E1 serine phosphorylation and nuclear localization. Together, these mechanisms contribute significantly to decreased eNOS expression and activity in chronic hypoxia. ii Secondly, we reveal an important functional relationship between the microRNA pathway and the HIF-mediated cellular hypoxic response. Specifically, the down-regulation of Dicer and an important number of Dicer-dependent microRNAs in chronic hypoxia represents an important adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, with significant implications for the development of RNAi- based therapies. Finally, we provide evidence that the up-regulation of specific microRNAs in acute hypoxia is a potentially important mechanism that serves to suppress global translation initiation in order to conserve energy and ensure cellular survival. Collectively, the findings presented in this thesis provide important new mechanistic insight into the post-transcriptional regulation of eNOS, as well as the functional integration of the microRNA and the cellular hypoxic response pathways.

Cellular messenger RNAs (mRNAs) exist almost exclusively in the context of ribonucleoprotein complexes (RNPs), which are largely responsible for the coordinated regulation of mRNA fate, and in particular, the post-transcriptional regulation of mRNA stability and translation. RNA- binding proteins, antisense RNAs, and microRNAs represent three major classes of post- transcriptional regulatory factors that interact with target mRNAs. Significantly, these interactions are dynamically regulated under both basal and stress conditions, such as hypoxia. Given the prominent contributions of post-transcriptional regulation to overall gene expression, a more comprehensive understanding of the underlying mechanisms is required. In this thesis, we present exciting new evidence for the functional importance of post- transcriptional gene regulation, especially in the vascular endothelium. Firstly, we show that the formation of hnRNP E1-containing RNPs contributes significantly to the remarkable basal stability of endothelial nitric oxide synthase (eNOS) mRNAs in endothelial cells by protecting them from inhibitory post-transcriptional forces. However, hypoxia impairs such RNP formation through hnRNP E1 serine phosphorylation and nuclear localization. Together, these mechanisms contribute significantly to decreased eNOS expression and activity in chronic hypoxia. ii Secondly, we reveal an important functional relationship between the microRNA pathway and the HIF-mediated cellular hypoxic response. Specifically, the down-regulation of Dicer and an important number of Dicer-dependent microRNAs in chronic hypoxia represents an important adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, with significant implications for the development of RNAi- based therapies. Finally, we provide evidence that the up-regulation of specific microRNAs in acute hypoxia is a potentially important mechanism that serves to suppress global translation initiation in order to conserve energy and ensure cellular survival. Collectively, the findings presented in this thesis provide important new mechanistic insight into the post-transcriptional regulation of eNOS, as well as the functional integration of the microRNA and the cellular hypoxic response pathways.

Both microRNAs (miRNAs) and RNA-binding proteins (RBPs) regulate post- transcriptional events, but the post-transcriptional contribution to the global mammalian transcriptomes is still not well understood. In this study we study the synergistic interaction between microRNAs that inhibit gene production, and a special RBP, HuR, that positively regulates mRNA stability. We examined their relationship in terms of spatial, conservational and expressional perspective. We show comprehensive mapping of HuR binding sites by combination of its structural and sequential preferences; and cross-platform normalization method within a process of refining miRNA and HuR binding site mapping. Finally, we observed co-evolution of miRNA and HuR binding sites by looking at their proximity and conservation levels. Collectively, our data suggest that mammalian microRNAs and HuR, with seemingly opposing regulatory effects, cooperatively regulate their mutual targets.

<p>RNA localization provides the blueprint for compartmentalized protein synthesis in eukaryotic cells. Current paradigms indicate that RNAs encoding secretory and membrane proteins are recruited to the endoplasmic reticulum (ER), via positive selection of a `signal peptide' tag encoded in the protein. Thus RNA sorting to the ER follows protein sorting and the RNA is considered a passive player. However, RNAs have been shown to access the ER independent of the signal peptide and display a wide range of affinities to the ER that does not correlate with signal peptide strength. How and why mRNAs localize to the ER to varying extents and whether such localization serves a purpose besides protein sorting is poorly understood. To establish the cause and consequence of RNA binding to the ER membrane, I pose three primary questions: 1. How are mRNAs targeted to the ER? 2. Once targeted, how are mRNAs anchored to the ER membrane? 3. Are ER localized mRNAs subject to transcript-specific regulation? </p><p>I address cytosolic mRNA targeting to the ER by comparing the partitioning profiles of cytosolic/nuclear protein-encoding mRNA population (mRNACyto) to that of mRNAs encoding a signal peptide (mRNAER). I show that, at a population level, mRNACyto display a mean ER enrichment that is proportional to the amount of ER-bound ribosomes. Thus, I propose that targeting of mRNACyto to the ER is stochastic and over time, the specific interactions engaged by an individual mRNACyto with the ER determines its steady state partitioning profile between the cytoplasm and the ER. </p><p>To address the modes of direct binding of mRNA to the ER, I examined the association of various RNA populations with the ER after disrupting membrane-bound ribosome's interaction with its ER receptor. mRNACyto and most of mRNAs encoding secretory proteins (mRNACargo) are released upon disruption of ribosome-receptor interactions, indicating no direct mRNA-ER interactions. However, the population of mRNAs that encode resident proteins of the endomembrane organelles such as the ER, lysosome, endosome and the Golgi apparatus (mRNARes) maintain their association with the ER despite the disruption of ribosome-receptor interactions. These results indicate direct binding of mRNARes to the ER, further suggesting that the function of the encoded proteins dictates the mode of association of corresponding mRNA with the ER. </p><p>To uncover the mode of mRNARes binding directly to ER, I performed differential proteomic analysis of cytosolic and membrane bound RNA-protein complexes, which revealed a network of RNA binding proteins that interact uniquely with the ER-anchored mRNAs. The anchoring of endomembrane resident protein-encoding RNAs to the ER through these RNA binding proteins may reflect an imprinting of the ER with the information necessary for the continued biogenesis of the endomembrane organelle system even in situations where translation-dependent ER targeting of an mRNA is compromised. </p><p>Finally, I address whether ER-bound mRNAs can be regulated differentially by comparing the fates of two signal peptide-encoding RNAs, B2M and GRP94, during the unfolded protein response (UPR). I show that in response to ER stress, GRP94 mRNA, but not B2M, relocates to stress-induced RNA granules, thus escaping an RNA decay program that operates at the ER membrane during the UPR. Hence, I propose that the mode of RNA association to the ER is subject to regulation and influences the fate of RNAs during cellular stress. Thus, by demonstrating diverse modes of mRNA localization to the ER and differential regulation of ER bound mRNAs during cellular stress, my work has helped establish an emerging role for the ER as a post-transcriptional gene regulatory platform.</p> / Dissertation

The RNA-binding protein Quaking (QKI) plays a fundamental role in post-transcriptional gene regulation during mammalian nervous system development. QKI is well known for advancing oligodendroglia differentiation and myelination, however, its functions in astrocytes and embryonic central nervous system (CNS) development remain poorly understood. Uncovering the complete spectrum of QKI molecular and functional repertoire is of additional importance in light of growing evidence linking QKI dysfunction with human disease, including schizophrenia and glioma. This thesis summarizes my contribution to fill this gap of knowledge.         In a first attempt to identify the QKI-mediated molecular pathways in astroglia, we studied the effects of QKI depletion on global gene expression in the human astrocytoma cell line. This work revealed a previously unknown role of QKI in regulating immune-related pathways. In particular, we identified several putative mRNA targets of QKI involved in interferon signaling, with possible implications in innate cellular antiviral defense, as well as tumor suppression. We next extended these investigations to human primary astrocytes, in order to more accurately model normal brain astrocytes. One of the most interesting outcomes of this analysis was that QKI regulates expression of transcripts encoding the Glial Fibrillary Acidic Protein, an intermediate filament protein that mediates diverse biological functions of astrocytes and is implicated in numerous CNS pathologies. We also characterized QKI splice variant composition and subcellular expression of encoded protein isoforms in human astrocytes. Finally, we explored the potential use of zebrafish as a model system to study neurodevelopmental functions of QKI in vivo. Two zebrafish orthologs, qkib and qki2, were identified and found to be widely expressed in the CNS neural progenitor cell domains. Furthermore, we showed that a knockdown of qkib perturbs the development of both neuronal and glial populations, and propose neural progenitor dysfunction as the primary cause of the observed phenotypes.        To conclude, the work presented in this thesis provides the first insight into understanding the functional significance of the human QKI in astroglia, and introduces zebrafish as a novel tool with which to further investigate the importance of this gene in neural development.

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.

Alzheimer’s disease (AD) is the most common neurodegenerative disease affecting the elderly population and is believed to be caused by the overproduction and accumulation of the toxic amyloid beta (Aβ) peptide in the brain. Aβ is produced by two separate enzymatic cleavage events of the larger membrane bound amyloid precursor protein, APP. The first, and rate-limiting, cleavage event is made by beta-secretase, or BACE1, and is thus an attractive therapeutic target. Our lab, as well as many others, has shown that BACE1 protein and activity are increased in late-stage sporadic AD. We have extended these findings to show that BACE1 is increased in the earliest stages of AD before the onset of significant Aβ accumulation, indicating a potential causal role in the disease. Interestingly, BACE1 mRNA levels are unchanged in AD, leading to reason that a post-transcriptional method of BACE1 regulation is altered in disease. To date, the mechanism for this aberrant post-transcriptional regulation has not been elucidated. This study has implicated the cellular nucleic acid binding protein (CNBP), a highly conserved RNA binding protein, as a positive regulator of BACE1 translation, with implications for the etiology of sporadic AD. CNBP overexpression in cultured cells or spiked into a cell-free in vitro translation system increased BACE1 protein expression without affecting BACE1 mRNA levels. Knockdown of CNBP reduced BACE1 protein and mRNA slightly. Furthermore, CNBP associated with BACE1 mRNA in cell lysates and bound directly to the BACE1 5’ UTR in vitro, which confers most of the regulatory activity. Importantly, CNBP was increased in the progression of AD and correlated with BACE1 expression. Cellular stressors (such as glucose deprivation and oxidative stress) that occur in the AD brain increase BACE1 translation and we have found that these stressors increased CNBP expression as well. Early experimental evidence suggests that CNBP may enhance BACE1 translation through a cap-independent mechanism, which is an alternative translational pathway activated by cell stress. These studies indicate that the RNA binding protein CNBP is a novel trans-acting factor important for the regulation of BACE1 protein production and may be a viable therapeutic target for AD.