Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 122-138).

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [126]-149).

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves 130-135).

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

In the cell, the genetic information encoded in the DNA is transcribed to RNA. All RNAs that are transcribed in the cell are initially produced as precursor RNAs, which have to undergo various steps of processing to obtain their mature form. The maturation and processing for all RNA classes requires the activity of multiple RNA binding proteins (RBPs). An important family of RBPs that is involved in RNA maturation and processing is the SR-protein family. SR proteins are important for the regulation of a multitude of processes that include: splicing, transcription, export, RNA stabilization, translation and ncRNA processing. As of yet, there have been no comprehensive studies that describe how SR proteins dynamically regulate the maturation of RNAs. The results presented in this thesis provide new insights into the function and activity of SR proteins during RNA maturation. My experiments greatly expand the knowledge surrounding the action of RNA-binding proteins in vivo and in different cell compartments. To study the action of two different SR proteins in different cell compartments, I developed a new technique that combines cell fractionation and iCLIP, which I named FRACKING. For the first time, this method allowed me to collect information regarding the subcellular location where the RNA-protein interactions are taking place, giving a dynamic picture of the in vivo binding of SR proteins and of RNA binding proteins (RBP) in general. By using FRACKING on two heavily shuttling SR proteins, SRSF3 and SRSF7, I showed that both SR proteins are very dynamic in their binding behavior with RNAs. My research showed that both SRSF3 and SRSF7 strongly associate with RNAs during transcription (co-transcriptionally) and that they often remain bound to these transcripts until they are exported to the cytoplasm. The functions of SRSF3 and SRSF7 are closely related to their binding location on the target RNAs. I identified a subset of highly conserved introns that associated with SR proteins and are retained in their transcripts. These intron-retaining isoforms, contrary to textbook knowledge, are exported to the cytoplasm. I showed, for the first time, that SRSF3 and SRSF7 strongly interact with snoRNAs in the chromatin, and that this snoRNA-SR-protein binding behavior is distinct between SRSF3 and SRSF7. SRSF3 binds to the mature snoRNA sequence, and also to the surrounding intronic sequences, pointing towards a possible activity in guiding snoRNA maturation. Whereas SRSF7 associates to mature snoRNA sequences. Taken together, my study identified a dynamic pool of interactions for two SR proteins, in different cell compartments and discovered new activities for the two SR proteins. Importantly, this study challenges textbook knowledge on splicing and export of mRNAs by identifying a subset of transcripts that can be exported even when they retain introns.

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.

Parkinson's Disease (PD) is a late-onset neurodegenerative disease characterized by progressive motor dysfunction caused by a loss of dopaminergic neurons for which there is no known cure. Among the most common genetic causes of PD are mutations in the leucine-rich repeat kinase 2 gene (LRRK2), encoding a multi-domain protein with kinase activity. The LRRK2 G2019S mutation causes hyperactivity of the kinase domain and is the most frequent LRRK2 mutation in patients with familial PD, though its role in PD pathology remains unclear. Preliminary data from the lab of our collaborator, Dr. David Park, demonstrated through a genetic screen in Drosophila melanogaster that the deletion of rbp9 encoding an RNA-binding protein prevented pathology induced by PD-relevant mutations in the LRRK2 kinase domain. The neuronal homolog of RBP9 in humans is HuD, a member of the Hu family of RNA-binding proteins that regulates the expression of many transcripts involved in neuronal development, plasticity, and survival. In addition, HuD has been shown to modify the age-at-onset or risk of developing PD. Here, we studied the effect of LRRK2 on the post-transcriptional regulation of mRNAs bound by HuD in the context of PD. Our findings showed that HuD is a substrate for LRRK2 phosphorylation in vitro, and that LRRK2 G2019S hyperphosphorylates HuD. We demonstrated that LRRK2 kinase activity is required for the binding of several transcripts by HuD that encode PD-relevant proteins such as α-synuclein and neuronal survival factor BDNF. Our findings in human neuroblastoma cells indicated that LRRK2 regulates the protein levels of HuD mRNA targets α-synuclein and BDNF in a mechanism that can by modified by HuD. Finally, we showed that the combination of HuD knockout with LRRK2 G2019S expression in mice rescues aberrant expression of HuD targets in mice with only the LRRK2 G2019S mutation or the knockout of HuD alone. Together, our findings demonstrate that LRRK2 affects the post-transcriptional regulation of HuD-bound mRNAs, and suggest the use of HuD as a potential therapeutic target in patients with PD caused by the LRRK2 G2019S mutation.

abstract: Exome sequencing was used to identify novel variants linked to amyotrophic lateral sclerosis (ALS), in a family without mutations in genes previously linked to ALS. A F115C mutation in the gene MATR3 was identified, and further examination of other ALS kindreds identified an additional three mutations in MATR3; S85C, P154S and T622A. Matrin 3 is an RNA/DNA binding protein as well as part of the nuclear matrix. Matrin 3 interacts with TDP-43, a protein that is both mutated in some forms of ALS, and found in pathological inclusions in most ALS patients. Matrin 3 pathology, including mislocalization and rare cytoplasmic inclusions, was identified in spinal cord tissue from a patient carrying a mutation in Matrin 3, as well as sporadic ALS patients. In an effort to determine the mechanism of Matrin 3 linked ALS, the protein interactome of wild-type and ALS-linked MATR3 mutations was examined. Immunoprecipitation followed by mass spectrometry experiments were performed using NSC-34 cells expressing human wild-type or mutant Matrin 3. Gene ontology analysis identified a novel role for Matrin 3 in mRNA transport centered on proteins in the TRanscription and EXport (TREX) complex, known to function in mRNA biogenesis and nuclear export. ALS-linked mutations in Matrin 3 led to its re-distribution within the nucleus, decreased co-localization with endogenous Matrin 3 and increased co-localization with specific TREX components. Expression of disease-causing Matrin 3 mutations led to nuclear mRNA export defects of both global mRNA and more specifically the mRNA of TDP-43 and FUS. Our findings identify ALS-causing mutations in the gene MATR3, as well as a potential pathogenic mechanism attributable to MATR3 mutations and further link cellular transport defects to ALS. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2018