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The overall objective of this study has been to address some of the longstanding questions concerning functional organization of the interphase nucleus. This was achieved by using recently developed high-resolution fluorescence in situ hybridization techniques for a precise localization of specific DNA and RNA sequences in conjunction with immunocytochemistry and biochemical fractionation. This study is based on the philosophy that new insights may be gained by an approach that attempts to interrelate genomic organization, spatial arrangement of RNA metabolism, and nuclear substructure within the mammalian cell nucleus. The nuclear distribution of an exogenous, viral RNA (Epstein-Barr Virus, EBV) within nuclear matrix preparations was studied by developing an approach which couples in situhybridization with biochemical fractionation procedures. EBV RNA molecules accumulate in highly localized foci or elongated tracks within the nucleus of lymphoma cells. These RNA tracks were retained with spatial and quantitative fidelity in nuclear matrix preparations even after biochemical fractionation which removes 95% of cellular protein, DNA, and phospholipid. This provided direct evidence that the primary transcripts are localized via their binding to, or comprising part of, a non-chromatin nuclear substructure. Then the nuclear distribution of RNA from an endogenous gene, fibronectin, was investigated using fluorescence techniques modified for more sensitive detection of endogenous RNAs within nuclear morphology. A series of in situhybridization experiments were performed using different combinations of intron, cDNA, and genomic probes for RNA/RNA or RNA/DNA analysis in intact cells. Fibronectin RNAs were highly localized in the nucleus, forming foci or tracks. Both intron and exon sequences were highly concentrated at the same site within the nucleus, indicating the presence of primary unspliced transcripts. Double-color hybridization using a nontranscribed 5' flanking sequence probe and a genomic DNA probe showed that the gene and RNA track for fibronectin were spatially overlapped, with the gene consistently towards one end of the track. These results provided evidence that the accumulation of RNA molecules occurs directly at or near the site of transcription, and further indicated a structural polarity to the RNA track formation with the gene towards one end. It was further discovered that within a single cell, cDNA probes produced longer tracks than those formed with intron probes, i.e. intron signals were generally confined to a smaller part of the track than the exon signals, indicating that splicing occurs within the RNA track. Additional experiments using poly(A) RNA hybridization or anti-SC-35 antibody staining combined with fibronectin RNA hybridization have shown that the fibronectin tracks were associated with recently discovered transcript domains enriched in poly(A) RNA and splicing factors. To further determine whether other specific genes and RNAs are functionally organized within the nucleus, the nuclear distribution of several active or inactive genes was analyzed in terms of their spatial relationship to transcript domains. The results indicated that in addition to fibronectin, the genes or their primary transcripts from two other active genes, collagen and actin, were also closely associated with the domains. For both of these, over 90% of the gene/RNA sites were either overlapping or directly contacting the domains. In contrast. for two inactive genes, cardiac myosin heavy chain and neurotensin, it was found that both genes were separated from the domains in the majority of nuclei. Histone genes, which have several unique features, showed a relatively complex result with about half of the gene signals extremely close to the domains. Therefore, three actively expressed genes were demonstrated to be tightly associated with the domains and, moreover, their RNAs showed distinct and characteristic spatial relationships with the domains. In contrast, two inactive genes were not associated with the domains. One potential implication of these finding is that active genes may be preferentially localized in and around these transcript domains. The nuclear localization of another RNA, XIST, standing for X-inactivation specific transcript, was studied because of its potentially unique biological role. XIST is the only gene which is known to be expressed from the inactive human X chromosome but not from the active X chromosome, and was believed to be important in X inactivation. Using fluorescence in situhybridization, it was found that XIST RNA was highly localized within the nucleus and always completely overlapped the Barr body which is the condensed, inactive X chromosome. The different fine distribution pattern of XIST RNA within the nucleus as compared to other protein coding RNAs suggested a unique function for this RNA, possibly involving a structural role in inactivating the X chromosome. The final area of my thesis research was to study and acquire expertise in the applications of fluorescence in situ hybridization in gene mapping and cancer genetics. A retinoblastoma (RB)-related putative tumor suppressor gene, p107, was mapped to human chromosome 20 in band q11.2. Localization of p107 to 20q11.2 was of particular interest because of the correlation of breakpoints in this area with specific myeloid disorders such as acute nonlymphocytic leukemia and myelodysplastic syndrome. Other applications of in situ hybridization including the search for unknown genes at a known chromosomal breakpoint, detection of deletions, translocations or other chromosomal rearrangements associated with specific tumors were also explored and reviewed.

Splicing of mRNA precursors (pre-mRNA) comprises a series of ATP-dependent steps, the first of which is the stable binding of U2 snRNP at the pre-mRNA branchpoint. The basis of ATP use in splicing is not well understood. Several yeast splicing factors belong to DEAD/H box family of RNA-dependent ATPase, and are implicated in dynamic RNA structure rearrangement during spliceosome assembly. In mammals, however, such information is conspicuously lacking. In fact, none of the known mammalian splicing factors has characteristics for ATP hydrolysis. In an attempt to identify mammalian splicing factors involved in ATP usage, we have developed a novel approach to identify and purify spliceosomal ATP binding proteins. Six spliceosomal ATP binding proteins were found, one of them, SAFp56, was purified and microsequenced, and found to be a DEAD box protein containing unique DECD motif instead of the canonical DEAD motif. During the course of this work, a new functional region in U2AF65, an essential splicing factor required for U2 snRNP entry into the spliceosome, was defined. This information was used to clone a human U2AF65 associated protein (UAP). UAP and SAFp56 are identical. We refer to this protein as hUAP56 (human 56 kDa U2AF65 associated protein). We present evidence that hUAP56 is an essential splicing factor required for the U2 snRNP binding to pre-mRNA. Interestingly, UAP56 is recruited to pre-mRNA in a polypyrimidine tract bound U2AF65-dependent fashion. This result underscores a new function of U2AF65, and provides the first description of how a specific DEAD box protein is directed to a pre-mRNA splicing signal, and/or, to the proximity of its substrate at a particular stage. Like an authentic DEAD box protein. hUAP56 has ATP binding, RNA-stimulated ATPase, as well as RNA binding activity. A particularly novel result is that the ATPase activity of UAP56 is stimulated by U2AF65. This observation strongly suggests the role of UAP56 in ATP dependent mechanism during U2 snRNP binding to the pre-mRNA branchpoint, and implies that UAP56 may function through a distinct mechanism. We identify yeast UAP (yUAP), a highly conserved S. cerevisiae homologue of hUAP56. yUAP is essential for viability, can be functionally substituted for by hUAP56, and like its human counterpart, is an essential pre-mRNA splicing factor required for spliceosome assembly. Furthermore, we show that yUAP is required for formation of the branchpoint dependent commitment complex, the precursor for U2 snRNP addition. Site-directed mutagenesis revealed that all DEAD box protein consensus motifs are required for yUAP function. Interestingly, a strain harboring a yUAP mutant in which the DECD sequence, characteristic of UAP members, was changed to canonical sequence, is inviable. Our results demonstrate that UAP is structurally and functionally conserved from yeast to man. In conjunction with previous studies, we conclude that at least two DEAD box proteins, Prp5p and yUAP, are required for the U2 snRNP-branchpoint interaction.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20999号 / 医博第4345号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 武田 俊一, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Over 90% of human genes produce precursor mRNA (pre-mRNA) that undergoes splicing, an RNA processing mechanism. Alternative splicing (AS) of pre-mRNA allows a gene to generate multiple coding and non-coding isoforms by removing introns and ligating distinct exonic combinations. It is a mechanism that plays a major role in driving molecular diversity in mammals. This process is tightly regulated to determine the types and levels of protein products expressed in specific cellular contexts. Cis-acting splicing regulatory elements (SREs) found within the pre-mRNA are recognized and bound by RNA-binding proteins that either assist or interfere with the recruitment of the spliceosome. In the field of splicing, a long-standing goal has been to develop a “splicing code”, or a set of rules to understand the splicing patterns of a gene in a predictable manner. It is essential to highlight the significance of sequence context for SREs and the potential impact that distal intronic elements can have on splice site selection to better understand splicing. Given the importance of sequence context and the involvement of distal intronic regions in splicing, future approaches aimed at identifying SREs should consider these factors.This thesis will describe the process of the identification and validation of two novel distal intronic SREs located in critical disease exons. Importantly, these findings were made by combining experimental and computational approaches and through the development of a high-throughput SRE screening methodology. Chapter 1 will provide a general context for splicing, in particular AS, as an important mechanism among a plethora of RNA regulatory functions. The significance of AS regulation will be explored, as it plays a key role in the occurrence of physiological events and incorrect regulation can trigger disease. I will also introduce several methods used to study SREs, with experimental efforts primarily focusing on exonic and proximal intronic sequences. Additionally, as mis-splicing is associated with disease, there is a high interest in modulating splicing with novel therapeutic interventions, the development of which benefits from an increased understanding of SREs. Specifically, I will provide a landscape view of the splicing research field for the genes spinal muscular atrophy 2 (SMN2) and microtubule-binding protein tau (MAPT), given their relevance to our discoveries. As there is currently a paucity of high-throughput methods for studying SREs, especially those that allow for analysis of SREs in a near-native sequence context, I will introduce the CRISPR-Cas system (dCas13d) as a potential splicing modulator. This system will form the foundation for developing a tool to help us understand splicing regulation. Chapter 2 will discuss the discovery of a distal SRE regulating MAPT exon 10 splicing divergence in the primate lineage. Our lineage-specific AS analysis found that MAPT exon 10 shows a two-step evolutionary shift in the Catarrhine and hominoid lineages. The previously identified splicing regulatory elements cannot explain this evolutionary shift. Instead, a key splicing factor, muscleblind-like (MBNL), was found to be a major contributor to the observed splicing pattern divergence. Further mechanistic dissection revealed divergent, distal regulatory sequences in intron 10 that are recognized by MBNL. Based on this finding, we also demonstrated the potential of developing a therapeutically compatible strategy to target the MBNL binding sites by a steric hindrance to modulate exon 10 splicing effectively. Chapter 3 will discuss the development of a method allowing for a more unbiased, high-throughput screening of SREs, including those in the distal intronic regions. This method relies on the nuclease-inactive dRfxCas13d to modulate splicing. We use a dual-color fluorescent splicing reporter to identify the impact of splicing in a high-throughput manner. For our proof of concept, we use SMN2 exon 7 to identify SREs that influence the splicing of this exon and corroborate our findings with known SMN2 SREs. We performed a screen on the SMN2 dual-color splicing reporter using a gRNA library and obtained highly reproducible results. The screen also correctly identified gRNAs targeting known SREs, including the well-studied exonic regions and the downstream ISS-N1 element, the target of the ASO therapeutic known as nusinersen. Importantly, this screen also discovered novel splicing inhibiting gRNAs in a more distal region of the downstream intron, suggesting that a robust splicing enhancer was targeted. Previous studies likely overlooked this region due to its distance from the exon. This novel approach allows for the simultaneous screening of sizeable genetic regions using a large-scale gRNA library.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24522号 / 医博第4964号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 萩原 正敏, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cell type-specific alternative splicing (AS) of pre-mRNA regulated by RNA-binding proteins (RBPs) is widespread, but particularly prominent in the brain, driving gene isoform differences between a diverse range of neuron types. While several AS programs have been shown to be critical to the function of particular neuron types, previous studies have usually been limited to one or a few RBPs and cell types, resulting in a piecemeal understanding of these regulatory patterns. Towards a comprehensive view of the neuron type-specific AS regulatory landscape, we apply current computational and experimental methods to survey neuronal AS, infer its regulation by hundreds of RBPs, and experimentally validate regulatory predictions. In Chapter 1, we examine AS in 133 transcriptomic cell types of mouse cortical neurons defined by single-cell RNA sequencing (scRNA-seq) and define neuron type-specific exons and some of their likely regulators. In Chapter 2, we leverage the rich transcriptomic dynamics of the cortical neuron dataset to systematically infer splicing regulatory network and predict RBP activity on the cell type level. We use the information theory-based method ARACNe to reverse engineer RBP-target regulatory networks and VIPER to infer differential RBP activity across neuron types in a workflow we call Master Regulator analysis of Alternative Splicing (MR-AS). RBP regulons predicted by MR-AS are consistent with high-confidence lists of RBP targets and are supported by motif and CLIP read distribution analyses. Estimation of cell type-specific RBP activity using the predicted regulons shows the expected decreases in RBP KO samples. Chapter 3 focuses on two neuron type-specific AS regulatory programs as case studies, which we validate in vitro using embryonic stem cell (ESC)-derived neuron types. Elavl2 was predicted to drive neurons towards an MGE interneuron-specific AS profile. Elavl2 knockout in ESC-derived MGE interneurons causes modulation of exon inclusion consistent with the predicted regulation of MGE interneuron AS, shifting their splicing profiles towards those of CGE interneurons. We also identified a module of exons that show consistent AS between long- and short-projection neurons across multiple neuronal classes, which are shifted in the expected direction when ESC-derived interneurons are transcriptionally reprogrammed to reflect a long-axon globus pallidus-like neuronal identity. In Chapter 4, we use the RBP regulons to predict RBP activity on a single-cell level and examine its variability, leading us to identify both neuron type-specific AS programs and a neuron type-orthogonal gradient of activity (NTOG). Exons associated with responses to neuronal depolarization and long-term potentiation show a gradient of inclusion across the NTOG, suggesting it may reflect differential activation of activity-dependent AS programs of the assayed neurons. Together, the results described in this thesis demonstrate the validity and broad utility of the inferred AS regulatory networks as a resource for elucidating RBP splicing regulation differences and their functional impact across neuron types.

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No description available.

Angiogenesis is the formation of new blood vessels from existing vasculature. Vascular Endothelial Growth Factor (VEGF), a known angiogenic protein, stimulates endothelial cell proliferation and migration via interactions with its receptors, KDR and Flt-1. A secreted form of Flt-1 (sFlt-1), derived from alternatively-spliced RNA, can inhibit actions of VEGF in vivo. It has been suggested that alterations in sFlt-1 expression could significantly change the angiogenic VEGF activity. This project focuses on characterizing intronic elements that regulate Flt-1 mRNA splicing. A "wild-type" construct (pFIN13), containing the first 13 exons, intron 13 and exons 14-30 of mouse Flt-1, was shown to produce both forms of Flt-1 mRNA after transfection into HEK293 cells. To gauge the strength of the native splicing signals in intron 13 of Flt-1, a series of point mutations were made in the polypyrimidine tract using pFIN13. After transient transfection, the levels of Flt-1 and sFlt-1 protein and mRNA were compared using quantitative PCR, RNA hybridization analysis, and protein immunoblotting. Results from quantitative PCR showed that purine substitutions were associated with 120 to 350 fold decreases in Flt-1 mRNA (normalized against neor), consistent with less efficient splicing. These large decreases in Flt-1 mRNA were accompanied by increases in sFlt-1 mRNA. Modest (20 to 100%) increases in Flt-1 mRNA, reflecting improved splicing, resulted from increasing the uridine complement in the polypyrimidine tract. These results suggest that the wild-type polypyrimidine tract is of intermediate strength and may be a regulatory locus for modulating Flt-1: sFlt-1 ratios. / Master of Science