Group I introns belong to a class of large RNAs that catalyze their own excision from precursor RNA through a two-step process called self-splicing reaction. These self-splicing introns have often been converted into ribozymes with the ability site specifically cleave RNA molecules. One such ribozyme, derived from a self-splicing Pneumocystis carinii group I intron, has subsequently been shown to sequence specifically excise a segment from an exogenous RNA transcript through trans excision-splicing reaction.The trans excision-splicing reaction requires that the substrate be cleaved at two positions called the 5' and 3' splice sites. The sequence requirements at these splice sites were studied. All sixteen possible base pair combinations at the 5' splice site and the four possible nucleotides at the 3' splice site were tested for reactivity. It was found that all base pair combinations at the 5' splice site allow the first reaction step and seven out of sixteen combinations allow the second step to occur. Moreover, it was also found that non-Watson-Crick base pairs are important for 5' splice site recognition and suppress cryptic splicing. In contrast to the 5' splice site, 3' splice site absolutely requires a guanosine.The pathway of the trans excision-splicing reaction is poorly understood. Therefore, as an initial approach, a kinetic framework for the first step (5' cleavage) was established. The framework revealed that substrate binds at a rate expected for RNA-RNA helix formation. The substrate dissociates with a rate constant (0.9 min-1), similar to that for substrate cleavage (3.9 min-1). Following cleavage, the product dissociation is slower than the cleavage, making this step rate limiting for multiple-turnover reactions. Furthermore, evidence suggests that P10 helix forms after the 5' cleavage step and a conformational change exists between the two reaction steps of trans excision-splicing reaction. Combining the data presented herein and the prior knowledge of RNA catalysis, provide a much more detailed view of the second step of the trans excision-splicing reaction.These studies further characterize trans excision-splicing reaction in vitro and provide an insight into its reaction pathway. In addition, the results describe the limits ofthe trans excision-splicing reaction and suggest how key steps can be targeted for improvement using rational ribozyme design approach.

Group I introns are catalytic RNAs capable of self-splicing out of RNA transcripts. Ribozymes derived from these group I introns are used to explore the molecular recognition properties involved in intron catalysis. New ribozyme reactions are designed based on the inherent ability of these ribozymes to perform site-specific nucleophilic attacks. This study explores the molecular recognition properties of group I intron-derived ribozyme reactions and describe a new ribozyme reaction involving molecular recognition properties previously not seen.We report the development, analysis, and use of a new combinatorial approach to analyze the substrate sequence dependence of suicide inhibition, cyclization, and reverse cyclization reactions catalyzed by a group I intron from the opportunistic pathogen Pneumocystis carinii. We demonstrate that the sequence specificity of these Internal Guide Sequence (IGS) mediated reactions is not high, suggesting that RNA targeting strategies which exploit tertiary interactions could have low specificity due to the tolerance of mismatched base pairs.A group I intron-derived ribozyme from P. carinii has been previously shown to bind an exogenous RNA substrate, splice-out an internal segment, and then ligate the two ends back together (the trans excision-splicing reaction). We now report that a group I intron derived ribozyme from the ciliate Tetrahymena thermophila can also perform the trans excision-splicing reaction, although not nearly as well as the P. carinii ribozyme.In addition, we discovered a new ribozyme reaction called trans insertion-splicing where the P. carinii ribozyme binds two exogenous RNA substrates and inserts one directly into the other. Although this reaction gives the reverse products of the trans excision-splicing reaction, the trans insertion-splicing reaction is not simply the reverse reaction. The ribozyme recognizes two exogenous substrates through more complex molecular recognition interactions than what has been previously seen in group I intron-derived ribozyme reactions. We give evidence for this new reaction mechanism composed of three steps, with intermediates attached to the ribozyme.

OATP1B3 belongs to the OATP (organic anion transporting polypeptides) superfamily, responsible for mediating the transport of various endogenous and xenobiotic substrates. OATP1B3 was initially reported to be expressed exclusively in the hepatocytes where it mediates the uptake of numerous endogenous substrates (e.g. bile acids, steroid hormone conjugates) and several clinically relevant drugs including anticancer drugs. Later, a number of studies reported that OATP1B3 is also frequently expressed in multiple types of cancers and may be associated with differing clinical outcomes. However, a detailed investigation on the expression, localization and functions of OATP1B3 expressed in cancer has been lacking. In this thesis work, we confirmed that colon and pancreatic cancer cells express a cancer-specific OATP1B3 variant (csOATP1B3), different from OATP1B3 wild-type (WT) expressed in the normal liver. The csOATP1B3 utilizes an alternative transcription initiation site and the translated product of csOATP1B3 lacks the first 28 amino acids at the N-terminus of OATP1B3 WT. Our results show that csOATP1B3 has modest uptake transporter functions and reduced plasma membrane localization compared to OATP1B3 WT. In our efforts to investigate the regulatory mechanism underlying the expression of csOATP1B3, we found that hypoxia inducible factor-1α (HIF-1α) may play a key role in the regulation of csOATP1B3 in colon and pancreatic cancer cells. In a separate study, we tested whether the N-terminal sequence of OATP1B3 WT plays an important role in the membrane trafficking. This is based on the observation that csOATP1B3 lacking the first 28 amino acids at N-terminus of OATP1B3 WT displays a predominantly cytoplasmic localization pattern. Using the constructs with N-terminal truncations and point mutations, we verified that the N-terminus of OATP1B3 WT contains important motifs in its membrane trafficking. In particular, the amino acids within a putative β-turn-forming tetrapeptide appear to be important in regulating the membrane trafficking of OATP1B3 WT. The findings from this thesis work provide important insights into the functional and clinical significance of OATP1B3 in cancer and normal liver.

Transcription elongation by RNA polymerase II is regulated by an array of protein complexes. Among various elongation factors, Spt5 is conserved in the three kingdoms of life. I investigated functional interactions of its C-terminal repeats (CTR) domain with several elongation protein complexes in Saccharomyces cerevisiae. By using genetics and molecular biology methods, I established two major pathways in this thesis. The first describes how BUR kinase-mediated phosphorylation of CTR domain leads to co-transcriptional recruitment of the PAF complex to regulate histone modifications on active genes. The second describes how CTR phosphorylation facilitates recruitment of capping enzymes to enhance gene splicing. Finally, several Spt5-associated protein complexes were studied, and potential molecular mechanisms underlying these observations are proposed and discussed.

Histone deacetylases (HDAC) 1 and 2 play crucial role in chromatin remodeling and gene expression regimes, as part of multiprotein corepressor complexes. Protein kinase CK2-driven phosphorylation of HDAC1 and 2 regulates their catalytic activities and is required to form the corepressor complexes. Phosphorylation-mediated differential distributions of HDAC1 and 2 complexes in regulatory and coding regions of transcribed genes catalyze the dynamic protein acetylation of histones and other proteins, thereby influence gene expression. During mitosis, highly phosphorylated HDAC1 and 2 heterodimers dissociate and displace from mitotic chromosomes. Our goal was to identify the kinase involved in mitotic phosphorylation of HDAC1 and 2. We postulated that CK2-mediated increased phosphorylation of HDAC1 and 2 leads to dissociation of the heterodimers, and, the mitotic chromosomal exclusions of HDAC1 and 2 are largely due to the displacement of HDAC-associated proteins and transcription factors, which recruit HDACs, from chromosomes during mitosis. We further explored the role of un- or monomodified HDAC1 and 2 complexes in immediate-early genes (IEGs), FOSL1 (FOS-like antigen-1) and MCL1 (Myeloid cell leukemia-1), regulation. Dynamic histone acetylation is an important regulator of these genes that are overexpressed in a number of diseases and cancers. We hypothesized that transcription dependent recruitment of HDAC1 and 2 complexes over the gene body regions plays a regulatory role in transcription and splicing regulation of these genes. We present evidence that CK2-catalyzed increased phosphorylation of HDAC1 and 2 regulates the formation of distinct corepressor complexes containing either HDAC1 or HDAC2 homodimers during mitosis, which might target cellular factors. Furthermore, the exclusion of HDAC-recruiting proteins is the major factor for their displacement from mitotic chromosomes. We further demonstrated that un- or monophosphorylated HDAC1 and 2 are associated with gene body of FOSL1 in a transcription dependent manner. However, HDAC inhibitors prevented FOSL1 activation independently of the nucleosome response pathway, which is required for IEG induction. Interestingly, our mass spectrometry results revealed that HDAC1 and 2 interact with a number of splicing proteins, in particular, with serine/arginine-rich splicing factor 1 (SRSF1). HDAC1 and 2 are co-occupied with SRSF1 over gene body regions of FOSL1 and MCL1, regardless of underlying splicing mechanisms. Using siRNA-mediated knockdown approaches and HDAC inhibitors, we demonstrated that alternative splicing of MCL1 is regulated by RNA-directed localized changes in the histone acetylation levels at the alternative exon. The change in histone acetylation levels correlates with the increased transcription elongation and results in change in MCL1 splicing by exon skipping mechanism. Taken together, our results contribute to further understanding of how the multi-faceted HDAC1 and 2 complexes can be regulated and function in various processes, including, but not limited to, transcription regulation and alternative splicing. This can be an exciting area of future research for therapeutic interventions.

Transcription elongation by RNA polymerase II is regulated by an array of protein complexes. Among various elongation factors, Spt5 is conserved in the three kingdoms of life. I investigated functional interactions of its C-terminal repeats (CTR) domain with several elongation protein complexes in Saccharomyces cerevisiae. By using genetics and molecular biology methods, I established two major pathways in this thesis. The first describes how BUR kinase-mediated phosphorylation of CTR domain leads to co-transcriptional recruitment of the PAF complex to regulate histone modifications on active genes. The second describes how CTR phosphorylation facilitates recruitment of capping enzymes to enhance gene splicing. Finally, several Spt5-associated protein complexes were studied, and potential molecular mechanisms underlying these observations are proposed and discussed.

Conditional protein splicing (CPS) is an intein-mediated post-translational modification. Inteins are intervening protein elements that autocatalytically excise themselves from precursor proteins to ligate flanking protein sequences, called exteins, with a native peptide bond. Artificially split inteins can mediate the same process by splicing proteins in trans, when intermolecular reconstitution of split intein fragments occurs. An established CPS model utilizes an artificially split Saccharomyces cerevisiae intein, called VMA. In this model, VMA intein fragments are fused to the heterodimerization domains, FKBP and FRB, which selectively form a complex with the immunosuppressive drug, rapamycin. Treatment with rapamycin, therefore, heterodimerizes FKBP and FRB, and triggers trans-splicing activity by proximity association of intein fragments. Here, we engineered a CPS model to assemble inert fragments of the potent fungal ribotoxin, alpha (α)-sarcin, in vivo. Using this model, we demonstrate rapamycin-dependent protein splicing of α-sarcin fragments and a corresponding induction of cytotoxicity in HeLa cells. We further show that permissive extein context and incubation temperature are critical factors regulating the splicing of active target proteins. Ultimately, this technology could have potential applications in the fields of developmental biology and anti-tumour therapy.

Hereditary myopathy with lactic acidosis (HML, OMIM#255125) is an autosomal recessive disorder which originates from Västerbotten and Ångermanland in the Northern part of Sweden. HML is characterized by severe exercise intolerance which manifests with tachycardia, dyspnea, muscle pain, cramps, elevated lactate and pyruvate levels, weakness and myoglobinuria. The symptoms arise from malfunction of the energy metabolism in skeletal muscles with defects in several important enzymes involved in the TCA cycle and the electron transport chain. All affected proteins contain iron-sulfur (Fe-S) clusters, which led to the suggestion that the disease was caused by malfunctions in either the transportation, assembly or processing of Fe-S clusters. The aim of my thesis was to identify the disease causing gene of HML and to investigate the underlying disease-mechanisms. In paper I we identified a disease-critical region on chromosome 12; a region containing 16 genes. One of the genes coded for the Fe-S cluster assembly protein ISCU and an intronic base pair substitution (g.7044G>C) was identified in the last intron of this gene. The mutation gave rise to the insertion of intron sequence into the mRNA, leading to a protein containing 15 abberant amino acids and a premature stop. In paper II we investigated why a mutation in an evolutionary well conserved protein with a very important cellular role, which in addition is expressed in almost all tissues, gives rise to a muscle-restricted phenotype. Semi-quantitative RT-PCR analysis showed that the mutant transcript constituted almost 80% of total ISCU mRNA in muscle, while in both heart and liver the normal splice form was dominant. We could also show that, in mice, complete absence of Iscu protein was coupled with early embryonic death, further emphasizing the importance of the protein in all tissues. These data strongly suggested that tissue-specific splicing was the main mechanism responsible for the muscle-specific phenotype of HML. In paper III the splicing mechanisms that give rise to the mutant ISCU transcript was further investigated. We identified three proteins; PTBP1, IGF2BP1 and RBM39, that could bind to the region containing the mutation and could affect the splicing pattern of ISCU in an in vitro system. PTBP1 repressed the inclusion of the intronic sequence, while IGF2BP1 and RBM39 repressed the total ISCU mRNA level though the effect was more pronounced for the normal transcript. Moreover, IGF2BP1 and RBM39 were also able to reverse the effect of PTBP1. IGF2BP1, though not a splicing factor, had higher affinity for the mutant sequence. This suggested that the mutation enables IGF2BP1 binding, thereby preventing the PTBP1 induced repression seen in the normal case. In conclusion, we have determined the genetic cause of HML, identifying a base pair substitution in the last intron of the ISCU gene that gives rise to abnormally spliced transcript. The muscle-specific phenotype was also analyzed and tissue-specific splicing was identified as the main disease-mechanism. Furthermore, nuclear factors with ability to affect the splicing pattern of the mutant ISCU gene were identified. This work has thoroughly investigated the fundamental disease mechanisms, thus providing deeper understanding for this hereditary myopathy.

An investigation of the expression profile of mRNA encoding Calpain 3, the causative agent in the inherited human muscular disease Limb Girdle Muscular Dystrophy Type 2A, was conducted in two representative mammalian species, human and mouse. Transcripts encoding Calpain 3 were identified from mammalian tissues other than skeletal muscle. In human Peripheral Blood Mononuclear Cells (PBMCs) these transcripts were identified in both the T-cell and B-cell compartments and in a number of human blood cell lines representing different haematopoietic lineages. Calpain 3 transcripts encoding the murine homologue were also described from mouse PBMCs and from murine tissues involved in haematopoiesis. In addition to the confirmation of Calpain 3 expression in non-skeletal muscle tissues in both these species, transcripts were identified with precise and defined deletions, which mapped to known exon-exon boundaries in the Calpain 3 gene from both species. These deletions constituted the removal by alternative splicing of skeletal muscle-specific components of the Calpain 3 protein known to regulate its function in this tissue. Monoclonal antibodies to the Calpain 3 protein were used to confirm the presence of Calpain 3 protein in non-skeletal muscle tissues of both human and mouse. In humans the expression of Calpain 3 protein was confirmed in PBMCs and in the mouse, Calpain 3 expression was confirmed in tissues of the haematopoietic compartment. In both species the Calpain 3 protein expressed correlated with translation from a transcript lacking the skeletal muscle-specific components generated by alternative splicing. An attempt was made using a Yeast Two Hybrid assay to identify potential regulatory molecules of Calpain 3 in human PBMCs, but without a definitive candidate molecule being found. A developmental model of muscle differentiation (murine C2C12 myoblast cells) was used to ascertain the expression profile of Calpain 3 in the early stages of myofibrillogenesis. Using Quantitative Real Time PCR the expression profile of Calpain 3 was assessed in differentiating C2C12 cells. These results showed that the absolute levels of Calpain 3 transcription were elevated during differentiation and that a temporal Calpain 3 isoform shift occurred during this process. This temporal shift in expression was from transcripts having identical deletions to those seen in the haematopoietic tissues, to full length transcripts representative of skeletal muscle-specific Calpain 3. The identification of Calpain 3 expression outside skeletal muscle tissue is novel and the isoforms expressed in these tissues are structurally more analogous to the ubiquitously expressed calpains. This has implications for LGMD2A where a loss of function of Calpain 3 in non-skeletal muscle tissue could be compensated for by the ubiquitous calpains, thus explaining the lack of any non-muscle tissue pathology in LGMD2A patients.

An investigation of the expression profile of mRNA encoding Calpain 3, the causative agent in the inherited human muscular disease Limb Girdle Muscular Dystrophy Type 2A, was conducted in two representative mammalian species, human and mouse. Transcripts encoding Calpain 3 were identified from mammalian tissues other than skeletal muscle. In human Peripheral Blood Mononuclear Cells (PBMCs) these transcripts were identified in both the T-cell and B-cell compartments and in a number of human blood cell lines representing different haematopoietic lineages. Calpain 3 transcripts encoding the murine homologue were also described from mouse PBMCs and from murine tissues involved in haematopoiesis. In addition to the confirmation of Calpain 3 expression in non-skeletal muscle tissues in both these species, transcripts were identified with precise and defined deletions, which mapped to known exon-exon boundaries in the Calpain 3 gene from both species. These deletions constituted the removal by alternative splicing of skeletal muscle-specific components of the Calpain 3 protein known to regulate its function in this tissue. Monoclonal antibodies to the Calpain 3 protein were used to confirm the presence of Calpain 3 protein in non-skeletal muscle tissues of both human and mouse. In humans the expression of Calpain 3 protein was confirmed in PBMCs and in the mouse, Calpain 3 expression was confirmed in tissues of the haematopoietic compartment. In both species the Calpain 3 protein expressed correlated with translation from a transcript lacking the skeletal muscle-specific components generated by alternative splicing. An attempt was made using a Yeast Two Hybrid assay to identify potential regulatory molecules of Calpain 3 in human PBMCs, but without a definitive candidate molecule being found. A developmental model of muscle differentiation (murine C2C12 myoblast cells) was used to ascertain the expression profile of Calpain 3 in the early stages of myofibrillogenesis. Using Quantitative Real Time PCR the expression profile of Calpain 3 was assessed in differentiating C2C12 cells. These results showed that the absolute levels of Calpain 3 transcription were elevated during differentiation and that a temporal Calpain 3 isoform shift occurred during this process. This temporal shift in expression was from transcripts having identical deletions to those seen in the haematopoietic tissues, to full length transcripts representative of skeletal muscle-specific Calpain 3. The identification of Calpain 3 expression outside skeletal muscle tissue is novel and the isoforms expressed in these tissues are structurally more analogous to the ubiquitously expressed calpains. This has implications for LGMD2A where a loss of function of Calpain 3 in non-skeletal muscle tissue could be compensated for by the ubiquitous calpains, thus explaining the lack of any non-muscle tissue pathology in LGMD2A patients.