Magnocellular neurosecretory cells (MNCs) are neuroendocrine cells with somata located in the hypothalamus and nerve terminals in the posterior pituitary. They receive neuronal inputs from the brain and release vasopressin and oxytocin into the blood to regulate many important functions such as water balance, lactation, and parturition. The process of hormone release depends on Ca2+ influx mediated by voltage-gated Ca2+ channels (VGCCs) on the plasma membranes of neuroendocrine cells. To better understand the cellular and molecular components that are involved in regulating secretory vesicle exocytosis, this thesis work was conducted to investigate the expression and function of different subtypes of VGCCs in MNCs and pituicytes (the glial cells surrounding MNC nerve terminals).<p> Molecular biology, immunohistochemistry and cellular biology were used to detect expression and alternative splicing of different VGCC subtypes in MNCs, neurons, and pituicytes. First, the presence of CaV2.2 and CaV2.3 channels were detected on the pituicytes in situ. When the pituicytes were isolated and cultured for 14 days, more VGCC subtypes were expressed including CaV1.2 channels. Regulation of VGCC expression was measured in normal and dehydrated rats, and CaV1.2 channels were found to be selectively up-regulated in pituicytes after 24 hours of dehydration.<p> Second, two splice variants of CaV2.1 channels (CaV2.1Ä1 and Ä2) that lack a large portion of the synprint (synaptic protein interaction) site were detected in the rat brain. To determine whether the splice variants were expressed in MNCs, we did immunocytochemistry using two antibodies (the selective and the inclusive antibody) that recognized the carboxyl-terminus of channels and the synprint site, respectively, in different cell types. We found that vasopressin MNCs, but not the oxytocin MNCs, and one type of endocrine cell (the melanotropes of the pituitary gland) expressed the synprint site deleted variants, whereas the hippocampal neurons mainly expressed the full-length isoform. The splice variants were properly distributed on the plasma membrane of the somata and nerve terminals of the MNCs, suggesting the synprint site is not essential for CaV2.1 channel targeting into the nerve terminals of neuroendocrine cells.<p> Third, expression and distribution of CaV2.2 channels were studied in the MNCs. All CaV2.2 isoforms we detected contained the full-length synprint site. To test the importance of the CASK/Mint1 binding site for CaV2.2 channel targeting, we over-expressed a peptide that inhibits the interaction between CaV2.2 channels and CASK/Mint1 in differentiated PC12 cells (a neuroendocrine cell line). We found that the distribution of CaV2.2 channels in the growth cones of PC12 cells were significantly decreased, suggesting that the CASK/Mint1 interaction is important for CaV2.2 channel targeting into the neuroendocrine terminals.<p> In conclusion, these results provide insights of VGCC expression in neuroendocrine cells, and also give rise to a better understanding of the molecular components that are involved in forming the exocytotic machinery in these cells.

Alternative splicing functions to generate proteomic diversity and to regulate gene expression in higher eukaryotes. Genome-wide analyses suggest that alternative splicing and transcription typically regulate different gene sets to achieve cell- and tissue-type specificity. However, within individual cell-types, most alternative splicing events occur co-transcriptionally and are impacted by the transcriptional machinery. Despite many focused studies on co-transcriptional regulation of alternative splicing, its mechanisms and functions in regulation of gene expression are still poorly understood. To investigate relationships between transcription and alternative splicing, I performed microarray profiling of alternative splicing and transcript levels during activation of a T cell line. This experiment revealed that different sets of genes and associated functional categories are regulated by alternative splicing and transcription during T cell activation. I next employed inhibitors of RNA polymerase II (Pol II) elongation and microarray profiling to identify genes with coupled changes in splicing and transcript levels when transcription is impeded in activated T cell. Genes that were affected at both levels were significantly enriched in RNA binding and processing functions, and generally displayed increased alternative exon inclusion and decreased transcript levels when transcription elongation was disrupted. Similar effects were observed when transcription was driven by mutant polymerases with reduced elongation activity, and when cells were subjected to stress treatments. Many of the elongation inhibition-sensitive exons from the affected genes introduce premature termination codons into the mRNA, resulting in spliced mRNAs that are substrates of the nonsense-mediated decay pathway and further reduction in mRNA levels. ChIP-Seq experiment demonstrated that Pol II occupancy specifically increased in introns flanking the affected exons. These results provide evidence that a physiological function of transcription elongation-coupled alternative splicing regulation is to regulate the levels of RNA processing factors under conditions that reduce elongation activity, including cell stress. In summary, my thesis research has provided new insights into the integration of transcription and splicing control. While these two regulatory levels can control different gene sets during the activation of T cells, within a given cell type, they are closely coupled to control specific alternative splicing events that appear to coordinate mRNA and RNA processing factors levels.

Alternative splicing functions to generate proteomic diversity and to regulate gene expression in higher eukaryotes. Genome-wide analyses suggest that alternative splicing and transcription typically regulate different gene sets to achieve cell- and tissue-type specificity. However, within individual cell-types, most alternative splicing events occur co-transcriptionally and are impacted by the transcriptional machinery. Despite many focused studies on co-transcriptional regulation of alternative splicing, its mechanisms and functions in regulation of gene expression are still poorly understood. To investigate relationships between transcription and alternative splicing, I performed microarray profiling of alternative splicing and transcript levels during activation of a T cell line. This experiment revealed that different sets of genes and associated functional categories are regulated by alternative splicing and transcription during T cell activation. I next employed inhibitors of RNA polymerase II (Pol II) elongation and microarray profiling to identify genes with coupled changes in splicing and transcript levels when transcription is impeded in activated T cell. Genes that were affected at both levels were significantly enriched in RNA binding and processing functions, and generally displayed increased alternative exon inclusion and decreased transcript levels when transcription elongation was disrupted. Similar effects were observed when transcription was driven by mutant polymerases with reduced elongation activity, and when cells were subjected to stress treatments. Many of the elongation inhibition-sensitive exons from the affected genes introduce premature termination codons into the mRNA, resulting in spliced mRNAs that are substrates of the nonsense-mediated decay pathway and further reduction in mRNA levels. ChIP-Seq experiment demonstrated that Pol II occupancy specifically increased in introns flanking the affected exons. These results provide evidence that a physiological function of transcription elongation-coupled alternative splicing regulation is to regulate the levels of RNA processing factors under conditions that reduce elongation activity, including cell stress. In summary, my thesis research has provided new insights into the integration of transcription and splicing control. While these two regulatory levels can control different gene sets during the activation of T cells, within a given cell type, they are closely coupled to control specific alternative splicing events that appear to coordinate mRNA and RNA processing factors levels.

Poly(A) polymerase (PAP) is the enzyme catalyzing the synthesis of the adenine tail to the 3’-end of mRNA. This A-tail is present on the majority of the primary RNA transcripts of protein-coding genes, and is important for mRNA stability, export to the cytoplasm and translation. Therefore, PAP is a key regulator of eukaryotic gene expression. This thesis describes the heterogeneity of PAP and the functional significance of multiple isoforms of PAP. PAP exists in many different isoforms generated by three different mechanisms, gene duplication, alternative mRNA processing and post-translational modification. In HeLa cell extracts three different forms of PAP being 90, 100 and 106 kDa in size have been detected, where the 106 kDa isoform is a phosphorylated version of the 100 kDa species. It is shown that the N-terminal region of PAP contains a region required for catalysis, while the C-terminal end is important for the interaction with the cleavage and polyadenylation specificity factor (CPSF). Interestingly, it was found that also the extreme N-terminal end is important for the interaction with CPSF. This region is post-translationally modified by phosphorylation. Five alternatively spliced forms of PAP mRNAs are encoded by the PAPOLA gene while one unique species is encoded by the PAPOLG gene. The analysis showed that the exact structure of the alternatively spliced C-terminal end of PAP played an important role for catalytic efficiency. Thus, the C-terminal end contains a region important for modulating the catalytic efficiency of PAP. Aminoglycoside antibiotics inhibit PAP activity, most likely by displacement of catalytically important divalent metal ions. Data shows that different aminoglycosides inhibit PAP activity by different mechanisms suggesting that the binding sites for the different aminoglycosides do not completely overlap. It is concluded that aminoglycosides interfere with enzymes important for housekeeping functions in mammalian cell, which may explain some of the toxic side effects caused by aminoglycoside antibiotics in clinical practice.

Human papillomavirus type 16 (HPV-16) is the major causative agent of cervical cancer. The life cycle of this oncogenic DNA tumour virus is strictly associated with the differentiation program of the infected epithelial cells. Expression of the viral capsid genes L1 and L2 can only be detected in the terminally differentiated epithelial cells. The studies here focus on the regulation of HPV-16 late gene expression, which is under tight regulation. Our experimental system consisted of almost the full length HPV-16 genome driven by a strong CMV promoter. This plasmid and mutants thereof could be transfected into HeLa cells and RNA levels monitored. Using this system, we identified an hnRNP A1-dependent splicing silencer between positions 178 and 226 of the L1 gene. This silencer inhibited the use of the 3' splice site, located immediately upstream of the L1 AUG. We speculate that this splicing silencer plays an essential role in preventing late gene expression at an early stage of the viral life cycle. We subsequently identified a splicing enhancer located in the first 17 nucleotides of L1 that may be needed to counteract the multiple hnRNP A1 dependent splicing silencers in the L1 coding region. A 55kDa protein specifically bound to this splicing enhancer. We also demonstrated that binding of the cellular factors to the splicing silencer in the L1 coding region had an inhibitory effect on expression from L1 cDNA expression plasmids. The HPV-16 genome is divided into the early region and the late region, separated by the early poly(A) signal (pAE). pAE is used preferentially early in infection, thereby efficiently blocking late gene expression. We demonstrated that a 57 nucleotide U-rich region of the early 3’untranslated region (3’eUTR) acted as an enhancing upstream element on the usage of pAE. We demonstrated that this U-rich region specifically interacts with hFip1, CstF-64, hnRNP C1/C2 and PTB, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 pAE. In conclusion, two regulatory RNA elements that both act to prevent late gene expression at an early stage in the viral life cycle and in proliferating cells were identified: a splicing silencer in the late region and an upstream u-rich element at the pAE.

Plants are sessile organisms and thus more exposed to stressful environments. By changing the expression of stress related genes, plants are able to cope with stress. Alternative splicing (AS) of pre-mRNA is a major contributor to proteome diversity in eukaryotes. It has been shown that different abiotic stresses affect AS patterns, suggesting a functional role of AS in stress tolerance. The Serine/Arginine-rich proteins (SR proteins) are a conserved family of splicing regulators in eukaryotes. SR proteins are essential for AS and studies have shown that they are themselves subjects to AS after stress exposure which means that they can control their own splicing. In this study, the aim was to characterize the different SR-proteins in the SR subfamily in P. patens, analyze their phylogeny and measure the change in expression of the genes after exposure to five types of stress; osmotic, salinity, dehydration, cold and hormonal. The result showed both individual and overlapping changes in their expression profiles of the three genes. Furthermore, there was an alteration in the alternative splicing pattern for two genes during three of the stresses which resulted in intron retention and possibly a premature termination codon and subseqent non-sense mediated decay.

Transcription elongation regulator 1 (TCERG1) is a nuclear protein involved in transcriptional elongation and splicing events, suggesting these two activities may be connected. Moreover, TCERG1 was recently identified as a novel interactor and co-repressor of CCAAT/Enhancer Binding Protein &alpha; (C/EBP&alpha;) transcriptional activity, suggesting TCERG1 has additional biological roles. Interestingly, TCERG1 also inhibits the growth arrest activity of C/EBP&alpha;. Additionally, the original clone found to interact with C/EBP&alpha; consisted of only the amino-terminal domain of TCERG1 and functional analysis of this clone indicated that it retained the ability to repress both C/EBP&alpha; mediated growth arrest and transcriptional activity. Furthermore, a TCERG1 mutant whose amino-terminal region was deleted was unable to interact with or repress the transcriptional and growth arrest activities of C/EBP&alpha;, suggesting the functional domain(s) lie elsewhere. In this study, domains of TCERG1 were examined for the ability to inhibit C/EBP&alpha;-mediated growth arrest and the mechanism whereby this effect occurs. By exploiting fluorescent properties of expressed proteins fused with green fluorescent protein, the extent to which each TCERG1 mutant was able to reverse C/EBP&alpha;-mediated growth arrest of cultured cells was assessed. Our analyses suggest that the inhibitory activity of TCERG1 lies within the amino-terminal region and may involve WWI and WWII domains within this region. Additionally, laser scanning confocal microscopy (LCSM) was used to visualize the subnuclear localization of fluorescent proteins fused to TCERG1 and C/EBP&alpha;. When expressed alone, TCERG1 localized to splicing factor-rich nuclear speckles while C/EBP&alpha; was found to reside in discrete punctate foci, both localization patterns being distinct and different from each other. Results from co-localization studies after co-expressing both proteins indicate an alteration in the subnuclear distribution of TCERG1. Furthermore, TCERG1 co-localizes with C/EBP&alpha;, suggesting a possible mechanism whereby TCERG1 inhibits the growth arrest and transcriptional activities mediated by C/EBP&alpha;.

Magnocellular neurosecretory cells (MNCs) are neuroendocrine cells with somata located in the hypothalamus and nerve terminals in the posterior pituitary. They receive neuronal inputs from the brain and release vasopressin and oxytocin into the blood to regulate many important functions such as water balance, lactation, and parturition. The process of hormone release depends on Ca2+ influx mediated by voltage-gated Ca2+ channels (VGCCs) on the plasma membranes of neuroendocrine cells. To better understand the cellular and molecular components that are involved in regulating secretory vesicle exocytosis, this thesis work was conducted to investigate the expression and function of different subtypes of VGCCs in MNCs and pituicytes (the glial cells surrounding MNC nerve terminals).<p> Molecular biology, immunohistochemistry and cellular biology were used to detect expression and alternative splicing of different VGCC subtypes in MNCs, neurons, and pituicytes. First, the presence of CaV2.2 and CaV2.3 channels were detected on the pituicytes in situ. When the pituicytes were isolated and cultured for 14 days, more VGCC subtypes were expressed including CaV1.2 channels. Regulation of VGCC expression was measured in normal and dehydrated rats, and CaV1.2 channels were found to be selectively up-regulated in pituicytes after 24 hours of dehydration.<p> Second, two splice variants of CaV2.1 channels (CaV2.1Ä1 and Ä2) that lack a large portion of the synprint (synaptic protein interaction) site were detected in the rat brain. To determine whether the splice variants were expressed in MNCs, we did immunocytochemistry using two antibodies (the selective and the inclusive antibody) that recognized the carboxyl-terminus of channels and the synprint site, respectively, in different cell types. We found that vasopressin MNCs, but not the oxytocin MNCs, and one type of endocrine cell (the melanotropes of the pituitary gland) expressed the synprint site deleted variants, whereas the hippocampal neurons mainly expressed the full-length isoform. The splice variants were properly distributed on the plasma membrane of the somata and nerve terminals of the MNCs, suggesting the synprint site is not essential for CaV2.1 channel targeting into the nerve terminals of neuroendocrine cells.<p> Third, expression and distribution of CaV2.2 channels were studied in the MNCs. All CaV2.2 isoforms we detected contained the full-length synprint site. To test the importance of the CASK/Mint1 binding site for CaV2.2 channel targeting, we over-expressed a peptide that inhibits the interaction between CaV2.2 channels and CASK/Mint1 in differentiated PC12 cells (a neuroendocrine cell line). We found that the distribution of CaV2.2 channels in the growth cones of PC12 cells were significantly decreased, suggesting that the CASK/Mint1 interaction is important for CaV2.2 channel targeting into the neuroendocrine terminals.<p> In conclusion, these results provide insights of VGCC expression in neuroendocrine cells, and also give rise to a better understanding of the molecular components that are involved in forming the exocytotic machinery in these cells.

During the maturation of pre-mRNA introns are removed and exons are spliced together, to form a primary transcript, a reaction that is catalyzed by the spliceosome. Alternative splicing is a complex reaction that mainly utilizes one of four mechanisms; exon skipping, 5’ splice site choice, 3’ splice site choice and intron retention. To achieve accurate splicing four sequence elements are essential, two of which are located in the splice sites themselves; 5’ splice sites and 3’ splice sites, but also the polypyrimidine tract and the branch point sequence. Alternative splicing can be regulated by histone or chromatin modulations, siRNA, transcription efficiency and various proteins, many of which belong to either the SR protein family or the hnRNP family of proteins. SR proteins usually promote exon inclusion, while hnRNP proteins usually promote exon skipping. There are also regulatory elements that are called exonic splicing enhancers or silencers depending on if they promote or inhibit the inclusion of the exon they reside in. These elements also exist in introns and are then called intronic splicing enhancers or silencers. The enhancer elements are most commonly targeted by SR proteins and the silencer elements are usually targeted by hnRNP proteins. This paper will mainly focus on the regulation of alternative splicing and the role of alternative splicing under abnormal conditions, such as when mutations cause disease.

Alternativ splicing är en process som ger upphov till att olika mRNA-sekvenser bildas från en enda gen, vilket bidrar till en ökad proteindiversitet hos organismen. Olika mRNA-sekvenser kan uppstå eftersom att det förekommer olika varianter av alternativ splicing som även kan kombineras på flera olika sätt: cassette exon (inkludering/exkludering av exon), intron retention (intronet behålls), alternative 5´splice-site choice (olika 5´ splice sites kan väljas) och slutligen alternative 3´ splice-site choice (andra 3´ splice sites kan väljas). För att alternativ splicing ska äga rum i olika pre-mRNA måste den regleras av cis-reglerande element. De cis-reglerande elementen utgörs av fyra grupper: exonic splicing enhancers (ESE), exonic splicing silencers (ESS), intronic splicing enhancers (ISE) samt intronic splicing silencers (ISS). Som namnen förtäljer finns de antingen i exoner eller introner, där de interagerar med transagerande faktorer, SR-proteiner (aktiverare) eller hnRNPs (hämmare). Alternativ splicing förekommer både i djur och i växter. Hos Homo sapiens genomgår över 74 % av de 25,000 gener som finns hos organismen, alternativ splicing. Däremot i växten Arabidopsis thaliana, genomgår endast 22 %, av den totala mängden på cirka 26,000 gener, alternativ splicing. Eftersom att processen bidrar till en ökad proteindiversitet, kommer det medföra att olika processer i organismerna påverkas, exempelvis celltillväxt, celldöd samt utvecklingen av olika sjukdomar, såsom Parkinson och cystisk fibros. Många studier har gjorts som bekräftar dess betydelse för organismerna men på grund av processens komplexitet är det fortfarande ett ämne som ständigt måste utforskas.