Im Rahmen dieser Arbeit wurde Organisation, Expression und Funktion des humanen Peroxisomal-Testis-Spezifisch-1(PXT1)-Gens untersucht. Die mRNA des humanen PXT1-Gens enthält nicht wie bisher bekannt zwei Exons, sondern fünf Exons. Die Expression von drei putativen Exons stromaufwärts konnte in dieser Arbeit bestätigt werden. Die Ergebnisse qualitativer und quantitativer Real Time-PCR zeigen, dass sich das Exon 1 aus drei unterschiedlich gespleißten Einheiten (Exons 1a, 1b und 1c) zusammensetzt. Das humane PXT1-Gen unterliegt dem alternativen Spleißen, wovon die Exons 1b, 1c, 2 und 4 betroffen sind, was Sequenzanalysen zeigen. Sechs Transkripte konnten insgesamt identifiziert werden. Die zusätzlichen Exons haben Auswirkungen auf die Proteinstruktur aufgrund der Verlängerung des ORF, kodierend für einst 51 Aminosäuren, auf 134. Im längeren Protein wird die BH3 interacting domain (BID) nachgewiesen, von der eine proapoptotische Funktion bekannt ist. Aufgrund des alternativ gespleißten Exon 4 und der daraus resultierenden Leserasterverschiebung existiert ein verkürztes Protein, in dessen mRNA sich ein vorzeitiges Stopkodon befindet. Die proapoptotische Domäne ist nicht mehr nachweisbar. In silico-Analysen zeigen, dass die Sequenzen der Exons 1a und 1b von PXT1 sich mit dem KCTD20-Gen überlappen, das für einen Kaliumkanal kodiert.  Im Unterschied zum murinen, testisspezifischen Pxt1-Gen, ist das humane Homolog trotz Prädominanz im Testis auch schwächer in anderen Geweben nachweisbar.  Zur weiteren Klärung der proapoptotischen Funktion von Pxt1 in Keimzellen wurde am Mausmodell (Pxt1-Knockout-Maus) die Anzahl an DNA-Strangbrüchen untersucht. Im Vergleich zu den Kontrolltieren (C57BL/6J) zeigt die Pxt1-Knockout-Maus eine signifikant erhöhte Anzahl an Spermien mit DNA-Strangbrüchen. Dieses Ergebnis bestätigt die Annahme, dass das PXT1/Pxt1-Gen eine Art Entsorgungsfunktion für beschädigte Spermien ausübt. Im zeitlichen Verlauf zeigte sich aber, dass die Spermien der Knockout-Tiere nicht sensibler als die Wildtyp-Tiere auf DNaseI reagieren.  Als mögliches Kandidatengen für Mutationsanalysen bei Männern mit Fertilitätsstörungen wurden 55 Patienten mit Fertilitätsstörungen (Azoo- oder Oligozoospermie) auf Punktmutationen im PXT1 untersucht. Eine Mutation konnte nicht identifiziert werden. Des Weiteren wurde die DNA der Patienten auf Copy Number Variations analysiert. Sowohl heterozygote als auch homozygote Duplikationen konnten im Exon1, bestätigt mithilfe der arraybasierten Comparativen Genomischen Hybridisierung (aCGH), vereinzelt auch in Exon2 und Exon3 nachgewiesen werden. Zusätzlich konnte bei einem Patienten eine Deletion nachgewiesen werden. Die bestätigte Duplikation im Exon 1 besitzt aber keinen Krankheitswert, da sie in einem Kontrollkollektiv eine Prävalenz von 41% in heterozygoter und 10% in homozygoter Form besitzt.

RNA Binding Motif (RBM) domain proteins RBM5 and RBM10 have been shown to influence apoptosis, cell cycle arrest and splicing in transformed cells. In this study, RBM5 and RBM10 were examined in non-transformed cells in order to gain a wider range of knowledge regarding their function. Expression of Rbm5 and Rbm10, as well as select splice variants, was examined at the mRNA and protein level throughout H9c2 skeletal and cardiac myoblast differentiation. Results suggest that Rbm5 and Rbm10 may (a) be involved in regulating cell cycle arrest and apoptosis during skeletal myoblast differentiation and (b) undergo post-transcriptional or translational regulation throughout myoblast differentiation. All in all, the expression profiles obtained in the course of this study will help to suggest a role for Rbm5 and Rbm10 in differentiation, as well as possible differentiation-specific target genes with which they may interact.

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.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of proteins intitially characterised in the late 1980’s by their presence in complexes that form on nascent RNA transcripts. This definition was purely operational, and was based on protein isolation techniques available at that time. Since then, the tendency to refer to and view the hnRNPs as a protein family has become increasingly prevalent, although there has been no systematic sequence- or structure-based study of their evolutionary history. While the hnRNPs share some structural characteristics (modular structure, presence of RNA-binding domains) and functional properties (binding to RNA, involvement in multiple steps of RNA processing), these criteria also apply to other types of RNA binding proteins (RBPs), such as the SR and ELAV families of proteins. Thus, we have adopted a more methodical and rigorous approach to the classification of hnRNPs and other RBPs, through the phylogenetic analysis of their sequences and domains. Besides establishing phylogenetic relationships and simplifying nomenclature, studying the evolutionary divergence of the hnRNPs is important for understanding their functional features. The hnRNP A/B subfamily is comprised of paralogues A1, A2/B1, A3 and A0, which exhibit a high level of similarity at both the sequence and structural level. While they are often treated as functional homologues, they are not functionally identical. Hence, we undertook a detailed comparison of their sequences, and found that the introduction of novel splicing signals or mutation of existing sequence elements has led to changes in alternative splicing patterns between the paralogues, which may affect the regulation of their expression and their RNA binding properties. In addition, we also investigated species-specific alternative splicing of the hnRNPs A/B, which has significant implications for the interpretation of current research, since different research groups tend to use different model organisms in their experiments. Hence, exploration of the sequence divergence of the hnRNPs A/B has provided some clues as to how their functional differences arose, and also highlighted the need to take species-specific splicing into consideration. Alternative splicing can create functional variation not only between paralogues, but also between splice variants. hnRNP A2/B1, which has a well-established role in mRNA trafficking in neuronal cells, has four spliceoforms. In order to study the contribution of each isoform to this process, we investigated isoform-specific variations in intracellular localisation, and expression in different developmental stages and species. We found that in rat, minor isoform A2b was the predominant isoform in the cytoplasm, and may be the key player in mRNA trafficking. These findings demonstrate the importance of considering individual isoforms (including those expressed in low abundance) when studying the function of alternatively spliced proteins, especially when the function is restricted to a particular subcellular compartment. In addition to its cytoplasmic role in mRNA trafficking, hnRNP A2/B1, and the other hnRNPs A/B, have multiple nuclear functions, including packaging of nascent transcripts, nuclear export of mRNA, regulation of alternative splicing and telomere maintenance. These processes take place in discrete regions within the nucleus, and thus we examined the subnuclear distribution of the hnRNPs A/B. We found that hnRNP A1 had a localisation pattern distinct from that of A2/B1 and A3, and that these patterns were spatially and temporally regulated. Hence, the evolutionary divergence of the hnRNPs A/B has affected the localisation, expression and splicing patterns of these proteins, which we have examined at multiple levels, including comparisons across all hnRNPs, within the hnRNP A/B paralogues, and between the hnRNP A2/B1 splice variants. As the hnRNPs A/B are involved in almost every step in RNA processing, this functional diversity has significant implications for transcriptomic complexity. Furthermore, our findings highlight the importance of taking species- and isoform-specific differences into account when investigating protein function. In conclusion, this study of the hnRNPs A/B provides a conceptual framework for exploring the relationships between sequence, structural and functional divergence, which may be applicable to protein families in general.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of proteins intitially characterised in the late 1980’s by their presence in complexes that form on nascent RNA transcripts. This definition was purely operational, and was based on protein isolation techniques available at that time. Since then, the tendency to refer to and view the hnRNPs as a protein family has become increasingly prevalent, although there has been no systematic sequence- or structure-based study of their evolutionary history. While the hnRNPs share some structural characteristics (modular structure, presence of RNA-binding domains) and functional properties (binding to RNA, involvement in multiple steps of RNA processing), these criteria also apply to other types of RNA binding proteins (RBPs), such as the SR and ELAV families of proteins. Thus, we have adopted a more methodical and rigorous approach to the classification of hnRNPs and other RBPs, through the phylogenetic analysis of their sequences and domains. Besides establishing phylogenetic relationships and simplifying nomenclature, studying the evolutionary divergence of the hnRNPs is important for understanding their functional features. The hnRNP A/B subfamily is comprised of paralogues A1, A2/B1, A3 and A0, which exhibit a high level of similarity at both the sequence and structural level. While they are often treated as functional homologues, they are not functionally identical. Hence, we undertook a detailed comparison of their sequences, and found that the introduction of novel splicing signals or mutation of existing sequence elements has led to changes in alternative splicing patterns between the paralogues, which may affect the regulation of their expression and their RNA binding properties. In addition, we also investigated species-specific alternative splicing of the hnRNPs A/B, which has significant implications for the interpretation of current research, since different research groups tend to use different model organisms in their experiments. Hence, exploration of the sequence divergence of the hnRNPs A/B has provided some clues as to how their functional differences arose, and also highlighted the need to take species-specific splicing into consideration. Alternative splicing can create functional variation not only between paralogues, but also between splice variants. hnRNP A2/B1, which has a well-established role in mRNA trafficking in neuronal cells, has four spliceoforms. In order to study the contribution of each isoform to this process, we investigated isoform-specific variations in intracellular localisation, and expression in different developmental stages and species. We found that in rat, minor isoform A2b was the predominant isoform in the cytoplasm, and may be the key player in mRNA trafficking. These findings demonstrate the importance of considering individual isoforms (including those expressed in low abundance) when studying the function of alternatively spliced proteins, especially when the function is restricted to a particular subcellular compartment. In addition to its cytoplasmic role in mRNA trafficking, hnRNP A2/B1, and the other hnRNPs A/B, have multiple nuclear functions, including packaging of nascent transcripts, nuclear export of mRNA, regulation of alternative splicing and telomere maintenance. These processes take place in discrete regions within the nucleus, and thus we examined the subnuclear distribution of the hnRNPs A/B. We found that hnRNP A1 had a localisation pattern distinct from that of A2/B1 and A3, and that these patterns were spatially and temporally regulated. Hence, the evolutionary divergence of the hnRNPs A/B has affected the localisation, expression and splicing patterns of these proteins, which we have examined at multiple levels, including comparisons across all hnRNPs, within the hnRNP A/B paralogues, and between the hnRNP A2/B1 splice variants. As the hnRNPs A/B are involved in almost every step in RNA processing, this functional diversity has significant implications for transcriptomic complexity. Furthermore, our findings highlight the importance of taking species- and isoform-specific differences into account when investigating protein function. In conclusion, this study of the hnRNPs A/B provides a conceptual framework for exploring the relationships between sequence, structural and functional divergence, which may be applicable to protein families in general.

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.

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.