The contribution of exon-skipping events on chromosome 22 to protein coding diversity

Completion of the human genome sequence provides evidence for a gene count with lower bound 30,000–40,000. Significant protein complexity may derive in part from multiple transcript isoforms. Recent EST based studies have revealed that alternate transcription, including alternative splicing, polyadenylation and transcription start sites, occurs within at least 30–40% of human genes. Transcript form surveys have yet to integrate the genomic context, expression, frequency, and contribution to protein diversity of isoform variation. We determine here the degree to which protein coding diversity may be influenced by alternate expression of transcripts by exhaustive manual confirmation of genome sequence annotation, and comparison to available transcript data to accurately associate skipped exon isoforms with genomic sequence. Relative expression levels of transcripts are estimated from EST database representation. The rigorous in silico method accurately identifies exon skipping using verified genome sequence. 545 genes have been studied in this first hand-curated assessment of exon skipping on chromosome 22

Violating the splicing rules: TG dinucleotides function as alternative 3' splice sites in U2-dependent introns

TG dinucleotides functioning as alternative 3' splice sites were identified and experimentally verified in 36 human genes

Developmental gene expression profile of Vmo1 in the mouse auditory system

Hearing loss (HL) is a sensory disorder that affects an estimated 250 million people worldwide and can greatly affect quality of life. In New Zealand, more than 10% of the population is affected by HL with the Māori population being overrepresented among all age groups. Therefore, understanding the mechanism of HL is extremely important for the development of new pharmaceuticals for the prevention or treatment of HL disorders. The main aim of the research undertaken in this thesis was to characterise the function of the Mus musculus (mouse) vitelline membrane outer layer one (Vmo1) gene. This gene is considered an excellent candidate for being involved in human HL and/or balance disorders. Our hypothesis is based on its restricted gene localisation within the mouse inner ear and the postulated function of Reissner’s membrane. Two methods were used to address this aim. Firstly, comparative genomics was used to determine the level of nucleotide and amino acid conservation of VMO1 across mammalian species, and to search for DNA motifs that may imply a biological function. Secondly, molecular biology and histochemical techniques were used to DNA sequence the Vmo1 gene, detect the expression of 22 kDa VMO1 protein within mouse tissues, and to localise the expression of VMO1 protein within the mouse inner ear. Comparative genomics results showed VMO1 to be highly conserved across 36 species. An in-depth analysis of the differences and similiarites between the mouse, human and chicken indicated a high level of gene conservation with an even greater degree of identity and similarity seen at a proteomic level. In addition, a high level of conservation across amino acids involved in the formation and stabilisation of the three dimensional structure. Thus, results suggest an important function for the VMO1 protein. Two commerical VMO1 antibodies were purchased to determine the localisation of the mouse VMO1 protein. They were validated for specificity using western blot analysis of protein lysates dissected from postnatal day 28 mice (P28). VMO1 was identified within the inner ear protein lysate and tear gland protein lysate of an expected molecular weight size of 20-37kDa with additional binding observed in the ear sample at 250kDa. Immunohistochemistry detected high concentrations of VMO1 protein within the tectorial membrane (TM) and inner pillar cells (IPC) in inner ear sections from the mouse at P5. In agreement with the comparative genomics analysis, VMO1 is a secreted protein. The movement of the hair cells (HC) relative to the TM is is essential for the transduction of sound into electrical signals. The IPC act as supporting cells for the hair cells, and help to couple movement of the basilar membrane to the HC. In conclusion, the importance of the TM and IPC in hearing function, and the localisation of the VMO1 protein within these structures implies an important role for VMO1 in hearing function. We recommend further studies to examine the specificity of the VMO1 antibody, and the development of a Vmo1 knockout mouse to support the functional analysis of Vmo1 in the auditory system

Identification of a novel human E-Cadherin splice variant andassessment of its effects upon EMT-related events

Epithelial Cadherin (E-cadherin) is involved in calcium-dependent cell-cell adhesion and signal transduction. The E-cadherin decrease/loss is a hallmark of Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. The underlying molecular mechanisms that trigger E-cadherin loss and consequent EMT have not been completely elucidated. This study reports the identification of a novel human E-cadherin variant mRNA produced by alternative splicing. A bioinformatics evaluation of the novel mRNA sequence and biochemical verifications suggest its regulation by Nonsense-Mediated mRNA Decay (NMD). The novel E-cadherin variant was detected in 29/42 (69%) human tumor cell lines, expressed at variable levels (E-cadherin variant expression relative to the wild type mRNA = 0.05-11.6%). Stable transfection of the novel E-cadherin variant in MCF-7 cells (MCF7Ecadvar) resulted in downregulation of wild type E-cadherin expression (transcript/protein) and EMT-related changes, among them acquisition of a fibroblastic-like cell phenotype, increased expression of Twist, Snail, Zeb1, and Slug transcriptional repressors and decreased expression of ESRP1 and ESRP2 RNA binding proteins. Moreover, loss of cytokeratins and gain of vimentin, N-cadherin and Dysadherin/FXYD5 proteins was observed. Dramatic changes in cell behavior were found in MCF7Ecadvar, as judged by the decreased cell-cell adhesion (Hanging-drop assay), increased cell motility (Wound Healing) and increased cell migration (Transwell) and invasion (Transwell w/Matrigel). Some changes were found in MCF-7 cells incubated with culture medium supplemented with conditioned medium from HEK-293 cells transfected with the E-cadherin variant mRNA. Further characterization of the novel E-cadherin variant will help understanding the molecular basis of tumor progression and improve cancer diagnosis.Fil: Matos, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Lapyckyj, Lara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Rosso, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Besso, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Mencucci, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Marin Briggiler, Clara Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Giustina, Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Furlong, Laura Ines. Universitat Pompeu Fabra; EspañaFil: Vazquez, Monica Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Beiträge zu Verbreitung und Analytik des subtilen alternativen Spleißens

Das alternative Spleißen (AS) ist ein Hauptakteur der Diversifizierung von Transkriptom und Proteom eines eukaryotischen Organismus. Die Studien dieser Dissertationsschrift thematisieren das erst kürzlich entdeckte subtile AS, das die Einführung kleiner Variationen im Transkript und in vielen Fällen auch im kodierten Protein bewirkt. Im Arabidopsis thaliana-Genom konnte eine häufige Präsenz von NAGNAG Tandem-Motiven nachgewiesen werden, die in den Spleißfaktor-kodierenden SR-Protein-Genen überrepräsentiert sind. Ausgewählte experimentell analysierte Fälle zeigten ähnliche organ- und bedingungsspezifische Änderungen der Splei߬varianten-Verhältnisse. Im Mensch wurde ein völlig neuer, seltener Typus des subtilen AS entdeckt. Eine Population von 36 Introns verwendet TG Dinukleotide als alternative 3’ Splei߬stellen und widerspricht damit den etablierten Spleißregeln. TG-3‘-Splei߬stellen wurden ausschließlich im Kontext einer AG Splei߬stelle mit einer maximalen Distanz von 28 nt gefunden. In deren orthologen 3’ Splei߬stellen sind TG-Dinukleotid und flankierende Intronsequenz zwischen Säuge¬tieren auffällig stark konserviert. Deren Verwendungshäufigkeit steigt mit der Konservierung von Splei߬stelle und flankierender Intronsequenz und wird höchstwahrscheinlich durch cis- und/oder trans-Elemente vermittelt. Zur quantitativen Ermittlung der Splei߬variantenverhältnisse wurden die Pyrosequenzierung und die Fluoreszenz-basierte Kapillarelektro¬phorese verwendet. Beide Methoden wurden hinsichtlich Reproduzierbarkeit und Genauigkeit, Experimentaufbau und Datenanalyse im Vergleich zur häufig verwendeten Polyacrylamid-Gelelektrophorese mit Ethidiumbromid-vermittelter Densitometrie analysiert. CE-LIF erzielte dabei die höchste Genauigkeit und Reproduzierbarkeit und stellte gleichzeitig die arbeits- und zeiteffizienteste Methode dar

The development and application of informatics-based systems for the analysis of the human transcriptome

Philosophiae Doctor - PhDDespite the fact that the sequence of the human genome is now complete it has become clear that the elucidation of the transcriptome is more complicated than previously expected. There is mounting evidence for unexpected and previously underestimated phenomena such as alternative splicing in the transcriptome. As a result, the identification of novel transcripts arising from the genome continues. Furthermore, as the volume of transcript data grows it is becoming increasingly difficult to integrate expression information which is from different sources, is stored in disparate locations, and is described using differing terminologies. Determining the function of translated transcripts also remains a complex task. Information about the expression profile – the location and timing of transcript expression – provides evidence that can be used in understanding the role of the expressed transcript in the organ or tissue under study, or in developmental pathways or disease phenotype observed. In this dissertation I present novel computational approaches with direct biological applications to two distinct but increasingly important areas of research in gene expression research. The first addresses detection and characterisation of alternatively spliced transcripts. The second is the construction of an hierarchical controlled vocabulary for gene expression data and the annotation of expression libraries with controlled terms from the hierarchies. In the final chapter the biological questions that can be approached, and the discoveries that can be made using these systems are illustrated with a view to demonstrating how the application of informatics can both enable and accelerate biological insight into the human transcriptome.South Afric

Complex genetic approaches to neurodegenerative diseases.

Neurodegenerative diseases are fatal disorders in which disease pathogenesis results in the progressive degeneration of the central and/or the peripheral nervous systems. These diseases currently affect -2% of the population but are expected to increase in prevalence as average life expectancy increases. The majority of these diseases have a complex genetic basis. The work presented in this thesis aimed to investigate the genetic basis of two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and the human prion diseases kuru and sporadic Creutzfeldt-Jakob disease (sCJD), using novel complex genetic approaches. ALS is a fatal neurodegenerative disease in which motor neurons are seen to degenerate. It is a complex disease with 10% of individuals having a family history and the remaining 90% of non-familial cases having some genetic component. The gene DYNC1H1 is involved in retrograde axonal transport and is a good candidate for ALS. In this thesis the genetic architecture of DYNC1H1 was elucidated and a mutation screen of exons 8, 13 and 14 was undertaken in familial forms of ALS and other motor neuron diseases. No mutations were found. A linkage disequilibrium (LD) based association study was conducted using two tagging single nucleotide polymorphisms (tSNPs) which were identified as sufficient to represent genetic variation across DYNC1HI. These tSNPs were tested for an association with sporadic ALS (SALS) in 261 cases and 225 matched controls but no association was identified. Kuru is a devastating epidemic prion disease which affected a highly geographically restricted area of the Papua New Guinea highlands, predominantly affected adult women and children. Its incidence has steadily declined since the cessation of its route of transmission, endocannibalism, in the late 1950's. Kuru imposed strong balancing selection on codon 129 of the prion gene (PRNP). Analysis of kuru-exposed and unexposed populations showed significant deviations from Hardy-Weinberg equilibrium (HWE) consistent with the known protective effect of codon 129 heterozygosity. Signatures of selection were investigated in the surviving populations, such as deviations from HWE and an increasing cline in codon 129 valine allele frequency, which covaried with disease exposure. A novel PRNP G127V polymorphism was detected which, while common in the area of highest kuru incidence, was absent from kuru patients and unexposed population groups. Genealogical analysis revealed that the heterozygous PRNP G127V genotype confers strong prion disease resistance, which has been selected by the kuru epidemic. Finally, PRNP copy number was investigated as a possible genetic mechanism for susceptibility to kuru and sCJD. No conclusive copy number changes were identified

Identification of Interacting Protein Partners of TOPORS in the Retina

Retinitis pigmentosa (RP, MIM#268000) is a heterogeneous disease characterised by loss of rod photoreceptors and pigment deposits in the retina. Historically, genes linked to RP were associated with rod-specific functions. Recently, a novel class of ubiquitously expressed causative genes has emerged including splicing factor genes and TOPORS (NM_005802). To date, studies show TOPORS is expressed in all tested human tissues, including the retina. However, mutations in this ubiquitously expressed gene only cause RP without any systemic symptoms. The purpose of this work was to understand why mutations in TOPORS, which encodes a multifunctional protein, cause a retina-only disease by identifying protein interacting partner(s) of TOPORS, using a yeast-two hybrid (Y2H) screen. In case the interacting partner(s) turn out to be retina specific, it may explain the retina-only phenotype. Human retinal cDNA library was constructed from total retinal cDNA directly in the Y187 Saccharomyces cerevisiae yeast strain. Retina-specificity of the cDNA library was validated by sequencing, leading to identification of several retina-specific genes, including rhodopsin (RHO; NM_000539). The library was screened for protein interacting partners of TOPORS, using MatchmakerTM Gold Yeast Two-Hybrid System (Clontech, CA, USA). Over 10^7 cDNA clones were screened, leading to isolation of 53 potential interactions. The identified interacting partners were prioritised for further evaluation, based on literature and database searches, and re-tested in yeast leading to identification of three candidates for further functional studies: a soluble fragment of integral membrane protein 2B (ITM2B; NM_0219999), previously linked to neurodegenerative disorders, and more recently associated with an inherited retinal dystrophy; a brain prostaglandin D2 synthase (PTGDS; NM_000954), highly expressed in the retina, previously suggested to play a role in retinal homeostasis; a regulatory subunit 4 of the 26 S protease (PSMC1; NM_002802), conferring substrate specificity to the proteasome complex during degradation of ubiquitinated proteins. The outcomes suggest several scenarios for why mutations in TOPORS result only in RP; however, further studies are essential to elucidate the role of TOPORS and its interacting partners in the aetiology of this debilitating disease