The function and evolution of introns have been topics of great interest since introns were discovered in the 1970s. Introns that interrupt protein-coding regions have the most obvious potential to affect coding sequences; therefore, their evolution have been studied most intensively. Splicing of introns within untranslated regions does not contribute directly to the diversity of proteins, yet ~35% of human transcripts contain introns within the 5' untranslated region (UTR). The evolution and possible functions of 5'UTR introns (5UIs) remain largely unexplored. Here we undertook a genome-wide functional analysis of 5UIs. Our main results are as follows: First, the distribution of these introns in the human genome is nonrandom. While genes with regulatory roles are enriched in having 5UIs, genes encoding proteins that are targeted to the endoplasmic reticulum and mitochondria are surprisingly depleted of these introns. Second, we offered and supported a model whereby gene encoding secretory and nuclear-encoded mitochondrial proteins share a common regulatory mechanism at the level of mRNA export, which is dependent on the absence of 5'UTR introns. Specifically, the upstream element in a given transcript, be it an intron or RNA elements near the 5' end of coding sequences (CDS), dictates the mRNA export pathway used. Finally, we discovered a strong correlation between existence of 5'UTR introns and sequence features near the 5' end of CDS. We developed an integrated machine-learning framework that can predict absence of 5UIs using solely the sequence near the 5' end of CDS. Our model achieved >80% accuracy when validated against nuclear-encoded mitochondrial transcripts. Specific RNA elements predictive of 5UI absence are found in ~40% of human transcripts spanning a wide spectrum of functions. By analyzing hundreds of large-scale datasets, we functionally characterized the transcripts with these RNA elements; revealing their association with translational regulation. These RNA elements were bound by proteins interacting with the Exon Junction Complex in vivo suggesting a molecular mechanism that links these elements to their downstream effects in mRNA export and translational regulation. While some 5'UTR introns might be evolving neutrally, our results, taken together, suggest that complex evolutionary forces are acting on this distinct class of introns.

<p>The action of Mung Bean Nuclease (MBN) on DNA makes it possible to clone intact gene fragments from genes of the malaria parasite, Plasmodium. This &ldquo / genease&rdquo / activity has provided a foundation for further investigation of the coding elements of the Plasmodium genome. MBN has been reported to cleave genomic DNA of Plasmodium preferentially at positions before and after genes, but not within gene coding regions. This mechanism has overcome the difficulty encountered in obtaining genes with low expression levels because the cleavage mechanism of the enzyme yields sequences of genes from genomic DNA rather than mRNA. However, as potentially useful as MBN may be, evidence to support its genease activity comes from analysis of a limited number of genes. It is not clear whether this mechanism is specific to certain genes or species of Plasmodia or whether it is a general cleavage mechanism for Plasmodium DNA .There have also been some projects (Nomura et al., 2001 / van Lin, Janse, and Waters, 2000) which have identified MBN generated fragments which contain fragments of genes with both introns and exons, rather than the intact genes expected from MBN-digestion of genomic DNA, which raises concerns about the efficiency of the MBN mechanism in generating complete genes.</p> <p><br /> Using a large-scale, whole genome mapping approach, 7242 MBN generated genome survey sequences (GSSs) have been mapped to determine their position relative to coding sequences within the complete genome sequences of the human malaria parasite Plasmodium falciparum and the incomplete genome of a rodent malaria parasite Plasmodium berghei. The location of MBN cleavage sites was determined with respect to coding regions in orthologous genes, non-coding /intergenic regions and exon-intron boundaries in these two species of Plasmodium. The survey illustrates that for P. falciparum 79% of GSSs had at least one terminal mapping within an ortholog coding sequence and 85% of GSSs which overlapped coding sequence boundaries mapped within 50 bp of the start or end of the gene. Similarly, despite the partial nature of P.berghei genome sequence information, 73% of P.berghei GSSs had at least one terminal mapping within an ortholog coding sequence and 37% of these mapped between 0-50 bp of the start or end of the gene. This indicates that a larger percentage of cleavage sites in both P.falciparum and P.berghei were found proximal to coding regions. Furthermore, 86% of P.falciparum GSSs had at least one terminal mapping within a coding exon and 85% of GSSs which overlapped exon-intron boundaries mapped within 50bp of the exon start and end site. The fact that 11% of GSSs mapped completely to intronic regions, suggests that some introns contain specific cleavage sites sensitive to cleavage and this also indicates that MBN cleavage of Plasmodium DNA does not always yield complete exons.</p> <p><br /> Finally, the results presented herein were obtained from analysis of several thousand Plasmodium genes which have different coding sequences, in different locations on individual chromosomes/contigs in two different species of Plasmodium. Therefore it appears that the MBN mechanism is neither species specific nor is it limited to specific genes.</p>

O sistema complemento participa da imunidade natural e da imunidade adquirida. Este sistema pode ser ativado por três diferentes vias: a via clássica, a via alternativa e a via das lectinas, desencadeando uma cascata proteolítica que irá resultar na possível eliminação de microorganismos e, por sua vez, no restabelecimento da homeostasia do indivíduo. A proteína C3 possui um papel central participando das três vias deste sistema. Sua clivagem gera fragmentos que estão associados a várias funções biológicas como opsonização, quimiotaxia, anafilatoxinas, além da formação da C3-convertases, que amplificam a via de ativação efetora contra patógenos. A completa deficiência de C3 é rara, está comumente associada a repetidas infecções, ao desenvolvimento de doenças mediadas por imunocomplexos e glomerulonefrite. Neste trabalho nós investigamos as bases moleculares desta deficiência em uma paciente com histórico de consanguinidade. Nosso estudo identificou uma mutação genética responsável pela completa deleção do éxon 27, implicando na perda de 99 nucleotídeos (região 3450 até 3549 correspondente ao cDNA do C3). O sequenciamento do gene C3 da região 6690313 até 6690961 mostrou uma troca de nucleotídeos T por um C (T &rarr; C) na posição 6690626, causando a exclusão do o éxon 27 e deficiência da proteína C3 do complemento humano nessa paciente. Também confirmamos o padrão de herança autossômica recessiva desta deficiência. Verificamos ainda que os fibloblastos da paciente estimulados com LPS por 24 h foram incapazes de secretar a proteína C3, o que nos leva a suspeitar que esta proteína mutante seja rapidamente degradada ainda dentro da célula, impedindo sua liberação para o meio extracelular. / The complement system participates in the natural immunity and acquired immunity. This system can be activated by three different pathways: the classical pathway, the alternative pathway and the lectin pathway, triggering a proteolytic cascade that will possibly result in the elimination of microorganisms and, in turn, in restoring the homeostasis of the individual. The C3 protein has a central role in the activation of all pathways. Its cleavage produces fragments that are associated with various biological functions such as opsonization, chemotaxis, and production of anaphylatoxins. In addition, the C3 participates in formation of C3 convertase of the alternative pathway which amplifies the activation of effector against pathogens. The complete C3 deficiency is rare and commonly associated with recurrent infections, development of diseases mediated by immune complexes and glomerulonephritis. In this work, we investigated the molecular basis of this deficiency in a patient family with a history of consanguinity. Our study has identified a genetic mutation responsible for the complete deletion of exon 27, resulting in the loss of 99 nucleotides (region 3450 to 3549 of the cDNA corresponding to C3). The sequencing of the C3 region 6690313 to 6690961 showed a T nucleotide exchange to C (T &rarr; C) at position 6690626 causing deletion of exon 27 leading to deficiency of the human complement protein C3 in this patient. We also confirmed the autosomal recessive patters of this patient deficiency in the family. We observed that patient fibroblasts stimulated with LPS for 24 h were unable to secrete C3, which leads us to suspect that this protein is degraded within the cell preventing its release into the extracellular.

Les Epidermolyses Bulleuses Dystrophiques (EBD) sont des génodermatoses rares et sévères transmises sur un mode autosomique récessif (EBDR) ou dominant (EBDD), dues à une perte de l’adhésion dermo-épidermique. Les patients atteints d’EBD souffrent de décollements bulleux cutanéo-muqueux qui menacent le pronostic fonctionnel et vital dans les formes les plus graves. Toutes les formes d’EBD sont dues à des mutations du gène COL7A1 codant pour le collagène VII qui est le constituant des fibres d’ancrage assurant l’adhésion de l’épiderme au derme. Il n’existe actuellement pas de thérapie satisfaisante des EBD. La première partie de ma thèse visait à démontrer la faisabilité d’une approche thérapeutique par saut d’exon des EBDR. Cette stratégie consiste à exciser l’exon porteur de la mutation durant le processus d’épissage, afin de restaurer l’expression d’une protéine fonctionnelle. Les exons 73 et 80 de COL7A1 sont particulièrement intéressants car ils sont le siège de mutations récurrentes et que leur excision préserve le cadre ouvert de lecture. Nous avons dans un premier temps démontré le caractère non indispensable des séquences codées par ces exons in vivo en utilisant un modèle de xénogreffe de peau humaine EBDR reconstruite, génétiquement modifiée à l’aide de vecteurs rétroviraux exprimant l'ADNc de COL7A1 délété des séquences des exons 73 ou 80. Puis, j’ai pu établir que la transfection d’oligoribonucléotides antisens (AONs) dirigés contre certaines séquences régulatrices de l’épissage permettait d’induire le saut en phase de ces exons dans des cellules primaires de patients EBDR, avec une efficacité atteignant 90% de saut d’exon. Les analyses par western blot et immunocytofluorescence après transfection ont permis de mettre en évidence une réexpression significative du collagène VII (jusqu’à 25%) dans les cellules de trois patients EBDR. Enfin, j’ai pu démontrer la réexpression du collagène VII in vivo, après injection de différentes doses d’AONs dans des peaux équivalentes générées avec des cellules de patients et greffées sur des souris immunodéficientes. Dans la seconde partie de ma thèse, j’ai étudié une famille EBD particulière, dont les deux enfants atteints présentaient une EBD beaucoup plus sévère que leur mère et leur grand père maternel, atteints d’une forme modérée d’EBDD. Le séquençage des 118 exons de COL7A1 et des régions d’épissage adjacentes a permis d’identifier une seule mutation dominante c.6698G>A (p.Gly2233Asp) dans l’exon 84, à l’état hétérozygote chez les quatre sujets. A partir de l’étude des transcrits paternels, j’ai pu identifier une nouvelle mutation c.2587+40G>A dans l’intron 19 de COL7A1, qui active un site donneur cryptique dans l’intron 19, entraînant sa rétention partielle et la formation d’un codon stop prématuré. La confirmation de la présence de cette seconde mutation, récessive, dans l’ADN des deux enfants a ainsi permis d’expliquer les différences phénotypiques observées, les deux enfants atteints étant hétérozygotes composites pour une mutation dominante et une mutation récessive de COL7A1. Cette mutation récessive constitue la mutation intronique la plus distante des sites consensus d’épissage de COL7A1 et souligne l’importance de l’étude des ARNm pour la recherche de mutations dans le cadre des EBD. Dans une dernière partie de ma thèse, j’ai débuté la caractérisation d’un modèle murin knock-in d’EBDR développé par notre laboratoire, qui mime certaines des caractéristiques phénotypiques des patients EBDR. Mon travail a permis de démontrer in vivo la faisabilité de l’approche par saut d’exons pour COL7A1. Cette première étape importante conduit à développer des études de preuve de principe et de toxicologie dans des modèles animaux, dans la perspective d’une transition vers la clinique. Il illustre également les variations pathologiques d’épissage pouvant faire l’objet d’approches thérapeutiques similaires. / Dystrophic Epidermolysis Bullosa (DEB) is a group of rare and severe genetic skin disorders, inherited in a dominant (DDEB) or recessive (RDEB) manner, and characterised by loss of adhesion between the epidermis and the underlying dermis. DEB patients suffer from severe blistering of the skin and mucosae after mild traumas, and in the most severe forms, DEB can be life-threatening. DEB is caused by mutations in the COL7A1 gene encoding type VII collagen that assembles into anchoring fibrils forming key dermo-epidermal adhesion structures. To date, there is no specific treatment for DEB. The first part of my thesis was to develop exon skipping as a therapeutic approach for RDEB. In this work, exon skipping strategy consists in modulating the splicing of a premessenger RNA to induce the skipping of a mutated exon and lead to the synthesis of a shorter but functional protein. Exons 73 and 80 of COL7A1 are of particular interest since they carry many recurrent mutations and their excision preserves the open reading frame. In first instance, we have demonstrated the dispensability of these exons for type VII collagen function in an in vivo xenograft model using RDEB cells transduced with retroviral vectors containing COL7A1 cDNAs, deleted of the sequences of exon 73 or 80. I have subsequently transfected primary RDEB keratinocytes and fibroblasts with antisense oligoribonucleotides (AONs) targeting key splicing regulatory elements of these exons, and achieved efficient skipping of these exons (up to 90%). Western blot and immunocytofluorescence experiments demonstrated significant type VII collagen re-expression (up to 25% of the normal amount) in cells from three RDEB patients. Finally, I have generated skin equivalents with cells of these patients, grafted them on immunodeficient mice and injected different doses of AONs in the grafts, and I have demonstrated type VII collagen re-expression in vivo. In the second part of my thesis, I have studied the case of a particular DEB family, in which two affected children presented a DEB much more severe than their mother and maternal grandfather, suffering from a mild form of DDEB. Sequencing of the 118 exons ofCOL7A1 and of their flanking splice sites, lead to the identification of a single dominant mutation c.6698G>A (p.Gly2233Asp) in exon 84, at the heterozygous state in the four individuals. By carrying out analyses on the paternal transcripts, I have identified a novel c.2587+40G>A recessive mutation in intron 19, which activates a cryptic donor splice site in this intron, leading to its partial retention and to the formation of a premature termination codon. I confirmed the presence of this second, recessive, mutation in the DNA of the two affected children, thus providing an explanation for the observed intrafamilial phenotypic variability: the two affected offsprings being compound heterozygotes for a dominant mutation and a recessive mutation in COL7A1. This novel mutation is the deepest intronic mutation found in COL7A1 so far, and emphasizes the importance of studying COL7A1 at the transcripts level to unravel intronic mutations, understand their molecular consequences and their involvement in the development of the disease. In the last part of my thesis, I have started the characterisation of a knock-in murine model of RDEB generated by our laboratory, which mimics some of the phenotypic characteristics of RDEB patients. My thesis work provided the in vivo demonstration of the feasibility of an exon skipping therapeutic approach for COL7A1. This first important step leads to development of proof of concept studies and toxicological studies in different animal models, with the aim of a clinical translation. It also illustrates the pathological splicing alterations that could benefit from similar approaches.

Magister Scientiae - MSc / The action of Mung Bean Nuclease (MBN) on DNA makes it possible to clone intact gene fragments from genes of the malaria parasite, Plasmodium. This &ldquo;genease&rdquo; activity has provided a foundation for further investigation of the coding elements of the Plasmodium genome. MBN has been reported to cleave genomic DNA of Plasmodium preferentially at positions before and after genes, but not within gene coding regions. This mechanism has overcome the difficulty encountered in obtaining genes with low expression levels because the cleavage mechanism of the enzyme yields sequences of genes from genomic DNA rather than mRNA. However, as potentially useful as MBN may be, evidence to support its genease activity comes from analysis of a limited number of genes. It is not clear whether this mechanism is specific to certain genes or species of Plasmodia or whether it is a general cleavage mechanism for Plasmodium DNA .There have also been some projects (Nomura et al., 2001;van Lin, Janse, and Waters, 2000) which have identified MBN generated fragments which contain fragments of genes with both introns and exons, rather than the intact genes expected from MBN-digestion of genomic DNA, which raises concerns about the efficiency of the MBN mechanism in generating complete genes.Using a large-scale, whole genome mapping approach, 7242 MBN generated genome survey sequences (GSSs) have been mapped to determine their position relative to coding sequences within the complete genome sequences of the human malaria parasite Plasmodium falciparum and the incomplete genome of a rodent malaria parasite Plasmodium berghei. The location of MBN cleavage sites was determined with respect to coding regions in orthologous genes, non-coding intergenic regions and exon-intron boundaries in these two species of Plasmodium. The survey illustrates that for P. falciparum 79% of GSSs had at least one terminal mapping within an ortholog coding sequence and 85% of GSSs which overlapped coding sequence boundaries mapped within 50 bp of the start or end of the gene. Similarly, despite the partial nature of P.berghei genome sequence information, 73% of P.berghei GSSs had at least one terminal mapping within an ortholog coding sequence and 37% of these mapped between 0-50 bp of the start or end of the gene. This indicates that a larger percentage of cleavage sites in both P.falciparum and P.berghei were found proximal to coding regions. Furthermore, 86% of P.falciparum GSSs had at least one terminal mapping within a coding exon and 85% of GSSs which overlapped exon-intron boundaries mapped within 50bp of the exon start and end site. The fact that 11% of GSSs mapped completely to intronic regions, suggests that some introns contain specific cleavage sites sensitive to cleavage and this also indicates that MBN cleavage of Plasmodium DNA does not always yield complete exons. Finally, the results presented herein were obtained from analysis of several thousand Plasmodium genes which have different coding sequences, in different locations on individual chromosomes/contigs in two different species of Plasmodium. Therefore it appears that the MBN mechanism is neither species specific nor is it limited to specific genes. / South Africa

Deep sequencing of mRNAs (RNA-Seq) is now the preferred method for transcriptome-wide quantification of gene expression. Yet many mRNA isoforms, such as those eliminated by nonsense-mediated decay (NMD), are inherently unstable. Thus a significant drawback of steady-state RNA-Seq is that it provides marginal information on the flux through alternative splicing pathways. Measurement of such flux necessitates capture of newly made species prior to mRNA decay. One means to capture nascent mRNAs is affinity purifying either the exon junction complex (EJC) or activated spliceosomes. Late-stage spliceosomes deposit the EJC upstream of exon-exon junctions, where it remains associated until the first round of translation. As most mRNA decay pathways are translation-dependent, these EJC- or spliceosome-associated, pre-translational mRNAs should provide an accurate record of the initial population of alternate mRNA isoforms. Previous work has analyzed the protein composition and structure of pre- translational mRNPs in detail. While in the Moore lab, my project has focused on exploring the diversity of mRNA isoforms contained within these complexes. As expected, known NMD isoforms are more highly represented in pre-translational mRNPs than in RNA-Seq libraries. To investigate whether pre-translational mRNPs contain novel mRNA isoforms, we created a bioinformatics pipeline that identified thousands of previously unannotated splicing events. Though many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis.

No description available.

No description available.

No description available.

Limnephilidae is a large family within Trichoptera, consisting of 4 subfamilies (Dicosmoecinae, Ecclisomyiinae, Limnephilinae, and Philocascinae), 98 genera, and 1178 species. It is among the most diverse families within Trichoptera. It is also ecologically diverse, occupying more habitats than any other family in the order. There are currently no published generic phylogenies of Limnephilidae based on molecular data. Here we used anchored hybrid enrichment to capture and sequence 922 loci for 57 species taken from what have been considered the full range of genera in the family. We expanded the taxon sampling by adding supplementary species with DNA barcodes, 28S sequences, or containing both from other sources. We present a favored tree from the collected data. We examine the evolutionary patterns associated with larval habitat transitions and highlight instances where our preferred evolutionary tree is incongruent with current limnephilid classification.