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No description available.

No description available.

Cystic fibrosis is an autosomal recessive disease caused by mutations in the CFTR gene. A significant number of mutations (~13%) alter the correct splicing of the CFTR gene, causing the transcription of aberrant transcripts resulting in the production of a non-functional CFTR channel. We focus our research on two intronic CF causing mutations, 3272-26A>G and 3849+10kbC>T that create a new acceptor and donor splice site, respectively, generating in the inclusion of intronic portions into the mRNA. We developed a new genome editing approach to permanently correct the abovementioned mutations by means of CRISPR nucleases. We exploited the use of either Streptococcus pyogenes Cas9, SpCas9, or Acidaminococcus sp. BV3L6, AsCas12a, to edit the aberrant splicing sites and restore the production of the correct transcript, avoiding modifications of the CFTR coding sequence. A comparative analysis between SpCas9 and AsCas12a revealed that the use of AsCas12a with a single crRNA efficiently edits the target loci, producing correctly spliced mRNAs in both 3272-26A>G and 3849+10kbC>T mutations. Furthermore, this genetic repair strategy proved to be highly specific, exhibiting a strong discrimination between the mutated and the wild-type allele and no detectable off-target activity with genome-wide analysis. The selected crRNAs were tested in patients derived primary airway cells and intestinal organoids compound heterozygous for the 3272-26A>G or 3849+10kbC>T mutations, that are considered relevant CF models for translational research. The efficient splicing repair and the complete recovery of CFTR channel activity observed confirmed the goodness of the proposed gene editing strategy. These results demonstrated that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.

Group I intron-derived ribozymes are catalytic RNAs that have been engineered to catalyze a variety of different reactions, in addition to the native self-splicing reaction. One such ribozyme, derived from a group I intron of Pneumocystis carinii, can modify RNA transcripts through either the excision or insertion of RNA sequences. These reactions are mediated through the trans excision-splicing (TES) or trans insertionsplicing (TIS) reaction pathways. To increase our current understanding of these reactions, as well as their general applicability, a mechanistic and kinetic framework for the TES reaction was established. Furthermore, additional ribozymes were investigated for their ability to catalyze the TES reaction. Lastly, the development of the TIS reaction into a viable strategy for the manipulation of RNA transcripts was investigated. The TES reaction proceeds through two reaction steps: substrate cleavage followed by exon ligation. Mechanistic studies revealed that substrate cleavage is catalyzed by the 3’ terminal guanosine of the Pneumocystis ribozyme. Moreover, kinetic studies suggest that a conformational change exists between the individual reaction steps. Intron-derived ribozymes from Tetrahymena thermophila and Candida albicans were also investigated for their propensity to catalyze the TES reaction. The results showed that each ribozyme could catalyze the TES reaction; however, Pneumocystis carinii is the most effective using the model constructs. Investigations of the TIS reaction focused on developing a new strategy for the insertion of modified oligonucleotides into an RNA substrate. These studies used oligonucleotides with modifications to the sugar, base, and backbone positions. Each of the modified oligonucleotides was shown to be an effective TIS substrate. These results demonstrate that TIS is a viable strategy for the incorporation of modified oligonucleotides, of varying composition, into an intended RNA target. The results from these studies show that group I introns are highly adaptable for catalyzing non-native reactions, including the TES and TIS reactions. Furthermore, group I introns are capable of catalyzing these unique reactions through distinct reaction pathways. Overall, these results demonstrate that group I introns are multi-faceted catalysts.

Découvert en 1977, l'épissage est une étape de maturation post-transcriptionnelle consistant à rabouter les exons et éliminer les introns d'un ARN pré-messager. Pour que l'épissage soit correctement pris en charge par l'épisome et ses protéines auxiliaires, différents signaux sont présents le long de la séquence de l'ARN pré-messager. Il est maintenant reconnu que près de la moitié des mutations pathogènes chez l'homme impactent l'épissage, aboutissant à un dysfonctionnement du gène. Il est ainsi indispensable pour les biologistes d'être capables de détecter ces signaux sur une séquence génomique.Cette thèse a donc pour but de concevoir de nouveaux algorithmes permettant d'apporter la puissance de calcul des ordinateurs au service de la biologie de l'épissage. La solution proposée, Human Splicing Finder (HSF), est capable de prédire les trois types de signaux d'épissage à partir d'une séquence quelconque extraite du génome humain. Nous avons évalué l'efficacité de prédiction d'HSF dans l'ensemble des situations associées à des mutations pathogènes pour lesquelles il a été démontré expérimentalement leur impact sur l'épissage et par rapport aux autres algorithmes de prédiction. Parallèlement à ces apports directs tant pour la connaissance des processus biologiques de l'épissage que pour le diagnostic, les nouvelles approches thérapeutiques génotype-spécifiques peuvent également bénéficier de ces nouveaux algorithmes. Ainsi HSF permet de mieux cibler les oligonucléotides anti-sens utilisés pour induire le saut d'exon dans la myopathie de Duchenne et les dysferlinopathies.La reconnaissance récente de l'intérêt majeur de l'épissage dans des domaines aussi variés que la recherche fondamentale, la thérapeutique et le diagnostic nécessitaient un point central d'accès aux signaux d'épissage. HSF a pour objet de remplir ce rôle, en étant régulièrement mis à jour pour intégrer de nouvelles connaissances, et est d'ores et déjà reconnu comme un outil de référence. / Discovered in 1977, splicing is a post-transcriptional maturation process that consists in link-ing exons together and removing introns from a pre-messanger RNA. For splicing to be cor-rectly undertaken by the spliceosome and its auxiliary proteins, several signals are located along the pre-messanger RNA sequence. Nearly half of pathogenous mutations in humans are now recognized to impact splicing and leading to a gene dysfunction. Therefore it is es-sential for biologists to detect those signals in any genomic sequence.Thus, the goals of this thesis were to conceive new algorithms: i) to identify splicing signals; ii) to predict the impact of mutations on these signals and iii) to give access to this information to researchers thanks to the power of bioinformatics. The proposed solution, Human Splicing Finder (HSF), is a web application able to predict all types of splicing signals hidden in any sequence extracted from the human genome. We demonstrated the prediction's efficiency of HSF for all situations associated with pathogenous mutations for which an impact on splicing has been experimentally demonstrated. Along with these direct benefits for the knowledge of biological processes for splicing and diagnosis, new genotype-specific therapeutic approaches can also benefit from these new algorithms. Thus, HSF allows to better target antisense olignucleotides used to induce exon skipping in Duchenne myopathy and dysferlinopathies.The recent recognition of the major interest of splicing in various domains such as fundamen-tal research, therapeutics and diagnosis needed a one stop shop for splicing signals. HSF has for object to fulfill this need, being regularly updated to integrate new knowledge and is already recognized as an international reference tool.

It was shown that 95 % of human multi-exon genes are alternatively spliced and the regulation of alternative splicing is extremely complex. Most pre-mRNA splicing events occur co- transcriptionally and there is increasing body of evidence, that chromatin modifications play an important role in the regulation of alternative splicing. Here we showed that inhibition of histone deacetylases (HDACs) modulates alternative splicing of ~700 genes via induction of histone H4 acetylation and increase of Pol II elongation rate along alternative region. We identified HDAC1 the catalytic activity of which is responsible for changes in alternative splicing. Then, we analyzed whether acetylhistone binding protein Brd2 regulates alternative splicing and showed that Brd2 occupies promoter regions of targeted genes and controls alternative splicing of ~300 genes. Later we showed that knockdown of histone acetyltransferase p300 promotes inclusion of the alternative fibronectin (FN1) EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as the p300 knockdown. Next we showed that p300 controls histone...

Alternative splicing (AS) is involved in expansion of transcriptome and proteome during cell growth, cell death, pluripotency, cell differentiation and development. There is increasing evidence to suggest that splicing decisions are made when the nascent RNA is still associated with chromatin. Here, I studied regulation of AS via chromatin modification with main focus on histone acetylation. First, we demonstrate that activity of histone deacetylases (HDACs) influences splice site selection in 700 genes. We provided evidence that HDAC inhibition induces histone H4 acetylation and increases RNA Polymerase II (RNA Pol II) processivity along an alternatively spliced element. In addition, HDAC inhibition reduces co-transcriptional association of the splicing regulator SRp40 with the target fibronectin exon. Further we showed that histone acetylation reader, Brd2 protein, affect transcription of 1450 genes. Besides, almost 290 genes change their AS pattern upon Brd2 depletion. We study distribution of Brd2 along the target and control genes and find that Brd2 is specifically localized at promoters of target genes only. Surprisingly, Brd2 interaction with chromatin cannot be explained solely by histone acetylation, which suggests that other protein-domains (in addition to bromodomains) are important for...

A proteína do retardo mental do X frágil (FMRP), codificada pelo gene do Retardo Mental do X Frágil (do inglês, Fragile Mental Retardation 1, FMR1) associa-se a RNA e transita entre o núcleo celular, grânulos citoplasmáticos e polirribossomos. No SNC, tem um importante papel como reguladora traducional atuando na maturação e eliminação sináptica. A exclusão do éxon 14 de transcritos do FMR1, associada ao uso do terceiro sítio de splicing do éxon 15 do FMR1, acarreta mudança da fase de leitura dos códons subsequentes, produzindo potencialmente isoformas da FMRP com nova região C-terminal, sem o sinal de localização nuclear e o domínio RGG, principal efetor de sua associação a RNAs. Sua importância funcional ainda não foi estudada. Neste trabalho, avaliamos por RT-qPCR a expressão dos transcritos que incluem e excluem o éxon 14 do Fmr1 no SNC de ratos e identificamos o córtex cerebral no décimo nono dia embrionário (E19) e no segundo dia pós-natal (P2) como tecido e fases do desenvolvimento em que são observados transcritos do Fmr1 com junção entre o éxon 13 e o terceiro sítio de splicing do éxon 15. Demonstramos que transcritos com essa junção exônica associam-se à proteína 4E de iniciação da tradução de eucarioto (eIF4E), indicando sua estabilidade no citoplasma. Geramos um anticorpo que identifica proteína de fusão com nova região C-terminal da FMRP. Paralelamente, a triagem por elementos em cis (transcritos) e fatores proteicos em trans trouxeram candidatos a inibirem o splicing do éxon 14 do FMR1 / Fragile X Mental Retardation Protein (FMRP), encoded by the Fragile Mental Retardation 1 (FMR1) gene, is an RNA-binding protein with nucleus-to-cytoplasm shuttling, and polyribosome association. In the central nervous system (CNS), FMRP is a translation regulator with important roles in synaptic maturation and elimination. In primary FMR1 transcripts, exon 14 skipping followed by selection of the exon 15 third splicing acceptor site, shifts the open reading frame of the downstream codons creating a putative FMRP isoform with a novel C-terminus, which lacks the nuclear localization signal and the major RNA binding domain, RGG box. The relevance of such isoform is as yet unknown. In the present study, we assessed, by RT-qPCR, the expression of rat Fmr1 transcripts in the CNS, relative to the inclusion of exon 14. We identified the cerebral cortex in the nineteenth embryonic day (E19) and the second postnatal day (P2) as the most prominent sources of transcripts bearing a splicing junction between exon 13 and the third splicing acceptor site in exon 15. Those transcripts are associated with the eukaryotic translation initiation factor 4E (eIF4E), indicating its cytoplasmic stability. We generated an antibody that recognizes a fusion protein carrying the novel FMRP C-terminus. Concurrently, a search for ci (transcript) and trans (protein) elements identified putative inhibitors of FMR1 exon 14 splicing

Os genes eucarióticos, em sua maioria, são divididos em exons e introns, requerendo processamento do RNAm para remover as sequências intrônicas e juntar os exons (splicing). As bordas exon/intron são definidas por sítios de splice que normalmente são reconhecidos com alta fidelidade, gerando os mesmos RNAms processados a cada vez. Apesar desse reconhecimento preciso, tem sido observada a junção de exons de maneiras alternativas (splicing alternativo), foco de muitos estudos recentes devido à sua importância em vários processos biológicos. Este processamento alternativo do RNAm pode ser principalmente de três tipos: exclusão de exon, no qual um exon pode ser incluído ou não no RNAm maduro; uso alternativo de sítios de splice, resultando em exons mais longos ou mais curtos e retenção de intron, o tipo menos estudado, no qual uma sequência intrônica é mantida no RNAm maduro. Um dos aspectos cruciais no entendimento de splicing alternativo é conhecer os mecanismos que levam à geração de diferentes transcritos. Coerente com a importância dos sítios de splice no splicing de RNAms, a retenção de intron parece ser causada por falha no reconhecimento daqueles que são sub-ótimos. Como os sítios de splice são reconhecidos aos pares ao se estabelecer uma ponte através de exons ou introns, dependendo de qual é mais curto, uma falha no reconhecimento de um exon ou de um intron leva a diferentes tipos de splicing alternativo (exclusão de exon ou retenção de intron, respectivamente). Desta forma, acredita-se que a ocorrência de retenção de intron esteja também associada a uma falha no reconhecimento de introns curtos. Embora estudos de introns retidos individuais tenham abordado estas questões, poucas análises sistemáticas de grandes quantidades de dados foram conduzidas sobre as características gerais que levam à retenção de intron. Para este fim, realizamos uma análise de bioinformática de sequências do genoma e transcriptoma (RNAm) humanos armazenadas em formato de computador. Para realizar as análises computacionais, desenvolvemos um sistema de anotação de splicing alternativo completo. Particionamos os eventos de retenção de intron identificados em sequências expressas pelo nosso sistema de anotação em dois grupos, com base na abundância relativa das duas isoformas (um grupo de eventos com <50% e outro com >50% de transcritos retendo o intron) e comparamos características relevantes. Verificamos que uma maior frequência de retenção de intron em humano está associada a sítios de splice mais fracos, genes com introns mais curtos e maior nível de expressão gênica, e menor densidade de um conjunto de elementos inibitórios exônicos e do promotor de splicing intrônico GGG. Os dois grupos apresentaram eventos conservados em camundongo, nos quais os introns retidos também eram curtos e apresentavam sítios de splice mais fracos. Embora nossos resultados tenham confirmado que sítios de splice mais fracos estão associados à retenção de intron, eles mostram que uma fração não-desprezível dos eventos não pode ser explicada apenas por esta característica. Nossa análise sugere que elementos reguladores em cis provavelmente têm um papel na regulação da retenção de intron e também revelou características previamente desconhecidas que parecem influenciar sua ocorrência. Estes resultados salientam a importância de considerar o compromisso entre estas características na regulação da frequência relativa de retenção de intron. / Most eukaryotic genes are split in exons and introns, requiring mRNA processing to remove intervening sequences and join exons (splicing). Exon/intron borders are defined by splice sites that are normally recognized with high fidelity, yielding the same processed mRNA each time. Notwithstanding such precise recognition, alternative joining of exons has been observed (alternative splicing) and is the focus of many recent studies, due to its importance in several biological processes. This alternative mRNA processing can be mainly of three types: exon skipping, whereby an exon may be included or not in the mature mRNA; alternative use of splice sites, resulting in longer or shorter exons and intron retention, the least studied type whereby an intronic sequence is maintained in the mature mRNA. One of the key aspects in understanding alternative splicing is to know the mechanisms that lead to the generation of different transcripts. Coherent with the importance of splice sites in mRNA splicing, intron retention seems to be caused by failure in the recognition of those that are sub-optimal. As splice sites are recognized in pairs by bridging either exons or introns, depending on which is the shortest, failure to recognize an exon or an intron leads to different types of alternative splicing (exon skipping or intron retention, respectively). This way, the occurrence of intron retention is believed to be associated to failure in recognition of short introns also. Although studies on individual retained introns have addressed such issues, few systematic surveys of large amounts of data have been conducted on the general features leading to intron retention. To this end, we performed a bioinformatics analysis of human genome and transcriptome (mRNA) sequences stored in computer format. To perform the computational analyses we developed a complete alternative splicing annotation system. We partitioned intron retention events identified in expressed sequences by our annotation system in two groups based on the relative abundance of both isoforms (one group of events with <50% and another with >50% of transcripts retaining the intron) and compared relevant features. We found that a higher frequency of intron retention in human is associated to weaker splice sites, genes with shorter intron lengths and higher expression level, and lower density of a set of exonic inhibitory elements and the intronic splicing enhancer GGG. Both groups of events presented conserved events in mouse, in which the retained introns were also short and presented weaker splice sites. Although our results confirmed that weaker splice sites are associated to intron retention, they showed that a non-negligible fraction of events can not be explained by this feature alone. Our analysis suggests that cis-regulatory elements are likely to play a crucial role in regulating intron retention and also revealed previously unknown features that seem to influence its occurrence. These results highlight the importance of considering the interplay among these features in the regulation of the relative frequency of intron retention.