Thesis (M.S.)--Miami University, Dept. of Zoology, 2005. / Title from first page of PDF document. Document formatted into pages; contains [1], ix, 75 p. : ill. Includes bibliographical references (p. 69-75).

Les eIF2B-pathies représentent un groupe de leucodystrophies de transmission autosomique récessive du à des mutations du facteur ubiquitaire eIF2B. Celui-ci intervient dans l’initiation de la traduction et ses régulations, particulièrement en cas de stress cellulaires, grâce à son activité d’échange de guanine (GEF). Un large spectre clinique et mutationnel a été décrit pour cette pathologie.La diminution de l’activité GEF a pu être validée comme marqueur diagnostique spécifique des eIF2B-pathies dans les lymphoblastes de patients atteints avec un seuil d’activité à 77,5% pour une spécificité de 100% et une sensibilité de 89%.La compréhension des mécanismes moléculaires en cause a ensuite été recherchée selon trois approches :- une première focalisée sur l’étude de la réponse au stress du réticulum endoplasmique (RE) dans les lymphoblastes de patients eIF2B-mutés. L’hyper-activation transcriptionnelle et traductionnelle des gènes de la réponse au stress du RE, observée dans d’autres études et sur d’autres types cellulaires n’a pas été retrouvée dans cette étude.- une approche globale d’étude transcriptomique différentielle dans des fibroblastes primaires de patients eIF2B-mutés soumis ou non à un stress cellulaire. La comparaison du transcriptome avec celui de contrôles sains et de patients porteurs d’une autre leucodystrophie n’a pas permis de mettre en évidence un effet spécifique du stress dans les fibroblastes eIF2B-mutés. En revanche, il a pu être montré une dérégulation de l’expression de 70 gènes spécifiquement dans ces fibroblastes ainsi que l’implication de voies métaboliques telles que l’épissage et la stabilité des ARNm, importantes au cours du développement du système nerveux central. Ces gènes trouvés dérégulés dans les fibroblastes, appartenant notamment à la famille des hnRNP, ont été ensuite validés dans les cerveaux de patients eIF2B-mutés et une anomalie d’épissage de certains transcrits importants pour les cellules gliales a également été identifiée.- enfin, pour valider l’hypothèse d’une anomalie développementale des cellules gliales, le modèle des cellules souches embryonnaires (ESC) a été utilisé et un défaut génétique a été introduit dans ces cellules afin de mimer les mutations eIF2B. Une anomalie de différentiation de ces ESC en cellules gliales a pu être mise en évidence dans ce modèle qui pourrait alors constituer un outil de choix pour tester des molécules pouvant potentiellement améliorer la différenciation de ces cellules, principales en cause dans cette pathologie. / EIF2B-related disorders are an autosomal recessive leukodystrophy caused by mutations in the ubiquitary eIF2B factor. This one is involved in the translation initiation step and its regulation, particularly upon cellular stresses, thanks to its guanine nucleotide exchange factor (GEF) activity. A wide continuum clinical and mutational spectrum has been described for this pathology.The decrease of eIF2B GEF activity has been validated as an eIF2B-pathies specific biomarker in affected patients’ lymphoblasts with 100% specificity and 89% sensibility using a threshold at 77.5%.Functional molecular mechanisms involved in the physiopathology of eIF2B-related disorders have been searched by three approaches:- the first one focalized on the study of the endoplasmic reticulum stress response in lymphoblasts from eIF2B-mutated patients. The translational hyper-induction of specific genes involved in the unfolded protein response, identified in other cell types, was not observed in this study.- a global approach using a differential transcriptomic study of primary fibroblasts from eIF2B-mutated patients submitted or not to a cellular stress. The comparison with the transcriptomic profile of fibroblasts from healthy controls and patients presenting with other types of leukodystrophies not allowed us to identify a specific stress effect in eIF2B-mutated fibroblasts. On the other hand, it has been shown 70 genes specifically differentially deregulated in eIF2B-mutated fibroblasts as well as metabolic pathways implication, like splicing and mRNA stability, that are critical during the central nervous system development. We then validated that these genes, belonging the the hnRNP family, were also deregulated in brains from eIF2B-mutated patients and a splice abnormality of genes implicated in glial cells network has also been identified.- finally, in order to validate the hypothesis of an abnormal glial cell development, the embryonic stem cells (ESC) model has been used and a genetic default has been introduced in these cells to mimic eIF2B mutations. We identified an abnormal differentiation of these ESC into glial cells. Therefore, this model would provide a unique tool to search therapeutic agents that would improve glial cell differentiation, the major cells implicated in this pathology.

BACKGROUND: The inactivation of the tumor suppressor gene BRCA1 is a predisposing factor for a breast/ovarian cancer development. Formation of cancer-specific alternative splicing variants with aberrant biological properties can represent additional mechanism decreasing the overall BRCA1 activity in DNA double strand break (DDSB) repair. In this study, we analyzed BRCA1 alternative splicing variants BRCA114-15 and 17-19 ascertained previously during the screening of high-risk breast cancer individuals. METHODS: We established a stable MCF-7 cell line-based model system for an in vitro analysis of BRCA1 variants. Using this system, we analyzed the impact of BRCA114-15 and 17-19 variants on DNA repair kinetics using comet assay and confocal immunomicroscopy. The capacity of DNA repair was assessed directly by an in vitro NHEJ assay and indirectly by a mitomycin C sensitivity test. The proliferation activities were determined by a clonogenic assay and growth curves. RESULTS: Overexpression of BRCA114-15 and 17-19 increases the endogenous level of DNA damage, slows down the DDSB repair, and decelerates the initial phase of radiation-induced foci formation and prolongs their persistence. Moreover, BRCA114-15 and 17-19 differentially influence the activity of HR and NHEJ and sensitivity of MCF-7 cells to ionizing...

JMJD6 or PSR (phosphatidyl serine receptor) was initially proposed to be a membrane receptor involved in apoptotic cell clearance by recognition of apoptotic cells. However, sequence analyses implied the presence of a jelly roll or double stranded beta helix (DSBH) structural domain in PSR/JMJD6 and similarity with JmjC family of enzymes which are involved in chromatin regulation. Subsequently, PSR was renamed as JMJD6 and was reported to be a histone arginine demethylase. Previous work from our group has shown that JMJD6 is a lysine hydroxylase that interacts with nuclear proteins including CROP and U2AF65 which are involved in mRNA splicing. Peptide screening and cell based assays led to the conclusion that JMJD6 catalyses lysine hydroxylation of splicing regulatory proteins containing arginine serine rich domains (SR proteins) including U2AF65 and Luc7like-2. Studies were carried out to investigate the putative arginine demethylation activity of JMJD6 using MS analysis of histone peptides and luminescence-based assays. New substrates from SR proteins were identified by immunoprecipitation of JMJD6 expressed in human cell lines followed by LC-MS/MS analysis and MALDI-MS based assays of synthesised peptide substrates. Work then focussed on studying the mechanism of lysyl-hydroxylation from substrate and enzyme perspective. A crystal structure of seleno-methionine labelled JMJD6 was obtained and it provided insights into the JMJD6 active site and its substrate interactions. Based on this data, single point variants of JMJD6 were prepared and their substrate binding properties were studied by MALDI-MS and 2OG turnover assays. Collagen lysyl-hydroxylases are also 2OG dependent oxygenases. Efforts to investigate the stereochemistry of JMJD6 catalysed hydroxylation, employing NMR and amino acid analyses were carried out. These studies led to the interesting finding that the C-5 stereochemistry of hydroxylysine in LUC7L2 peptide is opposite (2S,5S-hydroxylysine) to that present in collagen (2S,5R-hydroxylysine). It was found that JMJD6 undergoes autocatalytic self-hydroxylation. Lysine residues from both recombinant JMJD6 and that from HeLa cells at endogenous level were identified to be hydroxylated by amino acid and LC-MS/MS analyses. JMJD6 has a strong tendency to form aggregates and gel electrophoresis always reveals multimeric bands of various JMJD6 constructs. Characterisation and identification of oligomeric states of JMJD6 was carried out using Electron Microscopy. Studies were initiated to identify possible inhibitors by screening a set of 2OG analogues. The results from this preliminary inhibition studies have identified the tricarboxylic acid (TCA) cycle intermediates, succinate and fumarate to be JMJD6 inhibitors and form a basis of further studies aimed at identifying selective inhibitors.

BACKGROUND: The inactivation of the tumor suppressor gene BRCA1 is a predisposing factor for a breast/ovarian cancer development. Formation of cancer-specific alternative splicing variants with aberrant biological properties can represent additional mechanism decreasing the overall BRCA1 activity in DNA double strand break (DDSB) repair. In this study, we analyzed BRCA1 alternative splicing variants BRCA114-15 and 17-19 ascertained previously during the screening of high-risk breast cancer individuals. METHODS: We established a stable MCF-7 cell line-based model system for an in vitro analysis of BRCA1 variants. Using this system, we analyzed the impact of BRCA114-15 and 17-19 variants on DNA repair kinetics using comet assay and confocal immunomicroscopy. The capacity of DNA repair was assessed directly by an in vitro NHEJ assay and indirectly by a mitomycin C sensitivity test. The proliferation activities were determined by a clonogenic assay and growth curves. RESULTS: Overexpression of BRCA114-15 and 17-19 increases the endogenous level of DNA damage, slows down the DDSB repair, and decelerates the initial phase of radiation-induced foci formation and prolongs their persistence. Moreover, BRCA114-15 and 17-19 differentially influence the activity of HR and NHEJ and sensitivity of MCF-7 cells to ionizing...

Saccharomyces cerevisiae is an intron-poor organism with introns present in only 5% of its genes. The most prominent group of intron-containing genes are ribosomal protein (RP) genes. They are highly expressed and most of them are present as two paralogs. Parenteau et al. described the existence of intron- dependent intergenic regulatory circuits controlling expression ratios of RP paralogs. In this project, we did not confirm the regulation in 6 out of 7 tested regulatory circuits. We validated the regulation between RPL22 paralogs. We further showed that Rpl22 protein blocks the pre-mRNA splicing of both paralogs, with RPL22B paralog being more sensitive. Rpl22 protein binds to the stem-loop of RPL22B intron - disruption of the binding domain of Rpl22 proteins leads to loss of interaction. Moreover, the regulation seems to be working the same way in yeast Kluyveromyces lactis, which has only a single RPL22 copy. Overall, these results lead to better understanding of intergenic regulation, which adjusts the expression ratio between functionally different RPL22 paralogs. Key words introns, ribosomal protein genes, Rpl22, RPL22 paralogs, pre-mRNA splicing, Saccharomyces cerevisiae

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Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs. Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered. In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing. I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes: (1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs. (2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data. I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing. The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS. For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination. Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.

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