No description available.

Eukaryotic transcriptional activators stimulate transcription of genes otherwise expressed at low levels. The typical activator operates by binding to specific sites on DNA with its activating region contacting the multiprotein machinery that directs transcription. SBF/Staf is a transcriptional activator that binds to the SPH element found in the promoters of genes for snRNAs and genes that code for mRNAs. SBF/Staf binds to SPH through a reiterated zinc finger DNA binding domain and also contains two distinct activation domains, one for snRNA genes and one for mRNA genes. To test the role of SBF/Staf in vivo, morpholino antisense oligos were used to knock down SBF/Staf expression in zebrafish. A high percentage of developing zebrafish embryos exhibited abnormalities. Co-injection of a synthetic mRNA construct rescued the morpholino-induced knockdown. Furthermore, both the mRNA and snRNA activation domains have significant roles in the function of SBF/Staf because when each domain was removed separately, partial rescue of the knockdown phenotype was obtained. When both domains were removed, no rescue of the phenotype was observed. Unexpectedly, knockdown of SBF/Staf expression in zebrafish embryos caused an increase in steady-state levels of all endogenous mRNAs tested, as well as transcripts produced from co-injected U6 maxigenes. However, quantitative RT-PCR analysis showed a relatively smaller increase in the steady-state levels of several mRNAs from genes that contain a SPH element in their promoters. In zebrafish U6 genes, the SPH element is in the unique location of being next to the TATA box, instead of ~220 bp upstream of the start site as in mammals. To determine the significance of the proximally-located SPH element for transcription of the zebrafish U6 snRNA gene, the SPH element was mutated. Transcription of a zebrafish U6 maxigene was reduced to 20.6% in transfected ZF4 cells and 26.8% in injected embryos, compared to that of the U6 maxigene with a normal promoter. This work indicates a more global role of SBF/Staf in mRNA gene transcription, instead of only activating the transcription of snRNA and a few mRNA genes, leading to an increased importance of the role of SBF/Staf in transcriptional control.

Eukaryotic transcriptional activators stimulate transcription of genes otherwise expressed at low levels. The typical activator operates by binding to specific sites on DNA with its activating region contacting the multiprotein machinery that directs transcription. SBF/Staf is a transcriptional activator that binds to the SPH element found in the promoters of genes for snRNAs and genes that code for mRNAs. SBF/Staf binds to SPH through a reiterated zinc finger DNA binding domain and also contains two distinct activation domains, one for snRNA genes and one for mRNA genes. To test the role of SBF/Staf in vivo, morpholino antisense oligos were used to knock down SBF/Staf expression in zebrafish. A high percentage of developing zebrafish embryos exhibited abnormalities. Co-injection of a synthetic mRNA construct rescued the morpholino-induced knockdown. Furthermore, both the mRNA and snRNA activation domains have significant roles in the function of SBF/Staf because when each domain was removed separately, partial rescue of the knockdown phenotype was obtained. When both domains were removed, no rescue of the phenotype was observed. Unexpectedly, knockdown of SBF/Staf expression in zebrafish embryos caused an increase in steady-state levels of all endogenous mRNAs tested, as well as transcripts produced from co-injected U6 maxigenes. However, quantitative RT-PCR analysis showed a relatively smaller increase in the steady-state levels of several mRNAs from genes that contain a SPH element in their promoters. In zebrafish U6 genes, the SPH element is in the unique location of being next to the TATA box, instead of ~220 bp upstream of the start site as in mammals. To determine the significance of the proximally-located SPH element for transcription of the zebrafish U6 snRNA gene, the SPH element was mutated. Transcription of a zebrafish U6 maxigene was reduced to 20.6% in transfected ZF4 cells and 26.8% in injected embryos, compared to that of the U6 maxigene with a normal promoter. This work indicates a more global role of SBF/Staf in mRNA gene transcription, instead of only activating the transcription of snRNA and a few mRNA genes, leading to an increased importance of the role of SBF/Staf in transcriptional control.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18114号 / 理博第3992号 / 新制||理||1576(附属図書館) / 30972 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 大野 睦人, 教授 青山 卓史, 教授 高田 彰二 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Alors que le complexe snRNP est bien décrit chez les vertébrés, il nécessite plus d’études chez les invertébrés. Le snARN 7SK sert de maintient structural pour la fixation d’HEXIM à P-TEFb. En retour, HEXIM inhibe l’activité kinase de CDK9 via une fixation directe avec la Cycline T. En conséquence, les interactions entre le snARN 7SK et HEXIM va piéger le complexe P-TEFb sous une forme inactive qui conduit à inhiber l’élongation transcriptionnelle. Dans notre étude, nous montrons qu’un contrôle de l’activité P-TEFb existe aussi chez la Drosophile. Et la dynamique d’équilibre entre les deux formes de P-TEFb dépend également du snARN 7SK. Ce modèle est donc utilisé pour étudier le rôle biologique de la snRNP, et plus spécialement d’HEXIM, dans un contexte intégré. Nous avons donc analysé le profile d’expression d’HEXM durant le cycle de vie de la Drosophile et plus particulièrement pendant l’embryogenèse et l’organogenèse. L’expression permanente et ubiquitaire d’HEXIM suggère qu’elle est nécessaire au développement. Le fait que la perte de fonction d’HEXIM mène à de nombreux et sévères défauts confirme cette hypothèse. En utilisant le modèle des disques imaginaux de l’aile et de l’œil, nous avons étudié plus en profondeur le rôle d’HEXIM et nous avons montré qu’elle est essentielle pour la viabilité cellulaire. De plus, la perte de fonction d’HEXIM conduit à des changements du destin cellulaire et à des modifications des profiles d’expression de plusieurs gènes sélecteurs ou de morphogènes. De façon surprenante, la diminution d’HEXIM induit l’accumulation de Ci155 qui est requise pour activer l’expression de Ptc, ainsi que l’activation ectopique de la voie Hh. Cette accumulation notable de Ci155 est également détectée dans les cellules “immortelles” et dans les tissus en cours de régénération à la suite d’une ablation par voie génétique. Sur la base de ces données, nous proposons un rôle possible de l’accumulation de Ci155 dans le phénomène de prolifération compensatrice. Finalement, nous avons caractérisé un nouvel analogue du snARN 7SK chez la Drosophile, qui a été nommé dm7SK-like snARN. Ce dernier a une structure secondaire très similaire à celle de ces homologues vertébrés, alors que la séquence primaire est assez différente. De plus, presque tous les domaines structuraux importants pour les interactions avec HEXIM et les autres partenaires sont conservés chez cet ARN. Des interactions directes ont été démontrées entre HEXIM et cet ARN suggérant qu’il est un analogue structural du snARN 7SK. Ainsi, la présence de deux analogues du snARN 7SK suggère un autre niveau de régulation de l’expression des gènes, au moins chez la Drosophile. / Whereas 7SK snRNP complex has been well characterized in vertebrates, its activities still remain to be further elucidated in invertebrates. 7SK snRNA serves as a structural scaffold for the efficient binding of HEXIM to P-TEFb. HEXIM in turn inhibits the kinase activity of CDK9 via its direct binding to CyclinT. Consequently, the interaction between 7SK snRNA and HEXIM sequesters the active P-TEFb complex into the inactive form, thereby suppressing the transcription elongation. In this study, we first show that a similar P-TEFb control system exists in Drosophila. In addition, the dynamic equilibrium of the two complexes of P-TEFb in Drosophila also depends on 7SK snRNA. Thank to this similarity, we are able to examine the biological role of 7SK snRNP complex, especially HEXIM protein, in an integrative organism as Drosophila model. We next document the expression profile of HEXIM throughout the life cycle of Drosophila, especially during embryogenesis and organogenesis. The continuous and ubiquitous expression of HEXIM suggests its necessity during development. We demonstrate that HEXIM is indeed essential for the proper development of Drosophila, since its down-regulation results in numerous severe defects. By using wing and eye imaginal discs as study models, we further examine biological roles of HEXIM, and reveal that it is required for cell viability. Moreover, HEXIM knockdown leads to changes in cell fate commitments, and modifications in expression patterns of several selector genes and morphogens. Strikingly, down-regulation of HEXIM significantly induces the accumulation of Ci155, which is required for Ptc expression, and the ectopic activation of Hh signaling. This remarkable accumulation of Ci155 is also detected in “undead cells” and regenerated tissue upon genetic ablation. Given these findings, we thus propose a putative role of Ci155 accumulation in compensatory proliferation. Finally, we characterize a novel analog of 7SK snRNA in Drosophila, which is named dm7SK-like snRNA. This snRNA displays a very similar secondary structure with its vertebrate homologs, although the primary sequence is relatively different. More importantly, almost all of the structural elements crucial for the interaction with HEXIM and other partners are found conserved in this novel dm7SK-like snRNA. A direct interaction between dHEXIM and this snRNA also suggests that it is a functional analog of 7SK snRNA in Drosophila. Thus, the intriguing finding of the two analogs of 7SK snRNA would propose another regulation level of gene expression, at least in Drosophila.

In the cell we can find a lot of small noncoding RNAs, which are important for many processes. Among those RNAs are small nuclear RNA uridin rich, which with proteins create U snRNP.These particles play important role in pre-mRNA splicing. In this process are noncoding sequences (introns) removed and coding sequences (exons) are joined. It is catalyzed by spliceosome. The core of this spliceosome is created by U1, U2, U4, U5 and U6 snRNP. They are essential for this process. Some steps of U snRNP biogenesis proceed in nuclear structures called Cajal bodies (CB). In my thesis I focused on factors, which are important for targeting U snRNA into CB. I used U2 snRNA like a model. With the aid of microinjection of fluorescently labeled U2 snRNA mutants I found, that the Sm binding site on U2 snRNA is essential for targeting to CB. Knock down of Sm B/B'showed us, that Sm proteins are necessary for transport U2 snRNA to CB. Sm proteins are formed on U2 snRNA by SMN complex. Deletion of SMN binding site on U2 snRNA had the same inhibition effect. From these results we can see, that Sm proteins and SMN complex are important for U2 snRNA biogenesis espacially for targeting into CB. Key words: U snRNP, Cajal body, U snRNA, cell nucleus

The research of my thesis focused on the use of transcriptomic open discovery approaches to interrogate the molecular basis of two distinct yet related neurological disorders that are both associated with cognitive decline, Temporal Lobe Epilepsy and Alzheimer’s Disease. Interestingly, a potential role for compromised synaptogenesis early in disease was common to both, as was the direct role that neurons may play in brain inflammatory processes involving glia. Temporal lobe epilepsy (TLE) is a progressive disorder mediated by pathological changes in molecular cascades and hippocampal neural circuit remodeling that results in spontaneous seizures and cognitive dysfunction. Targeting these cascades may provide disease-modifying treatments for TLE patients. Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) inhibitors have emerged as potential disease-modifying therapies; however, a more detailed understanding of the contribution of JAK/STAT signaling to epileptogenesis is required to increase the potential therapeutic efficacy and reduce adverse effects associated with un-targeted JAK/STAT inhibition. With our collaborators, my lab developed a mouse line in which tamoxifen treatment conditionally abolishes STAT3 signaling from forebrain excitatory neurons (nSTAT3KO). Seizure frequency (continuous in vivo electroencephalography) and memory (contextual fear conditioning and motor learning) were analyzed in wildtype (Wt) and nSTAT3KO mice after intrahippocampal kainate (IHKA) injection as a model of TLE. Selective STAT3 KO in excitatory neurons reduced seizure progression and hippocampal memory deficits without reducing the extent of cell death or mossy fiber sprouting induced by IHKA injection. In my thesis, RNA was extracted from harvested hippocampi 24 h after IHKA and libraries were prepared for bulk RNA-sequencing (70–80 million reads/sample) using the NextSeq 500 Illumina system. 3190 genes were differentially expressed in Wt mice injected with KA vs saline (fold change |1.5|, FDR=<0.05). Ingenuity Pathway Analysis (IPA) revealed significant enrichment in 2 overarching sets of pathways: 1) those related to synaptic signaling and 2) those related to inflammation. As expected, many of the IHKA-induced genes were previously associated with epilepsy or seizure disorders (260 for Seizure Disorder, 267 for Epilepsy or Neurodevelopmental Disorder), and Seizure Disorder had the highest activation score in Neurological Disease based on gene expression patterns. Interestingly, a closer analysis of the IHKA-induced gene set revealed an enrichment of STAT3-associated genes (216), most of which were upregulated by IHKA. Compared to the 3190 Differentially Expressed Genes (DEGs) between IHKA and saline-injected Wt mice 24 hours after SE, more than half of these DEGs (1609) were rescued when comparing IHKA-injected nSTAT3KO mice and saline-injected Wt mice, indicating a significant rescue of gene expression when nSTAT3 is absent in excitatory neurons. While nSTAT3 KO influences the expression of genes in many different pathways, including the reversal of genes whose expression was inhibited in pathways of learning and memory by IHKA, the greatest surprise came from the predicted regulatory control over microglial function. nSTAT3KO mice displayed the greatest number of rescued DEGs compared to IHKA-injected WT mice in pathways that regulate inflammation and ion transport, and while inflammation was an expected response to IHKA, we were surprised to find evidence for its rescue in nSTAT3 KO mice. We also interrogated the expression of the Alzheimer’s disease genome as modeled using a rat model (TgF344-AD ) of familial AD that allows for behavioral and molecular characterization of AD, and expresses an endogenous pathogenic form of tau in addition to Abeta oligomers and plaques. AD is a neuropsychiatric disorder characterized initially by short term memory loss and disorientation, followed by declining cognitive functioning, and eventually, death. Widespread failure of 99% of AD drugs that make it to clinical trials has led to renewed interest in early signatures of disease in hopes of altering disease trajectory through early intervention. Key to such efforts is capturing a molecular window into AD at its earliest stages. The TgF344-AD rat shows overt pathology (including Aβ plaques, frank neuronal loss, and endogenous tau pathology) at 16 and 26 mo, but only to a very limited extent at 6 mo (Towne, 2013). Thus, in my thesis research, we set out to uncover any cell-type specific transcriptomic alterations that may be present in advance of major behavioral deficits or appearance of pathology, given that a strong body of literature suggests a long pre-symptomatic stage of illness in which subtle abnormalities may be present. 10x Genomics’ v3 gene expression assays were used to perform snRNA-seq on freshly dissected hippocampi from 6 mos, 9 mos and 19 mos littermate pairs of Tg and Wt rats (n=16 for 6 months and 9 months, with 8 for 19 months). ~2000 cells/subject were collected, and cDNA libraries were sequenced to a depth of ~120k reads/nuclei. Interestingly, data analysis revealed wide-scale gene changes in dentate granule cells (DGCs) and non-DGC excitatory neurons (Excit Ns) at 6 mos, suggestive of a significant decrease in synaptogenesis in Tg vs their Wt littermates, as well as small increases in cholesterol biosynthesis in the Tg rats in these cell types. By 9 months, some differentially expressed genes were observed across genotype in classes of glial cells, but the strongest impact on gene expression could still be seen in Excit Ns and DGCs, which continued to display evidence of decreased synaptogenesis, though to a lesser extent than at 6 mos. Interestingly, 9 mos Tg rats displayed an even stronger upregulation in genes related to cholesterol biosynthesis than 6 mos for both DGCs and Excit Ns. At 19 months, cholesterol and steroid biosynthesis were amongst the top biological pathways enriched for in Excit Ns and Inhibitory neurons of the Tg, to an even greater extent than changes in synaptogenesis. Altogether, our results suggest the transcriptional basis for a profound suppression of synapse formation or maintenance during early stages of illness in the TgF344-AD rat model, as well as abnormalities in neuronal cholesterol biosynthesis. Given that cholesterol is a key component of plasma membranes and lipid rafts, structures needed for the generation of new synapses and the stability of their receptor populations, it may be that deficiencies in the available cholesterol of Tg neuronal cells is leading to the impaired synaptogenesis in these cell types. Future work will focus on identifying whether these transcriptional alterations can be detected at even earlier time points, whether they are prescient for changes at the membrane in vivo that are correlated with memory impairment, and whether they are related to the alterations in the genome seen in our acquired epilepsy models, suggesting a common theme for the brain’s genomic response to injury of the hippocampus. / 2025-02-12T00:00:00Z

No description available.

Messenger RNA (mRNA) is transcribed by RNA polymerase II (Pol II) and has to undergo multiple processing events before it can be translated into a protein: a cap structure is added to its 5’ end, noncoding, intervening sequences (introns) are removed and coding exons are ligated together and a poly(A) tail is added to its 3’end. Splicing, the process of intron removal, is carried out in the spliceosome, a megacomplex comprehending up to 300 proteins. The core components of the spliceosome that directly interact with the pre-mRNA are the small nuclear ribonucleoprotein particles (snRNPs). They consist of one of the U-rich snRNAs U1, U2, U4, U5 or U6 together with several particle-specific proteins and core proteins. All mRNA processing events can occur co-transcriptionally, i.e. while the RNA is still attached to the gene via Pol II. The in vivo studies of co-transcriptional RNA processing events had been possible only in special biological systems by immunoelectron microscopy and only recently, Chromatin Immunoprecipitation (ChIP) made it possible to investigate cotranscriptional splicing factor assembly on genes. My thesis work is divided into two parts: Part I shows that the core components of the splicing machinery are recruited co-transcriptionally to mammalian genes in vivo by ChIP. The co-transcriptional splicing factor recruitment is dependent on active transcription and the presence of introns in genes. Furthermore, a new assay was developed that allows for the first time the direct monitoring of co-transcriptional splicing in human cells. The topoisomerase I inhibitor camptothecin increases splicing factor accumulation on the c-fos gene as well as co-transcriptional splicing levels, which provides direct evidence that co-transcriptional splicing events depend on the kinetics of RNA synthesis. Part II of the thesis is aimed to investigate whether Pol II has a functional role in the biogenesis of the U6 snRNA, which is the RNA part of the U6 snRNP involved in splicing. Pol III had been shown to transcribe the U6 snRNA gene, but ChIP experiments revealed that Pol II is associated with all the active U6 snRNA gene promoters. Pol II inhibition studies uncovered that U6 snRNA expression and probably 3’end formation is dependent on Pol II.

Les dysferlinopathies sont des dystrophies musculaires qui se manifestent par la dystrophie musculaire des ceintures de type 2B (LGMD2B) ou la myopathie de Miyoshi (MM). Elles sont causées par des mutations dans le gène dysferline. La dysferline est une protéine membranaire exprimée dans le muscle squelettique, responsable de la réparation des microlésions du sarcolemme. L’absence d’une telle réparation de la membrane entraîne une atrophie musculaire progressive. Ce travail de thèse explore le potentiel thérapeutique d'une stratégie de modulation d'épissage pour le traitement de la LGMD2B causée par la mutation faux-sens c4022T>C dans l'exon 38 du gène dysferline. Des oligonucléotides et des petits ARN U7 délivrés par un vecteur viral de type adéno-associé ont été utilisés comme outils antisens pour induire un saut d'exon in vitro et in vivo. Ce projet de thèse étudie également la capacité de la dysferline tronquée à se localiser de façon appropriée à la membrane et ainsi la réparer. / Dysferlinopathy is a muscular dystrophy that manifests as two major phenotypes: limb-girdle muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy (MM). It is caused by mutations in the dysferlin gene. Dysferlin is a membrane protein expressed in skeletal muscle. It is responsible for the repair of sarcolemma microlesions produced by muscle contractions. A compromised membrane repair leads to slowly progressing muscle wasting. This thesis explores the therapeutic potential of an antisense mediated splice switching strategy in LGMD2B caused by the missense mutation c4022T>C in the exon 38 of the dysferlin gene. Antisense oligonucleotides and U7 snRNAs delivered by an adeno-associated viral vector were used as antisense tools to trigger exon skipping in vitro and in vivo. The thesis investigates also if the truncated dysferlin maintainsa proper membrane localization and its membrane repair ability.