SR蛋白在前體信使核糖核酸(pre-mRNA)的組成性剪接和選擇性剪接中扮演者重要的角色，在這個過程中它需要被SR蛋白激酶(SRPK) 燐酸化才能正常行使功能。經典的SR蛋白是由N端一到二個RNA識別基序(RRM) 以及C端一串精氨酸－絲氨酸(RS) 二肽所構成。SR蛋白的燐酸化調控它的亞細胞定位以及生理功能。此外，SR 蛋白激酶1(SRPK1) 和SR蛋白原型ASF/SF2的復合物結構顯示底物的結合需要第二個非標準的RRM結構域以及在N端可以被燐酸化的RS結構域，但是，第一個標準的RRM結構域對於SR 蛋白激酶1的結合卻是可以或缺的。 / 在這裡，我們展示了SR蛋白激酶2(SRPK2) 結合並且燐酸化SRp20的RS結構域，SRp20是另外一個只包含一個RNA識別基序(RRM) 的SR蛋白。與ASF/SF2相似的是，SRp20中的標準RNA識別基序對於SRPK2的結合並不是必要的。與此同時，我們發現錨定槽對於底物的識別作用在SRPK2中也是保守的，因為，錨定槽中四個關鍵氨基酸的突變會削弱它對SRp20的結合。 / 此外，現在認為SRPK2的功能已經不限於對前體信使核糖核酸(pre-mRNA) 的剪接調控。最近發現，SRPK2也可以燐酸化Tau蛋白並且介導阿爾茨海默疾病中的認知性缺陷。組成性的激活是SR蛋白激酶中的一個固有特性，然而人們對於它的調控機制還不是很清楚。因此, 為了更好的瞭解SRPK2，我們采用酵母雙雜交的方法並且最終發現一個新的SRPK2相互作用蛋白: ZNF187。 / ZNF187是一個可以結合血清反應元件(SRE) 的轉綠因子。我們的研究發現，它可以正向調控SRE的轉錄激活。然而，SRPK2在EGF的刺激下卻起着抑制的效果，其中EGF的刺激會促使SRPK2進入細胞核。進一步證實，通過RNAi干擾的方法敲掉SRPK2可以增加ZNF187誘導的SRE活性。在共轉染實驗中，SRPK2可以把ZNF187誘導的SRE活性逆轉到本底水平。對於可以和EGF刺激的SRPK2有着相似細胞定位的缺失或者突變研究發現，它們都可以產生相一致的抑制現象。於此相反，對於和SRPK2有着不同細胞定位的突變，它卻不能產生抑制效果。因此，我們認為在EGF的刺激下，SRPK2進入細胞核並且負向的調控ZNF187激活的SRE。令人驚訝的是，如果細胞在FBS的刺激下，SRPK2卻上調SRE活性，並且它可以協同增加ZNF187對於SRE的誘導。這些結果表明SRPK2對於ZNF187誘導的SRE轉綠調控是刺激物依賴的。 / SR proteins are critical players in regulating both constitutive and alternative pre-mRNA splicing, during which the phosphorylation by SR Protein Kinases (SRPKs) is required. Classical SR proteins contain one or two RNA Recognition Motifs (RRM) in their N terminus and a stretch of Arginine-Serine (RS) dipeptides in C terminus. Phosphorylation status of SR proteins regulates their subcellular localization as well as physiological function. In addition, complex structure of SRPK1 with ASF/SF2, a prototype of SR protein, shows that substrate binding requires non-canonicalRRM2 domain and RS domain, which can be extensivelyphosphorylated. However, the canonical RRM1 domain is dispensable for such interaction. / Here we show that SRPK2 binds and phosphorylates SRp20, a classical single RRM domain-containing SR protein, at its RS domain. Similarly with ASF/SF2, the canonical RRM domain of SRp20 is dispensable for interacting with SRPK2. Meanwhile, we also find that a docking groove that iscritical for substrate binding in SRPK1 is also conserved in SRPK2, since mutations on four key residues in docking groove impair its binding affinity with SRp20. / In addition, SRPK2 is now known to function more then regulating mRNA splicing, such as cell proliferation and cell apoptosis. Recently, SRPK2 is also shown to be a kinase phosphorylating Tau and mediate the cognitive defects in Alzheimer’s disease (AD). Besides, an intrinsic character of SRPKs lies in that they are constitutively active, but the regulation mechanism is not well understood. Therefore, in order to obtain a better recognition about SRPK2, we applied yeast two-hybrid assay and eventually anew interaction partner called ZNF187 was identified. / ZNF187 is a transcriptional factor that binds with Serum Response Element (SRE). Our studies showed that it isa positive regulator of SRE activity. However, SRPK2 showed inhibiting effect on SRE activation with the treatment of EGF, which could induce its nucleus entry, when co-transfected, it reversed the stimulating effect on SRE by ZNF187 to basal level. Furthermore, knockdown of SRPK2 by RNAi would enhance ZNF187-stimuated SRE activation. Studies on truncation and mutations that have the similar effect with EGF-induced subcellular localization of SRPK2 also generated the same inhibiting phenomenon. In contrast, mutant that has distinct localization with SRPK2 wild type failed to exert suppression. Therefore, we conclude that with the treatment of EGF, SRPK2 moves into nucleus and negatively regulates ZNF187-stimulated transactivation of SRE. Surprisingly, when cells were treated with FBS, SRPK2 showed stimulation on SRE activity and it synergized ZNF187-stimulated effect on SRE, indicating that transcriptional regulation of SRPK2 on ZNF187-stimulated SRE activity is stimuli-dependent. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Shang, Yong. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 113-137). / Abstracts also in Chinese.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 96-98.

Le muscle squelettique adulte est un tissu avec une grande plasticité étant donné qu’il adapte sa taille suite à la surcharge fonctionnelle et il régénère suite à une lésion. La base de cette plasticité est la myofibre et les cellules souches associées, les cellules satellites (CS). Suite aux stimuli, les CS sortent de la quiescence, elles s’activent, proliférent, s’engagent dans la voie myogénique et fusionnent entre elles ou bien avec la fibre pre-éxistante. Une partie des CS retourne à la quiescence afin de maintenir le « pool » de progéniteurs. Ce projet a pour objectif de mieux caractériser des voies de signalisation responsables des adaptations des CS au cours de la régénération et le l’hypertrophie compensatoire. Srf est un facteur de transcription, particulièrement exprimé dans les muscles. Les gènes cibles de Srf sont des gènes qui participent à la régulation de la prolifération cellulaire et des gènes codant des protéines sarcomériques du muscle ou bien des gènes ayant un rôle dans l’adhésion cellulaire, la migration et l’organisation du cytosquelette. Il a été montré que la perte de fonction de Srf dans la lignée de cellules musculaire C2C12 inhibe leur prolifération et leur différenciation et que Srf contrôle l’expression de MyoD qui est un gène de détermination myogénique. Aucune donnée n’est disponible à ce jour concernant la fonction de Srf dans les CS in vivo. Nous avons généré des souris dépourvues de Srf spécifiquement dans les CS adultes. Les CS ont été recruitées par l’hypertrophie et la régénération musculaire. En parallèle des études ex vivo ont été menées afin de préciser si les phénotypes observés sont cellule-autonomes et afin de disséquer les mécanismes sous-jacents. Nous montrons que la perte de Srf dans les CS affecte fortement les processus de régénération et d’hypertrophie suggérant un rôle de Srf dans le contrôle du destin cellulaire de CS. Nos études montrent que la perte le Srf dans les SC n’affecte pas leur prolifération et leur engagement dans la différenciation myogénique. Par contre, leur motilité et leur capacité de fusion sont fortement réduites. Afin d’identifier les effecteurs de Srf impliqués dans la motilité et le défaut de fusion des CS mutantes, nous avons réalisé des études transcriptomiques et identifié le set de gènes dont l’expression est altérée par la perte de Srf dans des conditions de prolifération et de différenciation. L’analyse des fonctions altérées nous a indiqué que la voie de signalisation du cytosquelette d’actine était perturbée. En effet les CS dépourvues de Srf expriment moins d’actine et présentent une organisation du cytosquelette d’actine perturbée. Des expériences de sauvetage utilisant un modèle de souris permettant la surexpression inductible d’actine alpha dans les CS dépourvues de Srf ont montré que la surexpression d’actine chez les mutants Srf était suffisante pour rétablir partiellement l’organisation du cytosquelette et améliorer les capacités de fusion des CS. De manière intéressante, seule la fusion hétérotypique (entre une cellule contrôle et une cellule mutante), et pas la fusion homotypique (entre deux cellules mutantes), est rétablie par l’expression de l’actine. In vivo, le rétablissement de la fusion hétérotypique restaure la croissance hypertrophique des muscles alors que l’altération de la régénération chez les mutants Srf n’est que faiblement améliorée par la surexpression de l’actine. Cette étude nous a permis d’avoir une vision d’ensemble et mécanistique de la contribution du facteur de transcription Srf dans la biologie des CS et de mettre en évidence l’importance structurale du maintien du cytosquelette d’actine pour la fusion des cellules musculaires. / The adult skeletal muscle is a high plastic tissue as it adapts its size upon overload and it is capable of regeneration upon muscle lesion. The skeletal muscle is composed of a specialized syncytium, the myofiber, which is the functional unit of the muscle and a small population of myogenic progenitors, residing adjacent to the myofibers, termed as satellite cells (SCs). SCs are the muscle-specific stem cells which endow the skeletal muscle with its remarkable capacity to repair and to maintain homeostasis during muscle turnover. In resting adult muscles, SCs are quiescent but they activate upon exposure to stimuli. The activated SCs (myoblasts) proliferate extensively and subsequently differentiate and fuse between them or pre-existing myofibers, a series of cellular events called myogenesis. In parallel to the myogenesis, a reserve population of SCs escapes the myogenic program and self-renews to replenish the SC pool. The current project aims to further characterize the signalling pathways involved in SC functions during muscle regeneration and compensatory hypertrophy (CH). Srf is a muscle-enriched transcription factor with Srf-target genes implicated in cell proliferation, differentiation (sarcomeric proteins), adhesion, migration and cellular cytoskeleton. Studies in C2C12 mouse myogenic cell line showed that Srf loss prevent the myoblast proliferation and differentiation by down-regulating the expression of the myogenic determinant MyoD gene. We used a genetic murine model for adult SC-specific Srf-loss in order to conduct in vivo and ex vivo studies for the Srf role in SCs. Compensatory hypertrophy and regeneration are the two means by which SCs were recruited. We show that loss of Srf in SCs affects the regeneration process and the CH suggesting the Srf role in the SC fate. Srf-depleted SCs display probably no defect in their proliferation and differentiation but reduced capacity in motility and fusion. Transcriptomic analysis revealed altered actin cytoskeleton and signalling. Srf-depleted SCs show reduced actin expression and altered actin cytoskeleton. Rescue of actin expression in Srf-depleted SCs partially restored the cytoskeleton organization and the fusion process. Interestingly by actin overexpression only the heterotypic/asymmetric fusion was established but not the homotypic/symmetric fusion. Therefore actin overexpression restored the hypertrophic growth in the CH (in vivo model of heterotypic fusion) but failed to do so in the regeneration (in vivo model of homotypic fusion). This study contributed to the in vivo investigation of the Srf mechanistic role in adult SCs and underlined the importance of actin cytoskeleton maintenance in the fusion of myogenic cells.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2001. / Vita. Bibliography: 91-102.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: References located at the end of each chapter.

Background Promoters are key players in gene regulation. They receive signals from various sources (e.g. cell surface receptors) and control the level of transcription initiation, which largely determines gene expression. In vertebrates, transcription start sites and surrounding regulatory elements are often poorly defined. To support promoter analysis, we present CORG http://corg.molgen.mpg.de, a framework for studying upstream regions including untranslated exons (5' UTR). Description The automated annotation of promoter regions integrates information of two kinds. First, statistically significant cross-species conservation within upstream regions of orthologous genes is detected. Pairwise as well as multiple sequence comparisons are computed. Second, binding site descriptions (position-weight matrices) are employed to predict conserved regulatory elements with a novel approach. Assembled EST sequences and verified transcription start sites are incorporated to distinguish exonic from other sequences. As of now, we have included 5 species in our analysis pipeline (man, mouse, rat, fugu and zebrafish). We characterized promoter regions of 16,127 groups of orthologous genes. All data are presented in an intuitive way via our web site. Users are free to export data for single genes or access larger data sets via our DAS server http://tomcat.molgen.mpg.de:8080/das. The benefits of our framework are exemplarily shown in the context of phylogenetic profiling of transcription factor binding sites and detection of microRNAs close to transcription start sites of our gene set. Conclusion The CORG platform is a versatile tool to support analyses of gene regulation in vertebrate promoter regions. Applications for CORG cover a broad range from studying evolution of DNA binding sites and promoter constitution to the discovery of new regulatory sequence elements (e.g. microRNAs and binding sites).

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20701号 / 工博第4398号 / 新制||工||1683(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 小寺 秀俊, 教授 中部 主敬, 教授 安達 泰治, 准教授 横川 隆司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Neuronal plasticity deficits underlie many of the neurobehavioral problems seen in Fetal Alcohol Spectrum Disorders (FASD). Recently, we showed that third trimester alcohol exposure lead to a persistent disruption in ocular dominance (OD) plasticity. For instance, few days of monocular deprivation results in a robust reduction of cortical regions responsive to the deprived eye in normal animals, but not in ferrets exposed early to alcohol. This plasticity deficit can be reversed if alcohol-exposed animals are treated with a phosphodiesterase type 1 (PDE1) inhibitor during the period of monocular deprivation. PDE1 inhibition can increase cAMP and cGMP levels, activating transcription factors such as the cAMP response element binding protein (CREB) and the Serum response factor (SRF). SRF is important for many plasticity processes such as LTP, LTD, spine motility and axonal pathfinding. Here we attempt to rescue OD plasticity in alcohol-treated ferrets using a Sindbis viral vector to express a constitutively active form of SRF during the period of monocular deprivation. Using optical imaging of intrinsic signals and single unit recordings we observed that overexpression of a constitutively active form of SRF (Sindbis SRF+), but neither its dominant negative (SRF-) nor GFP, restored OD plasticity in alcohol-treated animals. Surprisingly, this restoration was observed throughout the extent of the primary visual cortex and most cells infected by the virus were positive for GFAP rather than NeuN. Hence, we further tested whether overexpression of SRF exclusively in astrocytes is sufficient to restore OD plasticity in alcohol-exposed ferrets. To accomplish that, first we exposed cultured astrocytes to the SRF+, SRF- or control GFP viruses. After 24h, these astrocytes were implanted in the visual cortex of alcohol-exposed animals or saline controls one day before MD. Optical imaging of intrinsic signals showed that alcohol-exposed animals that were implanted with astrocytes expressing SRF, but not SRF- or GFP, showed robust restoration of OD plasticity in all visual cortex. These findings suggest that overexpression of SRF exclusively in astrocytes can improve neuronal plasticity in FASD.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

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