The adaptor protein CT10-Regulator of Kinase (CRK) and the closely related CRK-Like (CRKL) are adaptor proteins that play important roles in many signaling pathways regulating cell proliferation and cell motility. A notable example is their required role in Reelin signaling during development of the laminated structures of the vertebrate central nervous system, including the cerebral cortex, cerebellum, hippocampus, and retina. As adaptors, CRK/CRKL are important in coupling phosphotyrosine signaling to G protein activity to regulate both cell proliferation and changes in the actin cytoskeleton, thereby exerting control over cell motility, and migration. While many proteins that interact with CRK/CRKL have been identified, the diverse roles of these molecules suggest that more remain to be found. Herein is described a novel CRK/CRKL interacting protein, Src Homology 2 domain-containing protein D (SHD), which demonstrates a phosphorylation-dependent interaction with the CRK/CRKL SH2 domain in HEK 293 cells stimulated with hydrogen peroxide, which globally boosts tyrosine phosphorylation by inhibiting tyrosine phosphatases. Treatment with an inhibitor for Src family kinases (SFKs), Src-1, or an inhibitor of Abl/Arg kinases, STI571, reduces peroxide-induced binding of the CRKL-SH2 domain to SHD. We show that overexpression of Abl kinase, but not the SFK Fyn is sufficient to induce binding of the CRKL-SH2 to SHD and that this interaction requires at least one of the five tyrosines in YxxP motifs found in SHD. Using mass spectrometry, we found that Abl phosphorylates SHD on Y144, which is located in a YxxP motif. Mutation of this site to phenylalanine reduces, but does not prevent, Abl-induced binding of SHD to the CRKL-SH2 domain, suggesting that other YxxP sites also facilitate the interaction. A discussion of the cellular consequences of the interaction between SHD and CRK/CRKL is presented. To explore the biological role of SHD, we used the zebrafish to study shdb, a putative ortholog of human SHD. The expression of shdb was unknown and so we performed in situ hybridization and determined that shdb was expressed in the developing nervous system. To study the function of this gene, we used a morpholino to knock down expression of shdb which resulted in significantly reduced eye size. Possible roles of Shdb in eye development are discussed as is future research aimed to elucidate the cellular and developmental mechanisms by which Shdb functions in the developing eye.

Indiana University-Purdue University Indianapolis (IUPUI) / Cell shape changes and signaling pathways are essential for the development and function of the lens. During lens development proliferating epithelial cells will migrate down to the equator of the lens, differentiate into lens fiber cells, and begin to elongate along the lens capsule. The Fibroblast Growth Factor (FGF) signaling pathway has been extensively studied for its role in lens fiber cell differentiation and elongation. However, the main mediators of FGF stimulated lens fiber cell elongation have not been identified. Adaptor proteins Crk and CrkL are SH2- and SH3-containing proteins that transduce signals from upstream tyrosine phosphorylated proteins to downstream effectors, including Ras, Rac1 and Rap1, which are important for cell proliferation, adhesion and migration. Underlying their diverse function, these two adaptor proteins have been implicated in receptor tyrosine kinase signaling, focal adhesion assembly, and cell shape. To explore the role of Crk and CrkL in FGF signaling-dependent lens development and fiber elongation, we employed Cre/LoxP system to generate a lens specific knockout of Crk/CrkL. This led to extracellular matrix defects, disorganization of the lens fiber cells, and a defect in lens fiber cell elongation. Deletion of Crk and CrkL in the lens also mitigated the gain-of-function phenotype caused by overexpression of FGF3, indicating an epistatic relationship between Crk/CrkL and FGF signaling during lens fiber cell elongation. Further studies, revealed that the activity of Crk and CrkL in FGF signaling is controlled by the phosphatase Shp2 and the defect observed in lens fiber cell elongation can be rescued by constitutive activation of the GTPases Ras and Rac1 in the Crk and CrkL mutant lens. Interestingly, the deletion of the GTPases Rap1 in the lens showed no obvious phenotype pertaining to lens fiber cell elongation. These findings suggest that Crk and CrkL play an important role in integrating FGF signaling and mediating lens fiber cell elongation during lens development.

Ziel dieser Arbeit war die Entwicklung neuer Methoden zur Untersuchung zirkulärer RNA. Das erste Projekt dieser Arbeit beschäftigte sich mit der Erstellung eines universell einsetzbaren Protokolls zur Generierung einer funktionalisierten zirkulären RNA. Hierbei konnte zunächst erfolgreich eine Vorschrift zur Herstellung unmodifizierter circRNA etabliert werden. Im zweiten Schritt gelang auch die Generierung einer zirkulären RNA mit Alkin-Funktionalisierung. Geringe Ausbeuten gaben Anlass zur Entwicklung eines alternativen Verfahrens, bei dem die Zyklisierung von Kopf-Schwanz modifizierter RNA durch CuAAC vorgenommen werden sollte. Dabei konnte zunächst eine 5‘-azidmodifizierte RNA durch in vitro Transkription gebildet werden, die anschließend am 3‘-Terminus mit einem 3‘ alkinmodifizierten Baustein mit Aminfunktionalität versehen wurde. Daraufhin konnte erfolgreich eine Zyklisierung mittels CuAAC vorgenommen werden. Ein grundlegendes Problem bei diesen Arbeiten war der Nachweis, dass die gebildete RNA tatsächlich in zirkulärer Form vorlag. Im Rahmen des zweiten Projektes dieser Arbeit wurde ein Assay zur direkten Unterscheidung von zirkulären und linearen Transkripten etabliert. Mittels reverser Transkription konnte ein rolling circle Mechanismus mit dem zirkulären Transkript durchgeführt werden, was in einer multimeren cDNA resultierte. Nach Amplifizierung über qPCR ermöglichteeine Gelanalyse den Nachweis eines spezifischen Bandenmusters für das circRNA-Transkript, wohingegen das lineare Transkript lediglich eine monomere Bande generierte. Anschließend erfolgte die Weiterentwicklung des Assays zu einer spezifischen Nachweismethode für zirkuläre RNA in biologischen Proben.Dabei kann eine abschließende Gelanalyse zur Identifizierung von falsch-positiven Ergebnissen genutzt werden. Die hier etablierte Methode ermöglicht künftig einen schnellen und einfachen Nachweis von circRNA beim Screeningvon biologischen Proben. / Circular RNAs belong to the group of long, non-coding RNAs and have gene regulating functions, comparable to miRNA. However, the field of circRNA research is proceeding slowly due to the lack of efficient analytical methods. That‘s the reason why the development of new analytical methods plays a keyrole within characterisation and identification of circRNAs. This thesis comprises two projects dealing on one hand with the creation of a protocol for the generation of functional circRNA on and the other hand, an assay to differentiate circular and linear RNA. For the generation of circRNA a non-modified circRNA was produced as positiv control by using T4 RNA ligase 2. After the addition of a modification step with T4 RNA ligase 1, it was possible to generate circRNA with alkyne functionalization. Due to limited yields of modified circRNA, the protocol was adapted and a protocol for chemical ligation was established. In this new procedure a RNA with 5‘-azido modification was generated by in vitro transcription, followed by incorporation of a 3‘-alkyne modified building block with additional amine funktionality at the RNA-3‘-end. Consecutively, it was possible to perform a cyclization with the double modified RNA by CuAAC. The second project comprises the establishment of an assay in order to differentiate circular and linear RNA. A rolling circle mechanism was utilized by reverse transcription of a circular RNA transcript, resulting in a multimeric cDNA. Following DNA amplification by qPCR, a specific fragmentation pattern for circRNA was verified by gel electrophoresis. In contrast to this, for linear RNA, a monomeric DNA pattern was seen. Subsequently the assay was advanced to a detection method for circular RNA in biological samples. A final gel electrophoresis allows the identification of false-positive results. In the future, the here developed method can be applied for fast and easy detection of circRNAs in biological samples.

No description available.

The development of new high throughput technologies able to multiplex disease biomarkers as well as advances in medical treatments has lead to the recent expansion of the newborn screening panel to include DNA-based targets. Four rare disorders; deletion 22q11.2 syndrome and Spinal Muscular Atrophy (SMA), Severe Combined Immunodeficiency (SCID) and Congenital Cytomegalovirus (CMV), are potential candidates for inclusion to the newborn screening panel within the next few years. The major focus of this study was to determine whether 5’-hydrolysis assays developed for the four distinct disorders with specific detection needs and analytical ranges could be combined on the OpenArray system and in multiplexed qPCR reactions. SNP detection of homozygous SMN1 deletions in SMA, CNV detection in the 22q11.2 critical region, and quantification of the SCID biomarker, T-cell receptor excision circles (TRECs) and CMV were all required for disease confirmation. SMA and 22q11.2 gene deletions were accurately detected using the OpenArray system, a first for the technology. The medium density deletion 22q11.2 multiplex successfully identified deletion carriers having either the larger 3 Mb deletion or the smaller 1.5 Mb deletions. Both TREC and CMV targets were detected but with a decrease in sensitivity when compared to their singleplex counterparts. Lastly, copy number detection of the TBX1 was performed when multiplexed with the TREC assay, without a decrease in detection limit of either assay. Here, we provide proof of principal that qPCR multiplexing technologies are amenable to implementation with a newborn screening laboratory.