Recent genomic data indicate that RNA polymerase II (Pol II) function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III). Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP) and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated ∼300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was α-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon α-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome.

Recent genomic data indicate that RNA polymerase II (Pol II) function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III). Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP) and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated ∼300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was α-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon α-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome.

Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ0 cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases. By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ0 cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.

Genetisch kodierte Photorezeptoren in Modellorganismen begründen die Optogenetik. Sie ermöglicht die nicht-invasive, reversible und räumlich-zeitlich präzise Perturbation von zellulären und physiologischen Signalprozessen durch Licht. Natürliche photoaktivierte Adenylylzyklasen (PACs) steigern die intrazelluläre Konzentration des Botenstoffs zyklischen Adenosinmonophosphats (cAMP) durch Blaulicht. Damit erlauben sie die optogenetische Analyse von cAMP-abhängigen Signalwegen. Diese Arbeit komplementiert PACs durch die synthetische rotlichtaktivierte Phosphodiesterase LAPD zur Degradation von cAMP und zyklischem Guanosinmonophosphat (cGMP). LAPD ist eine Chimäre aus dem photosensorischen Modul von Deinococcus radiodurans Bakteriophytochrom (DrBPhy) und der Effektordomäne der cAMP/cGMP-spezifischen H. sapiens Phosphodiesterase 2A (HsPDE2A). Die Fusionsstelle wurde von den helikalen Linkern zwischen Sensor- und Effektormodulen durch strukturelle Überlagerung abgeleitet. LAPD inkorporierte den Chromophor Biliverdin (BV) nach Expression in E. coli und Reinigung vollständig und entsprach spektral und photochemisch dem Wildtyp-DrBPhy. Durch Bestrahlung mit Rot- und Fernrotlicht (R bzw. FR) wurde LAPD in die metastabilen photochemischen Zustände Pfr (fernrot) bzw. Pr (rot) umgewandelt. Vollständig aktivierte LAPD katalysierte die Hydrolyse von cGMP und cAMP in derselben Größenordnung wie Wildtyp-HsPDE2A. LAPD degradierte cGMP und cAMP bei 6- bzw. 4-facher Steigerung von vmax unter R im Vergleich zu dunkeladaptiertem Enzym. Die Aktivität von R-adaptierter LAPD wurde durch FR reduziert. Die enzymatische Aktivität und Lichtregulation von LAPD-Linkervarianten waren abhängig von der Linkerlänge. LAPD degradierte lichtabhängig cGMP in einer PDE-Reporterzelle. Dabei genügte die endogene BV-Konzentration der Säugerzelle zur Sättigung des Lichteffekts. / Genetically encoded photoreceptors in model organisms establish optogenetics. It enables non-invasive, reversible, and spatio-temporally precise perturbation of cellular and physiological signalling by light. Natural photoactivated adenylate cyclases (PACs) increase the intracellular concentration of the second messenger cyclic adenosine monophosphate (cAMP) under blue light. Hence, PACs allow the optogenetic analysis of cAMP-dependent signalling. This work complements PACs with the synthetic red-light-activated phosphodiesterase LAPD for degradation of cAMP and cyclic guanosine monophosphate (cGMP). LAPD is a chimera made up of the photosensory module of Deinococcus radiodurans bacteriophytochrome (DrBPhy) and the effector domain of cAMP/cGMP-specific H. sapiens Phosphodiesterase 2A (HsPDE2A). The fusion site was derived from the helical linkers between sensor and effector modules via structural superposition. LAPD incorporated the chromophor biliverdin (BV) after expression in E. coli and purification quantitatively, and spectrally and photochemically resembled the wildtype DrBPhy. Upon irradiation with red and far-red light (R and FR, resp.), LAPD was converted to the metastable photochemical states Pfr (far-red) and Pr (red), respectively. Fully activated LAPD catalized the hydrolysis of cGMP and cAMP with rates similar to wildtype HsPDE2A. LAPD degraded cGMP and cAMP with 6- and 4-fold increase of vmax under R, respectively, as compared to the dark state. The activity of R-adapted LAPD was reduced upon irradiation with FR. Enzymatic activity and light regulation of LAPD linker variants depended on the linker length. LAPD light-dependently degraded cGMP in a PDE reporter cell line. Endogenous BV concentrations were sufficient to saturate the light effect in the mammalian cell, which enables a true optogenetic approach.

Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ0 cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases. By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ0 cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.