HelF, a suppressor of RNAi mediated gene silencing in Dictyostelium discoideum

RNA interference (RNAi) is a recently discovered process, in which double stranded RNA (dsRNA) triggers the homology-dependant degradation of cognate messenger RNA (mRNA). In a search for new components of the RNAi machinery in Dictyostelium, a new gene was identified, which was called helF. HelF is a putative RNA helicase, which shows a high homology to the helicase domain of Dicer, to the helicase domain of Dictyostelium RdRP and to the C. elegans gene drh-1, that codes for a dicer related DExH-box RNA helicase, which is required for RNAi. The aim of the present Ph.D. work was to investigate the role of HelF in PTGS, either induced by RNAi or asRNA. A genomic disruption of the helF gene was performed, which resulted in a distinct mutant morphology in late development. The cellular localization of the protein was elucidated by creating a HelF-GFP fusion protein, which was found to be localized in speckles in the nucleus. The involvement of HelF in the RNAi mechanism was studied. For this purpose, RNAi was induced by transformation of RNAi hairpin constructs against four endogenous genes in wild type and HelF- cells. The silencing efficiency was strongly enhanced in the HelF K.O. strain in comparison with the wild type. One gene, which could not be silenced in the wild type background, was successfully silenced in HelF-. When the helF gene was disrupted in a secondary transformation in a non-silenced strain, the silencing efficiency was strongly improved, a phenomenon named here “retrosilencing”. Transcriptional run-on experiments revealed that the enhanced gene silencing in HelF- was a posttranscriptional event, and that the silencing efficiency depended on the transcription levels of hairpin RNAs. In HelF-, the threshold level of hairpin transcription required for efficient silencing was dramatically lowered. The RNAi-mediated silencing was accompanied by the production of siRNAs; however, their amount did not depend on the level of hairpin transcription. These results indicated that HelF is a natural suppressor of RNAi in Dictyostelium. In contrast, asRNA mediated gene silencing was not enhanced in the HelF K.O, as shown for three tested genes. These results confirmed previous observations (H. Martens and W. Nellen, unpublished) that although similar, RNAi and asRNA mediated gene silencing mechanisms differ in their requirements for specific proteins. In order to characterize the function of the HelF protein on a molecular level and to study its interactions with other RNAi components, in vitro experiments were performed. Besides the DEAH-helicase domain, HelF contains a double-stranded RNA binding domain (dsRBD) at its N-terminus, which showed high similarity to the dsRBD domain of Dicer A from Dictyostelium. The ability of the recombinant dsRBDs from HelF and Dicer A to bind dsRNA was examined and compared. It was shown by gel-shift assays that both HelF-dsRBD and Dicer-dsRBD could bind directly to long dsRNAs. However, HelF-dsRBD bound more efficiently to dsRNA with imperfect matches than to perfect dsRNA. Both dsRBDs bound specifically to a pre-miRNA substrate (pre-let-7). The results suggested that most probably there were two binding sites for the proteins on the pre-miRNA substrate. Moreover, it was shown that HelF-dsRBD and Dicer-dsRBD have siRNA-binding activity. The affinities of the two dsRBDs to the pre-let-7 substrate were also examined by plasmon surface resonance analyses, which revealed a 9-fold higher binding affinity of the Dicer-dsRBD to pre-let-7 compared to that of the HelF-dsRBD. The binding of HelF-dsRBD to the pre-let-7 was impaired in the presence of Mg2+, while the Dicer-dsRBD interaction with pre-let-7 was not influenced by the presence of Mg2+. The results obtained in this thesis can be used to postulate a model for HelF function. In this, HelF acts as a nuclear suppressor of RNAi in wild type cells by recognition and binding of dsRNA substrates. The protein might act as a surveillance system to avoid RNAi initiation by fortuitous dsRNA formation or low abundance of dsRNA trigger. If the protein acts as an RNA helicase, it could unwind fold-back structures in the nucleus and thus lead to decreased RNAi efficiency. A knock-out of HelF would result in initiation of the RNAi pathway even by low levels of dsRNA. The exact molecular function of the protein in the RNAi mechanism still has to be elucidated. RNA interferenz (RNAi) ist ein in jüngster Zeit entdeckter Mechanismus, bei dem doppelsträngige RNA Moleküle (dsRNA) eine Homologie-abhängige Degradation einer verwandten messenger-RNA (mRNA) auslösen. Auf der Suche nach neuen Komponenten der RNAi-Maschinerie in Dictyostelium konnte ein neues Gen (helF) identifiziert werden. HelF ist eine putative RNA-Helikase mit einer hohen Homologie zur Helikasedomäne der bekannten Dicerproteine, der Helikasedomäne der Dictyostelium RdRP und zu dem C. elegans Gen drh-1, welches für eine Dicer-bezogene DExH-box RNA Helikase codiert, die am RNAi-Mechanismus beteiligt ist. Das Ziel dieser Arbeit war es, die Funktion von HelF im Zusammenhang des RNAi oder asRNA induzierten PTGS zu untersuchen. Es wurde eine Unterbrechung des helF-Gens auf genomischer Ebene (K.O.) vorgenommen, was bei den Mutanten zu einer veränderten Morphologie in der späten Entwicklung führte. Die Lokalisation des Proteins in der Zelle konnte mit Hilfe einer GFP-Fusion analysiert werden und kleinen Bereichen innerhalb des Nukleus zugewiesen werden. Im Weiteren wurde der Einfluss von HelF auf den RNAi-Mechanismus untersucht. Zu diesem Zweck wurde RNAi durch Einbringen von RNAi Hairpin-Konstrukten gegen vier endogene Gene im Wiltypstamm und der HelF--Mutante induziert. Im Vergleich zum Wildtypstamm konnte im HelF--Mutantenstamm eine stark erhöhte „Silencing“-Effizienz nachgewiesen werden. Ein Gen, welches nach RNAi Initiation im Wildtypstamm unverändert blieb, konnte im HelF--Mutantenstamm erfolgreich stillgelegt werden. Durch sekundäres Einführen einer Gendisruption im helF-Locus in einen Stamm, in welchem ein Gen nicht stillgelegt werden konnte, wurde die Effizienz des Stilllegens deutlich erhöht. Dieses Phänomen wurde hier erstmals als „Retrosilencing“ beschrieben. Mit Hilfe von transkriptionellen run-on Experimenten konnte belegt werden, dass es sich bei dieser erhöhten Stilllegungseffizienz um ein posttranskriptionelles Ereignis handelte, wobei die Stillegungseffizienz von der Transkriptionsstärke der Hairpin RNAs abhängt. Für die HelF--Mutanten konnte gezeigt werden, dass der Schwellenwert zum Auslösen eines effizienten Stillegens dramatisch abgesenkt war. Obwohl die RNAi-vermittelte Genstilllegung immer mit der Produktion von siRNAs einhergeht, war die Menge der siRNAs nicht abhängig von dem Expressionsniveau des Hairpin-Konstruktes. Diese Ergebnisse legen nahe, dass es sich bei der HelF um einen natürlichen Suppressor des RNAi-Mechanismus in Dictyostelium handelt. Im Gegensatz hierzu war die as-vermittelte Stilllegung von drei untersuchten Genen im HelF-K.O. im Vergleich zum Wildyp unverändert. Diese Ergebnisse bestätigten frühere Beobachtungen (H. Martens und W. Nellen, unveröffentlicht), wonach die Mechanismen für RNAi und asRNA-vermittelte Genstilllegung unterschiedliche spezifische Proteine benötigen. Um die Funktion des HelF-Proteins auf der molekularen Ebene genauer zu charakterisieren und die Interaktion mit anderen RNAi-Komponenten zu untersuchen, wurden in vitro Versuche durchgeführt. Das HelF-Protein enthält, neben der DEAH-Helikase-Domäne eine N-terminale Doppelstrang RNA bindende Domäne (dsRBD) mit einer hohen Ähnlichkeit zu der dsRBD des Dicer A aus Dictyostelium. Die dsRNA-Bindungsaktivität der beiden dsRBDs aus HelF und Dicer A wurde analysiert und verglichen. Es konnte mithilfe von Gel-Retardationsanalysen gezeigt werden, dass sowohl HelF-dsRBD als auch Dicer-dsRBD direkt an lange dsRNAs binden können. Hierbei zeigte sich, dass die HelF-dsRBD eine höhere Affinität zu einem imperfekten RNA-Doppelstrang besitzt, als zu einer perfekt gepaarten dsRNA. Für beide dsRBDs konnte eine spezifische Bindung an ein pre-miRNA Substrat nachgewiesen werden (pre-let-7). Dieses Ergebnis legt nah, dass es zwei Bindestellen für die Proteine auf dem pre-miRNA Substrat gibt. Überdies hinaus konnte gezeigt werden, dass die dsRBDs beider Proteine eine siRNA bindende Aktivität besitzen. Die Affinität beider dsRBDs an das pre-let-7 Substrat wurde weiterhin mit Hilfe der Plasmon Oberflächen Resonanz untersucht. Hierbei konnte eine 9-fach höhere Bindeaffinität der Dicer-dsRBD im Vergleich zur HelF-dsRBD nachgewiesen werden. Während die Bindung der HelF-dsRBD an das pre-let-7 durch die Anwesenheit von Mg2+ beeinträchtigt war, zeigte sich kein Einfluß von Mg2+ auf das Bindeverhalten der Dicer-dsRBD. Mit Hilfe der in dieser Arbeit gewonnen Ergebnisse lässt sich ein Model für die Funktion von HelF postulieren. In diesem Model wirkt HelF durch Erkennen und Binden von dsRNA Substraten als Suppressor von der RNAi im Kern. Das Protein kann als Überwachungsystem gegen eine irrtümliche Auslösung von RNAi wirken, die durch zufällige dsRNA Faltungen oder eine zu geringe Häufigkeit der siRNAs hervorgerufen sein könnte. Falls das Protein eine Helikase-Aktivität besitzt, könnte es rückgefaltete RNA Strukturen im Kern auflösen, was sich in einer verringerten RNAi-Effizienz wiederspiegelt. Durch Ausschalten des helF-Gens würde nach diesem Modell eine erfolgreiche Auslösung von RNAi schon bei sehr geringer Mengen an dsRNA möglich werden. Das Modell erlaubt, die exakte molekulare Funktion des HelF-Proteins im RNAi-Mechanismus weiter zu untersuchen.

Effect of dressing on high-order harmonic generation in vibrating H2 molecules

Wir entwickeln die Starkfeldnäherung für die Erzeugung hoher Harmonischer in Wasserstoffmolekülen, wobei die Vibrationsbewegung berücksichtigt wird, sowie die laserinduzierte Kopplung zwischen den beiden untersten Born-Oppenheimer-Zuständen im Molekülion, das durch die anfängliche Ionisation des Moleküls erzeugt wird. Wir zeigen, dass die Kopplung bei längeren Laserwellenlängen (≈ 2 μm) wichtig wird und zu einer Reduzierung der Erzeugung von Harmonischen führt, sowie zu einer Änderung des Verhältnisses von Harmonischen in verschiedenen Isotopen. ----------------------------------------------------------------------- We develop the strong-field approximation for high-order harmonic generation in hydrogen molecules, including the vibrational motion and the laser-induced coupling of the lowest two Born-Oppenheimer states in the molecular ion that is created by the initial ionization of the molecule. We show that the field dressing becomes important at long laser wavelengths (≈ 2 μm), leading to an overall reduction of harmonic generation and modifying the ratio of harmonic signals from different isotopes.

The PHD: A Planar, Harmonic Drive Robot for Joint Torque Control

This thesis details the development of a model of a seven degree of freedom manipulator for position control. Then, it goes on to discuss the design and construction of a the PHD, a robot built to serve two purposes: first, to perform research on joint torque control schemes, and second, to determine the important dynamic characteristics of the Harmonic Drive. The PHD, is a planar, three degree of freedom arm with torque sensors integral to each joint. Preliminary testing has shown that a simple linear spring model of the Harmonic Drive's flexibility is suitable in many situations.

Counterpoise-corrected geometries and harmonic frequencies of N-body clusters: application to (HF)n (n=3,4)

A study was conducted on the methods of basis set superposition error (BSSE)-free geometry optimization and frequency calculations in clusters larger than a dimer. In particular, three different counterpoise schemes were critically examined. It was shown that the counterpoise-corrected supermolecule energy can be easily obtained in all the cases by using the many-body partitioning of energy

Harmonic force fields from scaled SCF calculations: Program ASYM40

We report an extended version of our normal coordinate program ASYM40, which may be used to transform Cartesian force constants from ab initio calculations to a force field in nonredundant internal (symmetry) coordinates. When experimental data are available, scale factors for the theoretical force field may then be optimized by least-squares refinement. The alternative of refining an empirical force field to fit a wide variety of data, as with the previous version ASYM20, has been retained. We compare the results of least-squares refinement of the full harmonic force field with least-squares refinement of only the scale factors for an SCF calculated force field and conclude that the latter approach may be useful for large molecules where more sophisticated calculations are impractical. The refinement of scale factors for a theoretical force field is also useful when there are only limited spectroscopic data. The program will accept ab initio calculated force fields from any program that presents Cartesian force constants as output. The program is available through Quantum Chemistry Program Exchange.

Analytical potentials for triatomic molecules IX. The prediction of anharmonic force constants from potential energy surfaces based on harmonic force fields and dissociation energies for SO2 and O3

Analytical potential energy functions which are valid at all dissociation limits have been derived for the ground states of SO2 and O3. The procedure involves minimizing the errors between the observed vibrational spectra and spectra calculated by a variational procedure. Good agreement is obtained between the observed and calculated spectra for both molecules. Comparisons are made between anharmonic force fields, previously determined from the spectral data, and the force fields obtained by differentiating the derived analytical functions at the equilibrium configurations.

Microwave spectrum of CHD2CN and CHD2NC, and the harmonic force field and rz structure of methyl cyanide and isocyanide

The microwave spectra of CHD2CN and CHD2NC have been measured from 18 to 40 GHz; about 20 type A and 30 type C transitions have been observed for each molecule. These have been fitted to a Hamiltonian using 3 rotational constants, and 5 quartic and 4 sextic distortion constants, in the IrS reduction of Watson [in “Vibrational spectra and structure” Vol. 6 (1977)]; the standard error of the fit is 26 kHz. For methyl cyanide the 5 quartic distortion constants have been used to further refine the recent harmonic force field of Duncan et al. [J. Mol. Spectrosc. 69, 123 (1978)], but the changes are small. Finally, for both molecules, the harmonic force field has been used to determine zero point average moments of inertia Iz from the ground state rotational constants for many isotopic species, and these have been used to determine an rz structure. The results are compared with rs structure calculations.

Redundant coordinates in harmonic force-field calculations

Redundancy relations between vibrational coordinates may be linear (as for rectilinear coordinates used in deriving a G matrix), or non-linear (as for curvilinear coordinates used in formulating model force fields). It is shown that geometrically defined internal coordinates are necessarily curvilinear. Hence it is shown that linear force constants can occur in model force field calculations involving redundant coordinates, in disagreement with the recent proposal of Gussoni and Zerbi.

HMS - Harmonic Motion by Shadows

A problem is discussed which is generated by shadows and which is a generalization of simple harmonic motion.

Vibration-rotation spectra and the harmonic force field of HOCl

Vibration-rotation spectra of HOCl have been measured at a resolution of 0.05 cm−1 to determine vibration rotation constants, and 35–37 Cl isotope shifts in the vibration frequencies. The spectrum of DOCl has also been recorded, and a preliminary analysis for the band origins has been made. The vibrational frequency data and centrifugal distortion constants have been used to determine the harmonic force field in a least-squares refinement; the force field obtained also gives a good fit to data on the vibrational contributions to the inertial defect. The equilibrium rotational constants of HOCl have been obtained, and an equilibrium structure has been estimated.

Nitrous acid: vibrational frequency shifts due to 15N substitution, and the harmonic force field

Vibration rotation spectra of HO15 NO and DO15 NO have been measured at a resolution of 0•04 cm-1 to determine the isotopic shifts in the vibrational band origins. These have been used together with recently determined data on the vibrational band origins, Coriolis constants, and centrifugal distorition constants, to determine the harmonic force field of both cis and trans nitrous acid in least squares refinement calculations. The results are discussed in relation to recent ab initio calculations, the inertia defects, and the torsional potential function.

The harmonic force field and rz structure of HNCO

The presently available microwave, millimeter wave, and far-infrared data of five isotopic species of isocyanic acid, namely, HNCO, H15NCO, HN13CO, HNC18O, and DNCO, have been used to obtain improved values of the ground-state rotational constants, the five quartic distortion constants, and some higher-order distortion constants in the Ir S reduced Hamiltonian of Watson. The appropriate planarity relation among the quartic centrifugal distortion constants has been imposed in the fitting procedure. The general harmonic force field of isocyanic acid has been determined using all existing data, and assuming a trans bent equilibrium geometry of the molecule with an NCO angle of 170°. Finally an rz structure has been obtained using the Az, Bz, and Cz rotational constants of five isotopic species. The bending of the NCO chain is found to be 8° in the trans configuration.

The infrared spectrum, equilibrium structure and harmonic and anharmonic force field of thioborine, HBS

Infrared spectra of the two stretching fundamentals of both HBS and DBS have been observed, using a continuous flow system through a multiple reflection long path cell at a pressure around 1 Torr and a Nicolet Fourier Transform spectrometer with a resolution of about 0•1 cm-1. The v3 BS stretching fundamental of DBS, near 1140 cm-1, is observed in strong Fermi resonance with the overtone of the bend 2v2. The bending fundamental v2 has not been observed and must be a very weak band. The analysis of the results in conjunction with earlier work gives the equilibrium structure (re(BH) = 1•1698(12) , re(BS) = 1•5978(3) ) and the harmonic and anharmonic force field.

Intensities in the v7, v8 and v9 bands of CH2NH, and the harmonic force field of methyleneimine

A high resolution Fourier transform infrared spectrum of methyleneimine, HN=CH2, has been obtained in the gas phase in the region 700 to 1300 cm−1. The rovibrational line intensities of the three lowest fundamentals ν7 (A′), ν8 (A″), and ν9 (A″) have been simulated including all Coriolis interactions between the three bands, and by fitting the observed spectrum the relative signs and magnitudes of the vibrational transition moments have been determined. All of the available spectroscopic data have been used to determine the harmonic force field of methyleneimine.