Regulation of Prp43-mediated disassembly of spliceosomes by its cofactors Ntr1 and Ntr2.

The DEAH-box NTPase Prp43 disassembles spliceosomes in co-operation with the cofactors Ntr1/Spp382 and Ntr2, forming the NTR complex. How Prp43 is regulated by its cofactors to discard selectively only intron-lariat spliceosomes (ILS) and defective spliceosomes and to prevent disassembly of earlier and properly assembled/wild-type spliceosomes remains unclear. First, we show that Ntr1's G-patch motif (Ntr1GP) can be replaced by the GP motif of Pfa1/Sqs1, a Prp43's cofactor in ribosome biogenesis, demonstrating that the specific function of Ntr1GP is to activate Prp43 for spliceosome disassembly and not to guide Prp43 to its binding site in the spliceosome. Furthermore, we show that Ntr1's C-terminal domain (CTD) plays a safeguarding role by preventing Prp43 from disrupting wild-type spliceosomes other than the ILS. Ntr1 and Ntr2 can also discriminate between wild-type and defective spliceosomes. In both type of spliceosomes, Ntr1-CTD impedes Prp43-mediated disassembly while the Ntr1GP promotes disassembly. Intriguingly, Ntr2 plays a specific role in defective spliceosomes, likely by stabilizing Ntr1 and allowing Prp43 to enter a productive interaction with the GP motif of Ntr1. Our data indicate that Ntr1 and Ntr2 act as 'doorkeepers' and suggest that both cofactors inspect the RNP structure of spliceosomal complexes thereby targeting suboptimal spliceosomes for Prp43-mediated disassembly

Structural insights into how Prp5 proofreads the pre-mRNA branch site

During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1-4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2-BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2-BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2-BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing

Structural insights into the mechanism of the DEAH-box RNA helicase Prp43.

The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43ATP-analogRNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a ?-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases

Mesure ultra-rapide à distance et sans fil de la surpression aérienne en environnement extrême

National audienceCette communication propose une solution sans fil et analogique pour la mesure de surpression aérienne en environnement extrême. La solution sans fil proposée apporte de nombreux avantages en termes de simplicité, compacité et robustesse de mesure par rapport à la solution filaire classiquement utilisée. Elle offre également la possibilité de réaliser des mesures de pression dans la boule de feu lors d'une explosion. Après une étude système de la liaison sans fil proposée, le transducteur du capteur de pression dynamique sera présenté. Ses performances seront évaluées sous tube à choc

PVDF Sensors for Dynamic Pressure Metrology in Extreme Environment Capteurs PVDF pour la mesure de pression dynamique en environnement extrême

International audienceHigh pressure range, extreme temperature and fast dynamic transient create an extreme environment for accurate pressure measurement. Military applications have a real need for dynamic pressure sensor in order to measure pressure in air blast experiments. Such experiment is a typical extreme pressure measurement application with high temperature and pressure and fast dynamic transient [1]. Structure vulnerability and explosive optimisation need accurate experimental value to improve the accuracy of numerical models [2]. Most of known pressure sensors present a full-scale pressure measurement and bandwidth that are not sufficient for sensing fast pressure variation with high amplitude. The high cutoff frequency of these sensors creates highly undesirable harmonics distortion on the signal and a low (microsecond) rise time. This communication proposes the model and the design of dynamic pressure sensor with nanosecond rise time working in the GPa range in extreme temperature conditions. Based on well-known piezoelectric polymer technologies PVDF [3], key issues related to these sensors such as the packaging, impact of cables and role of the conditioning electronic are addressed in this communication. Based on acoustic/electrical equations analogies [4] the circuit model implementable in Spice software is reported for simulating the sensor response for different excitations such as pulse, unit step or sine function. Thermal protection layers, backing materials, measurement cables and conditioning electronics are taking into account in the sensor simulation and their impacts on the sensor performances are discussed. Moreover the bandwidth, rise time and sensitivity are derived from the proposed simulation model and design rules are given. Finally a PolyVinylidene DiFluoride (PVDF) sensor prototype is presented and shock tube experiment is described [5]. Different shock wave amplitude with nanosecond rise time has been recorded by the designed sensor and rise time, bandwidth and sensitivity have been measured and compared with ones provided by various commercial pressure sensors. The proposed sensor has been calibrated with this shock tube method. Dynamic uncertainties of the sensor have been evaluated based on the work of the EURAMET project [6]

Structural and functional analysis of the RNA helicase Prp43 from the thermophilic eukaryote Chaetomium thermophilum

RNA helicases are indispensable for all organisms in each domain of life and have implications in numerous cellular processes. The DEAH-box RNA helicase Prp43 is involved in pre-mRNA splicing as well as rRNA maturation. Here, the crystal structure of Chaetomium thermophilum Prp43 at 2.9 angstrom resolution is revealed. Furthermore, it is demonstrated that Prp43 from C. thermophilum is capable of functionally replacing its orthologue from Saccharomyces cerevisiae in spliceosomal disassembly assays

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system.

The spliceosome is a single-turnover enzyme that needs to be dismantled after catalysis to both release the mRNA and recycle small nuclear ribonucleoproteins (snRNPs) for subsequent rounds of pre-mRNA splicing. The RNP remodeling events occurring during spliceosome disassembly are poorly understood, and the composition of the released snRNPs are only roughly known. Using purified components in vitro, we generated post-catalytic spliceosomes that can be dissociated into mRNA and the intron-lariat spliceosome (ILS) by addition of the RNA helicase Prp22 plus ATP and without requiring the step 2 proteins Slu7 and Prp18. Incubation of the isolated ILS with the RNA helicase Prp43 plus Ntr1/Ntr2 and ATP generates defined spliceosomal dissociation products: the intron-lariat, U6 snRNA, a 20–25S U2 snRNP containing SF3a/b, an 18S U5 snRNP, and the “nineteen complex” associated with both the released U2 snRNP and intron-lariat RNA. Our system reproduces the entire ordered disassembly phase of the spliceosome with purified components, which defines the minimum set of agents required for this process. It enabled us to characterize the proteins of the ILS by mass spectrometry and identify the ATPase action of Prp43 as necessary and sufficient for dissociation of the ILS without the involvement of Brr2 ATPase

Incident pressure measurement in air blast using wireless sensors

International audienceThis paper presents the wireless measurement of the incident pressure inside the fire ball of an air blast shock wave. The wireless sensor setup and the experimental configuration are described. The wired and wireless experimental results are compared and analyszd. To our best knowledge, it is the first time that a wireless device takes place inside the fireball of an explosion for the measurement of the incident pressure in such harsh environment