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Bertram K.,MPI for Biophysical Chemistry | Agafonov D.E.,MPI for Biophysical Chemistry | Liu W.-T.,MPI for Biophysical Chemistry | Dybkov O.,MPI for Biophysical Chemistry | And 7 more authors.
Nature | Year: 2017

Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C∗). The architecture of the catalytic U2-U6 ribonucleoprotein (RNP) core of the human C∗ spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C∗ the branched intron region is separated from the catalytic centre by approximately 20 Å, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 Å from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C∗ complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.


Uzgun S.,Ludwig Maximilians University of Munich | Uzgun S.,Free University of Berlin | Akdemir O.,Fraunhofer Institute for Applied Polymer Research | Hasenpusch G.,Ludwig Maximilians University of Munich | And 12 more authors.
Biomacromolecules | Year: 2010

Oligo(ethylene glycol) methyl ether methacrylates (OEGMA) of various chain lengths (i.e., 9, 23, or 45 EG units) and N,N-dimethylaminoethyl methacrylate (DMAEMA) were copolymerized by atom transfer radical polymerization (ATRP), yielding well-defined P(DMAEMA-co-OEGMA) copolymers with increasing OEGMA molar fractions (FOEGMA) but a comparable degree of polymerization (DP ∼ 120). Increase of both FOEGMA and OEGMA chain lengths correlated inversely with gene vector size, morphology, and zeta potential. P(DMAEMAco-OEGMA) copolymers prevented gene vector aggregation at high plasmid DNA (pDNA) concentrations in isotonic solution and did not induce cytotoxicity even at high concentrations. Transfection efficiency of the most efficient P(DMAEMA-co-OEGMA) copolymers was found to be > 10-fold lower compared with branched polyethylenimine (PEI) 25 kDa. Although OEGMA copolymerization largely reduced gene vector binding with the cell surface, cellular internalization of the bound complexes was less affected. These observations suggest that inefficient endolysosomal escape limits transfection efficiency of P(DMAEMA-co-OEGMA) copolymer gene vectors. Despite this observation, optimized p(DMAEMA-co-OEGMA) gene vectors remained stable under conditions for in vivo application leading to 7-fold greater gene expression in the lungs compared with PEI. Tailor-made P(DMAEMA-co-OEGMA) copolymers are promising nonviral gene transfer agents that fulfill the requirements for successful in vivo gene delivery. © 2010 American Chemical Society.


Agafonov D.E.,MPI for Biophysical Chemistry | Van Santen M.,MPI for Biophysical Chemistry | Kastner B.,MPI for Biophysical Chemistry | Dube P.,3D Electronic Cryomicroscopy Group | And 4 more authors.
RNA | Year: 2016

The ATP analog ATPγS inhibits pre-mRNA splicing in vitro, but there have been conflicting reports as to which step of splicing is inhibited by this small molecule and its inhibitory mechanism remains unclear. Here we have dissected the effect of ATPγS on pre-mRNA splicing in vitro. Addition of ATPγS to splicing extracts depleted of ATP inhibited both catalytic steps of splicing. At ATPγS concentrations =0.5 mM, precatalytic B complexes accumulate, demonstrating a block prior to or during the spliceosome activation stage. Affinity purification of the ATPγS-stalled B complexes (BATPγS) and subsequent characterization of their abundant protein components by 2D gel electrophoresis revealed that BATPγS complexes are compositionally more homogeneous than B complexes previously isolated in the presence of ATP. In particular, they contain little or no Prp19/CDC5L complex proteins, indicating that these proteins are recruited after assembly of the precatalytic spliceosome. Under the electron microscope, BATPγS complexes exhibit a morphology highly similar to B complexes, indicating that the ATPγS-induced block in the transformation of the B to Bact complex is not due to a major structural defect. Likely mechanisms whereby ATPγS blocks spliceosome assembly at the activation stage, including inhibition of the RNA helicase Brr2, are discussed. Given their more homogeneous composition, B complexes stalled by ATPγS may prove highly useful for both functional and structural analyses of the precatalytic spliceosome and its conversion into an activated Bact spliceosomal complex. © 2016 Domanski et al.


Samwer M.,MPI for Biophysical Chemistry | Dehne H.-J.,MPI for Biophysical Chemistry | Spira F.,Austrian Academy of Sciences | Kollmar M.,Systems Biology of Motor Proteins Group | And 3 more authors.
EMBO Journal | Year: 2013

Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-β and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis. © 2013 European Molecular Biology Organization.


News Article | October 6, 2016
Site: phys.org

The assembly of proteins to form larger macromolecular structures within cells is linked to ribosomes and thus to their synthesis through the process of translation. This is the result of recent studies by scientists from the University of Würzburg and the Max Planck Institute (MPI) for Biophysical Chemistry in Göttingen. Ribosomes adopt the role of a quality "checkpoint" in this context: They make sure that newly built proteins are directly fed into the production lines of macromolecular complexes. The researchers have published their results in the journals Cell Reports and the EMBO Journal. "Think of it as LEGO bricks at the molecular level: One brick is attached to the next until the product is finished. If only one defective or wrong brick is used, the entire building may be compromised as a result." Professor Utz Fischer holds the Chair for Biochemistry at the University of Würzburg. For many years, he has been researching how the so-called "macromolecular machines" are assembled inside cells. His research focus is spliceosomes: these large RNA-protein complexes are an essential part of gene expression within cells. Their job is to remove those sections in the messenger RNA that do not contain any protein-encoding information and unite the relevant sections carrying the information. In their latest work, Fischer's team with colleagues from Göttingen have figured out the entire production sequence of so-called snRNPs, the subunits that make up the spliceosomes – from the synthesis of the single components to their assembly and combination into the functioning machine. They identified a hitherto unexpected player in this process: the ribosome. Ribosomes are the entities where genetic information (in the form of mRNA) is translated into proteins. How these single proteins subsequently assemble to form macromolecular machines had not been fully deciphered until recently. One thing was however certain: The notion that ribosomes release individual proteins into the cell interior where they roam about in search of the matching counterpart could definitely not be true. "The interior of cells is much too crowded for this", says Ashwin Chari, project group leader at the MPI for Biophysical Chemistry. It would take the proteins far too long to form complexes; they would get stuck forming erroneous structures and aggregate as a result causing severe diseases, such as Alzheimer´s, in the worst case. "Therefore, a mechanism has to exist in living cells, which protects the newly synthesised proteins at the ribosome and only allows them to associate with their correct counterpart," says Elham Paknia, who experimentally headed-up the entire project. The scientists have been able to prove that this assumption is actually true for the first time. Accordingly, the ribosome does not randomly release the proteins into the cytosol after synthesis, but holds them back until specific helpers, so-called chaperones, deliver the matching counterparts. In doing so, the ribosome assures that only the one intended structure is formed and therefore adopts the role of a "quality inspector" in addition to production. "Extremely high quality criteria" are a basic principle of cellular function according to the scientists. They were able to demonstrate that often more chaperones are involved in assembling the macromolecular machines than building blocks. This also becomes evident when looking at the cell's energy balance: "The catalysis itself requires much less resources than the regulation and control," says Utz Fischer. The huge effort is justified: Errors during spliceosome assembly, for instance, trigger spinal muscular atrophy. The disorder is characterised by the loss of motor neurons especially in the spinal cord causing muscle wasting and paralysis of affected individuals. Protein misfolding is also believed to cause various other diseases from diabetes to Alzheimer's. Even though Fischer and his colleagues in Göttingen have elucidated the ribosome's role in assembling macromolecules by using the building blocks of the spliceosome as a model system, the researchers are convinced that this is not an isolated case. "We have good reason to believe that this is a general principle", Chari says. After all, other macromolecules, too, need to be synthesised under the same crowded circumstances while maintaining the highest safety standards. Explore further: Research suggests new direction for tissue engineering and cancer therapeutics More information: Elham Paknia et al. The Ribosome Cooperates with the Assembly Chaperone pICln to Initiate Formation of snRNPs, Cell Reports (2016). DOI: 10.1016/j.celrep.2016.08.047


Rauhut R.,MPI for Biophysical Chemistry | Fabrizio P.,MPI for Biophysical Chemistry | Dybkov O.,MPI for Biophysical Chemistry | Hartmuth K.,MPI for Biophysical Chemistry | And 9 more authors.
Science | Year: 2016

The activated spliceosome (Bact) is in a catalytically inactive state and is remodeled into a catalytically active machine by the RNA helicase Prp2, but the mechanism is unclear. Here we describe a 3D electron cryomicroscopy structure of the S. cerevisiae Bact complex at 5.8 Å resolution. Our model reveals that in Bact the catalytic U2/U6 RNA-Prp8 ribonucleoprotein core is already established, and the 5′ splice site (ss) is oriented for step 1 catalysis but occluded by protein. The first step nucleophile - the branchsite adenosine - is sequestered within the Hsh155 HEAT domain and is held 50 Å away from the 5′ss. Our structure suggests that Prp2 ATPase-mediated remodeling leads to conformational changes in Hsh155’s HEAT domain that liberate the first step reactants for catalysis. © 2016 American Association for the Advancement of Science.


PubMed | University of Gottingen, 3D Electronic Cryomicroscopy Group, Bioanalytical Mass Spectrometry and MPI for Biophysical Chemistry
Type: | Journal: Nature | Year: 2017

Spliceosome rearrangements facilitated by RNA helicase Prp16 before catalytic step 2 of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after Prp16 action (C*). The architecture of the catalytic U2-U6 RNP core of the human C* spliceosome is highly similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated (by ~20) from the catalytic centre, and its position close to the U6 snRNA ACAGA box is stabilised by interactions with the Prp8 RNase H-like and Prp17 WD40 domains. RNA helicase Prp22 is located about 100 from the catalytic centre, suggesting that it destabilises the spliced mRNA after step 2 from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by Prp16, including a large-scale movement of the U2 snRNP.


Stark H.,MPI for Biophysical Chemistry | Stark H.,University of Gottingen
Methods in Enzymology | Year: 2010

Here, we review the GraFix (Gradient Fixation) method to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy (cryo-EM). During GraFix, macromolecules undergo a weak, intramolecular chemical cross-linking while being purified by density gradient ultracentrifugation. GraFix-stabilized particles can be used directly for negative-stain cryo-EM or, after a brief buffer-exchange step, for unstained cryo-EM. This highly reproducible method has proved to dramatically reduce problems in heterogeneity due to particle dissociation during EM grid preparation. Additionally, there is often an appreciable increase in particles binding to the carbon support film. This and the fact that binding times can be drastically increased, with no apparent disruption of the native structures of the macromolecules, makes GraFix a method of choice when preparing low-abundance complexes for cryo-EM. The higher sample quality following GraFix purification is evident when examining raw images, which usually present a low background of fragmented particles, good particle dispersion, and high-contrast, well-defined particles. Setting up the GraFix method is straightforward, and the resulting improvement in sample homogeneity has been beneficial in successfully obtaining the 3D structures of numerous macromolecular complexes by cryo-EM in the past few years. © 2010 Elsevier Inc.


PubMed | University of Gottingen, 3D Electronic Cryomicroscopy Group and MPI for Biophysical Chemistry
Type: Journal Article | Journal: RNA (New York, N.Y.) | Year: 2016

The ATP analog ATPS inhibits pre-mRNA splicing in vitro, but there have been conflicting reports as to which step of splicing is inhibited by this small molecule and its inhibitory mechanism remains unclear. Here we have dissected the effect of ATPS on pre-mRNA splicing in vitro. Addition of ATPS to splicing extracts depleted of ATP inhibited both catalytic steps of splicing. At ATPS concentrations 0.5 mM, precatalytic B complexes accumulate, demonstrating a block prior to or during the spliceosome activation stage. Affinity purification of the ATPS-stalled B complexes (B(ATPS)) and subsequent characterization of their abundant protein components by 2D gel electrophoresis revealed that B(ATPS) complexes are compositionally more homogeneous than B complexes previously isolated in the presence of ATP. In particular, they contain little or no Prp19/CDC5L complex proteins, indicating that these proteins are recruited after assembly of the precatalytic spliceosome. Under the electron microscope, B(ATPS) complexes exhibit a morphology highly similar to B complexes, indicating that the ATPS-induced block in the transformation of the B to B(act) complex is not due to a major structural defect. Likely mechanisms whereby ATPS blocks spliceosome assembly at the activation stage, including inhibition of the RNA helicase Brr2, are discussed. Given their more homogeneous composition, B complexes stalled by ATPS may prove highly useful for both functional and structural analyses of the precatalytic spliceosome and its conversion into an activated B(act) spliceosomal complex.


Westphal V.,MPI for Biophysical Chemistry | Hell S.W.,MPI for Biophysical Chemistry
Optics InfoBase Conference Papers | Year: 2011

Diffraction-unlimited imaging is one of the emerging fields in microscopy. In all of these techniques, fluorophore switching is key. The first technique developed is STED, recent advances will be shown. © 2011 OSA.

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