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

Nature ◽  
2021 ◽  
Author(s):  
Zhenwei Zhang ◽  
Norbert Rigo ◽  
Olexandr Dybkov ◽  
Jean-Baptiste Fourmann ◽  
Cindy L. Will ◽  
...  

AbstractDuring 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.

1999 ◽  
Vol 32 (3) ◽  
pp. 241-284 ◽  
Author(s):  
William G. Scott

1. How do ribozymes work? 2412. The hammerhead RNA as a prototype ribozyme 2422.1 RNA enzymes 2422.2 Satellite self-cleaving RNAs 2422.3 Hammerhead RNAs and hammerhead ribozymes 2443. The chemical mechanism of hammerhead RNA self-cleavage 2463.1 Phosphodiester isomerization via an SN2(P) reaction 2473.2 The canonical role of divalent metal ions in the hammerhead ribozyme reaction 2513.3 The hammerhead ribozyme does not actually require metal ions for catalysis 2543.4 Hammerhead RNA enzyme kinetics 2574. Sequence requirements for hammerhead RNA self-cleavage 2604.1 The conserved core, mutagenesis and functional group modifications 2604.2 Ground-state vs. transition-state effects 2615. The three-dimensional structure of the hammerhead ribozyme 2625.1 Enzyme–inhibitor complexes 2625.2 Enzyme–substrate complex in the initial state 2645.3 Hammerhead ribozyme self-cleavage in the crystal 2645.4 The requirement for a conformational change 2655.5 Capture of conformational intermediates using crystallographic freeze-trapping 2665.6 The structure of a hammerhead ribozyme ‘early’ conformational intermediate 2675.7 The structure of a hammerhead ribozyme ‘later’ conformational intermediate 2685.8 Is the conformational change pH dependent? 2695.9 Isolating the structure of the cleavage product 2715.10 Evidence for and against additional large-scale conformation changes 2745.11 NMR spectroscopic studies of the hammerhead ribozyme 2786. Concluding remarks 2807. Acknowledgements 2818. References 2811. How do ribozymes work? 241The discovery that RNA can be an enzyme (Guerrier-Takada et al. 1983; Zaug & Cech, 1986) has created the fundamental question of how RNA enzymes work. Before this discovery, it was generally assumed that proteins were the only biopolymers that had sufficient complexity and chemical heterogeneity to catalyze biochemical reactions. Clearly, RNA can adopt sufficiently complex tertiary structures that make catalysis possible. How does the three- dimensional structure of an RNA endow it with catalytic activity? What structural and functional principles are unique to RNA enzymes (or ribozymes), and what principles are so fundamental that they are shared with protein enzymes?


Author(s):  
Bo Li ◽  
Ruihong Qiao ◽  
Zhizhi Wang ◽  
Weihong Zhou ◽  
Xin Li ◽  
...  

Telomere repeat factor 1 (TRF1) is a subunit of shelterin (also known as the telosome) and plays a critical role in inhibiting telomere elongation by telomerase. Tankyrase 1 (TNKS1) is a poly(ADP-ribose) polymerase that regulates the activity of TRF1 through poly(ADP-ribosyl)ation (PARylation). PARylation of TRF1 by TNKS1 leads to the release of TRF1 from telomeres and allows telomerase to access telomeres. The interaction between TRF1 and TNKS1 is thus important for telomere stability and the mitotic cell cycle. Here, the crystal structure of a complex between the N-terminal acidic domain of TRF1 (residues 1–55) and a fragment of TNKS1 covering the second and third ankyrin-repeat clusters (ARC2-3) is presented at 2.2 Å resolution. The TNKS1–TRF1 complex crystals were optimized using an `oriented rescreening' strategy, in which the initial crystallization condition was used as a guide for a second round of large-scale sparse-matrix screening. This crystallographic and biochemical analysis provides a better understanding of the TRF1–TNKS1 interaction and the three-dimensional structure of the ankyrin-repeat domain of TNKS.


2019 ◽  
Author(s):  
Sushant Kumar ◽  
Arif Harmanci ◽  
Jagath Vytheeswaran ◽  
Mark B. Gerstein

AbstractA rapid decline in sequencing cost has made large-scale genome sequencing studies feasible. One of the fundamental goals of these studies is to catalog all pathogenic variants. Numerous methods and tools have been developed to interpret point mutations and small insertions and deletions. However, there is a lack of approaches for identifying pathogenic genomic structural variations (SVs). That said, SVs are known to play a crucial role in many diseases by altering the sequence and three-dimensional structure of the genome. Previous studies have suggested a complex interplay of genomic and epigenomic features in the emergence and distribution of SVs. However, the exact mechanism of pathogenesis for SVs in different diseases is not straightforward to decipher. Thus, we built an agnostic machine-learning-based workflow, called SVFX, to assign a “pathogenicity score” to somatic and germline SVs in various diseases. In particular, we generated somatic and germline training models, which included genomic, epigenomic, and conservation-based features for SV call sets in diseased and healthy individuals. We then applied SVFX to SVs in six different cancer cohorts and a cardiovascular disease (CVD) cohort. Overall, SVFX achieved high accuracy in identifying pathogenic SVs. Moreover, we found that predicted pathogenic SVs in cancer cohorts were enriched among known cancer genes and many cancer-related pathways (including Wnt signaling, Ras signaling, DNA repair, and ubiquitin-mediated proteolysis). Finally, we note that SVFX is flexible and can be easily extended to identify pathogenic SVs in additional disease cohorts.


Author(s):  
Yuya Hamaguchi ◽  
Yukari N. Takayabu

AbstractIn this study, the statistical relationship between tropical upper-tropospheric troughs (TUTTs) and the initiation of summertime tropical-depression type disturbances (TDDs) over the western and central North Pacific is investigated. By applying a spatiotemporal filter to the 34-year record of brightness temperature and using JRA-55 reanalysis products, TDD-event initiations are detected and classified as trough-related (TR) or non-trough-related (non-TR). The conventional understanding is that TDDs originate primarily in the lower-troposphere; our results refine this view by revealing that approximately 30% of TDDs in the 10°N-20°N latitude ranges are generated under the influence of TUTTs. Lead-lag composite analysis of both TR- and non-TR-TDDs clarifies that TR-TDDs occur under relatively dry and less convergent large-scale conditions in the lower-troposphere. This result suggests that TR-TDDs can form in a relatively unfavorable low-level environment. The three-dimensional structure of the wave activity flux reveals southward and downward propagation of wave energy in the upper troposphere that converges at the mid-troposphere around the region where TR-TDDs occur, suggesting the existence of extratropical forcing. Further, the role of dynamic forcing associated with the TUTT on the TR-TDD-initiation is analyzed using the quasi-geostrophic omega equation. The result reveals that moistening in the mid-to-upper troposphere takes place in association with the sustained dynamical ascent at the southeast side of the TUTT, which precedes the occurrence of deep convective heating. Along with a higher convective available potential energy due to the destabilizing effect of TUTTs, the moistening in the mid-to-upper troposphere also helps to prepare the environment favorable to TDDs initiation.


1993 ◽  
Vol 16 (12) ◽  
pp. 843-846 ◽  
Author(s):  
J.C. Gerlach ◽  
K. Klöppel ◽  
C. MÜller ◽  
N. Schnoy ◽  
M.D. Smith ◽  
...  

Utilizing a modified culture technique for hepatocytes, a high performance suspension culture is possible in which hepatocytes spontaneously form cell aggregates. The aggregates of 20-100 cells have been histologically confirmed to hold a three-dimensional structure, they show a long-term external metabolism and a survival time comparable with standard adhesion cultures. This technique has several advantages in the construction of large scale bioreactors for hybrid liver support systems.


2009 ◽  
Vol 284 (24) ◽  
pp. 16126-16134 ◽  
Author(s):  
Sarah L. Greig ◽  
Mazdak Radjainia ◽  
Alok K. Mitra

Colicin Ia is a soluble, harpoon-shaped bacteriocin which translocates across the periplasmic space of sensitive Escherichia coli cell by parasitizing an outer membrane receptor and forms voltage-gated ion channels in the inner membrane. This process leads to cell death, which has been thought to be caused by a single colicin Ia molecule. To directly visualize the three-dimensional structure of the channel, we generated two-dimensional crystals of colicin Ia inserted in lipid-bilayer membranes and determined a ∼17 three-dimensional model by electron crystallography. Supported by velocity sedimentation, chemical cross-linking and single-particle image analysis, the three-dimensional structure is a crown-shaped oligomer enclosing a ∼35 Å-wide extrabilayer vestibule. Our study suggests that lipid insertion instigates a global conformational change in colicin Ia and that more than one molecule participates in the channel architecture with the vestibule, possibly facilitating the known large scale peptide translocation upon channel opening.


2010 ◽  
Vol 6 (S275) ◽  
pp. 392-395
Author(s):  
Fabio De Colle

AbstractTwo-dimensional emission line images of the HH30 jet were recently used (De Colle et al. 2010) to recover the three-dimensional structure of the jet by applying standard tomographic technique (“Tikhonov regularization techniques”). In this paper I show that it is possible to determine the ejection history of the HH30 jet by directly comparing the outcome of numerical simulations with the results of the tomographic inversion. In particular, it is shown that the HH30 jet electron density map is best reproduced by assuming a velocity variation at the base of the jet with a large scale periodicity (with a period of ~3 yrs) added to small scales velocity variation (with periods ≲months).


2011 ◽  
Vol 89 (2) ◽  
pp. 246-255 ◽  
Author(s):  
German Reyes ◽  
Nicole M.I. Nivillac ◽  
Maria Chalsev ◽  
Imogen R. Coe

Nucleoside transporters (NTs) are integral membrane proteins necessary for the cellular entry of nucleoside analog drugs used in chemotherapeutic treatment of conditions such as cancer and viral or parasitic infections. NTs are also the targets of certain drugs used in the treatment of various cardiovascular conditions. Because of the importance of NTs in drug uptake, determination of the three-dimensional structure of these proteins, particularly hENT1, has the potential to improve these treatments through structure-based design of more specifically targeted and transported drugs. In this paper, we use NMR spectroscopy to investigate the structure of the large intracellular loop between transmembrane domains 6 and 7 and we also describe a method for the successful overexpression of full-length hENT1 in a bacterial system. Recombinant tandem histidine-affinity (HAT) and 3×FLAG tagged hENT1 was overexpressed in E. coli, affinity purified, and functionally characterized by nitrobenzylthioinosine (NBTI) binding. Anti-3×FLAG immunodetection confirmed the expression of N-HAT-3×FLAG-hENT1, while increased NBTI binding (3.2-fold compared with controls) confirmed the conformational integrity of the recombinant hENT1 within the bacterial inner membrane. Yields of recombinant hENT1 using this approach were ∼15 µg/L of bacterial culture and this approach provides a basis for large-scale production of protein for a variety of purposes.


2009 ◽  
Vol 55 (191) ◽  
pp. 411-421 ◽  
Author(s):  
Hinrich Schaefer ◽  
Vasilii V. Petrenko ◽  
Edward J. Brook ◽  
Jeffrey P. Severinghaus ◽  
Niels Reeh ◽  
...  

AbstractHorizontal ice-core sites, where ancient ice is exposed at the glacier surface, offer unique opportunities for paleo-studies of trace components requiring large sample volumes. Following previous work at the Pâkitsoq ice margin in West Greenland, we use a combination of geochemical parameters measured in the ice matrix (δ18Oice) and air occlusions (δ18Oatm, δ15N of N2 and methane concentration) to date ice layers from specific climatic intervals. The data presented here expand our understanding of the stratigraphy and three-dimensional structure of ice layers outcropping at Pâkitsoq. Sections containing ice from every distinct climatic interval during Termination I, including Last Glacial Maximum, Bølling/Allerød, Younger Dryas and the early Holocene, are identified. In the early Holocene, we find evidence for climatic fluctuations similar to signals found in deep ice cores from Greenland. A second glacial–interglacial transition exposed at the extreme margin of the ice is identified as another outcrop of Termination I (rather than the onset of the Eemian interglacial as postulated in earlier work). Consequently, the main structural feature at Pâkitsoq is a large-scale anticline with accordion-type folding in both exposed sequences of the glacial–Holocene transition, leading to multiple layer duplications and age reversals.


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