scholarly journals Re-refinement of the spliceosomal U4 snRNP core-domain structure

2016 ◽  
Vol 72 (1) ◽  
pp. 131-146 ◽  
Author(s):  
Jade Li ◽  
Adelaine K. Leung ◽  
Yasushi Kondo ◽  
Chris Oubridge ◽  
Kiyoshi Nagai
Keyword(s):  
Domain Structure ◽  
Early Model ◽  
Molecular Replacement ◽  
Model Errors ◽  
Sm Proteins ◽  
Core Domain ◽  
Small Nuclear Ribonucleoprotein ◽  
Experimental Phasing ◽  
Using Data ◽  
Snrnp Core

The core domain of small nuclear ribonucleoprotein (snRNP), comprised of a ring of seven paralogous proteins bound around a single-stranded RNA sequence, functions as the assembly nucleus in the maturation of U1, U2, U4 and U5 spliceosomal snRNPs. The structure of the human U4 snRNP core domain was initially solved at 3.6 Å resolution by experimental phasing using data with tetartohedral twinning. Molecular replacement from this model followed by density modification using untwinned data recently led to a structure of the minimal U1 snRNP at 3.3 Å resolution. With the latter structure providing a search model for molecular replacement, the U4 core-domain structure has now been re-refined. The U4 Sm site-sequence AAUUUUU has been shown to bind to the seven Sm proteins SmF–SmE–SmG–SmD3–SmB–SmD1–SmD2in an identical manner as the U1 Sm-site sequence AAUUUGU, except in SmD1where the bound U replaces G. The progression from the initial to the re-refined structure exemplifies a tortuous route to accuracy: where well diffracting crystals of complex assemblies are initially unavailable, the early model errors are rectified by exploiting preliminary interpretations in further experiments involving homologous structures. New insights are obtained from the more accurate model.

scholarly journals Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing

Crystals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 273
Author(s):  
Yoshita Srivastava ◽  
Rachel Bonn-Breach ◽  
Sai Shashank Chavali ◽  
Geoffrey M. Lippa ◽  
Jermaine L. Jenkins ◽  
...  
Keyword(s):  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Molecular Replacement ◽  
Hydrophobic Core ◽  
Anomalous Diffraction ◽  
Recognition Motif ◽  
Determination Process ◽  
Experimental Phasing ◽  
Crystal Contacts ◽  
Apical Loop

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.

Identification and characterization of the small nuclear ribonucleoprotein particle D' core protein

1990 ◽  
Vol 10 (9) ◽  
pp. 4480-4485
Author(s):  
J Andersen ◽  
R J Feeney ◽  
G W Zieve
Keyword(s):  
Electrophoretic Mobility ◽  
Protein Complexes ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
Core Proteins ◽  
Nuclear Ribonucleoprotein ◽  
Identification And Characterization ◽  
Snrnp Core ◽  
Small Nuclear Ribonucleoprotein Particle

The addition of urea to sodium dodecyl sulfate (SDS)-polyacrylamide gels has allowed the identification and characterization of the small nuclear ribonucleoprotein particle (snRNP) D' protein and has also improved resolution of the E, F, and G snRNP core proteins. In standard SDS-polyacrylamide gels, the D' and D snRNP core proteins comigrate at approximately 16 kilodaltons. The addition of urea to the separating gel caused the D' protein to shift to a slower electrophoretic mobility that is distinct from that of the D protein. The shift to a slower electrophoretic mobility in the presence of urea suggests that the D' protein has extensive secondary structure that is not totally disrupted by SDS alone. Both N-terminal sequencing and partial peptide maps indicate that the D and D' proteins are distinct gene products, and the sequence data have identified the faster moving of the two proteins as the previously cloned D protein (L. A. Rokeach, J. A. Haselby, and S. O. Hoch, Proc. Natl. Acad. Sci. USA 85:4832-4836, 1988). In the cytoplasm, the D protein is found primarily in the small-nuclear-RNA-free 6S protein complexes, while the D' protein is found primarily in the 20S protein complexes. Like the D protein, the D' protein is an autoantigen in patients with systemic lupus erythematosus and is recognized by some of the Sm class of autoimmune antisera.

scholarly journals Using a partial atomic model from medium-resolution cryo-EM to solve a large crystal structure

2021 ◽  
Vol 77 (1) ◽  
pp. 11-18
Author(s):  
Montserrat Fàbrega-Ferrer ◽  
Ana Cuervo ◽  
Francisco J. Fernández ◽  
Cristina Machón ◽  
Rosa Pérez-Luque ◽  
...  
Keyword(s):  
Molecular Replacement ◽  
Large Crystal ◽  
Rotation Matrices ◽  
Cryo Electron Microscopy ◽  
Partial Models ◽  
Medium Resolution ◽  
Experimental Phasing ◽  
Crystallographic Structures ◽  
Direct Use ◽  
Rotation Function

Medium-resolution cryo-electron microscopy maps, in particular when they include a significant number of α-helices, may allow the building of partial models that are useful for molecular-replacement searches in large crystallographic structures when the structures of homologs are not available and experimental phasing has failed. Here, as an example, the solution of the structure of a bacteriophage portal using a partial 30% model built into a 7.8 Å resolution cryo-EM map is shown. Inspection of the self-rotation function allowed the correct oligomerization state to be determined, and density-modification procedures using rotation matrices and a mask based on the cryo-EM structure were critical for solving the structure. A workflow is described that may be applicable to similar cases and this strategy is compared with direct use of the cryo-EM map for molecular replacement.

scholarly journals Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins.

1995 ◽  
Vol 15 (1) ◽  
pp. 445-455 ◽  
Author(s):  
J Roy ◽  
B Zheng ◽  
B C Rymond ◽  
J L Woolford
Keyword(s):  
Protein Complexes ◽  
Mrna Splicing ◽  
Yeast Cells ◽  
Ribonucleoprotein Particle ◽  
Sm Proteins ◽  
Small Nuclear Ribonucleoprotein ◽  
Snrnp Protein ◽  
Nuclear Ribonucleoprotein ◽  
Precursor Mrna

Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3. The Smd3 protein is required for pre-mRNA splicing in vivo. Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis. Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1. Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other. Thus, these two proteins have distinct functions. A pool of Smd3 exists in the yeast cytoplasm. This is consistent with the possibility that snRNP assembly in S. cerevisiae, as in metazoans, is initiated in the cytoplasm from a pool of RNA-free core snRNP protein complexes.

Atomic resolution structure of a chimeric DNA–RNA Z-type duplex in complex with Ba2+ions: a case of complicated multi-domain twinning

2016 ◽  
Vol 72 (2) ◽  
pp. 211-223 ◽  
Author(s):  
Miroslaw Gilski ◽  
Pawel Drozdzal ◽  
Ryszard Kierzek ◽  
Mariusz Jaskolski
Keyword(s):  
Molecular Replacement ◽  
Higher Symmetry ◽  
Barium Ions ◽  
Space Groups ◽  
Left Handed ◽  
Crystal Twinning ◽  
Using Data ◽  
High Degree ◽  
Packing Analysis ◽  
Resolution Structure

The self-complementary dCrGdCrGdCrG hexanucleotide, in which not only the pyrimidine/purine bases but also the ribo/deoxy sugars alternate along the sequence, was crystallized in the presence of barium cations in the form of a left-handed Z-type duplex. The asymmetric unit of theP21crystal with a pseudohexagonal lattice contains four chimeric duplexes and 16 partial Ba2+sites. The chimeric (DNA–RNA)2duplexes have novel patterns of hydration and exhibit a high degree of discrete conformational disorder of their sugar-phosphate backbones, which can at least partly be correlated with the fractional occupancies of the barium ions. The crystals of the DNA–RNA chimeric duplex in complex with Ba2+ions and also with Sr2+ions exhibit complicated twinning, which in combination with structural pseudosymmetry made structure determination difficult. The structure could be successfully solved by molecular replacement in space groupsP1 andP21but not in orthorhombic or higher symmetry and, after scrupulous twinning and packing analysis, was refined in space groupP21to anRandRfreeof 11.36 and 16.91%, respectively, using data extending to 1.09 Å resolution. With the crystal structure having monoclinic symmetry, the sixfold crystal twinning is a combination of threefold and twofold rotations. The paper describes the practical aspects of dealing with cases of complicated twinning and pseudosymmetry, and compares the available software tools for the refinement and analysis of such cases.

scholarly journals Dynamic Calibration with an Ensemble Kalman Filter Based Data Assimilation Approach for Root-Zone Moisture Predictions

2007 ◽  
Vol 8 (4) ◽  
pp. 910-921 ◽  
Author(s):  
Nicola Montaldo ◽  
John D. Albertson ◽  
Marco Mancini
Keyword(s):  
Soil Moisture ◽  
Kalman Filter ◽  
Ensemble Kalman Filter ◽  
Root Zone ◽  
Sensitivity Analyses ◽  
Surface Model ◽  
Early Model ◽  
Model Errors ◽  
Model Bias ◽  
Near Surface

Abstract In the presence of uncertain initial conditions and soil hydraulic properties, land surface model (LSM) performance can be significantly improved by the assimilation of periodic observations of certain state variables, such as the near-surface soil moisture (θg), as observed from a remote platform. In this paper the possibility of merging observations and the model optimally for providing robust predictions of root-zone soil moisture (θ2) is demonstrated. An assimilation approach that assimilates θg through the ensemble Kalman filter (EnKF) and provides a physics-based update of θ2 is developed. This approach, as with other common soil moisture assimilation approaches, may fail when a key LSM parameter, for example, the saturated hydraulic conductivity (ks), is estimated poorly. This leads to biased model errors producing a violation of a main assumption (model errors with zero mean) of the EnKF. For overcoming this model bias an innovative assimilation approach is developed that accepts this violation in the early model run times and dynamically calibrates all the components of the ks ensemble as a function of the persistent bias in root-zone soil moisture, allowing one to remove the model bias, restore the fidelity to the EnKF requirements, and reduce the model uncertainty. The robustness of the proposed approach is also examined in sensitivity analyses.

scholarly journals The Methylosome, a 20S Complex Containing JBP1 and pICln, Produces Dimethylarginine-Modified Sm Proteins

2001 ◽  
Vol 21 (24) ◽  
pp. 8289-8300 ◽  
Author(s):  
Westley J. Friesen ◽  
Sergey Paushkin ◽  
Anastasia Wyce ◽  
Severine Massenet ◽  
G. Scott Pesiridis ◽  
...  
Keyword(s):  
Muscular Atrophy ◽  
Ring Structure ◽  
Survival Motor Neuron ◽  
Sm Proteins ◽  
Particle Assembly ◽  
Interacting Protein ◽  
Smn Complex ◽  
Snrnp Core

ABSTRACT snRNPs, integral components of the pre-mRNA splicing machinery, consist of seven Sm proteins which assemble in the cytoplasm as a ring structure on the snRNAs U1, U2, U4, and U5. The survival motor neuron (SMN) protein, the spinal muscular atrophy disease gene product, is crucial for snRNP core particle assembly in vivo. SMN binds preferentially and directly to the symmetrical dimethylarginine (sDMA)-modified arginine- and glycine-rich (RG-rich) domains of SmD1 and SmD3. We found that the unmodified, but not the sDMA-modified, RG domains of SmD1 and SmD3 associate with a 20S methyltransferase complex, termed the methylosome, that contains the methyltransferase JBP1 and a JBP1-interacting protein, pICln. JBP1 binds SmD1 and SmD3 via their RG domains, while pICln binds the Sm domains. JBP1 produces sDMAs in the RG domain-containing Sm proteins. We further demonstrate the existence of a 6S complex that contains pICln, SmD1, and SmD3 but not JBP1. SmD3 from the methylosome, but not that from the 6S complex, can be transferred to the SMN complex in vitro. Together with previous results, these data indicate that methylation of Sm proteins by the methylosome directs Sm proteins to the SMN complex for assembly into snRNP core particles and suggest that the methylosome can regulate snRNP assembly.

scholarly journals Parallel Detection of Autoantibodies with Microarrays in Rheumatoid Diseases

2004 ◽  
Vol 50 (2) ◽  
pp. 416-422 ◽  
Author(s):  
Yanfei Feng ◽  
Xue Ke ◽  
Rongshui Ma ◽  
Ying Chen ◽  
Gengxi Hu ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Topoisomerase I ◽  
Cytokeratin 19 ◽  
Serum Samples ◽  
Sm Proteins ◽  
Single Stranded Dna ◽  
Small Nuclear Ribonucleoprotein ◽  
Clinical Diagnoses ◽  
Polystyrene Support ◽  
A Charge

Abstract Background: Clinical needs often dictate testing for several autoantibodies in a single patient with evidence of autoimmune disease. We developed a microarray containing 15 autoantigens for the detection of autoantibodies in rheumatoid autoimmune diseases. Methods: We synthesized recombinant centromere protein B, cytokeratin 19, SSA 52-kDa antigen, SSA 60-kDa antigen, SSB antigen, and Jo-1 antigen and prepared anti-nuclear antibody antigens. Cyclic citrullinated peptide, histone, goat IgG for detection of rheumatoid factor, double-stranded DNA, and single-stranded DNA were purchased, as were recombinant small nuclear ribonucleoprotein U1, topoisomerase I, and Smith antigen (Sm). All 15 antigens were of human origin except calf thymus Sm. Proteins were printed on polystyrene. The arrays were incubated with serum samples and then with horseradish peroxidase-conjugated secondary antibodies and chemiluminescent substrates, and light signals were captured by a charge-coupled device camera-based chip reader. Antibodies were quantified by use of calibration curves. Positive samples were confirmed by commercially available methods. Results: The detection limit of the microarray system was 20 pg of IgG printed on the polystyrene support. More than 85% of the confirmed positive sera were detected as positive with the microarray system based on cutoff values established with the microarray system. The imprecision (CV) of the microarrays was <15% for all 15 autoantibody assays, with the exception of single-stranded DNA (18% and 23%) within and between batches. Characteristic autoantibody patterns were seen in patients with clinical diagnoses of rheumatoid arthritis (n = 83), systemic lupus erythematosus (n = 71), systemic sclerosis (n = 36), polymyositis (n = 38), and Sjogren syndrome (n = 20). Conclusions: This microarray system provides results similar to those by conventional methods. Assessment of the diagnostic accuracy of the system remains to be done.

scholarly journals Negative Cooperativity between Gemin2 and RNA provides Insights into RNA Selection and the SMN Complex’s Release in snRNP Assembly

2018 ◽  
Author(s):  
Hongfei Yi ◽  
Li Mu ◽  
Congcong Shen ◽  
Xi Kong ◽  
Yingzhi Wang ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Basic Mechanism ◽  
Negative Cooperativity ◽  
Sm Proteins ◽  
Early Step ◽  
Smn Complex ◽  
Narrow State ◽  
Snrnp Core

ABSTRACTThe assembly of snRNP cores, in which seven Sm proteins, D1/D2/F/E/G/D3/B, form a ring around the nonameric Sm site of snRNAs, is the early step of spliceosome formation and essential to eukaryotes. It is mediated by the PMRT5 and SMN complexes sequentially in vivo. SMN deficiency causes neurodegenerative disease spinal muscular atrophy (SMA). How the SMN complex assembles snRNP cores is largely unknown, especially how the SMN complex achieves high RNA assembly specificity and how it is released. Here we show, using crystallographic and biochemical approaches, that Gemin2 of the SMN complex enhances RNA specificity of SmD1/D2/F/E/G via a negative cooperativity between Gemin2 and RNA in binding SmD1/D2/F/E/G. Gemin2, independent of its N-tail, constrains the horseshoe-shaped SmD1/D2/F/E/G from outside in a physiologically relevant, narrow state, enabling high RNA specificity. Moreover, the assembly of RNAs inside widens SmD1/D2/F/E/G, causes the release of Gemin2/SMN allosterically and allows SmD3/B to join. The assembly of SmD3/B further facilitates the release of Gemin2/SMN. This is the first to show negative cooperativity in snRNP assembly, which provides insights into RNA selection and the SMN complex’s release. These findings reveal a basic mechanism of snRNP core assembly and facilitate pathogenesis studies of SMA.

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