392 research outputs found
Molecular principles underlying dual RNA specificity in the Drosophila SNF protein
The first RNA recognition motif of the Drosophila SNF protein is an example of an RNA binding protein with multi-specificity. It binds different RNA hairpin loops in spliceosomal U1 or U2 small nuclear RNAs, and only in the latter case requires the auxiliary U2AⲠprotein. Here we investigate its functions by crystal structures of SNF alone and bound to U1 stem-loop II, U2AⲠor U2 stem-loop IV and U2Aâ˛, SNF dynamics from NMR spectroscopy, and structure-guided mutagenesis in binding studies. We find that different loop-closing base pairs and a nucleotide exchange at the tips of the loops contribute to differential SNF affinity for the RNAs. U2AⲠimmobilizes SNF and RNA residues to restore U2 stem-loop IV binding affinity, while U1 stem-loop II binding does not require such adjustments. Our findings show how U2AⲠcan modulate RNA specificity of SNF without changing SNF conformation or relying on direct RNA contacts
Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9
In flowering plant plastids and mitochondria, multiple organellar RNA editing
factor (MORF/RIP) proteins are required at most sites for efficient C to U RNA
editing catalyzed by the RNA editosome. MORF proteins harbor a conserved
stretch of residues (MORF-box), form homo- and heteromers and interact with
selected PPR (pentatricopeptide repeat) proteins, which recognize each editing
site. The molecular function of the MORF-box remains elusive since it shares
no sequence similarity with known domains. We determined structures of the A.
thaliana mitochondrial MORF1 and chloroplast MORF9 MORF-boxes which both adopt
a novel globular fold (MORF domain). Our structures state a paradigmatic model
for MORF domains and their specific dimerization via a hydrophobic interface.
We cross-validate the interface by yeast two-hybrid studies and pulldown
assays employing structure-based mutants. We find a structural similarity of
the MORF domain to an N-terminal ferredoxin-like domain (NFLD), which confers
RNA substrate positioning in bacterial 4-thio-uracil tRNA synthetases,
implying direct RNA contacts of MORF proteins during RNA editing. With the
MORF1 and MORF9 structures we elucidate a yet unknown fold, corroborate MORF
interaction studies, validate the mechanism of MORF multimerization by
structure-based mutants and pave the way towards a complete structural
characterization of the plant RNA editosome
Elucidation of orientation relations between Fe-Cr alloys and corrosion products after high temperature SO2 corrosion
The early stages of corrosion of Fe-Cr-model alloys (2 and 9 % Cr) were investigated after exposure at 650 degrees C in 0.5 % SO2 containing gas by electron backscattered diffraction (EBSD) and transmission electron microscopy (TEM). The impact of the grain orientation of the base alloy on the orientation relations of the corrosion products is presented. After 2 min - 5 min exposure the formation of a multi-layered corrosion zone was discovered. A clear orientation relationship between ferrite and the (Fe,Cr)(3)O-4 spinel could be demonstrated. The obtained results show the importance of the grain orientation on oxidation resistance
Structural and functional investigation of the human snRNP assembly factor AAR2 in complex with the RNase H-like domain of PRPF8
Small nuclear ribonucleoprotein complexes (snRNPs) represent the main subunits of the spliceosome. While the assembly of the snRNP core particles has been well characterized, comparably little is known of the incorporation of snRNP-specific proteins and the mechanisms of snRNP recycling. U5 snRNP assembly in yeast requires binding of the the Aar2 protein to Prp8p as a placeholder to preclude premature assembly of the SNRNP200 helicase, but the role of the human AAR2 homolog has not yet been investigated in detail. Here, a crystal structure of human AAR2 in complex with the RNase H-like domain of the U5-specific PRPF8 (PRP8F RH) is reported, revealing a significantly different interaction between the two proteins compared with that in yeast. Based on the structure of the AAR2âPRPF8 RH complex, the importance of the interacting regions and residues was probed and AAR2 variants were designed that failed to stably bind PRPF8 in vitro. Protein-interaction studies of AAR2 with U5 proteins using size-exclusion chromatography reveal similarities and marked differences in the interaction patterns compared with yeast Aar2p and imply phosphorylation-dependent regulation of AAR2 reminiscent of that in yeast. It is found that in vitro AAR2 seems to lock PRPF8 RH in a conformation that is only compatible with the first transesterification step of the splicing reaction and blocks a conformational switch to the step 2-like, Mg2+-coordinated conformation that is likely during U5 snRNP biogenesis. These findings extend the picture of AAR2 PRP8 interaction from yeast to humans and indicate a function for AAR2 in the spliceosomal assembly process beyond its role as an SNRNP200 placeholder in yeast
Exploring Idea Convergence and Conceptual Combination in Open Innovative Crowdsourcing from a Cognitive Load Perspective
Open innovative crowdsourcing has received increasing attention. This study sets out to investigate idea convergence and generation in open innovative crowdsourcing communities from a cognitive load perspective to explore aspects of cognitive idea processing. We have conducted a laboratory experiment to investigate the effects of three manipulations (task complexity, idea presentation, and procedural guidance) on three types of cognitive load and the following idea convergence and generation quality. We have also examined the influencing mechanisms of cognitive loads on satisfaction with process and satisfaction with outcome. Our results show that the three cognitive loads have significant effects: Higher intrinsic cognitive load significantly leads to lower satisfaction with process and outcome. Higher extraneous cognitive load significantly leads to satisfaction with process. Higher germane cognitive load significantly leads to higher convergence quality and lower new idea generation quality
The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling
Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. The intrinsically disordered TSSC4 protein has been identified as a nuclear-localized U5 snRNP and U4/U6-U5 tri-snRNP assembly/recycling factor, but how TSSC4âs intrinsic disorder supports TSSC4 functions remains unknown. Using diverse interaction assays and cryogenic electron microscopy-based structural analysis, we show that TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites. It thereby inhibits SNRNP200 helicase activity, spatially aligns the proteins, coordinates formation of a U5 sub-module and transiently blocks premature interaction of SNRNP200 with at least three other spliceosomal factors. Guided by the structure, we designed a TSSC4 variant that lacks stable binding to the PRPF8 Jab1/MPN domain or SNRNP200 in vitro. Comparative immunoprecipitation/mass spectrometry from HEK293 nuclear extract revealed distinct interaction profiles of wild type TSSC4 and the variant deficient in PRPF8/SNRNP200 binding with snRNP proteins, other spliceosomal proteins as well as snRNP assembly/recycling factors and chaperones. Our findings elucidate molecular strategies employed by an intrinsically disordered protein to promote snRNP assembly, and suggest multiple TSSC4-dependent stages during snRNP assembly/recycling
A multi-factor trafficking site on the spliceosome remodeling enzyme BRR2 recruits C9ORF78 to regulate alternative splicing
The intrinsically unstructured C9ORF78 protein was detected in spliceosomes but its role in splicing is presently unclear. We find that C9ORF78 tightly interacts with the spliceosome remodeling factor, BRR2, in vitro. Affinity purification/mass spectrometry and RNA UV-crosslinking analyses identify additional C9ORF78 interactors in spliceosomes. Cryogenic electron microscopy structures reveal how C9ORF78 and the spliceosomal B complex protein, FBP21, wrap around the C-terminal helicase cassette of BRR2 in a mutually exclusive manner. Knock-down of C9ORF78 leads to alternative NAGNAG 3â˛-splice site usage and exon skipping, the latter dependent on BRR2. Inspection of spliceosome structures shows that C9ORF78 could contact several detected spliceosome interactors when bound to BRR2, including the suggested 3â˛-splice site regulating helicase, PRPF22. Together, our data establish C9ORF78 as a late-stage splicing regulatory protein that takes advantage of a multi-factor trafficking site on BRR2, providing one explanation for suggested roles of BRR2 during splicing catalysis and alternative splicing
Beyond Realism and Moralism: A Defense of Political Minimalism
"This is the pre-peer reviewed version of the following article: RodrĂguez-AlcĂĄzar, J. Beyond Realism and Moralism: A Defense of Political Minimalism. Metaphilosophy, 48(5): 727-744 (2017), which has been published in final form at http://dx.doi.org/10.1111/meta.12259 . This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."What is the relationship between morals and politics? What is the
relationship between moral philosophy and political philosophy? Defenders of
political moralism postulate moral aims or constraints for politics, and hence
they see political philosophy as a chapter of moral philosophy. Contrastingly,
advocates of political realism describe politics as an independent endeavor
aiming at providing order and security, and conceive of political philosophy as an
autonomous discipline. This article claims that political moralism and political
realism share the mistake of assuming that politics has substantial, permanent
goals or constraints. After criticizing political substantialism, the article explains
the main ingredients of its alternative, political minimalism: (1) the idea that
politics, understood as collective instrumental rationality, aims at providing
adequate means for the accomplishment of people s goals, whatever these are;
and (2) the conception of the relationship between morality and politics as one of
âreciprocal containment.â Finally, it addresses some foreseeable criticisms of
political minimalism.This article is a result of the research project FFI2016â79000-P, financed by the Spanish Ministry of Economy, Industry, and Competitiveness
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