scholarly journals Enrichment of Zα domains at cytoplasmic stress granules is due to their innate ability to bind nucleic acids

2021 ◽  
Author(s):  
Luisa Gabriel ◽  
Bharath Srinivasan ◽  
Krzysztof Kuś ◽  
João F. Mata ◽  
Maria João Amorim ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Stress Granules ◽  
Helical Conformation ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Binding Ability ◽  
Amino Terminal ◽  
Mobility Shift Assay

Zα domains recognize the left-handed helical conformation of double stranded nucleic acids. They are found in proteins involved in the nucleic acid sensory pathway of vertebrate innate immune system and host evasion by viral pathogens. Previously, it has been demonstrated that ADAR1 and DAI localize to the cytosolic stress granules mediated by their Zα domains. To investigate the mechanism, we determined the interactions and localization pattern for the amino-terminal region of human DAI harbouring two Zα domains (ZαβDAI) and a nucleic acid-binding deficient mutant. Electrophoretic mobility shift assay demonstrated the ability of ZαβDAI to bind to hyperedited nucleic acids which are enriched in stress granules. Further, using immunofluorescence and immunoprecipitation coupled with mass-spectrometry, we identified several interacting partners of the ZαβDAI-RNA complex in-vivo under conditions of arsenite-induced stress. These interactions are lost upon loss of nucleic acid binding ability or with RNase treatment. Thus, we posit that the mechanism for the translocation of Zα domain-containing proteins to stress granules is mainly mediated by the nucleic acid binding ability of their Zα domains.

scholarly journals Enrichment of Zα domains at cytoplasmic stress granules is due to their innate ability to bind nucleic acids

2021 ◽  
Author(s):  
Luisa Gabriel ◽  
Bharath Srinivasan ◽  
Krzysztof Kuś ◽  
João F. Mata ◽  
Maria João Amorim ◽  
...  
Keyword(s):  
Immune System ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Innate Immune System ◽  
Rna Binding ◽  
Stress Granules ◽  
Innate Immune ◽  
Nucleic Acid Binding ◽  
Binding Ability ◽  
Amino Terminal

AbstractZα domains are a subfamily of winged Helix-Turn-Helix (wHTH) domains found exclusively in proteins involved in the nucleic acids sensory pathway of vertebrate innate immune system and host evasion by viral pathogens. Interestingly, they are the only known protein domains that recognise the left-handed helical conformation of both dsDNA and dsRNA, known as Z-DNA and Z-RNA. Previously, it has been demonstrated that ADAR1 and ZBP1, two proteins possessing the Zα domains, localize to cytosolic stress granules. It was further speculated that such localization is principally mediated by Zα domains. To characterize and better understand such distinct and specific localization, we characterised the in vivo interactions and localization pattern for the amino terminal region of human DAI harbouring two Zα domains (ZαβDAI). Using immunoprecipitation and mass spectrometry, we identified several interacting partners that were components of the complex formed by Zα domains and RNAs. Differential interacting partners to wild-type Zα, relative to mutant proteins, demonstrated that most of the physiologically relevant interactions are mediated by the nucleic acid binding ability of the Zαβ. Further, we also show enrichment of selected complex components in cytoplasmic stress granules under conditions of stress. This ability is mostly lost in the mutants of ZαβDAI (ZαβDAI 4×mut) that lack nucleic-acid binding ability. Thus, we posit that the mechanism for the translocation of Zα domain-containing proteins to stress granules is mainly mediated by the nucleic acid binding ability of their Zα domains. Finally, we demonstrate that FUS and PSF/p54nrb, two RNA binding proteins with established roles in stress granules, interact with Zα, which provides strong evidence for a role of these proteins in the innate immune system.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

scholarly journals Impact of nucleic acid and methylated H3K9 binding activities of Suv39h1 on its heterochromatin assembly

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Atsuko Shirai ◽  
Takayuki Kawaguchi ◽  
Hideaki Shimojo ◽  
Daisuke Muramatsu ◽  
Mayumi Ishida-Yonetani ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Binding Affinity ◽  
Histone H3 ◽  
Pericentric Heterochromatin ◽  
Nucleic Acid Binding ◽  
Major Satellite ◽  
Satellite Rnas ◽  
Heterochromatin Assembly

SUV39H is the major histone H3 lysine 9 (H3K9)-specific methyltransferase that targets pericentric regions and is crucial for assembling silent heterochromatin. SUV39H recognizes trimethylated H3K9 (H3K9me3) via its chromodomain (CD), and enriched H3K9me3 allows SUV39H to target specific chromosomal regions. However, the detailed targeting mechanisms, especially for naïve chromatin without preexisting H3K9me3, are poorly understood. Here we show that Suv39h1’s CD (Suv39h1-CD) binds nucleic acids, and this binding is important for its function in heterochromatin assembly. Suv39h1-CD had higher binding affinity for RNA than DNA, and its ability to bind nucleic acids was independent of its H3K9me3 recognition. Suv39h1 bound major satellite RNAs in vivo, and knockdown of major satellite RNAs lowered Suv39h1 retention on pericentromere. Suv39h1 mutational studies indicated that both the nucleic acid–binding and H3K9me–binding activities of Suv39h1-CD were crucial for its pericentric heterochromatin assembly. These results suggest that chromatin-bound RNAs contribute to creating SUV39H’s target specificity.

Distinct domains of a nucleolar protein mediate protein kinase binding, interaction with nucleic acids and nucleolar localization

1998 ◽  
Vol 111 (17) ◽  
pp. 2615-2623 ◽  
Author(s):  
A. Das ◽  
J.H. Park ◽  
C.B. Hagen ◽  
M. Parsons
Keyword(s):  
Protein Kinase ◽  
Nucleic Acids ◽  
Protozoan Parasite ◽  
In Vitro Transcription ◽  
Nucleic Acid Binding ◽  
Binding Interaction ◽  
Amino Terminal ◽  
Amino Acid Region ◽  
Carboxy Terminal

Nopp44/46 is a phosphoprotein of the protozoan parasite Trypanosoma brucei that is localized to the nucleolus. Based on the primary sequence, Nopp44/46 appears to be a protein composed of distinct domains. This communication describes the relationship of these domains to the known functional interactions of the molecule and suggests that the amino-terminal region defines a novel homology region that functions in nucleolar targeting. We have previously shown that Nopp44/46 is capable of interacting with nucleic acids and associating with a protein kinase. Using in vitro transcription and translation, we now demonstrate that the nucleic acid binding function maps to the carboxy-terminal domain of the molecule, a region rich in arginine-glycine-glycine motifs. Our experiments reveal that a central region containing a high proportion of acidic residues is required for association with the protein kinase. Analysis of transfectants expressing epitope-tagged Nopp44/46 deletion constructs showed that the amino-terminal 96 amino acids allowed nuclear and nucleolar accumulation of the protein. This region of the molecule shows homology to several recently described nucleolar proteins. Deletion of a 27-amino-acid region within this domain abrogated nucleolar, but not nuclear, localization. These studies show that Nopp44/46 is composed of distinct modules, each of which plays a different role in molecular interactions. We suggest that this protein could facilitate interactions between sets of nucleolar molecules.

scholarly journals Barcoding chemical modifications into nucleic acids improves drug stability in vivo

2018 ◽  
Vol 6 (44) ◽  
pp. 7197-7203 ◽  
Author(s):  
Cory D. Sago ◽  
Sujay Kalathoor ◽  
Jordan P. Fitzgerald ◽  
Gwyneth N. Lando ◽  
Naima Djeddar ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Drug Stability ◽  
Chemical Modifications

The efficacy of nucleic acid therapies can be limited by unwanted degradation.

scholarly journals RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing

Molecules ◽  
2019 ◽  
Vol 24 (16) ◽  
pp. 3020 ◽  
Author(s):  
Alan Ann Lerk Ong ◽  
Jiazi Tan ◽  
Malini Bhadra ◽  
Clément Dezanet ◽  
Kiran M. Patil ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Nucleic Acids ◽  
Splice Site ◽  
Peptide Nucleic Acids ◽  
Amino Sugar ◽  
Hairpin Structure ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Antisense Peptide ◽  
Exon 10

Alternative splicing of tau pre-mRNA is regulated by a 5′ splice site (5′ss) hairpin present at the exon 10–intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5′ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10–15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 3′ arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5′ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5′ arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5′ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent.

scholarly journals Modified Nucleic Acids: Expanding the Capabilities of Functional Oligonucleotides

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4659 ◽  
Author(s):  
Steven Ochoa ◽  
Valeria T. Milam
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Chemical Structure ◽  
Chemical Diversity ◽  
Physiochemical Properties ◽  
Phosphate Backbone ◽  
Modified Nucleic Acids ◽  
Recent Developments ◽  
Diagnostic Applications

In the last three decades, oligonucleotides have been extensively investigated as probes, molecular ligands and even catalysts within therapeutic and diagnostic applications. The narrow chemical repertoire of natural nucleic acids, however, imposes restrictions on the functional scope of oligonucleotides. Initial efforts to overcome this deficiency in chemical diversity included conservative modifications to the sugar-phosphate backbone or the pendant base groups and resulted in enhanced in vivo performance. More importantly, later work involving other modifications led to the realization of new functional characteristics beyond initial intended therapeutic and diagnostic prospects. These results have inspired the exploration of increasingly exotic chemistries highly divergent from the canonical nucleic acid chemical structure that possess unnatural physiochemical properties. In this review, the authors highlight recent developments in modified oligonucleotides and the thrust towards designing novel nucleic acid-based ligands and catalysts with specifically engineered functions inaccessible to natural oligonucleotides.

The reaction of the psoralens with deoxyribonucleic acid

1984 ◽  
Vol 17 (1) ◽  
pp. 1-44 ◽  
Author(s):  
John E. Hearst ◽  
Stephen T. Isaacs ◽  
David Kanne ◽  
Henry Rapoport ◽  
Kenneth Straub
Keyword(s):  
Natural Selection ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Chemical Modification ◽  
Deoxyribonucleic Acid ◽  
Molecular Level ◽  
Virus Particles ◽  
Cross Links ◽  
High Concentrations

Psoralen photochemistry is specific for nucleic acids and is better understood at the molecular level than are all other methods of chemical modification of nucleic acids. These compounds are used both for in vivo structure analysis and for photochemotherapy since they easily penetrate both cells and virus particles. Apparently, natural selection has selected for membrane and virus penetrability during the evolution of these natural products. Most cells are unaffected by relatively high concentrations of psoralens in the absence of ultraviolet light, and the metabolites of the psoralens have thus far not created a problem. Finally, psoralens form both monoadduct and cross-links in nucleic acid helices, the yield of each being easily controlled by the conditions used during the photochemistry.

scholarly journals Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (12) ◽  
pp. 4522-4534 ◽  
Author(s):  
R Ng ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
In Vitro Assays ◽  
Binding Studies ◽  
Mobility Shift ◽  
Sequence Element ◽  
Sequence Elements ◽  
Mobility Shift Assay

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

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