scholarly journals Nucleic-acid-binding properties of the C2-L1Tc nucleic acid chaperone encoded by L1Tc retrotransposon

2009 ◽  
Vol 424 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Sara R. Heras ◽  
M. Carmen Thomas ◽  
Francisco Macias ◽  
Manuel E. Patarroyo ◽  
Carlos Alonso ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Zinc Finger ◽  
Rna Binding ◽  
Nuclear Localization Sequence ◽  
Nucleic Acid Binding ◽  
Binding Model ◽  
Nucleic Acid Chaperone ◽  
Localization Sequence ◽  
Cysteine Motifs

It has been reported previously that the C2-L1Tc protein located in the Trypanosoma cruzi LINE (long interspersed nuclear element) L1Tc 3′ terminal end has NAC (nucleic acid chaperone) activity, an essential activity for retrotransposition of LINE-1. The C2-L1Tc protein contains two cysteine motifs of a C2H2 type, similar to those present in TFIIIA (transcription factor IIIA). The cysteine motifs are flanked by positively charged amino acid regions. The results of the present study show that the C2-L1Tc recombinant protein has at least a 16-fold higher affinity for single-stranded than for double-stranded nucleic acids, and that it exhibits a clear preference for RNA binding over DNA. The C2-L1Tc binding profile (to RNA and DNA) corresponds to a non-co-operative-binding model. The zinc fingers present in C2-L1Tc have a different binding affinity to nucleic acid molecules and also different NAC activity. The RRR and RRRKEK [NLS (nuclear localization sequence)] sequences, as well as the C2H2 zinc finger located immediately downstream of these basic stretches are the main motifs responsible for the strong affinity of C2-L1Tc to RNA. These domains also contribute to bind single- and double-stranded DNA and have a duplex-stabilizing effect. However, the peptide containing the zinc finger situated towards the C-terminal end of C2-L1Tc protein has a slight destabilization effect on a mismatched DNA duplex and shows a strong preference for single-stranded nucleic acids, such as C2-L1Tc. These results provide further insight into the essential properties of the C2-L1Tc protein as a NAC.

Binding Of Immune Serine Proteases To Nucleic Acids Enhances Their Nuclear Localization and Promotes Their Cleavage Of Nucleic Acid-Binding Protein Substrates

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3471-3471
Author(s):  
Jennifer Whangbo ◽  
Marshall Thomas ◽  
Geoffrey McCrossan ◽  
Aaron Deutsch ◽  
Kimberly Martinod ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nuclear Localization ◽  
Serine Proteases ◽  
Rna Binding ◽  
Nuclear Accumulation ◽  
Target Cells ◽  
Nucleic Acid Binding ◽  
High Affinity ◽  
Nucleic Acid Binding Proteins

Abstract When released from cytotoxic T lymphocytes and natural killer cells, Granzyme (Gzm) serine proteases induce programmed cell death of pathogen-infected cells and tumor cells. The Gzms rapidly accumulate in the target cell nucleus by an unknown mechanism. Many of the known substrates of GzmA and GzmB, the most abundant killer cell proteases, bind to DNA or RNA. Gzm substrates predicted by unbiased proteomics studies are also highly enriched for nucleic acid binding proteins. Here we show by fluorescence polarization assays that Gzms bind DNA and RNA with nanomolar affinity. We hypothesized that Gzm binding to nucleic acids enhances nuclear accumulation in target cells and facilitates their cleavage of nucleic acid-binding substrates. In fact, RNase treatment of cell lysates reduced cleavage of RNA binding protein (RBP) targets by GzmA and GzmB. Moreover, adding RNA to recombinant RBP substrates greatly enhanced in vitro cleavage by GzmB, but adding RNA to non-nucleic acid binding proteins did not. For example, exogenous RNA enhanced GzmB cleavage of recombinant hnRNP C1 (an RBP) but not LMNB1 (a non-RBP). In addition, GzmB cleaved the RNA-binding HuR protein efficiently only when it was bound to an HuR-binding RNA oligonucleotide, but not in the presence of an equal amount of non-binding RNA. Thus, nucleic acids facilitate Gzm cleavage of nucleic acid binding substrates. To evaluate whether nucleic acid binding influences Gzm trafficking in target cells, we incubated fixed target cells with RNase and then added Gzms. RNA degradation in target cells reduced Gzm cytosolic localization and increased nuclear accumulation. Similarly, pre-incubating Gzms with exogenous competitor DNA reduced Gzm nuclear localization. The Gzms form a monophyletic clade with other immune serine proteases including neutrophil elastase (NE) and cathepsin G (CATG). Upon neutrophil activation, NE translocates to the nucleus to drive the formation of neutrophil extracellular traps (NETs). NE and CATG, but not non-immune serine proteases such as trypsin and pancreatic elastase, also bind DNA with high affinity and localize to the nucleus of permeabilized cells. Consistent with this finding, competitor DNA also blocks the nuclear localization of NE. Moreover NE and CATG localization to NETs depends on DNA binding. Thus the antimicrobial activity of NETs may depend in part upon the affinity of these proteases for DNA. Our findings indicate that high affinity nucleic acid binding is a conserved and functionally important property of serine proteases involved in cell-mediated immunity. Disclosures: Lieberman: Alnylam Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals ProbeRating: a recommender system to infer binding profiles for nucleic acid-binding proteins

2020 ◽  
Vol 36 (18) ◽  
pp. 4797-4804
Author(s):  
Shu Yang ◽  
Xiaoxi Liu ◽  
Raymond T Ng
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Recommender System ◽  
Binding Proteins ◽  
Rna Binding ◽  
Supplementary Information ◽  
Sequence Information ◽  
Nucleic Acid Binding ◽  
Binding Data ◽  
Nucleic Acid Binding Proteins

Abstract Motivation The interaction between proteins and nucleic acids plays a crucial role in gene regulation and cell function. Determining the binding preferences of nucleic acid-binding proteins (NBPs), namely RNA-binding proteins (RBPs) and transcription factors (TFs), is the key to decipher the protein–nucleic acids interaction code. Today, available NBP binding data from in vivo or in vitro experiments are still limited, which leaves a large portion of NBPs uncovered. Unfortunately, existing computational methods that model the NBP binding preferences are mostly protein specific: they need the experimental data for a specific protein in interest, and thus only focus on experimentally characterized NBPs. The binding preferences of experimentally unexplored NBPs remain largely unknown. Results Here, we introduce ProbeRating, a nucleic acid recommender system that utilizes techniques from deep learning and word embeddings of natural language processing. ProbeRating is developed to predict binding profiles for unexplored or poorly studied NBPs by exploiting their homologs NBPs which currently have available binding data. Requiring only sequence information as input, ProbeRating adapts FastText from Facebook AI Research to extract biological features. It then builds a neural network-based recommender system. We evaluate the performance of ProbeRating on two different tasks: one for RBP and one for TF. As a result, ProbeRating outperforms previous methods on both tasks. The results show that ProbeRating can be a useful tool to study the binding mechanism for the many NBPs that lack direct experimental evidence. and implementation Availability and implementation The source code is freely available at <https://github.com/syang11/ProbeRating>. Supplementary information Supplementary data are available at Bioinformatics online.

Zinc finger proteins in the host-virus interplay: multifaceted functions based on their nucleic acid-binding property

2020 ◽  
Author(s):  
Guanming Wang ◽  
Chunfu Zheng
Keyword(s):  
Nucleic Acid ◽  
Zinc Finger ◽  
Rna Binding ◽  
Viral Infections ◽  
Zinc Finger Proteins ◽  
Zinc Ion ◽  
Research Progress ◽  
Binding Property ◽  
Nucleic Acid Binding ◽  
Transcription Profiles

Abstract Zinc finger proteins (ZFPs) are a huge family comprised of massive, structurally diverse proteins characterized by zinc ion coordinating. They engage in the host-virus interplay in-depth and occupy a significant portion of the host antiviral arsenal. Nucleic acid-binding is the basic property of certain ZFPs, which draws increasing attention due to their immense influence on viral infections. ZFPs exert multiple roles on the viral replications and host cell transcription profiles by recognizing viral genomes and host mRNAs. Their roles could be either antiviral or proviral and were separately discussed. Our review covers the recent research progress and provides a comprehensive understanding of ZFPs in antiviral immunity based on their DNA/RNA binding property.

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 The L1Tc C-Terminal Domain from Trypanosoma cruzi Non-Long Terminal Repeat Retrotransposon Codes for a Protein That Bears Two C2H2 Zinc Finger Motifs and Is Endowed with Nucleic Acid Chaperone Activity

2005 ◽  
Vol 25 (21) ◽  
pp. 9209-9220 ◽  
Author(s):  
Sara R. Heras ◽  
Manuel C. López ◽  
José Luis García-Pérez ◽  
Sandra L. Martin ◽  
M. Carmen Thomas
Keyword(s):  
Nucleic Acid ◽  
Trypanosoma Cruzi ◽  
Long Terminal Repeat ◽  
Zinc Finger ◽  
Chaperone Activity ◽  
Terminal Repeat ◽  
Duplex Dna ◽  
Long Terminal Repeat Retrotransposon ◽  
Nucleic Acid Chaperone ◽  
Zinc Finger Motifs

ABSTRACT L1Tc, a non-long terminal repeat retrotransposon from Trypanosoma cruzi, is a 4.9-kb actively transcribed element which contains a single open reading frame coding for the machinery necessary for its autonomous retrotransposition. In this paper, we analyze the protein encoded by the L1Tc 3′ region, termed C2-L1Tc, which contains two zinc finger motifs similar to those present in the TFIIIA transcription factor family. C2-L1Tc binds nucleic acids with different affinities, such that RNA > tRNA > single-stranded DNA > double-stranded DNA, without any evidence for sequence specificity. C2-L1Tc also exhibits nucleic acid chaperone activity on different DNA templates that may participate in the mechanism of retrotransposition of the element. C2-L1Tc promotes annealing of complementary oligonucleotides, prevents melting of perfect DNA duplexes, and facilitates the strand exchange between DNAs to form the most stable duplex DNA in competitive displacement assays. Mapping of regions of C2-L1Tc using specific peptides showed that nucleic acid chaperone activity required a short basic sequence accompanied by a zinc finger motif or by another basic region such as RRR. Thus, a short basic polypeptide containing the two C2H2 motifs promotes formation of the most stable duplex DNA at a concentration only three times higher than that required for C2-L1Tc.

scholarly journals Structural basis of nucleic acid binding byNicotiana tabacumglycine-rich RNA-binding protein: implications for its RNA chaperone function

2014 ◽  
Vol 42 (13) ◽  
pp. 8705-8718 ◽  
Author(s):  
Fariha Khan ◽  
Mark A. Daniëls ◽  
Gert E. Folkers ◽  
Rolf Boelens ◽  
S. M. Saqlan Naqvi ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Structural Basis ◽  
Nucleic Acid Binding ◽  
Rna Chaperone ◽  
Chaperone Function

Domain necessary for Drosophila ELAV nuclear localization: function requires nuclear ELAV

1999 ◽  
Vol 112 (24) ◽  
pp. 4501-4512 ◽  
Author(s):  
Y.M. Yannoni ◽  
K. White
Keyword(s):  
Nuclear Localization ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Localization Sequence ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Mutant Proteins ◽  
Localization Sequence ◽  
Recognition Motifs

The neuron specific Drosophila ELAV protein belongs to the ELAV family of RNA binding proteins which are characterized by three highly conserved RNA recognition motifs, an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. Despite their highly conserved RNA recognition motifs the ELAV family members are a group of proteins with diverse posttranscriptional functions including splicing regulation, mRNA stability and translatability and have a variety of subcellular localizations. The role of the ELAV hinge in localization and function was examined using transgenes encoding ELAV hinge deletions, in vivo. Subcellular localization of the hinge mutant proteins revealed that residues between amino acids 333–374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human ELAV family member, HuR. This defined sequence, however, was insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays revealed that mutant proteins that fail to localize to the nucleus are unable to provide ELAV vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag.

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