scholarly journals RNA and DNA G-quadruplexes bind to human dicer and inhibit its activity

2021 ◽  
Vol 78 (7) ◽  
pp. 3709-3724
Author(s):  
Natalia Koralewska ◽  
Agnieszka Szczepanska ◽  
Kinga Ciechanowska ◽  
Marta Wojnicka ◽  
Maria Pokornowska ◽  
...  
Keyword(s):  
Genome Instability ◽  
Detailed Knowledge ◽  
Biological Processes ◽  
Binding Assays ◽  
Vitro Binding ◽  
Protein Interactome ◽  
G Quadruplex ◽  
Quadruplex Formation ◽  
Rna And Dna

AbstractGuanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform–PAZ–Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.

scholarly journals RNA and DNA G-quadruplexes bind to human Dicer and inhibit its activity

2020 ◽  
Author(s):  
Natalia Koralewska ◽  
Agnieszka Szczepanska ◽  
Kinga Ciechanowska ◽  
Marta Wojnicka ◽  
Maria Pokornowska ◽  
...  
Keyword(s):  
Genome Instability ◽  
Detailed Knowledge ◽  
Biological Processes ◽  
Biological Functions ◽  
Binding Assays ◽  
Protein Interactome ◽  
G Quadruplex ◽  
Quadruplex Formation ◽  
Rna And Dna

AbstractGuanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform-PAZ-Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

scholarly journals Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

2007 ◽  
Vol 18 (11) ◽  
pp. 4317-4326 ◽  
Author(s):  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Rachel K. Miller ◽  
Kozo Kaibuchi ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Assays ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Lim Domains ◽  
Thick Filaments ◽  
Vitro Binding ◽  
Two Hybrid ◽  
Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

scholarly journals Hypertonicity-induced phosphorylation and nuclear localization of the transcription factor TonEBP

2001 ◽  
Vol 280 (2) ◽  
pp. C248-C253 ◽  
Author(s):  
Stephen C. Dahl ◽  
Joseph S. Handler ◽  
H. Moo Kwon
Keyword(s):  
Transcription Factor ◽  
Nuclear Localization ◽  
Kinase Inhibitors ◽  
Dependent Manner ◽  
Binding Assays ◽  
P38 Inhibitor ◽  
Vitro Binding ◽  
Compatible Osmolytes

The accumulation of compatible osmolytes during osmotic stress is observed in virtually all organisms. In mammals, the hypertonicity-induced expression of osmolyte transporters and synthetic enzymes is conferred by the presence of upstream tonicity-responsive enhancer (TonE) sequences. Recently, we described the cloning and initial characterization of TonE-binding protein (TonEBP), a transcription factor that translocates to the nucleus and associates with TonE sequences in a tonicity-dependent manner. We now report that hypertonicity induces an increase in TonEBP phosphorylation that temporally correlates with increased nuclear localization of the molecule. TonEBP phosphorylation is not affected by a number of kinase inhibitors, including the p38 inhibitor SB-203580. In addition, in vitro binding assays show that the association of TonEBP with TonE sequences is not affected by phosphorylation. Thus TonEBP phosphorylation is an early step in the response of cells to hypertonicity and may be required for nuclear import or retention.

scholarly journals Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

2010 ◽  
Vol 21 (4) ◽  
pp. 630-638 ◽  
Author(s):  
Yutaka Ogawa ◽  
Yoichi Miyamoto ◽  
Munehiro Asally ◽  
Masahiro Oka ◽  
Yoshinari Yasuda ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Time Lapse ◽  
Binding Assays ◽  
Vitro Binding ◽  
Importin Α ◽  
Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin α complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.

KPNA7, an oocyte- and embryo-specific karyopherin?subtype, is required for porcine embryo development

2012 ◽  
Vol 24 (2) ◽  
pp. 382 ◽  
Author(s):  
Xin Wang ◽  
Ki-Eun Park ◽  
Stephanie Koser ◽  
Shihong Liu ◽  
Luca Magnani ◽  
...  
Keyword(s):  
Binding Assays ◽  
Control Groups ◽  
Nuclear Localisation ◽  
Cytoplasmic Proteins ◽  
Vitro Binding ◽  
Intracellular Proteins ◽  
Porcine Embryo ◽  
Stage Embryo ◽  
Interfering Rna

Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherin α subtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherin α subtypes in porcine oocytes and embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherin α 7 (KPNA7), an oocyte and cleavage stage embryo-specific karyopherin α subtype. We hypothesised that knockdown of KPNA7 would negatively affect porcine cleavage development. To test this hypothesis, in vitro matured and fertilised porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P < 0.05). In vitro binding assays also suggest that KPNA7 may transport intracellular proteins that possess unique nuclear localisation signals. Our data suggest that embryos have differential requirements for individual karyopherin α subtypes and that these karyopherin α subtypes differentially transport intracellular cargo during cleavage development.

scholarly journals The Functional Consequences of Eukaryotic Topoisomerase 1 Interaction with G-Quadruplex DNA

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 193
Author(s):  
Alexandra Berroyer ◽  
Nayun Kim
Keyword(s):  
Topoisomerase I ◽  
Genome Instability ◽  
Strand Break ◽  
Dna Topology ◽  
Single Strand Break ◽  
Dna Structures ◽  
G4 Dna ◽  
G Quadruplex

Topoisomerase I in eukaryotic cells is an important regulator of DNA topology. Its catalytic function is to remove positive or negative superhelical tension by binding to duplex DNA, creating a reversible single-strand break, and finally religating the broken strand. Proper maintenance of DNA topological homeostasis, in turn, is critically important in the regulation of replication, transcription, DNA repair, and other processes of DNA metabolism. One of the cellular processes regulated by the DNA topology and thus by Topoisomerase I is the formation of non-canonical DNA structures. Non-canonical or non-B DNA structures, including the four-stranded G-quadruplex or G4 DNA, are potentially pathological in that they interfere with replication or transcription, forming hotspots of genome instability. In this review, we first describe the role of Topoisomerase I in reducing the formation of non-canonical nucleic acid structures in the genome. We further discuss the interesting recent discovery that Top1 and Top1 mutants bind to G4 DNA structures in vivo and in vitro and speculate on the possible consequences of these interactions.

scholarly journals Evaluation of Interactions of Human Cytomegalovirus Immediate-Early IE2 Regulatory Protein with Small Ubiquitin-Like Modifiers and Their Conjugation Enzyme Ubc9

2001 ◽  
Vol 75 (8) ◽  
pp. 3859-3872 ◽  
Author(s):  
Jin-Hyun Ahn ◽  
Yixun Xu ◽  
Won-Jong Jang ◽  
Michael J. Matunis ◽  
Gary S. Hayward
Keyword(s):  
Hcmv Infection ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Binding Assays ◽  
Yeast Two Hybrid ◽  
Vitro Binding ◽  
Lysine Residues ◽  
Infected Cells ◽  
Two Hybrid

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathioneS-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.

scholarly journals Junctional adhesion molecule (JAM) binds to PAR-3

2001 ◽  
Vol 154 (3) ◽  
pp. 491-498 ◽  
Author(s):  
Masahiko Itoh ◽  
Hiroyuki Sasaki ◽  
Mikio Furuse ◽  
Harunobu Ozaki ◽  
Toru Kita ◽  
...  
Keyword(s):  
Adhesion Molecule ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Spatial Relationship ◽  
Immunoglobulin Superfamily ◽  
Binding Assays ◽  
Adhesion Sites ◽  
Vitro Binding ◽  
Self Association

At tight junctions (TJs), claudins with four transmembrane domains are incorporated into TJ strands. Junctional adhesion molecule (JAM), which belongs to the immunoglobulin superfamily, is also localized at TJs, but it remains unclear how JAM is integrated into TJs. Immunoreplica electron microscopy revealed that JAM showed an intimate spatial relationship with TJ strands in epithelial cells. In L fibroblasts expressing exogenous JAM, JAM was concentrated at cell–cell adhesion sites, where there were no strand-like structures, but rather characteristic membrane domains free of intramembranous particles were detected. These domains were specifically labeled with anti-JAM polyclonal antibody, suggesting that JAM forms planar aggregates through their lateral self-association. Immunofluorescence microscopy and in vitro binding assays revealed that ZO-1 directly binds to the COOH termini of claudins and JAM at its PDZ1 and PDZ3 domains, respectively. Furthermore, another PDZ-containing polarity-related protein, PAR-3, was directly bound to the COOH terminus of JAM, but not to that of claudins. These findings led to a molecular architectural model for TJs: small aggregates of JAM are tethered to claudin-based strands through ZO-1, and these JAM aggregates recruit PAR-3 to TJs. We also discuss the importance of this model from the perspective of the general molecular mechanisms behind the recruitment of PAR proteins to plasma membranes.

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