Publications by authors named "Dmitry A Kretov"

12 Publications

  • Page 1 of 1

Ago2-Dependent Processing Allows miR-451 to Evade the Global MicroRNA Turnover Elicited during Erythropoiesis.

Mol Cell 2020 04 18;78(2):317-328.e6. Epub 2020 Mar 18.

Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA. Electronic address:

MicroRNAs (miRNAs) are sequentially processed by two RNase III enzymes, Drosha and Dicer. miR-451 is the only known miRNA whose processing bypasses Dicer and instead relies on the slicer activity of Argonaute-2 (Ago2). miR-451 is highly conserved in vertebrates and regulates erythrocyte maturation, where it becomes the most abundant miRNA. However, the basis for the non-canonical biogenesis of miR-451 is unclear. Here, we show that Ago2 is less efficient than Dicer in processing pre-miRNAs, but this deficit is overcome when miR-144 represses Dicer in a negative-feedback loop during erythropoiesis. Loss of miR-144-mediated Dicer repression in zebrafish embryos and human cells leads to increased canonical miRNA production and impaired miR-451 maturation. Overexpression of Ago2 rescues some of the defects of miR-451 processing. Thus, the evolution of Ago2-dependent processing allows miR-451 to circumvent the global repression of canonical miRNAs elicited, in part, by the miR-144 targeting of Dicer during erythropoiesis.
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http://dx.doi.org/10.1016/j.molcel.2020.02.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201373PMC
April 2020

Inhibition of Transcription Induces Phosphorylation of YB-1 at Ser102 and Its Accumulation in the Nucleus.

Cells 2019 Dec 31;9(1). Epub 2019 Dec 31.

Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Russia.

The Y-box binding protein 1 (YB-1) is an RNA/DNA-binding protein regulating gene expression in the cytoplasm and the nucleus. Although mostly cytoplasmic, YB-1 accumulates in the nucleus under stress conditions. Its nuclear localization is associated with aggressiveness and multidrug resistance of cancer cells, which makes the understanding of the regulatory mechanisms of YB-1 subcellular distribution essential. Here, we report that inhibition of RNA polymerase II (RNAPII) activity results in the nuclear accumulation of YB-1 accompanied by its phosphorylation at Ser102. The inhibition of kinase activity reduces YB-1 phosphorylation and its accumulation in the nucleus. The presence of RNA in the nucleus is shown to be required for the nuclear retention of YB-1. Thus, the subcellular localization of YB-1 depends on its post-translational modifications (PTMs) and intracellular RNA distribution.
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http://dx.doi.org/10.3390/cells9010104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016903PMC
December 2019

GPCR-independent activation of G proteins promotes apical cell constriction in vivo.

J Cell Biol 2019 05 4;218(5):1743-1763. Epub 2019 Apr 4.

Department of Biochemistry, Boston University School of Medicine, Boston, MA

Heterotrimeric G proteins are signaling switches that control organismal morphogenesis across metazoans. In invertebrates, specific GPCRs instruct G proteins to promote collective apical cell constriction in the context of epithelial tissue morphogenesis. In contrast, tissue-specific factors that instruct G proteins during analogous processes in vertebrates are largely unknown. Here, we show that DAPLE, a non-GPCR protein linked to human neurodevelopmental disorders, is expressed specifically in the neural plate of embryos to trigger a G protein signaling pathway that promotes apical cell constriction during neurulation. DAPLE localizes to apical cell-cell junctions in the neuroepithelium, where it activates G protein signaling to drive actomyosin-dependent apical constriction and subsequent bending of the neural plate. This function is mediated by a (GBA) motif that was acquired by DAPLE in vertebrates during evolution. These findings reveal that regulation of tissue remodeling during vertebrate development can be driven by an unconventional mechanism of heterotrimeric G protein activation that operates in lieu of GPCRs.
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http://dx.doi.org/10.1083/jcb.201811174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504902PMC
May 2019

YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis.

Nucleic Acids Res 2019 04;47(6):3127-3141

SABNP, University of Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France.

The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.
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http://dx.doi.org/10.1093/nar/gky1303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451097PMC
April 2019

Microtubules as platforms for probing liquid-liquid phase separation in cells - application to RNA-binding proteins.

J Cell Sci 2018 06 11;131(11). Epub 2018 Jun 11.

SABNP Lab, Univ Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France

Liquid-liquid phase separation enables compartmentalization of biomolecules in cells, notably RNA and associated proteins in the nucleus. Besides having critical functions in RNA processing, there is a major interest in deciphering the molecular mechanisms of compartmentalization orchestrated by RNA-binding proteins such as TDP-43 (also known as TARDBP) and FUS because of their link to neuron diseases. However, tools for probing compartmentalization in cells are lacking. Here, we developed a method to analyze the mixing and demixing of two different phases in a cellular context. The principle is the following: RNA-binding proteins are confined on microtubules and quantitative parameters defining their spatial segregation are measured along the microtubule network. Through this approach, we found that four mRNA-binding proteins, HuR (also known as ELAVL1), G3BP1, TDP-43 and FUS form mRNA-rich liquid-like compartments on microtubules. TDP-43 is partly miscible with FUS but immiscible with either HuR or G3BP1. We also demonstrate that mRNA is essential to capture the mixing and demixing behavior of mRNA-binding proteins in cells. Taken together, we show that microtubules can be used as platforms to understand the mechanisms underlying liquid-liquid phase separation and their deregulation in human diseases.
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http://dx.doi.org/10.1242/jcs.214692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031325PMC
June 2018

Assessing miR-451 Activity and Its Role in Erythropoiesis.

Methods Mol Biol 2018 ;1680:179-190

Department of Biochemistry, Boston University School of Medicine, 72 East Concord Street, Boston, MA, 02118, USA.

The ability to microinject small RNAs and mRNAs into zebrafish embryos, of different genetic backgrounds, allows for the precise dissection of microRNA processing pathways at the molecular level, while simultaneously provides insight into their physiologic role. Here, we apply such an approach to determine the impact of Argonaute 2 in the processing of miR-451, a vertebrate-specific microRNA required for terminal erythrocyte differentiation. This was achieved using fluorescent microRNA reporter sensor assays and phenotype rescue experiments.
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http://dx.doi.org/10.1007/978-1-4939-7339-2_12DOI Listing
May 2018

Nanoscale Analysis Reveals the Maturation of Neurodegeneration-Associated Protein Aggregates: Grown in mRNA Granules then Released by Stress Granule Proteins.

ACS Nano 2017 07 5;11(7):7189-7200. Epub 2017 Jul 5.

SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay , 91025 Evry, France.

TDP-43 and FUS are two mRNA-binding proteins associated with neurodegenerative diseases that form cytoplasmic inclusions with prion-like properties in affected neurons. Documenting the early stages of the formation of TDP-43 or FUS protein aggregates and the role of mRNA stress granules that are considered as critical intermediates for protein aggregation is therefore of interest to understand disease propagation. Here, we developed a single molecule approach via atomic force microscopy (AFM), which provides structural information out of reach by fluorescence microscopy. In addition, the aggregation process can be probed in the test tube without separating the interacting partners, which would affect the thermodynamic equilibrium. The results demonstrate that isolated mRNA molecules serve as crucibles to promote TDP-43 and FUS multimerization. Their subsequent merging results in the formation of mRNA granules containing TDP-43 and FUS aggregates. Interestingly, TDP-43 or FUS protein aggregates can be released from mRNA granules by either YB-1 or G3BP1, two stress granule proteins that compete for the binding to mRNA with TDP-43 and FUS. Altogether, the results indicate that age-related successive assembly/disassembly of stress granules in neurons, regulated by mRNA-binding proteins such as YB-1 and G3BP1, could be a source of protein aggregation.
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http://dx.doi.org/10.1021/acsnano.7b03071DOI Listing
July 2017

Probing protein interactions in living mammalian cells on a microtubule bench.

Sci Rep 2015 Nov 27;5:17304. Epub 2015 Nov 27.

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM U1204 and Université Evry-Val d'Essonne, Evry, 91025 France.

Microtubules are μm-long cylinders of about 25 nm in diameter which are present in the cytoplasm of eukaryotic cells. Here, we have developed a new method which uses these cylindrical structures as platforms to detect protein interactions in cells. The principle is simple: a protein of interest used as bait is brought to microtubules by fusing it to Tau, a microtubule-associated protein. The presence of a protein prey on microtubules then reveals an interaction between bait and prey. This method requires only a conventional optical microscope and straightforward fluorescence image analysis for detection and quantification of protein interactions. To test the reliability of this detection scheme, we used it to probe the interactions among three mRNA-binding proteins in both fixed and living cells and compared the results to those obtained by pull-down assays. We also tested whether the molecular interactions of Cx43, a membrane protein, can be investigated with this system. Altogether, the results indicate that microtubules can be used as platforms to detect protein interactions in mammalian cells, which should provide a basis for investigating pathogenic protein interactions involved in human diseases.
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http://dx.doi.org/10.1038/srep17304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661529PMC
November 2015

Poly(ADP-ribosyl)ation as a new posttranslational modification of YB-1.

Biochimie 2015 Dec 9;119:36-44. Epub 2015 Oct 9.

Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, 630090, Russia; Novosibirsk State University, Novosibirsk, 630090, Russia. Electronic address:

Multifunctional Y-box binding protein 1 (YB-1) is actively studied as one of the components of cellular response to genotoxic stress. However, the precise role of YB-1 in the process of DNA repair is still obscure. In the present work we report for the first time new posttranslational modification of YB-1 - poly(ADP-ribosyl)ation, catalyzed by one of the main regulatory enzymes of DNA repair - poly(ADP-ribose)polymerase 1 (PARP1) in the presence of model DNA substrate carrying multiple DNA lesions. Therefore, poly(ADP-ribosyl)ation of YB-1 catalyzed with PARP1, can be stimulated by damaged DNA. The observed property of YB-1 underlines its ability to participate in the DNA repair by its involvement in the regulatory cascades of DNA repair.
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http://dx.doi.org/10.1016/j.biochi.2015.10.008DOI Listing
December 2015

mRNA and DNA selection via protein multimerization: YB-1 as a case study.

Nucleic Acids Res 2015 Oct 13;43(19):9457-73. Epub 2015 Aug 13.

Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM U1204 and Université Evry-Val d'Essonne, Evry, 91025 France

Translation is tightly regulated in cells for keeping adequate protein levels, this task being notably accomplished by dedicated mRNA-binding proteins recognizing a specific set of mRNAs to repress or facilitate their translation. To select specific mRNAs, mRNA-binding proteins can strongly bind to specific mRNA sequences/structures. However, many mRNA-binding proteins rather display a weak specificity to short and redundant sequences. Here we examined an alternative mechanism by which mRNA-binding proteins could inhibit the translation of specific mRNAs, using YB-1, a major translation regulator, as a case study. Based on a cooperative binding, YB-1 forms stable homo-multimers on some mRNAs while avoiding other mRNAs. Via such inhomogeneous distribution, YB-1 can selectively inhibit translation of mRNAs on which it has formed stable multimers. This novel mechanistic view on mRNA selection may be shared by other proteins considering the elevated occurrence of multimerization among mRNA-binding proteins. Interestingly, we also demonstrate how, by using the same mechanism, YB-1 can form multimers on specific DNA structures, which could provide novel insights into YB-1 nuclear functions in DNA repair and multi-drug resistance.
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http://dx.doi.org/10.1093/nar/gkv822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627072PMC
October 2015

Inhibition of abasic site cleavage in bubble DNA by multifunctional protein YB-1.

J Mol Recognit 2015 Feb 21;28(2):117-23. Epub 2015 Jan 21.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk, 630090, Russia.

Y-box binding protein 1 (YB-1) is widely known to participate in a multiple DNA and RNA processing events in the living cell. YB-1 is also regarded as a putative component of DNA repair. This possibility is supported by relocalization of YB-1 into the nucleus following genotoxic stress. Increased affinity of YB-1 for damaged DNA, especially in its single-stranded form, and its functional interaction with proteins responsible for the initiation of apurinic/apyrimidinic (AP) site repair, namely, AP endonuclease 1 and DNA glycosylase NEIL1, suggest that YB-1 could be involved in the repair of AP sites as a regulatory protein. Here we show that YB-1 has a significant inhibitory effect on the cleavage of AP sites located in single-stranded DNA and in DNA bubble structures. Such interference may be considered as a possible mechanism to prevent single-stranded intermediates of DNA replication, transcription and repair from being converted into highly genotoxic DNA strand breaks, thus allowing the cell to coordinate different DNA processing mechanisms.
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http://dx.doi.org/10.1002/jmr.2435DOI Listing
February 2015

Formation of amyloid-like fibrils by Y-box binding protein 1 (YB-1) is mediated by its cold shock domain and modulated by disordered terminal domains.

PLoS One 2012 8;7(5):e36969. Epub 2012 May 8.

Group of Protein Biosynthesis Regulation, Institute of Protein Research, Russian Academy of Sciences, Pushchino, Russia.

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036969PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348147PMC
September 2012