Biochemical characterization of Yin Yang 1 – RNA complexes

2008 ◽  
Vol 86 (1) ◽  
pp. 31-36 ◽  
Author(s):  
Zachery R. Belak ◽  
Andrew Ficzycz ◽  
Nick Ovsenek
Keyword(s):  
Rna Binding ◽  
Biochemical Characterization ◽  
Ribonucleoprotein Complexes ◽  
Yin Yang 1 ◽  
Significant Difference ◽  
Yin Yang ◽  
Rna Interaction ◽  
Rna Complexes

YY1 (Yin Yang 1) is present in the Xenopus oocyte cytoplasm as a constituent of messenger ribonucleoprotein complexes (mRNPs). Association of YY1 with mRNPs requires direct RNA-binding activity. Previously, we have shown YY1 has a high affinity for U-rich RNA; however, potential interactions with plausible in vivo targets have not been investigated. Here we report a biochemical characterization of the YY1–RNA interaction including an investigation of the stability, potential 5′-methylguanosine affinity, and specificity for target RNAs. The formation of YY1–RNA complexes in vitro was highly resistant to thermal, ionic, and detergent disruption. The endogenous oocyte YY1–mRNA interactions were also found to be highly stable. Specific YY1–RNA interactions were observed with selected mRNA and 5S RNA probes. The affinity of YY1 for these substrates was within an order of magnitude of that for its cognate DNA element. Experiments aimed at determining the potential role of the 7-methylguanosine cap on RNA-binding reveal no significant difference in the affinity of YY1 for capped or uncapped mRNA. Taken together, the results show that the YY1–RNA interaction is highly stable, and that YY1 possesses the ability to interact with structurally divergent RNA substrates. These data are the first to specifically document the interaction between YY1 and potential in vivo targets.

scholarly journals PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactions

2016 ◽  
Author(s):  
Alex M. Tamburino ◽  
Ebru Kaymak ◽  
Shaleen Shrestha ◽  
Amy D. Holdorf ◽  
Sean P. Ryder ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Small Rna ◽  
Rna Binding ◽  
Mrna Translation ◽  
Single Experiment ◽  
Novel Interactions ◽  
Transcriptional Gene Regulation ◽  
Rna Interaction ◽  
Eukaryotic Genomes

SUMMARYInteractions between RNA binding protein (RBP) and mRNAs are critical to post-transcriptional gene regulation. Eukaryotic genomes encode thousands of mRNAs and hundreds of RBPs. However, in contrast to interactions between transcription factors (TFs) and DNA, the interactome between RBPs and RNA has been explored for only a small number of proteins and RNAs. This is largely because the focus has been on using ‘protein-centered’ (RBP-to-RNA) interaction mapping methods that identify the RNAs with which an individual RBP interacts. While powerful, these methods cannot as of yet be applied to the entire RBPome. Moreover, it may be desirable for a researcher to identify the repertoire of RBPs that can interact with an mRNA of interest – in a ‘gene-centered’ manner, yet few such techniques are available. Here, we present Protein-RNA Interaction Mapping Assay (PRIMA) with which an RNA ‘bait’ can be tested versus multiple RBP ‘preys’ in a single experiment. PRIMA is a translation-based assay that examines interactions in the yeast cytoplasm, the cellular location of mRNA translation. We show that PRIMA can be used with small RNA elements, as well as with full-length Caenorhabditis elegans 3′UTRs. PRIMA faithfully recapitulates numerous well-characterized RNA-RBP interactions and also identified novel interactions, some of which were confirmed in vivo. We envision that PRIMA will provide a complementary tool to expand the depth and scale with which the RNA-RBP interactome can be explored.

scholarly journals Morphological and Biochemical Characterization of Macrophages Activated by Carrageenan and Lipopolysaccharide In Vivo

2004 ◽  
Vol 29 (2) ◽  
pp. 27-34 ◽  
Author(s):  
Valéria Pereira Nacife ◽  
Maria de Nazaré Correia Soeiro ◽  
Rachel Novaes Gomes ◽  
Heloísa D’Avila ◽  
Hugo Caire Castro-Faria Neto ◽  
...  
Keyword(s):  
Biochemical Characterization

scholarly journals Astrocyte-specific deletion of the transcription factor Yin Yang 1 in murine substantia nigra mitigates manganese-induced dopaminergic neurotoxicity

2020 ◽  
Vol 295 (46) ◽  
pp. 15662-15676 ◽  
Author(s):  
Edward Pajarillo ◽  
James Johnson ◽  
Asha Rizor ◽  
Ivan Nyarko-Danquah ◽  
Getinet Adinew ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Substantia Nigra ◽  
Critical Role ◽  
Conditional Knockout ◽  
Motor Functions ◽  
Mn Toxicity ◽  
Protein Levels ◽  
Yin Yang 1 ◽  
Yin Yang

Manganese (Mn)-induced neurotoxicity resembles Parkinson's disease (PD), but the mechanisms underpinning its effects remain unknown. Mn dysregulates astrocytic glutamate transporters, GLT-1 and GLAST, and dopaminergic function, including tyrosine hydroxylase (TH). Our previous in vitro studies have shown that Mn repressed GLAST and GLT-1 via activation of transcription factor Yin Yang 1 (YY1). Here, we investigated if in vivo astrocytic YY1 deletion mitigates Mn-induced dopaminergic neurotoxicity, attenuating Mn-induced reduction in GLAST/GLT-1 expression in murine substantia nigra (SN). AAV5-GFAP-Cre-GFP particles were infused into the SN of 8-week–old YY1flox/flox mice to generate a region-specific astrocytic YY1 conditional knockout (cKO) mouse model. 3 weeks after adeno-associated viral (AAV) infusion, mice were exposed to 330 μg of Mn (MnCl2 30 mg/kg, intranasal instillation, daily) for 3 weeks. After Mn exposure, motor functions were determined in open-field and rotarod tests, followed by Western blotting, quantitative PCR, and immunohistochemistry to assess YY1, TH, GLAST, and GLT-1 levels. Infusion of AAV5-GFAP-Cre-GFP vectors into the SN resulted in region-specific astrocytic YY1 deletion and attenuation of Mn-induced impairment of motor functions, reduction of TH-expressing cells in SN, and TH mRNA/protein levels in midbrain/striatum. Astrocytic YY1 deletion also attenuated the Mn-induced decrease in GLAST/GLT-1 mRNA/protein levels in midbrain. Moreover, YY1 deletion abrogated its interaction with histone deacetylases in astrocytes. These results indicate that astrocytic YY1 plays a critical role in Mn-induced neurotoxicity in vivo, at least in part, by reducing astrocytic GLAST/GLT-1. Thus, YY1 might be a potential target for treatment of Mn toxicity and other neurological disorders associated with dysregulation of GLAST/GLT-1.

scholarly journals Human Immunodeficiency Virus Type 1 Vif Protein Is an Integral Component of an mRNP Complex of Viral RNA and Could Be Involved in the Viral RNA Folding and Packaging Process

2000 ◽  
Vol 74 (18) ◽  
pp. 8252-8261 ◽  
Author(s):  
Hui Zhang ◽  
Roger J. Pomerantz ◽  
Geethanjali Dornadula ◽  
Yong Sun
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Binding ◽  
Viral Rna ◽  
Immunodeficiency Virus ◽  
Mrnp Complex ◽  
Rna Interaction ◽  
Vif Protein ◽  
Hiv 1

ABSTRACT Virion infectivity factor (Vif) is a protein encoded by human immunodeficiency virus types 1 and 2 (HIV-1 and -2) and simian immunodeficiency virus, plus other lentiviruses, and is essential for viral replication either in vivo or in culture for nonpermissive cells such as peripheral blood lymphoid cells, macrophages, and H9 T cells. Defects in the vif gene affect virion morphology and reverse transcription but not the expression of viral components. It has been shown that Vif colocalizes with Gag in cells and Vif binds to the NCp7 domain of Gag in vitro. However, it seems that Vif is not specifically packaged into virions. The molecular mechanism(s) for Vif remains unknown. In this report, we demonstrate that HIV-1 Vif is an RNA-binding protein and specifically binds to HIV-1 genomic RNA in vitro. Further, Vif binds to HIV-1 RNA in the cytoplasm of virus-producing cells to form a 40S mRNP complex. Coimmunoprecipitation and in vivo UV cross-linking assays indicated that Vif directly interact with HIV-1 RNA in the virus-producing cells. Vif-RNA binding could be displaced by Gag-RNA binding, suggesting that Vif protein in the mRNP complex may mediate viral RNA interaction with HIV-1 Gag precursors. Furthermore, we have demonstrated that these Vif mutants that lose the RNA binding activity in vitro do not supportvif-deficient HIV-1 replication in H9 T cells, suggesting that the RNA binding capacity of Vif is important for its function. Further studies regarding Vif-RNA interaction in virus-producing cells will be important for studying the function of Vif in the HIV-1 life cycle.

Biochemical characterization of bona fide polycystin-1 in vitro and in vivo

2001 ◽  
Vol 38 (6) ◽  
pp. 1421-1429 ◽  
Author(s):  
Alessandra Boletta ◽  
Feng Qian ◽  
Luiz F. Onuchic ◽  
Alessandra Bragonzi ◽  
Marina Cortese ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Bona Fide

scholarly journals THE SEPARATION AND CHARACTERIZATION OF SUBMICROSCOPIC COMPONENTS OF HEPATIC CYTOPLASM

1959 ◽  
Vol 7 (2) ◽  
pp. 126-132 ◽  
Author(s):  
MITURU TAKANAMI
Keyword(s):  
Alkaline Phosphatase ◽  
Principal Components ◽  
Biochemical Properties ◽  
Sedimentation Rates ◽  
Differential Centrifugation ◽  
Ultrasonic Oscillation ◽  
Ribonucleoprotein Complexes ◽  
Acid And Alkaline Phosphatase

In an ultracentrifugal study on the cytoplasmic supernatant of rabbit liver, the following two principal components were separated from the supernatant by differential centrifugation and their biochemical properties investigated: (1) a granular substance sedimented at a rate of about 250s (250s component) and (2) a few macromolecular components the sedimentation rates of which were roughly in the range of from 40s to 100s (macromolecular components). The 250s component, which was rich in lipids and easily disintegrated into smaller units by treatments of ultrasonic oscillation and of Nadesoxycholate, exhibited much higher activities of dipeptidase, acid and alkaline phosphatase than the macromolecular components. By contrast, the latter macromolecular components which belonged to ribonucleoprotein complexes exhibited comparatively high activities of RNase and esterase. Uptake in vivo of radioactive phosphate (P32) by the RNA contained in the above two principal components markedly differed from each other. When the RNA contained in the 250s component was separated by the use of Nadesoxycholate into RNA in a non-sedimentable portion and that in a sedimentable portion corresponding to a ribonucleoprotein coplex, the RNA in the latter state showed an uptake rate extremely different from that of the macromolecular components. So it is emphasized that the ribonucleoprotein complex comprised in the 250s component and that existing free in the cytoplasm (i.e. macromolecular components) are metabolically different.

scholarly journals Yin Yang 1 is required for PHD finger protein 20-mediated myogenic differentiation in vitro and in vivo

2020 ◽  
Vol 27 (12) ◽  
pp. 3321-3336
Author(s):  
Hyunji Lee ◽  
Youngeun Hong ◽  
Gyeyeong Kong ◽  
Dong Hoon Lee ◽  
Minhee Kim ◽  
...  
Keyword(s):  
Myogenic Differentiation ◽  
Phd Finger ◽  
Yin Yang 1 ◽  
Finger Protein ◽  
Yin Yang

scholarly journals Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR–Cas immunity

2020 ◽  
Vol 48 (8) ◽  
pp. 4418-4434 ◽  
Author(s):  
Kawanda Foster ◽  
Sabine Grüschow ◽  
Scott Bailey ◽  
Malcolm F White ◽  
Michael P Terns
Keyword(s):  
Rna Binding ◽  
Pyrococcus Furiosus ◽  
Type Iii ◽  
Second Messenger Signaling ◽  
Biochemical Analyses ◽  
Rna Interaction ◽  
Dual Mechanism ◽  
Target Rna ◽  
Rna And Dna

Abstract Type III CRISPR–Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3′ protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.

scholarly journals RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

2009 ◽  
Vol 90 (7) ◽  
pp. 1748-1756 ◽  
Author(s):  
M. Gopinath ◽  
M. S. Shaila
Keyword(s):  
Biochemical Characterization ◽  
Covalent Bond ◽  
Rinderpest Virus ◽  
Viral Mrnas ◽  
L Protein ◽  
Rna Triphosphatase ◽  
Rnp Complex

Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L–P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of γ-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 5′ sequence. The L protein forms a covalent enzyme–guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717–2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.

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