Rapid Immunopurification of Ribonucleoprotein Complexes of Plants

2015 ◽  
pp. 209-219 ◽  
Author(s):  
Reed Sorenson ◽  
Julia Bailey-Serres
Keyword(s):  
Ribonucleoprotein Complexes

DEVELOPMENT OF APPROACHES FOR DETECTION OF GENOME-INTEGRATED PROVIRAL DNA OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV-1) IN ULTRA LOW CONCENTRATIONS USING THE CRISPR/CAS SYSTEM

2020 ◽  
Author(s):  
M.A. Tyumentseva ◽  
◽  
A.I. Tyumentsev ◽  
V.G. Akimkin ◽  
◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Health Monitoring ◽  
Biological Samples ◽  
Proviral Dna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Low Concentrations ◽  
Immunodeficiency Virus ◽  
Hiv 1

For the effective functioning of supervisory and health monitoring services, it is necessary to introduce modern molecular technologies into their practice. Therefore, the task of developing new effective methods for detecting pathogen, for example HIV, based on CRISPR/CAS genome editing systems, remains urgent. In the present work, guide RNAs and specific oligonucleotides were developed for preliminary amplification of highly conserved regions of the HIV-1 genome. The developed guide RNAs make it possible to detect single copies of HIV-1 proviral DNA in vitro as part of CRISPR/CAS ribonucleoprotein complexes in biological samples after preliminary amplification.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

scholarly journals The role of CPEB family proteins in the nervous system function in the norm and pathology

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Eugene Kozlov ◽  
Yulii V. Shidlovskii ◽  
Rudolf Gilmutdinov ◽  
Paul Schedl ◽  
Mariya Zhukova
Keyword(s):  
Nervous System ◽  
Gene Regulation ◽  
Neural Development ◽  
Fragile X ◽  
Autism Spectrum ◽  
Mrna Transport ◽  
Ribonucleoprotein Complexes ◽  
Posttranscriptional Gene Regulation ◽  
Nervous System Function ◽  
Functional Features

AbstractPosttranscriptional gene regulation includes mRNA transport, localization, translation, and regulation of mRNA stability. CPEB (cytoplasmic polyadenylation element binding) family proteins bind to specific sites within the 3′-untranslated region and mediate poly- and deadenylation of transcripts, activating or repressing protein synthesis. As part of ribonucleoprotein complexes, the CPEB proteins participate in mRNA transport and localization to different sub-cellular compartments. The CPEB proteins are evolutionarily conserved and have similar functions in vertebrates and invertebrates. In the nervous system, the CPEB proteins are involved in cell division, neural development, learning, and memory. Here we consider the functional features of these proteins in the nervous system of phylogenetically distant organisms: Drosophila, a well-studied model, and mammals. Disruption of the CPEB proteins functioning is associated with various pathologies, such as autism spectrum disorder and brain cancer. At the same time, CPEB gene regulation can provide for a recovery of the brain function in patients with fragile X syndrome and Huntington's disease, making the CPEB genes promising targets for gene therapy.

scholarly journals The noncoding MIR100HG RNA enhances the autocrine function of transforming growth factor β signaling

Oncogene ◽  
2021 ◽  
Author(s):  
Panagiotis Papoutsoglou ◽  
Dorival Mendes Rodrigues-Junior ◽  
Anita Morén ◽  
Andrew Bergman ◽  
Fredrik Pontén ◽  
...  
Keyword(s):  
Growth Factor ◽  
Target Genes ◽  
Rna Binding ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Tgfβ Signaling ◽  
Ribonucleoprotein Complexes ◽  
Expression Of Genes ◽  
Downstream Effectors ◽  
Human Carcinomas

AbstractActivation of the transforming growth factor β (TGFβ) pathway modulates the expression of genes involved in cell growth arrest, motility, and embryogenesis. An expression screen for long noncoding RNAs indicated that TGFβ induced mir-100-let-7a-2-mir-125b-1 cluster host gene (MIR100HG) expression in diverse cancer types, thus confirming an earlier demonstration of TGFβ-mediated transcriptional induction of MIR100HG in pancreatic adenocarcinoma. MIR100HG depletion attenuated TGFβ signaling, expression of TGFβ-target genes, and TGFβ-mediated cell cycle arrest. Moreover, MIR100HG silencing inhibited both normal and cancer cell motility and enhanced the cytotoxicity of cytostatic drugs. MIR100HG overexpression had an inverse impact on TGFβ signaling responses. Screening for downstream effectors of MIR100HG identified the ligand TGFβ1. MIR100HG and TGFB1 mRNA formed ribonucleoprotein complexes with the RNA-binding protein HuR, promoting TGFβ1 cytokine secretion. In addition, TGFβ regulated let-7a-2–3p, miR-125b-5p, and miR-125b-1–3p expression, all encoded by MIR100HG intron-3. Certain intron-3 miRNAs may be involved in TGFβ/SMAD-mediated responses (let-7a-2–3p) and others (miR-100, miR-125b) in resistance to cytotoxic drugs mediated by MIR100HG. In support of a model whereby TGFβ induces MIR100HG, which then enhances TGFβ1 secretion, analysis of human carcinomas showed that MIR100HG expression correlated with expression of TGFB1 and its downstream extracellular target TGFBI. Thus, MIR100HG controls the magnitude of TGFβ signaling via TGFβ1 autoinduction and secretion in carcinomas.

scholarly journals Autonomous splicing and complementation of in vivo-assembled spliceosomes.

1989 ◽  
Vol 108 (3) ◽  
pp. 765-777 ◽  
Author(s):  
S Zeitlin ◽  
R C Wilson ◽  
A Efstratiadis
Keyword(s):  
Nuclear Extract ◽  
Physiological Concentration ◽  
Ribonucleoprotein Complexes ◽  
Matrix Bound ◽  
The Relationship ◽  
Matrix Preparation ◽  
Cell Nuclear Extract ◽  
Biochemical Features

We have used an in vivo system generating assayable amounts of a specific pre-mRNA to study the relationship between splicing and an operationally defined nuclear matrix preparation (NM). When NM is prepared by extraction of DNase I-treated nuclei with an approximately physiological concentration of KCl (0.1 M), a portion of NM-associated precursor can be spliced in vitro in the presence of ATP and Mg2+ and in the absence of splicing extract ("autonomous splicing"). We propose that the autonomous reaction, which does not exhibit a temporal lag and is half-complete in 5 min, occurs in fully assembled, matrix-bound ribonucleoprotein complexes (in vivo spliceosomes). Extraction of the NM with concentrations of KCl greater than 0.4 M eliminates autonomous splicing but leaves behind preassembled complexes that can be complemented for splicing with HeLa cell nuclear extract. The splicing complementing factor, representing one or more activities present in the nuclear extract and also in the cytoplasmic S100 fraction, is relatively heat resistant, devoid of an RNA component, and does not bind to DEAE-Sepharose in 0.1 M KCl. It exists in the nucleus in two forms; bound to autonomous spliceosomes and free in the nucleoplasm. Biochemical features of the complementation reaction, and conditions for reversible uncoupling of the two splicing steps are described and discussed.

scholarly journals Voltage-Dependent Anion Channel 1 Interacts with Ribonucleoprotein Complexes To Enhance Infectious Bursal Disease Virus Polymerase Activity

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Chunyan Han ◽  
Xiangwei Zeng ◽  
Shuai Yao ◽  
Li Gao ◽  
Lizhou Zhang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Disease Virus ◽  
Infectious Bursal Disease Virus ◽  
Viral Proteins ◽  
Polymerase Activity ◽  
Infectious Bursal Disease ◽  
Cellular Factor ◽  
Ribonucleoprotein Complexes ◽  
Viral Yield ◽  
Bursal Disease

ABSTRACTInfectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus. Segment A contains two overlapping open reading frames (ORFs), which encode viral proteins VP2, VP3, VP4, and VP5. Segment B contains one ORF and encodes the viral RNA-dependent RNA polymerase, VP1. IBDV ribonucleoprotein complexes are composed of VP1, VP3, and dsRNA and play a critical role in mediating viral replication and transcription during the virus life cycle. In the present study, we identified a cellular factor, VDAC1, which was upregulated during IBDV infection and found to mediate IBDV polymerase activity. VDAC1 senses IBDV infection by interacting with viral proteins VP1 and VP3. This association is caused by RNA bridging, and all three proteins colocalize in the cytoplasm. Furthermore, small interfering RNA (siRNA)-mediated downregulation ofVDAC1resulted in a reduction in viral polymerase activity and a subsequent decrease in viral yield. Moreover, overexpression of VDAC1 enhanced IBDV polymerase activity. We also found that the viral protein VP3 can replace segment A to execute polymerase activity. A previous study showed that mutations in the C terminus of VP3 directly influence the formation of VP1-VP3 complexes. Our immunoprecipitation experiments demonstrated that protein-protein interactions between VDAC1 and VP3 and between VDAC1 and VP1 play a role in stabilizing the interaction between VP3 and VP1, further promoting IBDV polymerase activity.IMPORTANCEThe cellular factor VDAC1 controls the entry and exit of mitochondrial metabolites and plays a pivotal role during intrinsic apoptosis by mediating the release of many apoptogenic molecules. Here we identify a novel role of VDAC1, showing that VDAC1 interacts with IBDV ribonucleoproteins (RNPs) and facilitates IBDV replication by enhancing IBDV polymerase activity through its ability to stabilize interactions in RNP complexes. To our knowledge, this is the first report that VDAC1 is specifically involved in regulating IBDV RNA polymerase activity, providing novel insight into virus-host interactions.

scholarly journals Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

2000 ◽  
Vol 20 (15) ◽  
pp. 5516-5528 ◽  
Author(s):  
Žaklina Strezoska ◽  
Dimitri G. Pestov ◽  
Lester F. Lau
Keyword(s):  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Weight Fraction ◽  
Rnase A ◽  
Mutant Form ◽  
Rrna Processing ◽  
Ribonucleoprotein Complexes ◽  
Nuclear Extracts ◽  
Mouse Tissues ◽  
Mouse Cells

ABSTRACT We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G1 in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1Δ, which lacks 231 amino acids in the N- terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1Δ leads to cell growth arrest in the G1phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1Δ expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1Δ in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit.

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