scholarly journals MSY2 and MSY4 Bind a Conserved Sequence in the 3′ Untranslated Region of Protamine 1 mRNA In Vitro and In Vivo

2001 ◽  
Vol 21 (20) ◽  
pp. 7010-7019 ◽  
Author(s):  
Flaviano Giorgini ◽  
Holly G. Davies ◽  
Robert E. Braun
Keyword(s):  
Untranslated Region ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Binding Activity ◽  
Mobility Shift ◽  
Conserved Sequence ◽  
Mobility Shift Assay ◽  
Protamine 1

ABSTRACT Y-box proteins are major constituents of ribonucleoprotein particles (RNPs) which contain translationally silent mRNAs in gametic cells. We have recently shown that a sequence-specific RNA binding activity present in spermatogenic cells contains the two Y-box proteins MSY2 and MSY4. We show here that MSY2 and MSY4 bind a sequence, 5′-UCCAUCA-3′, present in the 3′ untranslated region of the translationally repressed protamine 1 (Prm1) mRNA. Using pre- and post-RNase T1-digested substrate RNAs, it was determined that MSY2 and MSY4 can bind an RNA of eight nucleotides containing the MSY2 and MSY4 binding site. Single nucleotide mutations in the sequence eliminated the binding of MSY2 and MSY4 in an electrophoretic mobility shift assay, and the resulting mutants failed to compete for binding in a competition assay. A consensus site of UACCACAUCCACU(subscripts indicate nucleotides which do not disrupt YRS binding by MSY2 and MSY4), denoted the Y-box recognition site (YRS), was defined from this mutational analysis. These mutations in the YRS were further characterized in vivo using a novel application of the yeast three-hybrid system. Experiments with transgenic mice show that disruption of the YRS in vivo relieves Prm1-like repression of a reporter gene. The conservation of the RNA binding motifs among Y-box protein family members raises the possibility that other Y-box proteins may have previously unrecognized sequence-specific RNA binding activities.

scholarly journals Oxalomalate, a competitive inhibitor of aconitase, modulates the RNA-binding activity of iron-regulatory proteins

2000 ◽  
Vol 348 (2) ◽  
pp. 315-320 ◽  
Author(s):  
Michela FESTA ◽  
Alfredo COLONNA ◽  
Concetta PIETROPAOLO ◽  
Alfredo RUFFO
Keyword(s):  
Iron Metabolism ◽  
Rna Binding ◽  
Competitive Inhibitor ◽  
Binding Activity ◽  
Regulatory Proteins ◽  
Mobility Shift ◽  
Prolonged Incubation ◽  
Iron Regulatory Proteins

We investigated the effect of oxalomalate (OMA, α-hydroxy-β-oxalosuccinic acid), a competitive inhibitor of aconitase, on the RNA-binding activity of the iron-regulatory proteins (IRP1 and IRP2) that control the post-transcriptional expression of various proteins involved in iron metabolism. The RNA-binding activity of IRP was evaluated by electrophoretic mobility-shift assay of cell lysates from 3T3-L1 mouse fibroblasts, SH-SY5Y human cells and mouse livers incubated in vitro with OMA, with and without 2-mercaptoethanol (2-ME). Analogous experiments were performed in vivo by prolonged incubation (72 h) of 3T3-L1 cells with OMA, and by injecting young mice with equimolar concentrations of oxaloacetate and glyoxylate, which are the precursors of OMA synthesis. OMA remarkably decreased the binding activity of IRP1 and, when present, of IRP2, in all samples analysed. In addition, the recovery of IRP1 by 2-ME in the presence of OMA was constantly lower versus control values. These findings suggest that the severe decrease in IRP1 RNA-binding activity depends on: (i) linking of OMA to the active site of aconitase, which prevents the switch to IRP1 and explains resistance to the reducing agents, and (ii) possible interaction of OMA with some functional amino acid residues in IRP that are responsible for binding to the specific mRNA sequences involved in the regulation of iron metabolism.

scholarly journals Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor Shiftless

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1230
Author(s):  
Sawsan Napthine ◽  
Chris H. Hill ◽  
Holly C. M. Nugent ◽  
Ian Brierley
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Stop Codon ◽  
Leukemia Virus ◽  
Size Exclusion ◽  
Human Immunodeficiency Virus Replication ◽  
Mobility Shift ◽  
Ribosomal Frameshifting ◽  
Stop Codon Readthrough

The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.

scholarly journals Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (12) ◽  
pp. 4522-4534 ◽  
Author(s):  
R Ng ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
In Vitro Assays ◽  
Binding Studies ◽  
Mobility Shift ◽  
Sequence Element ◽  
Sequence Elements ◽  
Mobility Shift Assay

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 by oxygen levels: a model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation

2009 ◽  
Vol 425 (1) ◽  
pp. 235-243 ◽  
Author(s):  
Raquel  Castro-Prego ◽  
Mónica Lamas-Maceiras ◽  
Pilar Soengas ◽  
Isabel Carneiro ◽  
Isabel González-Siso ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oxygen Availability ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Hypoxic Conditions ◽  
Cross Links ◽  
Mobility Shift Assay ◽  
Transcriptional Adaptation

Ixr1p from Saccharomyces cerevisiae has been previously studied because it binds to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. Ixr1p is also a transcriptional regulator of anaerobic/hypoxic genes, such as SRP1/TIR1, which encodes a stress-response cell wall manoprotein, and COX5B, which encodes the Vb subunit of the mitochondrial complex cytochrome c oxidase. However, factors controlling IXR1 expression remained unexplored. In the present study we show that IXR1 mRNA levels are controlled by oxygen availability and increase during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1–Ssn6. Ixr1p itself is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (nucleotides −557 to −376). EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is also required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H2O2. IXR1 expression is moderately activated by H2O2 and this induction is Yap1p-dependent. A model of IXR1 regulation as a relay for sensing different signals related to change in oxygen availability is proposed. In this model, transcriptional adaptation from aerobiosis to hypoxia depends on ROX1 and IXR1 cross-regulation.

scholarly journals Ouabain Modulates Zymosan-Induced Peritonitis in Mice

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Jacqueline Alves Leite ◽  
Anne Kaliery De Abreu Alves ◽  
José Guilherme Marques Galvão ◽  
Mariana Pires Teixeira ◽  
Luiz Henrique Agra Cavalcante-Silva ◽  
...  
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Potent Inhibitor ◽  
Binding Activity ◽  
Acute Inflammatory Response ◽  
Mobility Shift ◽  
Plasma Exudation ◽  
Mobility Shift Assay ◽  
Number Of Cells

Ouabain, a potent inhibitor of the Na+, K+-ATPase, was identified as an endogenous substance. Recently, ouabain was shown to affect various immunological processes. We have previously demonstrated the ability of ouabain to modulate inflammation, but little is known about the mechanisms involved. Thus, the aim of the present work is to evaluate the immune modulatory role of ouabain on zymosan-induced peritonitis in mice. Our results show that ouabain decreased plasma exudation (33%). After induction of inflammation, OUA treatment led to a 46% reduction in the total number of cells, as a reflex of a decrease of polymorphonuclear leukocytes, which does not appear to be due to cell death. Furthermore, OUA decreased TNF-α(57%) and IL-1β(58%) levels, without interfering with IL-6 and IL-10. Also,in vitroexperiments show that ouabain did not affect endocytic capacity. Moreover, electrophoretic mobility shift assay (EMSA) shows that zymosan treatment increased (85%) NF-κB binding activity and that ouabain reduced (30%) NF-κB binding activity induced by zymosan. Therefore, our data suggest that ouabain modulated acute inflammatory response, reducing the number of cells and cytokines levels in the peritoneal cavity, as well as NFκB activation, suggesting a new mode of action of this substance.

scholarly journals Fourteen Residues of the U1 snRNP-Specific U1A Protein Are Required for Homodimerization, Cooperative RNA Binding, and Inhibition of Polyadenylation

2000 ◽  
Vol 20 (6) ◽  
pp. 2209-2217 ◽  
Author(s):  
Jacqueline M. T. Klein Gunnewiek ◽  
Reem I. Hussein ◽  
Yvonne van Aarssen ◽  
Daphne Palacios ◽  
Rob de Jong ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Structural Data ◽  
Protein Molecule ◽  
Cooperative Binding ◽  
Mobility Shift ◽  
Small Nuclear Ribonucleoprotein ◽  
U1a Protein

ABSTRACT It was previously shown that the human U1A protein, one of three U1 small nuclear ribonucleoprotein-specific proteins, autoregulates its own production by binding to and inhibiting the polyadenylation of its own pre-mRNA. The U1A autoregulatory complex requires two molecules of U1A protein to cooperatively bind a 50-nucleotide polyadenylation-inhibitory element (PIE) RNA located in the U1A 3′ untranslated region. Based on both biochemical and nuclear magnetic resonance structural data, it was predicted that protein-protein interactions between the N-terminal regions (amino acids [aa] 1 to 115) of the two U1A proteins would form the basis for cooperative binding to PIE RNA and for inhibition of polyadenylation. In this study, we not only experimentally confirmed these predictions but discovered some unexpected features of how the U1A autoregulatory complex functions. We found that the U1A protein homodimerizes in the yeast two-hybrid system even when its ability to bind RNA is incapacitated. U1A dimerization requires two separate regions, both located in the N-terminal 115 residues. Using both coselection and gel mobility shift assays, U1A dimerization was also observed in vitro and found to depend on the same two regions that were found in vivo. Mutation of the second homodimerization region (aa 103 to 115) also resulted in loss of inhibition of polyadenylation and loss of cooperative binding of two U1A protein molecules to PIE RNA. This same mutation had no effect on the binding of one U1A protein molecule to PIE RNA. A peptide containing two copies of aa 103 to 115 is a potent inhibitor of polyadenylation. Based on these data, a model of the U1A autoregulatory complex is presented.

scholarly journals Transcript analysis reveals an extended regulon and the importance of protein–protein co-operativity for the Escherichia coli methionine repressor

2006 ◽  
Vol 396 (2) ◽  
pp. 227-234 ◽  
Author(s):  
Ferenc Marincs ◽  
Iain W. Manfield ◽  
Jonathan A. Stead ◽  
Kenneth J. Mcdowall ◽  
Peter G. Stockley
Keyword(s):  
Escherichia Coli ◽  
Gene Replacement ◽  
Dna Arrays ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Metabolic Role ◽  
Mobility Shift Assay

We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5′ promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein–protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein–protein and protein–DNA affinity.

scholarly journals RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein

2021 ◽  
Vol 22 (16) ◽  
pp. 9103
Author(s):  
Julita Gumna ◽  
Angelika Andrzejewska-Romanowska ◽  
David J. Garfinkel ◽  
Katarzyna Pachulska-Wieczorek
Keyword(s):  
Rna Structure ◽  
Rna Binding ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Mobility Shift ◽  
Gag Protein ◽  
Rna Dimerization ◽  
Electrophoretic Mobility Shift Assays

A universal feature of retroelement propagation is the formation of distinct nucleoprotein complexes mediated by the Gag capsid protein. The Ty1 retrotransposon Gag protein from Saccharomyces cerevisiae lacks sequence homology with retroviral Gag, but is functionally related. In addition to capsid assembly functions, Ty1 Gag promotes Ty1 RNA dimerization and cyclization and initiation of reverse transcription. Direct interactions between Gag and retrotransposon genomic RNA (gRNA) are needed for Ty1 replication, and mutations in the RNA-binding domain disrupt nucleation of retrosomes and assembly of functional virus-like particles (VLPs). Unlike retroviral Gag, the specificity of Ty1 Gag-RNA interactions remain poorly understood. Here we use microscale thermophoresis (MST) and electrophoretic mobility shift assays (EMSA) to analyze interactions of immature and mature Ty1 Gag with RNAs. The salt-dependent experiments showed that Ty1 Gag binds with high and similar affinity to different RNAs. However, we observed a preferential interaction between Ty1 Gag and Ty1 RNA containing a packaging signal (Psi) in RNA competition analyses. We also uncover a relationship between Ty1 RNA structure and Gag binding involving the pseudoknot present on Ty1 gRNA. In all likelihood, the differences in Gag binding affinity detected in vitro only partially explain selective Ty1 RNA packaging into VLPs in vivo.

scholarly journals Circ-CTNNB1 Drives Aerobic Glycolysis and Osteosarcoma Progression via m6A Modification Through Interacting With RBM15

2021 ◽  
Author(s):  
Hucheng Liu ◽  
Jun Xiao ◽  
Bo Li ◽  
Yajun Chen ◽  
Jin Zeng ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Rna Binding ◽  
Aerobic Glycolysis ◽  
Ectopic Expression ◽  
Phosphoglycerate Kinase ◽  
Binding Motif ◽  
Mobility Shift ◽  
Underlying Mechanisms

Abstract Background In a previous study, we have identified that circ-CTNNB1 (a circular RNA derived from CTNNB1) drives cancer progression through the activation of the Wnt/β-catenin signaling pathway in various tumors. However, the functions of circ-CTNNB1 in regulating osteosarcoma (OS, a highly malignant bone tumor in children and adolescents) remain unclear. In this study, we aimed to assess the role of circ-CTNNB1 in OS and identify the underlying mechanisms, which may contribute to the exploration of a potential therapeutic strategy for OS. Methods Circ-CTNNB1 was analyzed by qRT-PCR, and the results were confirmed by Sanger sequencing. The interaction and effects between circ-CTNNB1 and RNA binding motif protein 15 (RBM15) were analyzed through biotin-labeled RNA pull-down and mass spectrometry, in vitro binding, and RNA electrophoretic mobility shift assays. In vitro and in vivo experiments were performed to evaluate the biological functions and underlying mechanisms of circ-CTNNB1 and RBM15 in OS cells. Results Circ-CTNNB1 was highly expressed in OS tissues and predominantly detected in the nucleus of OS cells. Ectopic expression of circ-CTNNB1 promoted the growth, invasion, and metastasis of OS cells in vitro and in vivo. Mechanistically, circ-CTNNB1 interacted with RBM15 and subsequently promoted the expression of hexokinase 2 (HK2), glucose-6-phosphate isomerase (GPI), and phosphoglycerate kinase 1 (PGK1) through N6-methyladenosine (m6A) modification to facilitate the glycolysis process and activate OS progression. Conclusions These results indicate that oncogenic circ-CTNNB1 drives aerobic glycolysis and OS progression by facilitating RBM15-mediated m6A modification.

Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae

1987 ◽  
Vol 7 (12) ◽  
pp. 4522-4534
Author(s):  
R Ng ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
In Vitro Assays ◽  
Binding Studies ◽  
Mobility Shift ◽  
Sequence Element ◽  
Sequence Elements ◽  
Mobility Shift Assay

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

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