RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein

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.

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The ArgP Protein Stimulates the Klebsiella pneumoniae gdhA Promoter in a Lysine-Sensitive Manner

Journal of Bacteriology ◽

10.1128/jb.00295-08 ◽

2008 ◽

Vol 190 (12) ◽

pp. 4351-4359 ◽

Cited By ~ 11

Author(s):

Thomas J. Goss

Keyword(s):

Klebsiella Pneumoniae ◽

Upstream Region ◽

Mobility Shift ◽

Base Pairs ◽

Dnase I Footprinting ◽

Sensitive Factor ◽

Electrophoretic Mobility Shift Assays ◽

Knockout Mutation

ABSTRACT The lysine-sensitive factor that binds to the upstream region of the Klebsiella pneumoniae gdhA promoter and stimulates gdhA transcription during growth in minimal medium has been proposed to be the K. pneumoniae ArgP protein (M. R. Nandineni, R. S. Laishram, and J. Gowrishankar, J. Bacteriol. 186:6391-6399, 2004). A knockout mutation of the K. pneumoniae argP gene was generated and used to assess the roles of exogenous lysine and argP in the regulation of the gdhA promoter. Disruption of argP reduced the strength and the lysine-dependent regulation of the gdhA promoter. Electrophoretic mobility shift assays using crude extracts prepared from wild-type and argP-defective strains indicted the presence of an argP-dependent factor whose ability to bind the gdhA promoter was lysine sensitive. DNase I footprinting studies using purified K. pneumoniae ArgP protein indicated that ArgP bound the region that lies approximately 50 to 100 base pairs upstream of the gdhA transcription start site in a manner that was sensitive to the presence of lysine. Substitutions within the region bound by ArgP affected the binding of ArgP to the gdhA promoter region in vitro and the argP-dependent stimulation of the gdhA promoter in vivo. These observations suggest that elevated intracellular levels of lysine reduce the affinity of ArgP for its binding site at the gdhA promoter, preventing ArgP from binding to and stimulating transcription from the promoter in vivo.

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3,5-Diiodo-L-thyronine (T2) has selective thyromimetic effects in vivo and in vitro

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0190137 ◽

1997 ◽

Vol 19 (2) ◽

pp. 137-147 ◽

Cited By ~ 60

Author(s):

SG Ball ◽

J Sokolov ◽

WW Chin

Keyword(s):

Gh3 Cells ◽

Mrna Levels ◽

Thyroid Status ◽

Hormone Activity ◽

Mobility Shift ◽

Nuclear Extracts ◽

Electrophoretic Mobility Shift Assays ◽

Gh3 Cell

Recent data have suggested that the iodothyronine, 3,5-diiodo-l-thyronine (T2), has selective thyromimetic activity. In vivo, T2 has been shown to suppress TSH levels at doses that do not produce significant peripheral manifestations of thyroid hormone activity. Furthermore, T2 has been shown to produce smaller increments in peripheral indices of thyroid status than does T3, when doses resulting in equivalent suppression of circulating TSH are compared. We have assessed the selective thyromimetic activity of T2 in vivo and in vitro, and performed in vitro studies to assess the potential molecular basis for these selective properties. T2 was 100-fold less potent than T3 in stimulating GH mRNA levels in GH3 cells. In contrast, the iodothyronines were almost equivalent in their ability to downregulate TRbeta2 mRNA levels in this cell line. Both 3,3'-diiodo-L-thyronine and thyronine exhibited no significant thyromimetic effects on either process. In vivo, doses of T2 and T3 that were equivalent in their induction of hepatic malic enzyme (ME) mRNA did not produce equivalent suppression of circulating TSH, with T2 being only 27% as effective as T3. T2 was up to 500-fold less potent than T3 in displacing [125I]-T3 from in vitro translated specific nuclear receptors (TRs) and GH3 cell nuclear extracts. Electrophoretic mobility shift assays, assessing the ability of T2 to produce dissociation of TRbeta1 homodimers from inverted palindrome T3 response elements, indicated that T2 was also 1000-fold less potent than T3 in this respect. These data confirm that T2 has significant thyromimetic activity, and that this activity is selective both in vivo and in vitro. However, there are no data to support a selective central effect, T2 being relatively more potent in stimulating hepatic ME mRNA than in suppression of TSH in vivo. The basis for this differential thyromimetic activity is not selective affinity of the different TR isoforms for T2, or divergent properties of T2 in competitive binding and functional assays in vitro.

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

Molecular and Cellular Biology ◽

10.1128/mcb.20.6.2209-2217.2000 ◽

2000 ◽

Vol 20 (6) ◽

pp. 2209-2217 ◽

Cited By ~ 19

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.

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E2A expression, nuclear localization, and in vivo formation of DNA- and non-DNA-binding species during B-cell development.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7321 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7321-7333 ◽

Cited By ~ 34

Author(s):

Y Jacobs ◽

C Vierra ◽

C Nelson

Keyword(s):

B Cell ◽

Electrophoretic Mobility ◽

Cell Lines ◽

Nuclear Localization ◽

Cell Development ◽

B Cell Development ◽

Mobility Shift ◽

Electrophoretic Mobility Shift Assays

A monoclonal antibody (Yae) was characterized and shown to specifically recognize E2A proteins in vivo, including the E2A-Pbx1 fusion gene products, p77E2A-Pbx1 and p85E2A-Pbx1. E2A proteins of a predominant molecular mass of 72 kDa, which comigrated with in vitro-produced rat E12 and and rat E47, were detected in human pro-B, pre-B, mature B, and plasma cell lines. The Yae antibody detected an E2A-containing microE2 enhancer element-binding complex (BCF-1) in pre-B- and mature B-cell lines in electrophoretic mobility shift assays which displayed a migration rate similar to that of in vitro-produced rat E12 and rat E47. A new E2A-containing microE2-binding species (P-E2A) was identified in plasma cells by using electrophoretic mobility shift assays. E2A proteins were detected in pro-B cells but were unable to bind the microE2 site. These observations suggest that the microE2 site is the target of stage-specific E2A regulatory complexes during B-cell development. Immunostaining analyses demonstrated the predominant nuclear localization of E2A proteins. Finally, we have identified an E2A form, designated I-E2A, which is unable to bind DNA. Our observations demonstrate novel in vivo mechanisms for the regulation of transcription by E2A proteins during B-cell development.

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MdMYB3 helps regulate anthocyanin accumulation in apple calli under moderately acidic conditions

10.1101/429456 ◽

2018 ◽

Author(s):

Yi-Cheng Wang ◽

Jing-Jing Sun ◽

Yan-Fen Qiu ◽

Xiao-Jun Gong ◽

Li Ma ◽

...

Keyword(s):

Anthocyanin Biosynthesis ◽

Myb Transcription Factor ◽

Anthocyanin Content ◽

Anthocyanin Accumulation ◽

Mobility Shift ◽

In The Wild ◽

Acidic Conditions ◽

Electrophoretic Mobility Shift Assays

AbstractAnthocyanins are the key factors controlling the coloration of plant tissues. However, the molecular mechanism underlying the effects of environmental pH on the synthesis of apple anthocyanins is unclear. In this study, we analyzed the anthocyanin contents of apple calli cultured in media at different pHs (5.5, 6.0, and 6.5). The highest anthocyanin content was observed at pH 6.0. Additionally, the moderately acidic conditions up-regulated the expression of MdMYB3 as well as specific anthocyanin biosynthesis structural genes (MdDFR and MdUFGT). Moreover, the anthocyanin content was higher in calli overexpressing MdMYB3 than in the wild-type controls at different pHs. Yeast one-hybrid assay results indicated that MdMYB3 binds to the MdDFR and MdUFGT promoters in vivo. An analysis of the MdDFR and MdUFGT promoters revealed multiple MYB-binding sites. Meanwhile, electrophoretic mobility shift assays confirmed that MdMYB3 binds to the MdDFR and MdUFGT promoters in vitro. Furthermore, GUS promoter activity assays suggested that the MdDFR and MdUFGT promoter activities are enhanced by acidic conditions, and the binding of MdMYB3 may further enhance activity. These results implied that an acid-induced apple MYB transcription factor (MdMYB3) promotes anthocyanin accumulation by up-regulating the expression of MdDFR and MdUFGT under moderately acidic conditions.

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A Deep Learning Approach for Learning Intrinsic Protein-RNA Binding Preferences

10.1101/328633 ◽

2018 ◽

Author(s):

Ilan Ben-Bassat ◽

Benny Chor ◽

Yaron Orenstein

Keyword(s):

Neural Network ◽

Rna Structure ◽

Rna Binding ◽

Gene Expression Regulation ◽

Supplementary Information ◽

Joint Analysis ◽

Binding Prediction ◽

Binding Models

AbstractMotivationThe complexes formed by binding of proteins to RNAs play key roles in many biological processes, such as splicing, gene expression regulation, translation, and viral replication. Understanding protein-RNA binding may thus provide important insights to the functionality and dynamics of many cellular processes. This has sparked substantial interest in exploring protein-RNA binding experimentally, and predicting it computationally. The key computational challenge is to efficiently and accurately infer RNA-binding models that will enable prediction of novel protein-RNA interactions to additional transcripts of interest.ResultsWe developed DLPRB, a new deep neural network (DNN) approach for learning protein-RNA binding preferences and predicting novel interactions. We present two different network architectures: a convolutional neural network (CNN), and a recurrent neural network (RNN). The novelty of our network hinges upon two key aspects: (i) the joint analysis of both RNA sequence and structure, which is represented as a probability vector of different RNA structural contexts; (ii) novel features in the architecture of the networks, such as the application of RNNs to RNA-binding prediction, and the combination of hundreds of variable-length filters in the CNN. Our results in inferring accurate RNA-binding models from high-throughput in vitro data exhibit substantial improvements, compared to all previous approaches for protein-RNA binding prediction (both DNN and non-DNN based). A highly significant improvement is achieved for in vitro binding prediction, and a more modest, yet statistically significant,improvement for in vivo binding prediction. When incorporating experimentally-measured RNA structure compared to predicted one, the improvement on in vivo data increases. By visualizing the binding specificities, we can gain novel biological insights underlying the mechanism of protein RNA-binding.AvailabilityThe source code is publicly available at https://github.com/ilanbb/[email protected] informationSupplementary data are available at Bioinformatics online.

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

10.21203/rs.3.rs-991482/v1 ◽

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.

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E2A expression, nuclear localization, and in vivo formation of DNA- and non-DNA-binding species during B-cell development

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7321-7333.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7321-7333

Author(s):

Y Jacobs ◽

C Vierra ◽

C Nelson

Keyword(s):

B Cell ◽

Electrophoretic Mobility ◽

Cell Lines ◽

Nuclear Localization ◽

Cell Development ◽

B Cell Development ◽

Mobility Shift ◽

Electrophoretic Mobility Shift Assays

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Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3165-3175.1992 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3165-3175

Author(s):

M Bennett ◽

S Piñol-Roma ◽

D Staknis ◽

G Dreyfuss ◽

R Reed

Keyword(s):

Rna Polymerase Ii ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Packaging ◽

Splice Site Selection ◽

Spliceosome Assembly ◽

Hnrnp Proteins ◽

Heterogeneous Nuclear Ribonucleoproteins

We have investigated the composition of the earliest detectable complex (H) assembled on pre-mRNA during the in vitro splicing reaction. We show that most of the proteins in this complex correspond to heterogeneous nuclear ribonucleoproteins (hnRNP), a set of abundant RNA-binding proteins that bind nascent RNA polymerase II transcripts in vivo. Thus, these studies establish a direct parallel between the initial events of RNA processing in vitro and in vivo. In contrast to previous studies, in which total hnRNP particles were isolated from mammalian nuclei, we determined the hnRNP composition of complexes assembled on individual RNAs of defined sequence. We found that a unique combination of hnRNP proteins is associated with each RNA. Thus, our data provide direct evidence for transcript-dependent assembly of pre-mRNA in hnRNP complexes. The observation that pre-mRNA is differentially bound by hnRNP proteins prior to spliceosome assembly suggests the possibility that RNA packaging could play a central role in the mechanism of splice site selection, as well as other posttranscriptional events.

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Selective Packaging in Murine Coronavirus Promotes Virulence by Limiting Type I Interferon Responses

mBio ◽

10.1128/mbio.00272-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 8

Author(s):

Jeremiah Athmer ◽

Anthony R. Fehr ◽

Matthew E. Grunewald ◽

Wen Qu ◽

D. Lori Wheeler ◽

...

Keyword(s):

Innate Immune ◽

Type I ◽

Genomic Rna ◽

Rna Packaging ◽

Packaging Signal ◽

Subgenomic Rnas ◽

Immune Detection ◽

Negative Sense

ABSTRACTSelective packaging is a mechanism used by multiple virus families to specifically incorporate genomic RNA (gRNA) into virions and exclude other types of RNA. Lineage A betacoronaviruses incorporate a 95-bp stem-loop structure, the packaging signal (PS), into the nsp15 locus of ORF1b that is both necessary and sufficient for the packaging of RNAs. However, unlike other viral PSs, where mutations generally resulted in viral replication defects, mutation of the coronavirus (CoV) PS results in large increases in subgenomic RNA packaging with minimal effects on gRNA packagingin vitroand on viral titers. Here, we show that selective packaging is also required for viral evasion of the innate immune response and optimal pathogenicity. We engineered two distinct PS mutants in two different strains of murine hepatitis virus (MHV) that packaged increased levels of subgenomic RNAs, negative-sense genomic RNA, and even cellular RNAs. All PS mutant viruses replicated normallyin vitrobut caused dramatically reduced lethality and weight lossin vivo. PS mutant virus infection of bone marrow-derived macrophages resulted in increased interferon (IFN) production, indicating that the innate immune system limited the replication and/or pathogenesis of PS mutant virusesin vivo. PS mutant viruses remained attenuated in MAVS−/−and Toll-like receptor 7-knockout (TLR7−/−) mice, two well-known RNA sensors for CoVs, but virulence was restored in interferon alpha/beta receptor-knockout (IFNAR−/−) mice or in MAVS−/−mice treated with IFNAR-blocking antibodies. Together, these data indicate that coronaviruses promote virulence by utilizing selective packaging to avoid innate immune detection.IMPORTANCECoronaviruses (CoVs) produce many types of RNA molecules during their replication cycle, including both positive- and negative-sense genomic and subgenomic RNAs. Despite this, coronaviruses selectively package only positive-sense genomic RNA into their virions. Why CoVs selectively package their genomic RNA is not clear, as disruption of the packaging signal in MHV, which leads to loss of selective packaging, does not affect genomic RNA packaging or virus replication in cultured cells. This contrasts with other viruses, where disruption of selective packaging generally leads to altered replication. Here, we demonstrate that in the absence of selective packaging, the virulence of MHV was significantly reduced. Importantly, virulence was restored in the absence of interferon signaling, indicating that selective packaging is a mechanism used by CoVs to escape innate immune detection.

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