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 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 A Novel Regulatory Role for the Circadian Clock Protein TOC1 via RNA binding

2020 ◽  
Author(s):  
Yi Li ◽  
Yong-Gang Chang ◽  
Dawn H. Nagel ◽  
Tingjian Chen ◽  
Matias L Rugnone ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Sequence Similarity ◽  
Transcriptional Repressor ◽  
Titration Calorimetry ◽  
Mobility Shift ◽  
Rna Motif ◽  
Clock Protein

AbstractThe circadian clock enables plants to predict daily changes of external signals and synchronize them with internal processes, conferring enhanced fitness and growth vigor. The first described Arabidopsis circadian clock protein is TIMING OF CAB EXPRESSION 1 (TOC1), which functions in a transcriptional feedback loop with two myb transcription factors, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Previous studies have shown that TOC1 is a DNA-binding transcriptional repressor of CCA1 and LHY. However, the DNA motifs enriched amongst TOC1 targets share weak sequence similarity and lack consensus, suggesting that TOC1 regulates the expression of its targets through a novel mechanism. Here we show that the TOC1 protein binds directly to RNA via its conserved CCT domain. Using in vitro RNA selection, we identified an RNA motif that is recognized by the TOC1-CCT domain. The TOC1-CCT domain binds to this RNA sequence with nanomolar affinity determined by quantitative electrophoretic mobility shift assays (EMSAs) and isothermal titration calorimetry (ITC). NMR experiments showed that two CCT fragments, CCT533-547 and CCT550-565, use basic residues to bind the RNA motif. Mutational analysis confirmed that lysyl and arginyl residues bind to RNA in a cooperative manner. Furthermore, transiently expressed wildtype and mutant TOC1 in protoplasts demonstrated that RNA binding activity of TOC1 is required for its function as a transcriptional repressor in vivo. Our results reveal a novel regulatory mechanism for TOC1 through RNA binding, suggesting that TOC1 might play key roles as a multi-function protein.

scholarly journals Homozygous Inactivating Mutation inNANOS3in Two Sisters with Primary Ovarian Insufficiency

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Mariza G. Santos ◽  
Aline Z. Machado ◽  
Conceição N. Martins ◽  
Sorahia Domenice ◽  
Elaine M. F. Costa ◽  
...  
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Primordial Germ Cells ◽  
Embryonic Cell ◽  
Primary Ovarian Insufficiency ◽  
Primary Amenorrhea ◽  
Female Reproduction ◽  
Ovarian Insufficiency ◽  
Rna Interaction

Despite the increasing understanding of female reproduction, the molecular diagnosis of primary ovarian insufficiency (POI) is seldom obtained. The RNA-binding protein NANOS3 poses as an interesting candidate gene for POI since members of the Nanos family have an evolutionarily conserved function in germ cell development and maintenance by repressing apoptosis. We performed mutational analysis ofNANOS3in a cohort of 85 Brazilian women with familial or isolated POI, presenting with primary or secondary amenorrhea, and in ethnically-matched control women. A homozygous p.Glu120Lys mutation inNANOS3was identified in two sisters with primary amenorrhea. The substituted amino acid is located within the second C2HC motif in the conserved zinc finger domain of NANOS3 andin silicomolecular modelling suggests destabilization of protein-RNA interaction.In vitroanalyses of apoptosis through flow cytometry and confocal microscopy show that NANOS3 capacity to prevent apoptosis was impaired by this mutation. The identification of an inactivating missense mutation inNANOS3suggests a mechanism for POI involving increased primordial germ cells (PGCs) apoptosis during embryonic cell migration and highlights the importance of NANOS proteins in human ovarian biology.

scholarly journals Essential Amino Acids of the Hantaan Virus N Protein in Its Interaction with RNA

2005 ◽  
Vol 79 (15) ◽  
pp. 10032-10039 ◽  
Author(s):  
William Severson ◽  
Xiaolin Xu ◽  
Michaela Kuhn ◽  
Nina Senutovitch ◽  
Mercy Thokala ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Essential Amino Acids ◽  
Full Length ◽  
Hantaan Virus ◽  
Deletion Mapping ◽  
Mobility Shift ◽  
N Protein ◽  
Lysine Residues

ABSTRACT The nucleocapsid (N) protein of hantavirus encapsidates viral genomic and antigenomic RNAs. Previously, deletion mapping identified a central, conserved region (amino acids 175 to 217) within the Hantaan virus (HTNV) N protein that interacts with a high affinity with these viral RNAs (vRNAs). To further define the boundaries of the RNA binding domain (RBD), several peptides were synthesized and examined for the ability to bind full-length S-segment vRNA. Peptide 195-217 retained 94% of the vRNA bound by the HTNV N protein, while peptides 175-186 and 205-217 bound only 1% of the vRNA. To further explore which residues were essential for binding vRNA, we performed a comprehensive mutational analysis of the amino acids in the RBD. Single and double Ala substitutions were constructed for 18 amino acids from amino acids 175 to 217 in the full-length N protein. In addition, Ala substitutions were made for the three R residues in peptide 185-217. An analysis of protein-RNA interactions by electrophoretic mobility shift assays implicated E192, Y206, and S217 as important for binding. Chemical modification experiments showed that lysine residues, but not arginine or cysteine residues, contribute to RNA binding, which agreed with bioinformatic predictions. Overall, these data implicate lysine residues dispersed from amino acids 175 to 429 of the protein and three amino acids located in the RBD as essential for RNA binding.

scholarly journals Human U4/U6 snRNP Recycling Factor p110: Mutational Analysis Reveals the Function of the Tetratricopeptide Repeat Domain in Recycling

2004 ◽  
Vol 24 (17) ◽  
pp. 7392-7401 ◽  
Author(s):  
Jan Medenbach ◽  
Silke Schreiner ◽  
Sunbin Liu ◽  
Reinhard Lührmann ◽  
Albrecht Bindereif
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Protein A ◽  
Tetratricopeptide Repeat ◽  
Terminal Sequence ◽  
Recognition Motifs ◽  
Tpr Domain

ABSTRACT After each spliceosome cycle, the U4 and U6 snRNAs are released separately and are recycled to the functional U4/U6 snRNP, requiring in the mammalian system the U6-specific RNA binding protein p110 (SART3). Its domain structure is made up of an extensive N-terminal domain with at least seven tetratricopeptide repeat (TPR) motifs, followed by two RNA recognition motifs (RRMs) and a highly conserved C-terminal sequence of 10 amino acids. Here we demonstrate under in vitro recycling conditions that U6-p110 is an essential splicing factor. Recycling activity requires both the RRMs and the TPR domain but not the highly conserved C-terminal sequence. For U6-specific RNA binding, the two RRMs with some flanking regions are sufficient. Yeast two-hybrid assays reveal that p110 interacts through its TPR domain with the U4/U6-specific 90K protein, indicating a specific role of the TPR domain in spliceosome recycling. On the 90K protein, a short internal region (amino acids 416 to 550) suffices for the interaction with p110. Together, these data suggest a model whereby p110 brings together U4 and U6 snRNAs through both RNA-protein and protein-protein interactions.

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 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.

scholarly journals Effects of Bicyclomycin on RNA- and ATP-Binding Activities of Transcription Termination Factor Rho

1998 ◽  
Vol 42 (3) ◽  
pp. 571-578 ◽  
Author(s):  
Lucia Carrano ◽  
Cecilia Bucci ◽  
Roberto De Pascalis ◽  
Alfredo Lavitola ◽  
Filomena Manna ◽  
...  
Keyword(s):  
Rna Binding ◽  
Transcription Termination ◽  
Gel Filtration ◽  
Experimental System ◽  
Cross Linking ◽  
Atp Binding ◽  
Mobility Shift ◽  
Termination Factor ◽  
Transcription Termination Factor

ABSTRACT Bicyclomycin is a commercially important antibiotic that has been shown to be effective against many gram-negative bacteria. Genetic and biochemical evidence indicates that the antibiotic interferes with RNA metabolism in Escherichia coli by inhibiting the activity of transcription termination factor Rho. However, the precise mechanism of inhibition is not completely known. In this study we have used in vitro transcription assays to analyze the effects of bicyclomycin on the termination step of transcription. The Rho-dependent transcription termination region located within thehisG cistron of Salmonella typhimurium has been used as an experimental system. The possible interference of the antibiotic with the various functions of factor Rho, such as RNA binding at the primary site, ATP binding, and hexamer formation, has been investigated by RNA gel mobility shift, photochemical cross-linking, and gel filtration experiments. The results of these studies demonstrate that bicyclomycin does not interfere with the binding of Rho to the loading site on nascent RNA. Binding of the factor to ATP is not impeded, on the contrary, the antibiotic appears to decrease the apparent equilibrium dissociation constant for ATP in photochemical cross-linking experiments. The available evidence suggests that this decrease might be due to an interference with the correct positioning of ATP within the nucleotide-binding pocket leading b an inherent block of ATP hydrolysis. Possibly, as a consequence of this interference, the antibiotic also prevents ATP-dependent stabilization of Rho hexamers.

A study of mesoderm patterning through the analysis of the regulation of Xmyf-5 expression

Development ◽  
2002 ◽  
Vol 129 (12) ◽  
pp. 2917-2927
Author(s):  
Matthew Polli ◽  
Enrique Amaya
Keyword(s):  
Mutational Analysis ◽  
Model Organism ◽  
Essential Element ◽  
Positional Information ◽  
Loss Of Function ◽  
Mobility Shift ◽  
Endogenous Gene ◽  
Electrophoretic Mobility Shift Assays ◽  
Mesoderm Patterning

Xenopus laevis has been a particularly useful model organism for identifying factors involved in the induction and patterning of the mesoderm, however, much remains to be learned about how these factors interact. The myogenic transcription factor Xmyf-5 is the earliest known gene to be expressed specifically in the dorsolateral mesoderm of the gastrula, a domain that is established by the interaction of dorsal and ventral signals. For this reason, we have begun to investigate how the expression of Xmyf-5 is regulated. We have identified a 7.28 kb Xenopus tropicalis Xmyf-5 (Xtmyf-5) genomic DNA fragment that accurately recapitulates the expression of the endogenous gene. Deletion and mutational analysis has identified HBX2, an essential element, approximately 1.2 kb upstream from the start of transcription, which is necessary for both activation and repression of Xtmyf-5 expression, implying that positional information is integrated at this site. Electrophoretic mobility shift assays demonstrate that HBX2 specifically interacts with gastrula stage embryonic extracts and that in vitro translated Xvent-1 protein binds to one of its functional motifs. Combined with gain- and loss-of-function experiments, the promoter analysis described here suggests that Xvent-1 functions to repress Xmyf-5 expression in the ventral domain of the marginal zone. Furthermore, the identification of HBX2 provides a tool with which to identify other molecules involved in the regulation of Xmyf-5 expression during gastrulation.

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