scholarly journals Norovirus VPg Binds RNA through a Conserved N-Terminal K/R Basic Patch

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1282
Author(s):  
Alice M. McSweeney ◽  
Vivienne L. Young ◽  
Vernon K. Ward
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Terminal Region ◽  
Binding Potential ◽  
Binding Motif ◽  
Murine Norovirus ◽  
Functional Protein ◽  
Mobility Shift ◽  
Cycle Arrest ◽  
Electrophoretic Mobility Shift Assays

The viral protein genome-linked (VPg) of noroviruses is a multi-functional protein that participates in essential roles during the viral replication cycle. Predictive analyses indicate that murine norovirus (MNV) VPg contains a disordered N-terminal region with RNA binding potential. VPg proteins were expressed with an N-terminal spidroin fusion protein in insect cells and the interaction with RNA investigated by electrophoretic mobility shift assays (EMSA) against a series of RNA probes (pentaprobes) representing all possible five nucleotide combinations. MNV VPg and human norovirus (HuNV) VPg proteins were directly bound to RNA in a non-specific manner. To identify amino acids involved in binding to RNA, all basic (K/R) residues in the first 12 amino acids of MNV VPg were mutated to alanine. Removal of the K/R amino acids eliminated RNA binding and is consistent with a K/R basic patch RNA binding motif within the disordered N-terminal region of norovirus VPgs. Finally, we show that mutation of the K/R basic patch required for RNA binding eliminates the ability of MNV VPg to induce a G0/G1 cell cycle arrest.

scholarly journals The RNA Binding Domain of the Hantaan Virus N Protein Maps to a Central, Conserved Region

2002 ◽  
Vol 76 (7) ◽  
pp. 3301-3308 ◽  
Author(s):  
Xiaolin Xu ◽  
William Severson ◽  
Noah Villegas ◽  
Connie S. Schmaljohn ◽  
Colleen B. Jonsson
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Amino Acid Sequences ◽  
Hantaan Virus ◽  
Mobility Shift ◽  
Sequence Alignments ◽  
Competition Analysis ◽  
N Protein ◽  
Protein Amino Acids ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The nucleocapsid (N) protein of hantaviruses encapsidates both viral genomic and antigenomic RNAs, although only the genomic viral RNA (vRNA) is packaged into virions. To define the domain within the Hantaan virus (HTNV) N protein that mediates these interactions, 14 N- and C-terminal deletion constructs were cloned into a bacterial expression vector, expressed, and purified to homogeneity. Each protein was examined for its ability to bind the HTNV S segment vRNA with filter binding and gel electrophoretic mobility shift assays. These studies mapped a minimal region within the HTNV N protein (amino acids 175 to 217) that bound vRNA. Sequence alignments made from several hantavirus N protein sequences showed that the region identified has a 58% identity and an 86% similarity among these amino acid sequences. Two peptides corresponding to amino acids 175 to 196 (N1) and 197 to 218 (N2) were synthesized. The RNA binding of each peptide was measured by filter binding and competition analysis. Three oligoribonucleotides were used to measure binding affinity and assess specificity. The N2 peptide contained the major RNA binding determinants, while the N1 peptide, when mixed with N2, contributed to the specificity of vRNA recognition.

scholarly journals TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution

2005 ◽  
Vol 387 (2) ◽  
pp. 401-409 ◽  
Author(s):  
Jolanta KOPEC ◽  
Alexander BERGMANN ◽  
Gerhard FRITZ ◽  
Elisabeth GROHMANN ◽  
Walter KELLER
Keyword(s):  
Amino Acids ◽  
Broad Host Range ◽  
Mobility Shift ◽  
Gram Positive ◽  
Nick Site ◽  
Α Helix ◽  
Electrophoretic Mobility Shift Assays ◽  
Dna Complex ◽  
Β Sheet

TraA is the DNA relaxase encoded by the broad-host-range Grampositive plasmid pIP501. It is the second relaxase to be characterized from plasmids originating from Gram-positive organisms. Full-length TraA (654 amino acids) and the N-terminal domain (246 amino acids), termed TraAN246, were expressed as 6×His-tagged fusions and purified. Small-angle X-ray scattering and chemical cross-linking proved that TraAN246 and TraA form dimers in solution. Both proteins revealed oriTpIP501 (origin of transfer of pIP501) cleavage activity on supercoiled plasmid DNA in vitro. oriT binding was demonstrated by electrophoretic mobility shift assays. Radiolabelled oligonucleotides covering different parts of oriTpIP501 were subjected to binding with TraA and TraAN246. The KD of the protein–DNA complex encompassing the inverted repeat, the nick site and an additional 7 bases was found to be 55 nM for TraA and 26 nM for TraAN246. The unfolding of both protein constructs was monitored by measuring the change in the CD signal at 220 nm upon temperature change. The unfolding transition of both proteins occurred at approx. 42 °C. CD spectra measured at 20 °C showed 30% α-helix and 13% β-sheet for TraA, and 27% α-helix and 18% β-sheet content for the truncated protein. Upon DNA binding, an enhanced secondary structure content and increased thermal stability were observed for the TraAN246 protein, suggesting an induced-fit mechanism for the formation of the specific relaxase–oriT complex.

scholarly journals Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme

2006 ◽  
Vol 394 (1) ◽  
pp. 163-171 ◽  
Author(s):  
Sandra Müller ◽  
Jennifer Disse ◽  
Manuela Schöttler ◽  
Sylvia Schön ◽  
Christian Prante ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Binding Capacity ◽  
Functional Characterization ◽  
Terminal Region ◽  
Binding Motif ◽  
Heparin Binding ◽  
Loss Of Function ◽  
The Impact

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

scholarly journals The transcription factor PITX1 drives astrocyte differentiation by regulating the SOX9 gene

2020 ◽  
Vol 295 (39) ◽  
pp. 13677-13690
Author(s):  
Jeong Su Byun ◽  
Mihee Oh ◽  
Seonha Lee ◽  
Jung-Eun Gil ◽  
Yeajin Mo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Formation Control ◽  
Synapse Formation ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Mobility Shift ◽  
Differentiation Protocol ◽  
Mature Brain ◽  
Astrocyte Differentiation ◽  
Electrophoretic Mobility Shift Assays

Astrocytes perform multiple essential functions in the developing and mature brain, including regulation of synapse formation, control of neurotransmitter release and uptake, and maintenance of extracellular ion balance. As a result, astrocytes have been implicated in the progression of neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Despite these critical functions, the study of human astrocytes can be difficult because standard differentiation protocols are time-consuming and technically challenging, but a differentiation protocol recently developed in our laboratory enables the efficient derivation of astrocytes from human embryonic stem cells. We used this protocol along with microarrays, luciferase assays, electrophoretic mobility shift assays, and ChIP assays to explore the genes involved in astrocyte differentiation. We demonstrate that paired-like homeodomain transcription factor 1 (PITX1) is critical for astrocyte differentiation. PITX1 overexpression induced early differentiation of astrocytes, and its knockdown blocked astrocyte differentiation. PITX1 overexpression also increased and PITX1 knockdown decreased expression of sex-determining region Y box 9 (SOX9), known initiator of gliogenesis, during early astrocyte differentiation. Moreover, we determined that PITX1 activates the SOX9 promoter through a unique binding motif. Taken together, these findings indicate that PITX1 drives astrocyte differentiation by sustaining activation of the SOX9 promoter.

scholarly journals Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis.

1991 ◽  
Vol 11 (6) ◽  
pp. 2994-3000 ◽  
Author(s):  
K M Yao ◽  
K White
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rna Binding ◽  
Terminal Region ◽  
Drosophila Virilis ◽  
Functional Domain ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

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 Cleavage and polyadenylation specificity factor 30: An RNA-binding zinc-finger protein with an unexpected 2Fe–2S cluster

2016 ◽  
Vol 113 (17) ◽  
pp. 4700-4705 ◽  
Author(s):  
Geoffrey D. Shimberg ◽  
Jamie L. Michalek ◽  
Abdulafeez A. Oluyadi ◽  
Andria V. Rodrigues ◽  
Beth E. Zucconi ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Inductively Coupled Plasma ◽  
Rna Binding ◽  
Zinc Finger Protein ◽  
Mobility Shift ◽  
Inductively Coupled ◽  
Plasma Mass Spectrometry ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Electrophoretic Mobility Shift Assays

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a key protein involved in pre-mRNA processing. CPSF30 contains five Cys3His domains (annotated as “zinc-finger” domains). Using inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy, and UV-visible spectroscopy, we report that CPSF30 is isolated with iron, in addition to zinc. Iron is present in CPSF30 as a 2Fe–2S cluster and uses one of the Cys3His domains; 2Fe–2S clusters with a Cys3His ligand set are rare and notably have also been identified in MitoNEET, a protein that was also annotated as a zinc finger. These findings support a role for iron in some zinc-finger proteins. Using electrophoretic mobility shift assays and fluorescence anisotropy, we report that CPSF30 selectively recognizes the AU-rich hexamer (AAUAAA) sequence present in pre-mRNA, providing the first molecular-based evidence to our knowledge for CPSF30/RNA binding. Removal of zinc, or both zinc and iron, abrogates binding, whereas removal of just iron significantly lessens binding. From these data we propose a model for RNA recognition that involves a metal-dependent cooperative binding mechanism.

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 Characterization of the RNA-Binding Domains in the Replicase Proteins of Tomato Bushy Stunt Virus

2003 ◽  
Vol 77 (17) ◽  
pp. 9244-9258 ◽  
Author(s):  
K. S. Rajendran ◽  
Peter D. Nagy
Keyword(s):  
Rna Binding ◽  
Tomato Bushy Stunt Virus ◽  
Binding Motif ◽  
Mobility Shift ◽  
Terminal Domain ◽  
Binding Domains ◽  
Replicase Proteins ◽  
Rna Binding Domains ◽  
Gel Mobility Shift

ABSTRACT Tomato bushy stunt virus (TBSV), a tombusvirus with a nonsegmented, plus-stranded RNA genome, codes for two essential replicase proteins. The sequence of one of the replicase proteins, namely p33, overlaps with the N-terminal domain of p92, which contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its nonoverlapping C-terminal portion. In this work, we demonstrate that both replicase proteins bind to RNA in vitro based on gel mobility shift and surface plasmon resonance measurements. We also show evidence that the binding of p33 to single-stranded RNA (ssRNA) is stronger than binding to double-stranded RNA (dsRNA), ssDNA, or dsDNA in vitro. Competition experiments with ssRNA revealed that p33 binds to a TBSV-derived sequence with higher affinity than to other nonviral ssRNA sequences. Additional studies revealed that p33 could bind to RNA in a cooperative manner. Using deletion derivatives of the Escherichia coli-expressed recombinant proteins in gel mobility shift and Northwestern assays, we demonstrate that p33 and the overlapping domain of p92, based on its sequence identity with p33, contain an arginine- and proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). This motif is highly conserved among tombusviruses and related carmoviruses, and it is similar to the arginine-rich motif present in the Tat trans-activator protein of human immunodeficiency virus type 1. We also find that the nonoverlapping C-terminal domain of p92 contains additional RNA-binding regions. Interestingly, the location of one of the RNA-binding domains in p92 is similar to the RNA-binding domain of the NS5B RdRp protein of hepatitis C virus.

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