scholarly journals Bovine Coronavirus Nonstructural Protein 1 (p28) Is an RNA Binding Protein That Binds Terminal Genomic cis-Replication Elements

2009 ◽  
Vol 83 (12) ◽  
pp. 6087-6097 ◽  
Author(s):  
Kortney M. Gustin ◽  
Bo-Jhih Guan ◽  
Agnieszka Dziduszko ◽  
David A. Brian
Keyword(s):  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Coding Region ◽  
Bovine Coronavirus ◽  
Stem Loop ◽  
Nonstructural Protein 1 ◽  
Gel Shift ◽  
Rna Levels

ABSTRACT Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5′-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of ∼60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5′ untranslated region (UTR)- and one 3′ UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5′ UTR with ∼2.5 μM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.

scholarly journals U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Jagjit Singh ◽  
Kavleen Sikand ◽  
Heike Conrad ◽  
Cindy L. Will ◽  
Anton A. Komar ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Stem Loop

scholarly journals The Protease Lon and the RNA-Binding Protein Hfq Reduce Silencing of the Escherichia coli bgl Operon by H-NS

2004 ◽  
Vol 186 (9) ◽  
pp. 2708-2716 ◽  
Author(s):  
Sudhanshu Dole ◽  
Yvonne Klingen ◽  
V. Nagarajavel ◽  
Karin Schnetz
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Specific Effect ◽  
Receptor Protein ◽  
Coding Region ◽  
Genes Encoding ◽  
Pleiotropic Regulators

ABSTRACT The histone-like nucleoid structuring protein H-NS represses the Escherichia coli bgl operon at two levels. H-NS binds upstream of the promoter, represses transcription initiation, and binds downstream within the coding region of the first gene, where it induces polarity of transcription elongation. In hns mutants, silencing of the bgl operon is completely relieved. Various screens for mutants in which silencing of bgl is reduced have yielded mutations in hns and in genes encoding the transcription factors LeuO and BglJ. In order to identify additional factors that regulate bgl, we performed a transposon mutagenesis screen for mutants in which silencing of the operon is strengthened. This screen yielded mutants with mutations in cyaA, hfq, lon, and pgi, encoding adenylate cyclase, RNA-binding protein Hfq, protease Lon, and phosphoglucose isomerase, respectively. In cyaA mutants, the cyclic AMP receptor protein-dependent promoter is presumably inactive. The specific effect of the pgi mutants on bgl is low. Interestingly, in the hfq and lon mutants, the downstream silencing of bgl by H-NS (i.e., the induction of polarity) is more efficient, while the silencing of the promoter by H-NS is unaffected. Furthermore, in an hns mutant, Hfq has no significant effect and the effect of Lon is reduced. These data provide evidence that the specific repression by H-NS can (directly or indirectly) be modulated and controlled by other pleiotropic regulators.

The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1

1993 ◽  
Vol 13 (1) ◽  
pp. 703-710
Author(s):  
D P Toczyski ◽  
J A Steitz
Keyword(s):  
Fusion Protein ◽  
Small Rna ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Glutathione S Transferase ◽  
Stem Loop ◽  
Barr Virus ◽  
Epstein Barr

EAP (EBER-associated protein) is an abundant, 15-kDa cellular RNA-binding protein which associates with certain herpesvirus small RNAs. We have raised polyclonal anti-EAP antibodies against a glutathione S-transferase-EAP fusion protein. Analysis of the RNA precipitated by these antibodies from Epstein-Barr virus (EBV)- or herpesvirus papio (HVP)-infected cells shows that > 95% of EBER 1 (EBV-encoded RNA 1) and the majority of HVP 1 (an HVP small RNA homologous to EBER 1) are associated with EAP. RNase protection experiments performed on native EBER 1 particles with affinity-purified anti-EAP antibodies demonstrate that EAP binds a stem-loop structure (stem-loop 3) of EBER 1. Since bacterially expressed glutathione S-transferase-EAP fusion protein binds EBER 1, we conclude that EAP binding is independent of any other cellular or viral protein. Detailed mutational analyses of stem-loop 3 suggest that EAP recognizes the majority of the nucleotides in this hairpin, interacting with both single-stranded and double-stranded regions in a sequence-specific manner. Binding studies utilizing EBER 1 deletion mutants suggest that there may also be a second, weaker EAP-binding site on stem-loop 4 of EBER 1. These data and the fact that stem-loop 3 represents the most highly conserved region between EBER 1 and HVP 1 suggest that EAP binding is a critical aspect of EBER 1 and HVP 1 function.

scholarly journals The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1.

1993 ◽  
Vol 13 (1) ◽  
pp. 703-710 ◽  
Author(s):  
D P Toczyski ◽  
J A Steitz
Keyword(s):  
Fusion Protein ◽  
Small Rna ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Glutathione S Transferase ◽  
Stem Loop ◽  
Barr Virus ◽  
Epstein Barr

EAP (EBER-associated protein) is an abundant, 15-kDa cellular RNA-binding protein which associates with certain herpesvirus small RNAs. We have raised polyclonal anti-EAP antibodies against a glutathione S-transferase-EAP fusion protein. Analysis of the RNA precipitated by these antibodies from Epstein-Barr virus (EBV)- or herpesvirus papio (HVP)-infected cells shows that > 95% of EBER 1 (EBV-encoded RNA 1) and the majority of HVP 1 (an HVP small RNA homologous to EBER 1) are associated with EAP. RNase protection experiments performed on native EBER 1 particles with affinity-purified anti-EAP antibodies demonstrate that EAP binds a stem-loop structure (stem-loop 3) of EBER 1. Since bacterially expressed glutathione S-transferase-EAP fusion protein binds EBER 1, we conclude that EAP binding is independent of any other cellular or viral protein. Detailed mutational analyses of stem-loop 3 suggest that EAP recognizes the majority of the nucleotides in this hairpin, interacting with both single-stranded and double-stranded regions in a sequence-specific manner. Binding studies utilizing EBER 1 deletion mutants suggest that there may also be a second, weaker EAP-binding site on stem-loop 4 of EBER 1. These data and the fact that stem-loop 3 represents the most highly conserved region between EBER 1 and HVP 1 suggest that EAP binding is a critical aspect of EBER 1 and HVP 1 function.

scholarly journals Global cataloguing of variations in untranslated regions of viral genome and prediction of key host RNA binding protein-microRNA interactions modulating genome stability in SARS-CoV2

2020 ◽  
Author(s):  
Moumita Mukherjee ◽  
Srikanta Goswami
Keyword(s):  
Genome Stability ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Untranslated Regions ◽  
Global Changes ◽  
Sequence Information ◽  
Coding Region ◽  
High Coverage ◽  
Geographical Regions

AbstractBackgroundThe world is going through the critical phase of COVID-19 pandemic, caused by human coronavirus, SARS-CoV2. Worldwide concerted effort to identify viral genomic changes across different sub-types has identified several strong changes in the coding region. However, there have not been many studies focusing on the variations in the 5’ and 3’ untranslated regions and their consequences. Considering the possible importance of these regions in host mediated regulation of viral RNA genome, we wanted to explore the phenomenon.MethodsTo have an idea of the global changes in 5’ and 3’-UTR sequences, we downloaded 8595 complete and high-coverage SARS-CoV2 genome sequence information from human host in FASTA format from Global Initiative on Sharing All Influenza Data (GISAID) from 15 different geographical regions. Next, we aligned them using Clustal Omega software and investigated the UTR variants. We also looked at the putative host RNA binding protein (RBP) and microRNA binding sites in these regions by ‘RBPmap’ and ‘RNA22 v2’ respectively. Expression status of selected RBPs and microRNAs were checked in lungs tissue.ResultsWe identified 28 unique variants in SARS-CoV2 UTR region based on a minimum variant percentage cut-off of 0.5. Along with 241C>T change the important 5’-UTR change identified was 187A>G, while 29734G>C, 29742G>A/T and 29774C>T were the most familiar variants of 3’UTR among most of the continents. Furthermore, we found that despite of the variations in the UTR regions, binding of host RBP to them remains mostly unaltered, which further influenced the functioning of specific miRNAs.ConclusionOur results, shows for the first time in SARS-Cov2 infection, a possible cross-talk between host RBPs-miRNAs and viral UTR variants, which ultimately could explain the mechanism of escaping host RNA decay machinery by the virus. The knowledge might be helpful in developing anti-viral compounds in future.

Interaction of 68–kDa TAR RNA-binding protein and other cellular proteins with rpion protein-RNA stem-loop

1995 ◽  
Vol 1 (5-6) ◽  
pp. 391-398 ◽  
Author(s):  
Ute Scheffer ◽  
Takashi Okamoto ◽  
Jock MS Forrest ◽  
Peter G Rytik ◽  
Werner EG Müller ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Cellular Proteins ◽  
Stem Loop ◽  
Tar Rna

scholarly journals DDX56 Binds to Chikungunya Virus RNA To Control Infection

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Frances Taschuk ◽  
Iulia Tapescu ◽  
Ryan H. Moy ◽  
Sara Cherry
Keyword(s):  
Chikungunya Virus ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Immune Defense ◽  
Genomic Rna ◽  
Stem Loop ◽  
Evolutionarily Conserved ◽  
Alphavirus Infection ◽  
Viral Genomic Rna

ABSTRACT DEAD box RNA helicases regulate diverse facets of RNA biology. Proteins of this family carry out essential cellular functions, and emerging literature is revealing additional roles in immune defense. Using RNA interference screening, we identified an evolutionarily conserved antiviral role for the helicase DDX56 against the alphavirus Sindbis virus (SINV), a mosquito-transmitted pathogen that infects humans. Depletion of DDX56 enhanced infection in Drosophila and human cells. Furthermore, we found that DDX56 also controls the emerging alphavirus chikungunya virus (CHIKV) through an interferon-independent mechanism. Using cross-linking immunoprecipitation (CLIP-Seq), we identified a predicted stem-loop on the viral genomic RNA bound by DDX56. Mechanistically, we found that DDX56 levels increase in the cytoplasm during CHIKV infection. In the cytoplasm, DDX56 impacts the earliest step in the viral replication cycle by binding and destabilizing the incoming viral genomic RNA, thereby attenuating infection. Thus, DDX56 is a conserved antiviral RNA binding protein that controls alphavirus infection. IMPORTANCE Arthropod-borne viruses are diverse pathogens and include the emerging virus chikungunya virus, which is associated with human disease. Through genetic screening, we found that the conserved RNA binding protein DDX56 is antiviral against chikungunya virus in insects and humans. DDX56 relocalizes from the nucleus to the cytoplasm, where it binds to a stem-loop in the viral genome and destabilizes incoming genomes. Thus, DDX56 is an evolutionarily conserved antiviral factor that controls alphavirus infection.

scholarly journals Translational regulation of viral secretory proteins by the 5′ coding regions and a viral RNA-binding protein

2017 ◽  
Vol 216 (8) ◽  
pp. 2283-2293 ◽  
Author(s):  
Johan Nordholm ◽  
Jeanne Petitou ◽  
Henrik Östbye ◽  
Diogo V. da Silva ◽  
Dan Dou ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Regulatory Elements ◽  
Secretory Protein ◽  
Viral Rna ◽  
Secretory Proteins ◽  
Coding Region ◽  
Coding Regions ◽  
Er Targeting

A primary function of 5′ regions in many secretory protein mRNAs is to encode an endoplasmic reticulum (ER) targeting sequence. In this study, we show how the regions coding for the ER-targeting sequences of the influenza glycoproteins NA and HA also function as translational regulatory elements that are controlled by the viral RNA-binding protein (RBP) NS1. The translational increase depends on the nucleotide composition and 5′ positioning of the ER-targeting sequence coding regions and is facilitated by the RNA-binding domain of NS1, which can associate with ER membranes. Inserting the ER-targeting sequence coding region of NA into different 5′ UTRs confirmed that NS1 can promote the translation of secretory protein mRNAs based on the nucleotides within this region rather than the resulting amino acids. By analyzing human protein mRNA sequences, we found evidence that this mechanism of using 5′ coding regions and particular RBPs to achieve gene-specific regulation may extend to human-secreted proteins.

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