Gel Mobility Shift Assays for RNA Binding Viral RNAi Suppressors

2011 ◽  
pp. 245-252 ◽  
Author(s):  
Tibor Csorba ◽  
József Burgyán
Keyword(s):  
Rna Binding ◽  
Mobility Shift ◽  
Gel Mobility Shift

scholarly journals Identification of cis Elements Directing Termination of Yeast Nonpolyadenylated snoRNA Transcripts

2004 ◽  
Vol 24 (14) ◽  
pp. 6241-6252 ◽  
Author(s):  
Kristina L. Carroll ◽  
Dennis A. Pradhan ◽  
Josh A. Granek ◽  
Neil D. Clarke ◽  
Jeffry L. Corden
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Large Degree ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Nascent Transcript ◽  
Pol Ii ◽  
Gel Mobility Shift

ABSTRACT RNA polymerase II (Pol II) termination is triggered by sequences present in the nascent transcript. Termination of pre-mRNA transcription is coupled to recognition of cis-acting sequences that direct cleavage and polyadenylation of the pre-mRNA. Termination of nonpolyadenylated [non-poly(A)] Pol II transcripts in Saccharomyces cerevisiae requires the RNA-binding proteins Nrd1 and Nab3. We have used a mutational strategy to characterize non-poly(A) termination elements downstream of the SNR13 and SNR47 snoRNA genes. This approach detected two common RNA sequence motifs, GUA[AG] and UCUU. The first motif corresponds to the known Nrd1-binding site, which we have verified here by gel mobility shift assays. We also show that Nab3 protein binds specifically to RNA containing the UCUU motif. Taken together, our data suggest that Nrd1 and Nab3 binding sites play a significant role in defining non-poly(A) terminators. As is the case with poly(A) terminators, there is no strong consensus for non-poly(A) terminators, and the arrangement of Nrd1p and Nab3p binding sites varies considerably. In addition, the organization of these sequences is not strongly conserved among even closely related yeasts. This indicates a large degree of genetic variability. Despite this variability, we were able to use a computational model to show that the binding sites for Nrd1 and Nab3 can identify genes for which transcription termination is mediated by these proteins.

The suppression of testis-brain RNA binding protein and kinesin heavy chain disrupts mRNA sorting in dendrites

1999 ◽  
Vol 112 (21) ◽  
pp. 3691-3702 ◽  
Author(s):  
W.L. Severt ◽  
T.U. Biber ◽  
X. Wu ◽  
N.B. Hecht ◽  
R.J. DeLorenzo ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Antisense Oligonucleotides ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Neuronal Remodeling ◽  
Kinesin Heavy Chain ◽  
Gel Mobility Shift

Ribonucleoprotein particles (RNPs) are thought to be key players in somato-dendritic sorting of mRNAs in CNS neurons and are implicated in activity-directed neuronal remodeling. Here, we use reporter constructs and gel mobility shift assays to show that the testis brain RNA-binding protein (TB-RBP) associates with mRNPs in a sequence (Y element) dependent manner. Using antisense oligonucleotides (anti-ODN), we demonstrate that blocking the TB-RBP Y element binding site disrupts and mis-localizes mRNPs containing (alpha)-calmodulin dependent kinase II (alpha)-CAMKII) and ligatin mRNAs. In addition, we show that suppression of kinesin heavy chain motor protein alters only the localization of (alpha)-CAMKII mRNA. Thus, differential sorting of mRNAs involves multiple mRNPs and selective motor proteins permitting localized mRNAs to utilize common mechanisms for shared steps.

scholarly journals Viroplasm Protein P9-1 ofRice Black-Streaked Dwarf VirusPreferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

2013 ◽  
Vol 87 (23) ◽  
pp. 12885-12899 ◽  
Author(s):  
Jianyan Wu ◽  
Jia Li ◽  
Xiang Mao ◽  
Weiwu Wang ◽  
Zhaobang Cheng ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Rna Binding ◽  
Structure And Function ◽  
Interior Structure ◽  
Mobility Shift ◽  
Chromatography Analysis ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
And Function

The P9-1 protein ofRice black-streaked dwarf virus(RBSDV) is an essential part of the viroplasm. However, little is known about its nature or biological function in the viroplasm. In this study, the structure and function of P9-1 were analyzed forin vitrobinding to nucleic acids. We found that the P9-1 protein preferentially bound to single-stranded versus double-stranded nucleic acids; however, the protein displayed no preference for RBSDV versus non-RBSDV single-stranded ssRNA (ssRNA). A gel mobility shift assay revealed that the RNA gradually shifted as increasing amounts of P9-1 were added, suggesting that multiple subunits of P9-1 bind to ssRNA. By using discontinuous blue native gel and chromatography analysis, we found that the P9-1 protein was capable of forming dimers, tetramers, and octamers. Strikingly, we demonstrated that P9-1 preferentially bound to ssRNA in the octamer, rather than the dimer, form. Deletion of the C-terminal arm resulted in P9-1 no longer forming octamers; consequently, the deletion mutant protein bound to ssRNA with significantly lower affinity and with fewer copies bound per ssRNA. Alanine substitution analysis revealed that electropositive amino acids among residues 25 to 44 are important for RNA binding and map to the central interior structure that was formed only by P9-1 octamers. Collectively, our findings provide novel insights into the structure and function of RBSDV viroplasm protein P9-1 binding to RNA.

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.

scholarly journals Bacillus subtilis Aconitase Is Required for Efficient Late-Sporulation Gene Expression

2006 ◽  
Vol 188 (17) ◽  
pp. 6396-6405 ◽  
Author(s):  
Alisa W. Serio ◽  
Kieran B. Pechter ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Activation ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Binding Activity ◽  
High Catalytic Activity ◽  
Spore Coat ◽  
Mobility Shift ◽  
Gel Mobility Shift

ABSTRACT Bacillus subtilis aconitase, encoded by the citB gene, is homologous to the bifunctional eukaryotic protein IRP-1 (iron regulatory protein 1). Like IRP-1, B. subtilis aconitase is both an enzyme and an RNA binding protein. In an attempt to separate the two activities of aconitase, the C-terminal region of the B. subtilis citB gene product was mutagenized. The resulting strain had high catalytic activity but was defective in sporulation. The defect was at a late stage of sporulation, specifically affecting expression of σK-dependent genes, many of which are important for spore coat assembly and require transcriptional activation by GerE. Accumulation of gerE mRNA and GerE protein was delayed in the aconitase mutant strain. Pure B. subtilis aconitase bound to the 3′ untranslated region of gerE mRNA in in vitro gel mobility shift assays, strongly suggesting that aconitase RNA binding activity may stabilize gerE mRNA in order to allow efficient GerE synthesis and proper timing of spore coat assembly.

scholarly journals JNK activation decreases PP2A regulatory subunit B56α expression and mRNA stability and increases AUF1 expression in cardiomyocytes

2006 ◽  
Vol 291 (3) ◽  
pp. H1183-H1192 ◽  
Author(s):  
Nicole D. Glaser ◽  
Yevgeniya O. Lukyanenko ◽  
Yibin Wang ◽  
Gerald M. Wilson ◽  
Terry B. Rogers
Keyword(s):  
Gene Expression ◽  
Untranslated Region ◽  
Time Course ◽  
Rna Binding ◽  
Cardiac Cells ◽  
Regulatory Subunit ◽  
Central Feature ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
Jnk Activation

A central feature of heart disease is a molecular remodeling of signaling pathways in cardiac myocytes. This study focused on novel molecular elements of MAPK-mediated alterations in the pattern of gene expression of the protein phosphatase 2A (PP2A). In an established model of sustained JNK activation, a 70% decrease in expression of the targeting subunit of PP2A, B56α, was observed in either neonatal or adult cardiomyocytes. This loss in protein abundance was accompanied by a decrease of 69% in B56α mRNA steady-state levels. Given that the 3′-untranslated region of this transcript contains adenylate-uridylate-rich elements known to regulate mRNA degradation, experiments explored the notion that instability of B56α mRNA accounts for the response. mRNA time-course analyses with real-time PCR methods showed that B56α transcript was transformed from a stable (no significant decay over 1 h) to a labile form that rapidly degraded within minutes. These results were supported by complementary experiments that revealed that the RNA-binding protein AUF1, known to destabilize target mRNA, was increased fourfold in JNK-activated cells. A variety of other stress-related stimuli, such as p38 MAPK activation and phorbol ester, upregulated AUF1 expression in cultured cardiac cells as well. In addition, gel mobility shift assays demonstrated that p37AUF1 binds with nanomolar affinity to segments of the B56α 3′-untranslated region. Thus these studies provide new evidence that signaling-induced mRNA instability is an important mechanism that underlies the changes in the pattern of gene expression evoked by stress-activated pathways in cardiac cells.

scholarly journals Double-Stranded RNA Binding Proteins in Serum Contribute to Systemic RNAi Across Phyla—Towards Finding the Missing Link in Achelata

2020 ◽  
Vol 21 (18) ◽  
pp. 6967
Author(s):  
Thomas M. Banks ◽  
Tianfang Wang ◽  
Quinn P. Fitzgibbon ◽  
Gregory G. Smith ◽  
Tomer Ventura
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Limiting Factor ◽  
Mobility Shift ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Spiny Lobsters ◽  
Systemic Rnai ◽  
Gel Mobility Shift

RNA interference (RNAi) has become a widely utilized method for studying gene function, yet despite this many of the mechanisms surrounding RNAi remain elusive. The core RNAi machinery is relatively well understood, however many of the systemic mechanisms, particularly double-stranded RNA (dsRNA) transport, are not. Here, we demonstrate that dsRNA binding proteins in the serum contribute to systemic RNAi and may be the limiting factor in RNAi capacity for species such as spiny lobsters, where gene silencing is not functional. Incubating sera from a variety of species across phyla with dsRNA led to a gel mobility shift in species in which systemic RNAi has been observed, with this response being absent in species in which systemic RNAi has never been observed. Proteomic analysis suggested lipoproteins may be responsible for this phenomenon and may transport dsRNA to spread the RNAi signal systemically. Following this, we identified the same gel shift in the slipper lobster Thenus australiensis and subsequently silenced the insulin androgenic gland hormone, marking the first time RNAi has been performed in any lobster species. These results pave the way for inducing RNAi in spiny lobsters and for a better understanding of the mechanisms of systemic RNAi in Crustacea, as well as across phyla.

scholarly journals Asymmetric dimerization of adenosine deaminase acting on RNA facilitates substrate recognition

2020 ◽  
Vol 48 (14) ◽  
pp. 7958-7972 ◽  
Author(s):  
Alexander S Thuy-Boun ◽  
Justin M Thomas ◽  
Herra L Grajo ◽  
Cody M Palumbo ◽  
SeHee Park ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Structure ◽  
Rna Binding ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Sequence Elements ◽  
Dimerization Interface ◽  
Gel Mobility Shift ◽  
And Function

Abstract Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.

scholarly journals Double Stranded RNA Binding Proteins in the Serum Contribute to Systemic RNAi across Phyla – Towards Finding the Missing Link in Achelata

2020 ◽  
Author(s):  
Thomas Banks ◽  
Tianfang Wang ◽  
Quinn Fitzgibbon ◽  
Gregory Smith ◽  
Tomer Ventura
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Limiting Factor ◽  
Mobility Shift ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Spiny Lobsters ◽  
Systemic Rnai ◽  
Gel Mobility Shift

RNA interference (RNAi) has become a widely utilised method to study gene function, yet despite this, many of the mechanisms surrounding RNAi remain elusive. The core RNAi machinery is relatively well understood, however many of the systemic mechanisms, particularly double stranded RNA (dsRNA) transport, are not. Here, we demonstrate that dsRNA binding proteins in the serum contribute to systemic RNAi, and may be the limiting factor in RNAi capacity for species such as spiny lobsters where gene silencing is not functional. Incubating serum from a variety of species across phyla with dsRNA led to a gel mobility shift in species where systemic RNAi has been observed, with this response being absent in species where systemic RNAi has never been observed. Proteomic analysis suggested lipoproteins may be responsible for this phenomenon, and may transport dsRNA to spread the RNAi signal systemically. Following this, we identified the same gel shift in the slipper lobster Thenus australiensis and subsequently silenced the insulin androgenic gland hormone, marking the first time RNAi has been performed in any lobster species. These results pave the way for inducing RNAi in spiny lobsters, and better understanding the mechanisms of systemic RNAi in Crustacea, as well as across phyla.

IL-1 beta stabilizes COX II mRNA in renal mesangial cells: role of 3'-untranslated region

1994 ◽  
Vol 267 (3) ◽  
pp. F504-F508 ◽  
Author(s):  
S. K. Srivastava ◽  
T. Tetsuka ◽  
D. Daphna-Iken ◽  
A. R. Morrison
Keyword(s):  
Posttranscriptional Regulation ◽  
Untranslated Region ◽  
Rna Binding ◽  
Mesangial Cells ◽  
Rna Binding Proteins ◽  
Interleukin 1 ◽  
Mobility Shift ◽  
Phosphatase Treatment ◽  
Prostaglandin Endoperoxide Synthase ◽  
Gel Mobility Shift

We stimulated rat mesangial cells for different time intervals with interleukin-1 beta (IL-1 beta) and phorbol 12-myristate 13-acetate, prepared cytoplasmic extracts, and examined these extracts for the presence of RNA binding proteins by gel mobility shift assays. Here we report that the 3'-untranslated region (3'-UNTR) of the prostaglandin endoperoxide synthase II (COX II) gene is responsible for posttranscriptional regulation of the response to IL-1 beta. Two cytosolic transacting factors of 65 and 45 kDa, respectively, have been detected that bind to the 3'-UNTR. Competition with excess RNA and acid phosphatase treatment of the cytoplasmic extract suggest the binding is specific and that phosphorylation is required for these rapid binding events. These experiments suggest that IL-1 beta induces the phosphorylation of cytosolic factors, which bind to the 3'-UNTR of COX II mRNA, and stabilizes the message.

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