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.

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 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 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.

Abstract 217: Quaking Post-Transcriptionally Guides Monocyte Adhesion and Differentiation into the Pro-Inflammatory Macrophage

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Ruben G de Bruin ◽  
Lily Shiue ◽  
Anjana Djarmshi ◽  
Hetty C de Boer ◽  
Wai Yi Leung ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Rna Binding ◽  
Inflammatory Diseases ◽  
Human Monocyte ◽  
Human Macrophage ◽  
Monocyte Adhesion ◽  
Sequence Elements ◽  
Bound Sequence ◽  
Novel Target ◽  
And Function

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce new transcription necessary for macrophage identity, but post-transcriptional control of human macrophage differentiation is less well understood. Here, we detail our discovery that levels of the RNA-binding protein Quaking (QKI) are low in monocytes of early atherosclerotic lesions, but abundant in macrophages of advanced plaques. Specific depletion of QKI protein impaired monocyte adhesion, migration and differentiation into macrophages, and lesion formation. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal developmental changes in RNA levels and alternative splicing of RNA transcripts enriched in QKI-bound sequence elements. The importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in post-transcriptionally guiding macrophage identity and function. These studies implicate QKI as a novel target for therapeutic intervention in inflammatory diseases.

scholarly journals Transition State Regulator AbrB Inhibits Transcription of Bacillus amyloliquefaciens FZB45 Phytase through Binding at Two Distinct Sites Located within the Extended phyC Promoter Region

2008 ◽  
Vol 190 (19) ◽  
pp. 6467-6474 ◽  
Author(s):  
Oliwia Makarewicz ◽  
Svetlana Neubauer ◽  
Corinna Preusse ◽  
Rainer Borriss
Keyword(s):  
Bacillus Amyloliquefaciens ◽  
Promoter Region ◽  
Response Regulator ◽  
Dependent Manner ◽  
Coding Region ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Regulator Protein ◽  
Transition State Regulator ◽  
Gel Mobility Shift

ABSTRACT We have previously identified the phyC gene of Bacillus amyloliquefaciens FZB45, encoding extracellular phytase, as a member of the PhoP regulon, which is expressed only during phosphate starvation. Its σA-dependent promoter is positively and negatively regulated by the phosphorylated PhoP response regulator in a phosphate-dependent manner (O. Makarewicz, S. Dubrac, T. Msadek, and R. Borriss, J. Bacteriol. 188:6953-6965, 2006). Here, we provide experimental evidence that the transcription of phyC underlies a second control mechanism exerted by the global transient-phase regulator protein, AbrB, which hinders its expression during exponential growth. Gel mobility shift and DNase I footprinting experiments demonstrated that AbrB binds to two different regions in the phyC promoter region that are separated by about 200 bp. One binding site is near the divergently orientated yodU gene, and the second site is located downstream of the phyC promoter and extends into the coding region of the phyC gene. Cooperative binding to the two distant binding regions is necessary for the AbrB-directed repression of phyC transcription. AbrB does not affect the transcription of the neighboring yodU gene.

scholarly journals Molecular Characterization of MexL, the Transcriptional Repressor of the mexJK Multidrug Efflux Operon in Pseudomonas aeruginosa

2005 ◽  
Vol 49 (5) ◽  
pp. 1844-1851 ◽  
Author(s):  
Rungtip Chuanchuen ◽  
Jared B. Gaynor ◽  
RoxAnn Karkhoff-Schweizer ◽  
Herbert P. Schweizer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Mutant Protein ◽  
Mobility Shift ◽  
Promoter Sequences ◽  
Exclusion Chromatography ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Gel Mobility Shift ◽  
Carboxy Terminal

ABSTRACT The Pseudomonas aeruginosa mexJK efflux operon is constitutively expressed in mutants with defects in the upstream mexL gene, which encodes a repressor of the TetR family. MexL and a MexLA47D mutant protein were purified from Escherichia coli as fusion proteins with carboxy-terminal hexahistidine tags. Native polyacrylamide gel electrophoresis and size exclusion chromatography revealed that MexL is a tetramer in solution. MexL and MexLA47D oligomerization was confirmed using a genetic approach, and the MexLA47D mutant protein was not impaired in multimerization. Gel mobility shift and footprinting assays demonstrated that MexL, but not MexLA47D, binds specifically to the 94-bp mexL-mexJ intergenic region to sequences located between positions −84 and −20 from the mexJ initiation codon. MexL protected about 60 nucleotides on each strand, and the protected regions overlapped almost perfectly, a finding consistent with MexL regulating the expression of both mexL and mexJK, which was ascertained by gene fusion analyses. The protected region contains predicted −10 and −35 promoter sequences for both mexL and mexJ, with partially overlapping −10 regions. The mexL promoter assignment was verified by mapping the mexL transcription start site, and the mexJ promoter was localized to the predicted regions using lacZ fusions. The MexL-protected region contains two inverted GTATTT repeats, and their location in the protected region and overlap with the mexL and mexJ promoter sequences strongly support a role in MexL binding.

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 Hydrophobic-cationic peptides enhance RNA polymerase ribozyme activity by accretion

2021 ◽  
Author(s):  
Peiying Li ◽  
Philipp Holliger ◽  
Shunsuke Tagami
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Rna Binding ◽  
Hydrophobic Interactions ◽  
Hammerhead Ribozyme ◽  
Cationic Peptides ◽  
Local Concentration ◽  
Dependent Manner ◽  
Regulatory Effects ◽  
And Function

ABSTRACTAccretion and the resulting increase in local concentration to enhance target stability and function is a widespread mechanism in biology (for example in the liquid-liquid demixing phases and coacervates). It is widely believed that such macromolecular aggregates (formed through ionic and hydrophobic interactions) may have played a role in the origin of life. Here, we report on the behaviour of a hydrophobic-cationic RNA binding peptide selected by phage display (P43: AKKVWIIMGGS) that forms insoluble aggregates, accrete RNA on their surfaces in a size-dependent manner, and thus enhance the activities of various ribozymes. At low Mg2+ concentrations ([Mg2+]: 25 mM MgCl2), the activity of a small ribozyme (hammerhead ribozyme) was enhanced by P43, while larger ribozymes (RNA polymerase ribozyme (RPR), RNase P, F1* ligase) were inhibited. In contrast, at high [Mg2+] (≥200 mM), the RPR activity was enhanced. Another hydrophobic-cationic peptide with a simpler sequence (K2V6: KKVVVVVV) also exhibited similar regulatory effects on the RPR activity. Furthermore, inactive RPR captured on P43 aggregates at low [Mg2+] could be reactivated in a high [Mg2+] buffer. Therefore, in marked contrast to previously studied purely cationic peptides (like K10) that enhance RPR only at low ionic strength, hydrophobic-cationic peptides can reversibly concentrate RNA and enhance the RPR activity even at high ionic strength conditions such as in eutectic ice phases. Such peptides could have aided the emergence of longer and functional RNAs in a fluctuating environment (e.g., dry-wet / freeze-thaw cycles) on the prebiotic earth.

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