scholarly journals Structure of a bacterial OapB protein with its OLE RNA target gives insights into the architecture of the OLE ribonucleoprotein complex

2021 ◽  
Vol 118 (9) ◽  
pp. e2020393118
Author(s):  
Yang Yang ◽  
Kimberly A. Harris ◽  
Danielle L. Widner ◽  
Ronald R. Breaker
Keyword(s):  
Rna Binding ◽  
Protein A ◽  
Structural Features ◽  
Bacillus Halodurans ◽  
Uncharacterized Protein ◽  
Binding Interface ◽  
Protein Partners ◽  
Rnp Complex ◽  
High Conservation ◽  
Rna Interaction

The OLE (ornate, large, and extremophilic) RNA class is one of the most complex and well-conserved bacterial noncoding RNAs known to exist. This RNA is known to be important for bacterial responses to stress caused by short-chain alcohols, cold, and elevated Mg2+ concentrations. These biological functions have been shown to require the formation of a ribonucleoprotein (RNP) complex including at least two protein partners: OLE-associated protein A (OapA) and OLE-associated protein B (OapB). OapB directly binds OLE RNA with high-affinity and specificity and is believed to assist in assembling the functional OLE RNP complex. To provide the atomic details of OapB–OLE RNA interaction and to potentially reveal previously uncharacterized protein–RNA interfaces, we determined the structure of OapB from Bacillus halodurans alone and in complex with an OLE RNA fragment at resolutions of 1.0 Å and 2.0 Å, respectively. The structure of OapB exhibits a K-shaped overall architecture wherein its conserved KOW motif and additional unique structural elements of OapB form a bipartite RNA-binding surface that docks to the P13 hairpin and P12.2 helix of OLE RNA. These high-resolution structures elucidate the molecular contacts used by OapB to form a stable RNP complex and explain the high conservation of sequences and structural features at the OapB–OLE RNA-binding interface. These findings provide insight into the role of OapB in the assembly and biological function of OLE RNP complex and can guide the exploration of additional possible OLE RNA-binding interactions present in OapB.

scholarly journals Sequence diversity in the 3’ untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

2021 ◽  
Author(s):  
Sarah E. Hickson ◽  
Eden Brekke ◽  
Johannes Schwerk ◽  
Indraneel Saluhke ◽  
Shivam Zaver ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Rna Binding ◽  
Structural Features ◽  
Differential Sensitivity ◽  
Restriction Factor ◽  
Dependent Manner ◽  
Tetratricopeptide Repeats ◽  
Rna Interaction

ABSTRACTAlphaviruses (family Togaviridae) are a diverse group of positive-sense RNA (+ssRNA) viruses that are transmitted by arthropods and are the causative agent of several significant human and veterinary diseases. Interferon (IFN)-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding IFN stimulated genes (ISGs) that are highly upregulated following viral infection, and have been identified as potential restrictors of alphaviruses. The mechanism by which IFIT1 restricts RNA viruses is dependent on self and non-self-discrimination of RNA, and alphaviruses evade this recognition via their 5’UTR. However, the role of IFIT2 during alphavirus replication and the mechanism of viral replication inhibition is unclear. In this study, we identify IFIT2 as a restriction factor for Venezuelan equine encephalitis virus (VEEV) and show that IFIT2 binds the 3’ untranslated region (3’UTR) of the virus. We investigated the potential role of variability in the 3’UTR of the virus affecting IFIT2 antiviral activity by studying infection with VEEV. Comparison of recombinant VEEV clones containing 3’UTR sequences derived from epizootic and enzootic isolates exhibited differential sensitivity to IFIT2 restriction in vitro infection studies, suggesting that the alphavirus 3’UTR sequence may function in part to evade IFIT2 restriction. In vitro binding assays demonstrate that IFIT2 binds to the VEEV 3’UTR, however in contrast to previous studies VEEV restriction did not appear to be dependent on the ability of IFIT2 to inhibit translation of viral RNA, suggesting a novel mechanism of IFIT2 restriction. Our study demonstrates that IFIT2 is a restriction factor for alphaviruses and variability in the 3’UTR of VEEV can modulate viral restriction by IFIT2. Ongoing studies are exploring the biological consequences of IFIT2-VEEV RNA interaction in viral pathogenesis and defining sequence and structural features of RNAs that regulate IFIT2 recognition.

scholarly journals Bacillus halodurans OapB forms a high-affinity complex with the P13 region of the noncoding RNA OLE

2020 ◽  
Vol 295 (28) ◽  
pp. 9326-9334
Author(s):  
Danielle L. Widner ◽  
Kimberly A. Harris ◽  
Lukas Corey ◽  
Ronald R. Breaker
Keyword(s):  
Noncoding Rna ◽  
Structural Features ◽  
Bacillus Halodurans ◽  
Mobility Shift ◽  
Single Nucleotide ◽  
Cold Temperatures ◽  
Rnp Complex ◽  
Rna Fragments ◽  
Electrophoretic Mobility Shift Assays

Noncoding RNAs (ncRNAs) longer than 200 nucleotides are rare in bacteria, likely because bacterial genomes are under strong evolutionary pressures to maintain a small genome size. Of the long ncRNAs unique to bacteria, the OLE (ornate, large, extremophilic) RNA class is among the largest and most structurally complex. OLE RNAs form a ribonucleoprotein (RNP) complex by partnering with at least two proteins, OapA and OapB, that directly bind OLE RNA. The biochemical functions of the OLE RNP complex remain unknown, but are required for proper adaptation to certain environmental stresses, such as cold temperatures, short chain alcohols, and high magnesium concentrations. In the current study, we used electrophoretic mobility shift assays to examine the binding of OLE RNA fragments by OapB and found that OapB recognizes a small subregion of OLE RNA, including stem P13, with a dissociation constant (KD) of ∼700 pm. Analyses with mutated RNA constructs, and the application of in vitro selection, revealed that strong binding of OLE RNA by OapB requires a stem containing a precisely located single-nucleotide bulge and a GNRA tetraloop. Although the vast majority of bacteria with the ole gene also have the oapB gene, there are many whose genomes contain oapB but lack ole, suggesting that OapB has other RNA partners in some species that might exhibit similar structural features.

scholarly journals Association of Potent Human Antiviral Cytidine Deaminases with 7SL RNA and Viral RNP in HIV-1 Virions

2010 ◽  
Vol 84 (24) ◽  
pp. 12903-12913 ◽  
Author(s):  
Wenyan Zhang ◽  
Juan Du ◽  
Kevin Yu ◽  
Tao Wang ◽  
Xiong Yong ◽  
...  
Keyword(s):  
Rna Binding ◽  
Signal Recognition Particle ◽  
Viral Inhibition ◽  
Cytidine Deaminases ◽  
Amino Terminal ◽  
7Sl Rna ◽  
Rnp Complex ◽  
Rna Interaction ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACT 7SL RNA promotes the formation of the signal recognition particle that targets secretory and membrane proteins to the endoplasmic reticulum. 7SL RNA is also selectively packaged by many retroviruses, including HIV-1. Here, we demonstrate that 7SL RNA is an integral component of the viral ribonucleoprotein (RNP) complex containing Gag, viral genomic RNA, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{tRNA}_{3}^{Lys}\) \end{document} . Only the potent anti-HIV-1 cytidine deaminases can bind to 7SL RNA and target to HIV-1 RNP. A conserved motif in the amino-terminal region of A3G is important for 7SL RNA interaction. The weak anti-HIV-1 A3C did not interact with 7SL RNA and failed to target to viral RNPs, despite efficient virion packaging. However, a chimeric construct of A3C plus the 7SL-binding amino terminus of A3G did target to viral RNPs and showed enhanced anti-HIV-1 activity. 7SL RNA binding is a conserved feature of human anti-HIV-1 cytidine deaminases. Thus, potent anti-HIV-1 cytidine deaminases have evolved to possess a unique RNA-binding ability for precise HIV-1 targeting and viral inhibition.

scholarly journals Ewing Sarcoma Protein: A Key Player in Human Cancer

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Maria Paola Paronetto
Keyword(s):  
Ewing Sarcoma ◽  
Cancer Biology ◽  
Genome Stability ◽  
Rna Binding ◽  
Human Cancer ◽  
Neuromuscular Disorders ◽  
Regulation Of Gene Expression ◽  
Protein A ◽  
Structural Features

The Ewing sarcoma protein (EWS) is a well-known player in cancer biology for the specific translocations occurring in sarcomas. The EWS-FLI1 gene fusion is the prototypical translocation that encodes the aberrant, chimeric transcription factor, which is a landmark of Ewing tumors. In all described Ewing sarcoma oncogenes, the EWS RNA binding domains are completely missing; thus RNA binding properties are not retained in the hybrid proteins. However, it is currently unknown whether the absence of EWS function in RNA metabolism plays a role in oncogenic transformation or if EWS plays a role by itself in cancer development besides its contribution to the translocation. In this regard, recent reports have highlighted an essential role for EWS in the regulation of DNA damage response (DDR), a process that counteracts genome stability and is often deregulated in cancer cells. The first part of this review will describe the structural features of EWS and its multiple roles in the regulation of gene expression, which are exerted by coordinating different steps in the synthesis and processing of pre-mRNAs. The second part will examine the role of EWS in the regulation of DDR- and cancer-related genes, with potential implications in cancer therapies. Finally, recent advances on the involvement of EWS in neuromuscular disorders will be discussed. Collectively, the information reviewed herein highlights the broad role of EWS in bridging different cellular processes and underlines the contribution of EWS to genome stability and proper cell-cycle progression in higher eukaryotic cells.

scholarly journals A second RNA-binding protein is essential for ethanol tolerance provided by the bacterial OLE ribonucleoprotein complex

2018 ◽  
Vol 115 (27) ◽  
pp. E6319-E6328 ◽  
Author(s):  
Kimberly A. Harris ◽  
Zhiyuan Zhou ◽  
Michelle L. Peters ◽  
Sarah G. Wilkins ◽  
Ronald R. Breaker
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Ethanol Stress ◽  
Bacillus Halodurans ◽  
Conserved Sequence ◽  
Rnp Complex

OLE (ornate, large, extremophilic) RNAs comprise a class of structured noncoding RNAs (ncRNAs) found in many extremophilic bacteria species. OLE RNAs constitute one of the longest and most widespread bacterial ncRNA classes whose major biochemical function remains unknown. In the Gram-positive alkaliphile Bacillus halodurans, OLE RNA is abundant, and localizes to the cell membrane by association with the transmembrane OLE-associated protein called OapA (formerly OAP). These characteristics, along with the well-conserved sequence and structural features of OLE RNAs, suggest that the OLE ribonucleoprotein (RNP) complex performs important biological functions. B. halodurans strains lacking OLE RNA (∆ole) or OapA (∆oapA) are less tolerant of cold (20 °C) and short-chain alcohols (e.g., ethanol). Here, we describe the effects of a mutant OapA (called PM1) that more strongly inhibits growth under cold or ethanol stress compared with strains lacking the oapA gene, even when wild-type OapA is present. This dominant-negative effect of PM1 is reversed by mutations that render OLE RNA nonfunctional. This finding demonstrates that the deleterious PM1 phenotype requires an intact RNP complex, and suggests that the complex has one or more additional undiscovered components. A genetic screen uncovered PM1 phenotype suppressor mutations in the ybzG gene, which codes for a putative RNA-binding protein of unknown biological function. We observe that YbzG protein (also called OapB) selectively binds OLE RNA in vitro, whereas a mutant version of the protein is not observed to bind OLE RNA. Thus, YbzG/OapB is an important component of the functional OLE RNP complex in B. halodurans.

scholarly journals Trypanosoma brucei Translation Initiation Factor Homolog EIF4E6 Forms a Tripartite Cytosolic Complex with EIF4G5 and a Capping Enzyme Homolog

2014 ◽  
Vol 13 (7) ◽  
pp. 896-908 ◽  
Author(s):  
Eden R. Freire ◽  
Amaranta M. Malvezzi ◽  
Ajay A. Vashisht ◽  
Joanna Zuberek ◽  
Edwin A. Saada ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Rna Binding ◽  
Regulation Of Gene Expression ◽  
Protein A ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nucleoside Triphosphate ◽  
Rna Interaction

ABSTRACT Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene–one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5′ cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules.

scholarly journals Synthesis and Characterization of a Fullerenol Derivative for Potential Biological Applications

2020 ◽  
Vol 4 (1) ◽  
pp. 15
Author(s):  
Eduardo Ravelo-Nieto ◽  
Alvaro Duarte-Ruiz ◽  
Luis H. Reyes ◽  
Juan C. Cruz
Keyword(s):  
Phase Transfer ◽  
Protein A ◽  
Structural Features ◽  
Cellular Level ◽  
Cellulose Membrane ◽  
Cell Penetration ◽  
Covalent Functionalization ◽  
Dialysis Tubing ◽  
Surface Functionality

Several biological barriers are generally responsible for the limited delivery of cargoes at the cellular level. Fullerenols have unique structural features and possess suitable properties for interaction with the cells. This study aimed to synthesize and characterize a fullerenol derivative with desirable characteristics (size, charge, functionality) to develop cell penetration vehicles. Fullerenol was synthesized from fullerene (C60) solubilized in toluene, followed by hydroxylation with hydrogen peroxide and tetra-n-butylammonium hydroxide (TBAH) as a phase transfer catalyst. The obtained product was purified by a Florisil chromatography column (water as the eluent), followed by dialysis (cellulose membrane dialysis tubing) and freeze-drying (yield 66%). Subsequently, a silane coupling agent was conjugated on the fullerenol surface to render free amine functional groups for further covalent functionalization with other molecules. Characterization via UV–VIS, FTIR-ATR, Raman, DLS, and SEM techniques was conducted to evaluate the composition, size, morphology, surface functionality, and structural properties. We are currently working on the conjugation of the potent cell-penetrating agents Buforin II (BUFII) and the Outer Membrane Protein A (OmpA) on the surface of the fullerenol to estimate whether cell penetration and endosome escape are improved concerning conventional polymeric vehicles and our previous developments with iron oxide nanoparticles.

scholarly journals A distinct ssDNA / RNA binding interface in the Nsp9 protein from SARS‐CoV ‐2

2021 ◽  
Author(s):  
Serene El‐Kamand ◽  
Mar‐Dean Du Plessis ◽  
Natasha Breen ◽  
Lexie Johnson ◽  
Samuel Beard ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Interface

Biochemical characterization of Yin Yang 1 – RNA complexes

2008 ◽  
Vol 86 (1) ◽  
pp. 31-36 ◽  
Author(s):  
Zachery R. Belak ◽  
Andrew Ficzycz ◽  
Nick Ovsenek
Keyword(s):  
Rna Binding ◽  
Biochemical Characterization ◽  
Ribonucleoprotein Complexes ◽  
Yin Yang 1 ◽  
Significant Difference ◽  
Yin Yang ◽  
Rna Interaction ◽  
Rna Complexes

YY1 (Yin Yang 1) is present in the Xenopus oocyte cytoplasm as a constituent of messenger ribonucleoprotein complexes (mRNPs). Association of YY1 with mRNPs requires direct RNA-binding activity. Previously, we have shown YY1 has a high affinity for U-rich RNA; however, potential interactions with plausible in vivo targets have not been investigated. Here we report a biochemical characterization of the YY1–RNA interaction including an investigation of the stability, potential 5′-methylguanosine affinity, and specificity for target RNAs. The formation of YY1–RNA complexes in vitro was highly resistant to thermal, ionic, and detergent disruption. The endogenous oocyte YY1–mRNA interactions were also found to be highly stable. Specific YY1–RNA interactions were observed with selected mRNA and 5S RNA probes. The affinity of YY1 for these substrates was within an order of magnitude of that for its cognate DNA element. Experiments aimed at determining the potential role of the 7-methylguanosine cap on RNA-binding reveal no significant difference in the affinity of YY1 for capped or uncapped mRNA. Taken together, the results show that the YY1–RNA interaction is highly stable, and that YY1 possesses the ability to interact with structurally divergent RNA substrates. These data are the first to specifically document the interaction between YY1 and potential in vivo targets.

scholarly journals Structural Similarities and Differences between Two Functionally Distinct SecA Proteins, Mycobacterium tuberculosis SecA1 and SecA2

2015 ◽  
Vol 198 (4) ◽  
pp. 720-730 ◽  
Author(s):  
Stephanie Swanson ◽  
Thomas R. Ioerger ◽  
Nathan W. Rigel ◽  
Brittany K. Miller ◽  
Miriam Braunstein ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Signal Peptide ◽  
Protein A ◽  
Structural Difference ◽  
Structural Similarity ◽  
Structural Features ◽  
Dominant Negative ◽  
Content Type ◽  
Functional Regions ◽  
Suppressor Mutations

ABSTRACTWhile SecA is the ATPase component of the major bacterial secretory (Sec) system, mycobacteria and some Gram-positive pathogens have a second paralog, SecA2. In bacteria with two SecA paralogs, each SecA is functionally distinct, and they cannot compensate for one another. Compared to SecA1, SecA2 exports a distinct and smaller set of substrates, some of which have roles in virulence. In the mycobacterial system, some SecA2-dependent substrates lack a signal peptide, while others contain a signal peptide but possess features in the mature protein that necessitate a role for SecA2 in their export. It is unclear how SecA2 functions in protein export, and one open question is whether SecA2 works with the canonical SecYEG channel to export proteins. In this study, we report the structure ofMycobacterium tuberculosisSecA2 (MtbSecA2), which is the first structure of any SecA2 protein. A high level of structural similarity is observed between SecA2 and SecA1. The major structural difference is the absence of the helical wing domain, which is likely to play a role in howMtbSecA2 recognizes its unique substrates. Importantly, structural features critical to the interaction between SecA1 and SecYEG are preserved in SecA2. Furthermore, suppressor mutations of a dominant-negativesecA2mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG or the translocating polypeptide substrate. These results support a model in which the mycobacterial SecA2 works with SecYEG.IMPORTANCESecA2 is a paralog of SecA1, which is the ATPase of the canonical bacterial Sec secretion system. SecA2 has a nonredundant function with SecA1, and SecA2 exports a distinct and smaller set of substrates than SecA1. This work reports the crystal structure of SecA2 ofMycobacterium tuberculosis(the first SecA2 structure reported for any organism). Many of the structural features of SecA1 are conserved in the SecA2 structure, including putative contacts with the SecYEG channel. Several structural differences are also identified that could relate to the unique function and selectivity of SecA2. Suppressor mutations of asecA2mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG.

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