Bacteriophage MS2 Coat Protein Research Articles

RNA Aptamer-Mediated Gene Activation Systems for Inducible Transgene Expression in Animal Cells.

RNA expression analyses can be used to obtain various information from inside cells, such as physical conditions, the chemical environment, and endogenous signals. For detecting RNA, the system regulating intracellular gene expression has the potential for monitoring RNA expression levels in real time within living cells. Synthetic biology provides powerful tools for detecting and analyzing RNA inside cells. Here, we devised an RNA aptamer-mediated gene activation system, RAMGA, to induce RNA-triggered gene expression activation by employing an inducible complex formation strategy grounded in synthetic biology. This methodology connects DNA-binding domains and transactivators through target RNA using RNA-binding domains, including phage coat proteins. MS2 bacteriophage coat protein fused with a transcriptional activator and PP7 bacteriophage coat protein fused with the tetracycline repressor (tetR) can be bridged by target RNA encoding MS2 and PP7 stem-loops, resulting in transcriptional activation. We generated recombinant CHO cells containing an inducible GFP expression module governed by a minimal promoter with a tetR-responsive element. Cells carrying the trigger RNA exhibited robust reporter gene expression, whereas cells lacking it exhibited no expression. GFP expression was upregulated over 200-fold compared with that in cells without a target RNA expression vector. Moreover, this system can detect the expression of mRNA tagged with aptamer tags and modulate reporter gene expression based on the target mRNA level without affecting the expression of the original mRNA-encoding gene. The RNA-triggered gene expression systems developed in this study have potential as a new platform for establishing gene circuits, evaluating endogenous gene expression, and developing novel RNA detectors.

Investigating the Prevalence of RNA-Binding Metabolic Enzymes in E. coli.

An open research field in cellular regulation is the assumed crosstalk between RNAs, metabolic enzymes, and metabolites, also known as the REM hypothesis. High-throughput assays have produced extensive interactome data with metabolic enzymes frequently found as hits, but only a few examples have been biochemically validated, with deficits especially in prokaryotes. Therefore, we rationally selected nineteen Escherichia coli enzymes from such datasets and examined their ability to bind RNAs using two complementary methods, iCLIP and SELEX. Found interactions were validated by EMSA and other methods. For most of the candidates, we observed no RNA binding (12/19) or a rather unspecific binding (5/19). Two of the candidates, namely glutamate-5-kinase (ProB) and quinone oxidoreductase (QorA), displayed specific and previously unknown binding to distinct RNAs. We concentrated on the interaction of QorA to the mRNA of yffO, a grounded prophage gene, which could be validated by EMSA and MST. Because the physiological function of both partners is not known, the biological relevance of this interaction remains elusive. Furthermore, we found novel RNA targets for the MS2 phage coat protein that served us as control. Our results indicate that RNA binding of metabolic enzymes in procaryotes is less frequent than suggested by the results of high-throughput studies, but does occur.

Multiple RNA- and DNA-binding proteins exhibit direct transfer of polynucleotides with implications for target-site search

We previously demonstrated that the polycomb repressive complex 2 chromatin-modifying enzyme can directly transfer between RNA and DNA without a free-enzyme intermediate state. Simulations suggested that such a direct transfer mechanism may be generally necessary for RNA to recruit proteins to chromatin, but the prevalence of direct transfer capability is unknown. Herein, we used fluorescence polarization assays and observed direct transfer for several well-characterized nucleic acid-binding proteins: three-prime repair exonuclease 1, heterogeneous nuclear ribonucleoprotein U, Fem-3-binding factor 2, and MS2 bacteriophage coat protein. For TREX1, the direct transfer mechanism was additionally observed in single-molecule assays, and the data suggest that direct transfer occurs through an unstable ternary intermediate with partially associated polynucleotides. Generally, direct transfer could allow many DNA- and RNA-binding proteins to conduct a one-dimensional search for their target sites. Furthermore, proteins that bind both RNA and DNA might be capable of readily translocating between those ligands.

Improved extracellular vesicle-based mRNA delivery for familial hypercholesterolemia treatment.

Extracellular vesicle (EV)-based low-density lipoprotein receptor (Ldlr) mRNA delivery showed excellent therapeutic effects in treating familial hypercholesterolemia (FH). Nevertheless, the loading inefficiency of EV-based mRNA delivery presents a significant challenge. Recently, RNA-binding proteins (RBPs) have been fused to EV membrane proteins for selectively encapsulating targeted RNAs to promote loading efficiency. However, the strong interaction between therapeutic RNAs and RBPs prevents RNA release from endosomes to the cytosol in the recipient cells. In this study, an improved strategy was developed for efficient encapsulation of Ldlr mRNA into EVs in donor cells and controllable release in recipient cells. Methods: The MS2 bacteriophage coat protein (CD9-MCP) fusion protein, Ldlr mRNA, and a customized MS2 containing RNA aptamer base-pair matched with Ldlr mRNA were expressed in donor cells. Cells receiving the above therapeutic EVs were simultaneously treated with EVs containing "Ldlr releaser" with a sequence similar to the recognition sites in Ldlr mRNA. Therapeutic effects were analyzed in Ldlr-/- mice receiving EV treatments via the tail vein. Results: In vitro experiments demonstrated improved loading efficiency of Ldlr mRNA in EVs via MS2-MCP interaction. Treatment of "Ldlr releaser" competitively interacted with MS2 aptamer with higher affinity and released Ldlr mRNA from CD9-MCP for efficient translation. When the combinatory EVs were delivered into recipient hepatocytes, the robust LDLR expression afforded therapeutic benefits in Ldlr-/- mice. Conclusion: We proposed an EV-based mRNA delivery strategy for enhanced encapsulation of therapeutic mRNAs in EVs and RNA release into the cytosol for translation in recipient cells with great potential for gene therapy.

UPF1 promotes rapid degradation of m6A-containing RNAs

N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic mRNAs and affects RNA processing and metabolism. When YTHDF2, an m6A-recognizing protein, binds to m6A, it facilitates the destabilization of m6A-containing RNAs (m6A RNAs). Here, we demonstrate that upstream frameshift 1 (UPF1), a key factor for nonsense-mediated mRNA decay, interacts with YTHDF2, thereby triggering rapid degradation of m6A RNAs. The UPF1-mediated m6A RNA degradation depends on a specific interaction between UPF1 and N-terminal residues 101-168 of YTHDF2, UPF1 ATPase/helicase activities, and UPF1 interaction with proline-rich nuclear receptor coactivator 2 (PNRC2), a decapping-promoting factor preferentially involved in nonsense-mediated mRNA decay. Furthermore, transcriptome-wide analyses show that YTHDF2-bound mRNAs that are not substrates for HRSP12-RNase P/MRP-mediated endoribonucleolytic cleavage are destabilized with a higher dependency on UPF1. Collectively, our data indicate dynamic and multilayered regulation of the stability of m6A RNAs and highlight the multifaceted role of UPF1 in mRNA decay.

CRISPR-Based Transcriptional Activation in Drosophila.

Overexpression is one of the classical approaches to study pleiotropic functions of genes of interest. To achieve overexpression, we often increase the transcription by introducing genes on exogenous vectors or by using the CRISPR/dCas9-based transcriptional activation system. To date, the most efficient CRISPR/dCas9-based transcriptional activator is the Synergistic Activation Mediator (SAM) system whereby three different transcriptional activation domains are directly fused to dCas9 and MS2 phage Coat Protein (MCP), respectively, and the system in Drosophila is named flySAM. Here we describe the effective and convenient transcriptional activation system, flySAM, starting from vector construction, microinjection, and transgenic fly selection to the phenotypic analysis.

Simple Affinity-Based Method for Concentrating Viruses from Wastewater Using Engineered Curli Fibers.

Wastewater surveillance is a proven method for tracking community spread and prevalence of some infectious viral diseases. A primary concentration step is often used to enrich viral particles from wastewater prior to subsequent viral quantification and/or sequencing. Here, we present a simple procedure for concentrating viruses from wastewater using bacterial biofilm protein nanofibers known as curli fibers. Through simple genetic engineering, we produced curli fibers functionalized with single-domain antibodies (also known as nanobodies) specific for the coat protein of the model virus bacteriophage MS2. Using these modified fibers in a simple spin-down protocol, we demonstrated efficient concentration of MS2 in both phosphate-buffered saline (PBS) and in the wastewater matrix. Additionally, we produced nanobody-functionalized curli fibers capable of binding the spike protein of SARS-CoV-2, showing the versatility of the system. Our concentration protocol is simple to implement, can be performed quickly under ambient conditions, and requires only components produced through bacterial culture. We believe this technology represents an attractive alternative to existing concentration methods and warrants further research and optimization for field-relevant applications.

Understanding the Folding Mediated Assembly of the Bacteriophage MS2 Coat Protein Dimers

The capsids of RNA viruses such as MS2 are great models for studying protein self-assembly because they are made almost entirely of multiple copies of a single coat protein (CP). Although CP is the minimal repeating unit of the capsid, previous studies have shown that CP exists as a homodimer (CP2) even in an acid-disassembled system, indicating that CP2 is an obligate dimer. Here, we investigate the molecular basis of this obligate dimerization using coarse-grained structure-based models and molecular dynamics simulations. We find that, unlike monomeric proteins of similar size, CP populates a single partially folded ensemble whose "foldedness" is sensitive to denaturing conditions. In contrast, CP2 folds similarly to single-domain proteins populating only the folded and the unfolded ensembles, separated by a prominent folding free energy barrier. Several intramonomer contacts form early, but the CP2 folding barrier is crossed only when the intermonomer contacts are made. A dissection of the structure of CP2 through mutant folding simulations shows that the folding barrier arises both from the topology of CP and the interface contacts of CP2. Together, our results show that CP2 is an obligate dimer because of kinetic stability, that is, dimerization induces a folding barrier and that makes it difficult for proteins in the dimer minimum to partially unfold and access the monomeric state without completely unfolding. We discuss the advantages of this obligate dimerization in the context of dimer design and virus stability.

Rewiring of endogenous signaling pathways to genomic targets for therapeutic cell reprogramming

Rewiring cellular sensors to trigger non-natural responses is fundamental for therapeutic cell engineering. Current designs rely on engineered receptors that are limited to single inputs, and often suffer from high leakiness and low fold induction. Here, we present Generalized Engineered Activation Regulators (GEARs) that overcome these limitations by being pathway-specific rather than input-specific. GEARs consist of the MS2 bacteriophage coat protein fused to regulatory or transactivation domains, and work by rerouting activation of the NFAT, NFκB, MAPK or SMAD pathways to dCas9-directed gene expression from genomic loci. This system enables membrane depolarization-induced activation of insulin expression in β-mimetic cells and IL-12 expression in activated Jurkat cells, as well as IL-12 production in response to the immunomodulatory cytokines TGFβ and TNFα in HEK293T cells. Engineered cells with the ability to reinterpret the extracellular milieu have potential for applications in immunotherapy and in the treatment of metabolic diseases.

In Vivo Tethering System to Isolate RNA-Binding Proteins Regulating mRNA Decay in Leishmania.

RNA-binding proteins (RBPs) play key roles in many aspects of RNA metabolism. In Leishmania, a unicellular eukaryote that favors the posttranscriptional mode of regulation for controlling gene expression levels, the function of RBPs becomes even more critical. However, due largely to limited in vivo approaches available for identifying RBPs in these parasites, there have been no significant advances to our understanding of the role these proteins play in posttranscriptional control through binding to cis-acting elements in the 3' untranslated region (3'UTR) of mRNAs. Here we describe an optimized in vivo RNA tethering approach using the bacteriophage MS2 coat protein combined to immunoprecipitation and mass spectrometry analysis to identify RBPs specifically interacting with 3'UTR short interspersed degenerated retroposon elements (SIDERs). Members of the SIDER2 subfamily were shown previously to promote mRNA degradation through a novel mechanism of mRNA decay. Using this modified MS2 tethering approach, we have identifiedcandidate RBPs specifically interacting with SIDER2 elements and contributing to the decay mechanism.

Automated Design of Diverse Stand-Alone Riboswitches.

Riboswitches that couple binding of ligands to conformational changes offer sensors and control elements for RNA synthetic biology and medical biotechnology. However, design of these riboswitches has required expert intuition or software specialized to transcription or translation outputs; design has been particularly challenging for applications in which the riboswitch output cannot be amplified by other molecular machinery. We present a fully automated design method called RiboLogic for such “stand-alone” riboswitches and test it via high-throughput experiments on 2875 molecules using RNA-MaP (RNA on a massively parallel array) technology. These molecules consistently modulate their affinity to the MS2 bacteriophage coat protein upon binding of flavin mononucleotide, tryptophan, theophylline, and microRNA miR-208a, achieving activation ratios of up to 20 and significantly better performance than control designs. By encompassing a wide diversity of stand-alone switches and highly quantitative data, the resulting ribologic-solves experimental data set provides a rich resource for further improvement of riboswitch models and design methods.

Endoribonucleolytic Cleavage of m6A-Containing RNAs by RNase P/MRP Complex.

N6-methyladenosine (m6A) is the most abundant internal modification in RNAs and plays regulatory roles in a variety of biological and physiological processes. Despite its important roles, the molecular mechanism underlying m6A-mediated gene regulation is poorly understood. Here, we show that m6A-containing RNAs are subject to endoribonucleolytic cleavage via YTHDF2 (m6A reader protein), HRSP12 (adaptor protein), and RNase P/MRP (endoribonucleases). We demonstrate that HRSP12 functions as an adaptor to bridge YTHDF2 and RNase P/MRP, eliciting rapid degradation of YTHDF2-bound RNAs. Transcriptome-wide analyses show that m6A RNAs that are preferentially targeted for endoribonucleolytic cleavage have an HRSP12-binding site and a RNase P/MRP-directed cleavage site upstream and downstream of the YTHDF2-binding site, respectively. We also find that a subset of m6A-containing circular RNAs associates with YTHDF2 in an HRSP12-dependent manner and is selectively downregulated by RNase P/MRP. Thus, our data expand the known functions of RNase P/MRP to endoribonucleolytic cleavage of m6A RNAs.

MS2 Labeling of Endogenous Beta-Actin mRNA Does Not Result in Stabilization of Degradation Intermediates.

The binding of MS2 bacteriophage coat protein (MCP) to MS2 binding site (MBS) RNA stem-loop sequences has been widely used to label mRNA for live-cell imaging at single-molecule resolution. However, concerns have been raised recently from studies with budding yeast showing aberrant mRNA metabolism following the MS2-GFP labeling. To investigate the degradation pattern of MS2-GFP-labeled mRNA in mammalian cells and tissues, we used Northern blot analysis of β-actin mRNA extracted from the Actb-MBS knock-in and MBS×MCP hybrid mouse models. In the immortalized mouse embryonic cell lines and various organ tissues derived from the mouse models, we found no noticeable accumulation of decay products of β-actin mRNA compared with the wild-type mice. Our results suggest that accumulation of MBS RNA decay fragments does not always happen depending on the mRNA species and the model organisms used.

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification.

Site-specific protein modification is a widely used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically hindered N termini, such as virus-like particles (VLPs) composed of the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.

Quantitative characterization of all single amino acid variants of a viral capsid-based drug delivery vehicle

Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. Here, we report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. SyMAPS studies on the MS2 bacteriophage coat protein revealed a high-resolution fitness landscape that challenges some conventional assumptions of protein engineering. An additional round of selection identified a previously unknown variant (CP[T71H]) that is stable at neutral pH but less tolerant to acidic conditions than the wild-type coat protein. The capsids formed by this variant could be more amenable to disassembly in late endosomes or early lysosomes—a feature that is advantageous for delivery applications. In addition to providing a mutability blueprint for virus-like particles, SyMAPS can be readily applied to other self-assembling proteins.

Rational Design of pH Sensitive MS2 Virus‐Like Particles for Drug Delivery Applications

Virus‐like particles (VLPs) have emerged as promising drug delivery and imaging scaffolds. VLPs made from the MS2 Bacteriophage Coat Protein (CP) enter cells via receptor‐mediated endocytosis and are believed to accumulate in the endosomal pathway. This pathway involves a decrease in pH levels that can potentially trigger the disassembly of acid sensitive VLPs, leading to cargo release. However, MS2 CPWT VLPs are highly stable in the endosomal pH range (4.5–5.5), which could slow endosomal release of cargo within the cell. Here, we set out to identify an acid‐sensitive variant of the MS2 CP that could lead to faster cargo release in the acidic environment of the endosome. We identified a previously‐unknown double mutant, CPT71H/E76C, that exhibits significant acid sensitivity in vitro at the endosomal pH range, in contrast to MS2 CPWT. The population of CPT71H/E76C VLPs was measured after a 4 h incubation at 37°C in a pH range from 1.0 to 7.3 with High Performance Liquid Chromatography using a Size Exclusion Chromatography column (HPLC‐SEC). Acid screens showed CPT71H/E76C to have 50% less well‐formed VLP at pH 4.9. These mutations alter a key hydrogen bond interaction, which could be the reason for increased acid sensitivity. To explore this hypothesis, capsids were alkylated with N‐ethylmaleimide, a moiety that could restore the hydrogen bond between the residues, and screen for acid sensitivity. Results showed modified capsids had an acid sensitivity similar to CPT71H, suggesting the hydrogen bond is involved in this property. Acid sensitive CPT71H/E76C could potentially improve the delivery of drugs to cancer cells.Support or Funding InformationNIH‐ MARC (5T34GM007821‐38) and the Amgen Scholars FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A bacterial three-hybrid assay detects Escherichia coli Hfq-sRNA interactions in vivo.

The interaction of RNA molecules with proteins is a critical aspect of gene regulation across all domains of life. Here, we report the development of a bacterial three-hybrid (B3H) assay to genetically detect RNA–protein interactions. The basis for this three-hybrid assay is a transcription-based bacterial two-hybrid assay that has been used widely to detect and dissect protein–protein interactions. In the three-hybrid assay, a DNA-bound protein with a fused RNA-binding moiety (the coat protein of bacteriophage MS2 (MS2CP)) is used to recruit a hybrid RNA upstream of a test promoter. The hybrid RNA consists of a constant region that binds the tethered MS2CP and a variable region. Interaction between the variable region of the hybrid RNA and a target RNA-binding protein that is fused to a subunit of Escherichia coli RNA polymerase (RNAP) stabilizes the binding of RNAP to the test promoter, thereby activating transcription of a reporter gene. We demonstrate that this three-hybrid assay detects interaction between non-coding small RNAs (sRNAs) and the hexameric RNA chaperone Hfq from E. coli and enables the identification of Hfq mutants with sRNA-binding defects. Our findings suggest that this B3H assay will be broadly applicable for the study of RNA–protein interactions.

WIG1 is crucial for AGO2-mediated ACOT7 mRNA silencing via miRNA-dependent and -independent mechanisms.

RNA-binding proteins (RBPs) are involved in mRNA splicing, maturation, transport, translation, storage and turnover. Here, we identified ACOT7 mRNA as a novel target of human WIG1. ACOT7 mRNA decay was triggered by the microRNA miR-9 in a WIG1-dependent manner via classic recruitment of Argonaute 2 (AGO2). Interestingly, AGO2 was also recruited to ACOT7 mRNA in a WIG1-dependent manner in the absence of miR-9, which indicates an alternative model whereby WIG1 controls AGO2-mediated gene silencing. The WIG1–AGO2 complex attenuated translation initiation via an interaction with translation initiation factor 5B (eIF5B). These results were confirmed using a WIG1 tethering system based on the MS2 bacteriophage coat protein and a reporter construct containing an MS2-binding site, and by immunoprecipitation of WIG1 and detection of WIG1-associated proteins using liquid chromatography-tandem mass spectrometry. We also identified WIG1-binding motifs using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation analyses. Altogether, our data indicate that WIG1 governs the miRNA-dependent and the miRNA-independent recruitment of AGO2 to lower the stability of and suppress the translation of ACOT7 mRNA.

Tethering RNA to chromatin for fluorescence microscopy based analysis of nuclear organization.

Nuclear RNAs emerge as important factors to orchestrate the dynamic organization of the nucleus into functional subcompartments. By tethering RNAs to distinct genomic loci, RNA-dependent chromatin changes can be dissected by fluorescence microscopic analysis. Here we describe how this approach is implemented in mammalian cells. It involves two high-affinity protein-nucleic acid interactions that can be established with a number of different protein domains and DNA and RNA sequences. A prototypic system is described here in detail: It consists of the binding of MS2 bacteriophage coat protein to its RNA recognition sequence and the interaction between the bacterial LacI repressor protein to its target lacO operator DNA sequence. Via these interactions RNAs tagged with the MS2 recognition sequences can be recruited to a locus with integrated lacO repeats. By inducing RNA-chromatin binding a number of RNA-dependent activities can be dissected: (i) The RNA-induced compaction or decondensation of chromatin, (ii) identification of RNA-interacting chromatin modifiers that set epigenetic signals such as posttranslational histone modifications, and (iii) nuclear relocation of a genomic locus targeted by the tethered RNA. Thus, a variety of RNA-dependent activities can be evaluated with the MS2-LacI system, which are crucial for understanding how RNA shapes nuclear organization.

Correction to A Selection for Assembly Reveals That a Single Amino Acid Mutant of the Bacteriophage MS2 Coat Protein Forms a Smaller Virus-like Particle.

Virus-like particles are used to encapsulate drugs, imaging agents, enzymes, and other biologically active molecules in order to enhance their function. However, the size of most virus-like particles is inflexible, precluding the design of appropriately sized containers for different applications. Here, we describe a chromatographic selection for virus-like particle assembly. Using this selection, we identified a single amino acid substitution to the coat protein of bacteriophage MS2 that mediates a uniform switch in particle geometry from T = 3 to T = 1 icosahedral symmetry. The resulting smaller particle retains the ability to be disassembled and reassembled in vitro and to be chemically modified to load cargo into its interior cavity. The pair of 27 and 17 nm MS2 particles will allow direct examination of the effect of size on function in established applications of virus-like particles, including drug delivery and imaging.