Massively parallel dissection of RNA in RNA-protein interactions in vivo.

Many of the biological functions performed by RNA are mediated by RNA-binding proteins (RBPs), and understanding the molecular basis of these interactions is fundamental to biology. Here, we present massively parallel RNA assay combined with immunoprecipitation (MPRNA-IP) for in vivo high-throughput dissection of RNA-protein interactions and describe statistical models for identifying RNA domains and parsing the structural contributions of RNA. By using custom pools of tens of thousands of RNA sequences containing systematically designed truncations and mutations, MPRNA-IP is able to identify RNA domains, sequences, and secondary structures necessary and sufficient for protein binding in a single experiment. We show that this approach is successful for multiple RNAs of interest, including the long noncoding RNA NORAD, bacteriophage MS2 RNA, and human telomerase RNA, and we use it to interrogate the hitherto unknown sequence or structural RNA-binding preferences of the DNA-looping factor CTCF. By integrating systematic mutation analysis with crosslinking immunoprecipitation, MPRNA-IP provides a novel high-throughput way to elucidate RNA-based mechanisms behind RNA-protein interactions in vivo.

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded ß-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

Bacteriophage MS2 displays unreported capsid variability assembling T = 4 and mixed capsids.

Bacteriophage MS2 is a positive-sense, single-stranded RNA virus encapsulated in an asymmetric T = 3 pseudo-icosahedral capsid. It infects Escherichia coli through the F-pilus, in which it binds through a maturation protein incorporated into its capsid. Cryogenic electron microscopy has previously shown that its genome is highly ordered within virions, and that it regulates the assembly process of the capsid. In this study, we have assembled recombinant MS2 capsids with non-genomic RNA containing the capsid incorporation sequence, and investigated the structures formed, revealing that T = 3, T = 4 and mixed capsids between these two triangulation numbers are generated, and resolving structures of T = 3 and T = 4 capsids to 4 Å and 6 Å respectively. We conclude that the basic MS2 capsid can form a mix of T = 3 and T = 4 structures, supporting a role for the ordered genome in favouring the formation of functional T = 3 virions.

Nanoscale Structural Characterization of Individual Viral Particles Using Atomic Force Microscopy Infrared Spectroscopy (AFM-IR) and Tip-Enhanced Raman Spectroscopy (TERS).

Viruses are infections species that infect a large spectrum of living systems. Although displaying a wide variety of shapes and sizes, they are all composed of nucleic acid encapsulated into a protein capsid. After virions enter the host cell, they replicate to produce multiple copies of themselves. They then lyse the host, releasing virions to infect new cells. The high proliferation rate of viruses is the underlying cause of their fast transmission among living species. Although many viruses are harmless, some of them are responsible for severe diseases such as AIDS, viral hepatitis, and flu. Traditionally, electron microscopy is used to identify and characterize viruses. This approach is time- and labor-consuming, which is problematic upon pandemic proliferation of previously unknown viruses, such as H1N1 and COVID-19. Herein, we demonstrate a novel diagnosis approach for label-free identification and structural characterization of individual viruses that is based on a combination of nanoscale Raman and infrared spectroscopy. Using atomic force microscopy-infrared (AFM-IR) spectroscopy, we were able to probe structural organization of the virions of Herpes Simplex Type 1 viruses and bacteriophage MS2. We also showed that tip-enhanced Raman spectroscopy (TERS) could be used to reveal protein secondary structure and amino acid composition of the virus surface. Our results show that AFM-IR and TERS provide different but complementary information about the structure of complex biological specimens. This structural information can be used for fast and reliable identification of viruses. This nanoscale bimodal imaging approach can be also used to investigate the origin of viral polymorphism and study mechanisms of virion assembly.

A Multiscale Model for the Self-Assembly of Coat Proteins in Bacteriophage MS2.

The self-assembly of viral capsids is an essential step to the formation of infectious viruses. Elucidating the kinetic mechanisms of how a capsid or virus-like particle assembles could advance our knowledge about the viral lifecycle, as well as the general principles in self-assembly of biomaterials. However, current understanding of capsid assembly remains incomplete for many viruses due to the fact that the transient intermediates along the assembling pathways are experimentally difficult to be detected. In this paper, we constructed a new multiscale computational framework to simulate the self-assembly of virus-like particles. We applied our method to the coat proteins of bacteriophage MS2 as a specific model system. This virus-like particle of bacteriophage MS2 has a unique feature that its 90 sequence-identical dimers can be classified into two structurally various groups: one is the symmetric CC dimer, and the other is the asymmetric AB dimer. The homotypic interactions between AB dimers result in a 5-fold symmetric contact, while the heterotypic interactions between AB and CC dimers result in 6-fold symmetric contact. We found that the assembly can be described as a physical process of phase transition that is regulated by various factors such as concentration and specific stoichiometry between AB and CC dimers. Our simulations also demonstrate that heterotypic and homotypic interfaces play distinctive roles in modulating the assembling kinetics. The interaction between AB and CC dimers is much more dynamic than that between two AB dimers. We therefore suggest that the alternate growth of viral capsid through the heterotypic dimer interactions dominates the assembling pathways. This is, to the best of our knowledge, the first multiscale model to simulate the assembling process of coat proteins in bacteriophage MS2. The generality of this approach opens the door to its further applications in assembly of other viral capsids, virus-like particles, and novel drug delivery systems.

Mutational analysis of the MS2 lysis protein L.

Small single-stranded nucleic acid phages effect lysis by expressing a single protein, the amurin, lacking muralytic enzymatic activity. Three amurins have been shown to act like 'protein antibiotics' by inhibiting cell-wall biosynthesis. However, the L lysis protein of the canonical ssRNA phage MS2, a 75 aa polypeptide, causes lysis by an unknown mechanism without affecting net peptidoglycan synthesis. To identify residues important for lytic function, randomly mutagenized alleles of L were generated, cloned into an inducible plasmid and the transformants were selected on agar containing the inducer. From a total of 396 clones, 67 were unique single base-pair changes that rendered L non-functional, of which 44 were missense mutants and 23 were nonsense mutants. Most of the non-functional missense alleles that accumulated in levels comparable to the wild-type allele are localized in the C-terminal half of L, clustered in and around an LS dipeptide sequence. The LS motif was used to align L genes from ssRNA phages lacking any sequence similarity to MS2 or to each other. This alignment revealed a conserved domain structure, in terms of charge, hydrophobic character and predicted helical content. None of the missense mutants affected membrane-association of L. Several of the L mutations in the central domains were highly conservative and recessive, suggesting a defect in a heterotypic protein-protein interaction, rather than in direct disruption of the bilayer structure, as had been previously proposed for L.

The role of the loop 1 region in MazFbs mRNA interferase from Bacillus subtilis in recognition of the 3' end of the RNA substrate.

MazFbs is an mRNA interferase from Bacillus subtilis specifically recognizing UACAU. The X-ray structure of its complex with an RNA substrate has been also solved. When its amino acid sequence is compared with that of MazFhw, an mRNA interferase from a highly halophilic archaeon, recognizing UUACUCA, the 9-residue loop-1 region is highly homologous except that the V16V17 sequence in MazFbs is replaced with TK in MazFhw. Thus, we examined the role of the VV sequence in RNA substrate recognition by replacing it with TK, GG, AA or LL. The substitution mutants thus constructed showed significant differences in cleavage specificity using MS2 phage RNA. The primer extension analysis of the cleavage sites revealed that the VV sequence plays an important role in the recognition of the 3'-end base of the RNA substrate.

High-throughput single-molecule screen for small-molecule perturbation of splicing and transcription kinetics.

In eukaryotes, mRNA synthesis is catalyzed by RNA polymerase II and involves several distinct steps, including transcript initiation, elongation, cleavage, and transcript release. Splicing of RNA can occur during (co-transcriptional) or after (post-transcriptional) RNA synthesis. Thus, RNA synthesis and processing occurs through the concerted activity of dozens of enzymes, each of which is potentially susceptible to perturbation by small molecules. However, there are few, if any, high-throughput screening strategies for identifying drugs which perturb a specific step in RNA synthesis and processing. Here we have developed a high-throughput fluorescence microscopy approach in single cells to screen for inhibitors of specific enzymatic steps in RNA synthesis and processing. By utilizing the high affinity interaction between bacteriophage capsid proteins (MS2, PP7) and RNA stem loops, we are able to fluorescently label the intron and exon of a ß-globin reporter gene in human cells. This approach allows one to measure the kinetics of transcription, splicing and release in both fixed and living cells using a tractable, genetically encoded assay in a stable cell line. We tested this reagent in a targeted screen of molecules that target chromatin readers and writers and identified three compounds that slow transcription elongation without changing transcription initiation.

A Catalytic Nanoreactor Based on in Vivo Encapsulation of Multiple Enzymes in an Engineered Protein Nanocompartment.

Bacterial protein compartments concentrate and sequester enzymes, thereby regulating biochemical reactions. Here, we generated a new functional nanocompartment in Escherichia coli by engineering the MS2 phage capsid protein to encapsulate multiple cargo proteins. Sequestration of multiple proteins in MS2-based capsids was achieved by SpyTag/SpyCatcher protein fusions that covalently crosslinked with the interior surface of the capsid. Further, the functional two-enzyme indigo biosynthetic pathway could be targeted to the engineered capsids, leading to a 60 % increase in indigo production in vivo. The enzyme-loaded particles could be purified in their active form and showed enhanced long-term stability in vitro (about 95 % activity after seven days) compared with free enzymes (about 5 % activity after seven days). In summary, this engineered in vivo encapsulation system provides a simple and versatile way for generating highly stable multi-enzyme nanoreactors for in vivo and in vitro applications.

Imaging bacterial protein expression using genetically encoded RNA sensors.

The difficulties in imaging the dynamics of protein expression in live bacterial cells can be overcome by using fluorescent sensors based on Spinach, an RNA that activates the fluorescence of a small-molecule fluorophore. These RNAs selectively bind target proteins and exhibit fluorescence increases that enable protein expression to be imaged in living Escherichia coli. These sensors are key components of a generalizable strategy to image protein expression in a single bacterium in real time.

The allosteric switching mechanism in bacteriophage MS2.

We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we use all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates.

Co-expression of RNA-protein complexes in Escherichia coli and applications to RNA biology.

RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA-protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA-His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

Supraspliceosomes at defined functional states portray the pre-assembled nature of the pre-mRNA processing machine in the cell nucleus.

When isolated from mammalian cell nuclei, all nuclear pre-mRNAs are packaged in multi-subunit large ribonucleoprotein complexes-supraspliceosomes-composed of four native spliceosomes interconnected by the pre-mRNA. Supraspliceosomes contain all five spliceosomal U snRNPs, together with other splicing factors, and are functional in splicing. Supraspliceosomes studied thus far represent the steady-state population of nuclear pre-mRNAs that were isolated at different stages of the splicing reaction. To analyze specific splicing complexes, here, we affinity purified Pseudomonas aeruginosa phage 7 (PP7)-tagged splicing complexes assembled in vivo on Adenovirus Major Late (AdML) transcripts at specific functional stages, and characterized them using molecular techniques including mass spectrometry. First, we show that these affinity purified splicing complexes assembled on PP7-tagged AdML mRNA or on PP7-tagged AdML pre-mRNA are assembled in supraspliceosomes. Second, similar to the general population of supraspliceosomes, these defined supraspliceosomes populations are assembled with all five U snRNPs at all splicing stages. This study shows that dynamic changes in base-pairing interactions of U snRNA:U snRNA and U snRNA:pre-mRNA that occur in vivo during the splicing reaction do not require changes in U snRNP composition of the supraspliceosome. Furthermore, there is no need to reassemble a native spliceosome for the splicing of each intron, and rearrangements of the interactions will suffice.

Effect of coat-protein concentration on the self-assembly of bacteriophage MS2 capsids around RNA.

Self-assembly is a vital part of the life cycle of certain icosahedral RNA viruses. Furthermore, the assembly process can be harnessed to make icosahedral virus-like particles (VLPs) from coat protein and RNA in vitro. Although much previous work has explored the effects of RNA-protein interactions on the assembly products, relatively little research has explored the effects of coat-protein concentration. We mix coat protein and RNA from bacteriophage MS2, and we use a combination of gel electrophoresis, dynamic light scattering, and transmission electron microscopy to investigate the assembly products. We show that with increasing coat-protein concentration, the products transition from well-formed MS2 VLPs to "monster" particles consisting of multiple partial capsids to RNA-protein condensates consisting of large networks of RNA and partially assembled capsids. We argue that the transition from well-formed to monster particles arises because the assembly follows a nucleation-and-growth pathway in which the nucleation rate depends sensitively on the coat-protein concentration, such that at high protein concentrations, multiple nuclei can form on each RNA strand. To understand the formation of the condensates, which occurs at even higher coat-protein concentrations, we use Monte Carlo simulations with coarse-grained models of capsomers and RNA. These simulations suggest that the formation of condensates occurs by the adsorption of protein to the RNA followed by the assembly of capsids. Multiple RNA molecules can become trapped when a capsid grows from capsomers attached to two different RNA molecules or when excess protein bridges together growing capsids on different RNA molecules. Our results provide insight into an important biophysical process and could inform design rules for making VLPs for various applications.

PRR1 coat protein binding to its RNA translational operator.

In small RNA bacteriophages, the genomic RNA binds to the coat proteins when the viral capsid assembles. This is achieved through sequence-specific interactions between a coat-protein dimer and an RNA stem-loop that includes the start codon for the replicase gene. The structure of virus-like particles of the small RNA phage PRR1 bound to an RNA segment corresponding to this stem-loop has been solved and the binding was compared with the related, and better investigated, phage MS2. The overall conformation of the RNA is found to be similar and the residues that are involved in RNA binding in PRR1 are the same as in MS2. The arrangement of the nucleotide bases in the loop of the stem-loop is different, leading to a difference in the stacking at the conserved Tyr86, which is equivalent to Tyr85 in MS2.

PET Imaging and biodistribution of chemically modified bacteriophage MS2.

The fields of nanotechnology and medicine have merged in the development of new imaging and drug delivery agents based on nanoparticle platforms. As one example, a mutant of bacteriophage MS2 can be differentially modified on the exterior and interior surfaces for the concurrent display of targeting functionalities and payloads, respectively. In order to realize their potential for use in in vivo applications, the biodistribution and circulation properties of this class of agents must first be investigated. A means of modulating and potentially improving the characteristics of nanoparticle agents is the appendage of PEG chains. Both MS2 and MS2-PEG capsids possessing interior DOTA chelators were labeled with (64)Cu and injected intravenously into mice possessing tumor xenografts. Dynamic imaging of the agents was performed using PET-CT on a single animal per sample, and the biodistribution at the terminal time point (24 h) was assessed by gamma counting of the organs ex vivo for 3 animals per agent. Compared to other viral capsids of similar size, the MS2 agents showed longer circulation times. Both MS2 and MS2-PEG bacteriophage behaved similarly, although the latter agent showed significantly less uptake in the spleen. This effect may be attributed to the ability of the PEG chains to mask the capsid charge. Although the tumor uptake of the agents may result from the enhanced permeation and retention (EPR) effect, selective tumor imaging may be achieved in the future by using exterior targeting groups.

MS2 viruslike particles: a robust, semisynthetic targeted drug delivery platform.

We show that viruslike particles (VLPs) reassembled in vitro with the RNA bacteriophage MS2 coat protein and an RNA conjugate encompassing a siRNA and a known capsid assembly signal can be targeted to HeLa cells by covalent attachment of human transferrin. The siRNA VLPs protect their cargoes from nuclease, have a double-stranded conformation in the capsid and carry multiple drug and targeting ligands. The relative efficiency of VLP reassembly has been assessed, and conditions have been determined for larger scale production. Targeted VLPs have been purified away from unmodified VLPs for the first time allowing improved analysis of the effects of this synthetic virion system. The particles enter cells via receptor-mediated endocytosis and produce siRNA effects at low nanomolar concentrations. Although less effective than a commercial cationic lipid vector at siRNA delivery, the smaller amounts of internalized RNA with VLP delivery had an effect as good as if not better than the lipid transfection route. This implies that the siRNAs delivered by this route are more accessible to the siRNA pathway than identical RNAs delivered in complex lipid aggregates. The data suggest that the MS2 system continues to show many of the features that will be required to create an effective targeted drug delivery system. The fluorescence assays of siRNA effects described here will facilitate the combinatorial analysis of both future formulations and dosing regimes.

A genetic in vivo system to detect asymmetrically distributed RNA.

Many RNAs show polarized or otherwise non-random subcellular distributions. To create a method for genome-wide genetic screens for RNAs with asymmetric subcellular distributions, we have combined methods for gene tagging and live imaging of messenger RNA (mRNA). A pilot screen in a highly polarized, differentiated cell in the Drosophila larva, the branched terminal cell of the tracheal system, demonstrates the feasibility of the method for identifying new asymmetrically localized mRNAs in vivo.

A two-stage mechanism of viral RNA compaction revealed by single molecule fluorescence.

Long RNAs often exist as multiple conformers in equilibrium. For the genomes of single-stranded RNA viruses, one of these conformers must include a compacted state allowing the RNA to be confined within the virion. We have used single molecule fluorescence correlation spectroscopy to monitor the conformations of viral genomes and sub-fragments in the absence and presence of coat proteins. Cognate RNA-coat protein interactions in two model viruses cause a rapid collapse in the hydrodynamic radii of their respective RNAs. This is caused by protein binding at multiple sites on the RNA that facilitate additional protein-protein contacts. The collapsed species recruit further coat proteins to complete capsid assembly with great efficiency and fidelity. The specificity in RNA-coat protein interactions seen at single-molecule concentrations reflects the packaging selectivity seen for such viruses in vivo. This contrasts with many in vitro reassembly measurements performed at much higher concentrations. RNA compaction by coat protein or polycation binding are distinct processes, implying that defined RNA-coat protein contacts are required for assembly.

UV radiation induces genome-mediated, site-specific cleavage in viral proteins.

Much research has been dedicated to understanding the molecular basis of UV damage to biomolecules, yet many questions remain regarding the specific pathways involved. Here we describe a genome-mediated mechanism that causes site-specific virus protein cleavage upon UV irradiation. Bacteriophage MS2 was disinfected with 254 nm UV, and protein damage was characterized with ESI- and MALDI-based FT-ICR, Orbitrap, and TOF mass spectroscopy. Top-down mass spectrometry of the products identified the backbone cleavage site as Cys46-Ser47 in the virus capsid protein, a location of viral genome-protein interaction. The presence of viral RNA was essential to inducing backbone cleavage. The similar bacteriophage GA did not exhibit site-specific protein cleavage. Based on the major protein fragments identified by accurate mass analysis, a cleavage mechanism is proposed by radical formation. The mechanism involves initial oxidation of the Cys46 side chain followed by hydrogen atom abstraction from Ser47 C(α). Computational protein QM/MM studies confirmed the initial steps of the radical mechanism. Collectively, this study describes a rare incidence of genome-induced protein cleavage without the addition of sensitizers.