Integrating cryo-OrbiSIMS with computational modelling and metadynamics simulations enhances RNA structure prediction at atomic resolution.

The 3D architecture of RNAs governs their molecular interactions, chemical reactions, and biological functions. However, a large number of RNAs and their protein complexes remain poorly understood due to the limitations of conventional structural biology techniques in deciphering their complex structures and dynamic interactions. To address this limitation, we have benchmarked an integrated approach that combines cryogenic OrbiSIMS, a state-of-the-art solid-state mass spectrometry technique, with computational methods for modelling RNA structures at atomic resolution with enhanced precision. Furthermore, using 7SK RNP as a test case, we have successfully determined the full 3D structure of a native RNA in its apo, native and disease-remodelled states, which offers insights into the structural interactions and plasticity of the 7SK complex within these states. Overall, our study establishes cryo-OrbiSIMS as a valuable tool in the field of RNA structural biology as it enables the study of challenging, native RNA systems.

1H, 13C, and 15N resonance assignments of the La Motif of the human La-related protein 1.

Human La-related protein 1 (HsLARP1) is involved in post-transcriptional regulation of certain 5' terminal oligopyrimidine (5'TOP) mRNAs as well as other mRNAs and binds to both the 5'TOP motif and the 3'-poly(A) tail of certain mRNAs. HsLARP1 is heavily involved in cell proliferation, cell cycle defects, and cancer, where HsLARP1 is significantly upregulated in malignant cells and tissues. Like all LARPs, HsLARP1 contains a folded RNA binding domain, the La motif (LaM). Our current understanding of post-transcriptional regulation that emanates from the intricate molecular framework of HsLARP1 is currently limited to small snapshots, obfuscating our understanding of the full picture on HsLARP1 functionality in post-transcriptional events. Here, we present the nearly complete resonance assignment of the LaM of HsLARP1, providing a significant platform for future NMR spectroscopic studies.

Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

A Guanidinobenzol-Rich Polymer Overcoming Cascade Delivery Barriers for CRISPR-Cas9 Genome Editing.

The efficient cytosolic delivery of the CRISPR-Cas9 machinery remains a challenge for genome editing. Herein, we performed ligand screening and identified a guanidinobenzol-rich polymer to overcome the cascade delivery barriers of CRISPR-Cas9 ribonucleoproteins (RNPs) for genome editing. RNPs were stably loaded into the polymeric nanoparticles (PGBA NPs) by their inherent affinity. The polymer facilitated rapid endosomal escape of RNPs via a dynamic multiple-step cascade process. Importantly, the incorporation of fluorescence in the polymer helps to identify the correlation between cellular uptake and editing efficiency, increasing the efficiency up to 70% from the initial 30% for the enrichment of edited cells. The PGBA NPs efficiently deliver RNPs for in vivo gene editing via both local and systemic injections and dramatically reduce PCSK9 level. These results indicate that PGBA NPs enable the cascade delivery of RNPs for genome editing, showing great promise in broadening the therapeutic potential of the CRISPR-Cas9 technique.

Near-Infrared Light Activated Formulation for the Spatially Controlled Release of CRISPR-Cas9 Ribonucleoprotein for Brain Gene Editing.

The CRISPR/Cas9 system has emerged as a promising platform for gene editing; however, the lack of an efficient and safe delivery system to introduce it into cells continues to hinder clinical translation. Here, we report a rationally designed gene-editing nanoparticle (NP) formulation for brain applications: an sgRNA:Cas9 ribonucleoprotein complex is immobilized on the NP surface by oligonucleotides that are complementary to the sgRNA. Irradiation of the formulation with a near-infrared (NIR) laser generates heat in the NP, leading to the release of the ribonucleoprotein complex. The gene-editing potential of the formulation was demonstrated in vitro at the single-cell level. The safety and gene editing of the formulation were also demonstrated in the brains of reporter mice, specifically in the subventricular zone after intracerebral administration and in the olfactory bulb after intranasal administration. The formulation presented here offers a new strategy for the spatially controlled delivery of the CRISPR system to the brain.

RNA to Rule Them All: Critical Steps in Lassa Virus Ribonucleoparticle Assembly and Recruitment.

Lassa virus is a negative-strand RNA virus with only four structural proteins that causes periodic outbreaks in West Africa. The nucleoprotein (NP) encapsidates the viral genome, forming ribonucleoprotein complexes (RNPs) together with the viral RNA and the L protein. RNPs must be continuously restructured during viral genome replication and transcription. The Z protein is important for membrane recruitment of RNPs, viral particle assembly, and budding and has also been shown to interact with the L protein. However, the interaction of NP, viral RNA, and Z is poorly understood. Here, we characterize the interactions between Lassa virus NP, Z, and RNA using structural mass spectrometry. We identify the presence of RNA as the driver for the disassembly of ring-like NP trimers, a storage form, into monomers to subsequently form higher order RNA-bound NP assemblies. We locate the interaction site of Z and NP and demonstrate that while NP binds Z independently of the presence of RNA, this interaction is pH-dependent. These data improve our understanding of RNP assembly, recruitment, and release in Lassa virus.

Comprehensive mapping of exon junction complex binding sites reveals universal EJC deposition in Drosophila.

BACKGROUND: The exon junction complex (EJC) is involved in most steps of the mRNA life cycle, ranging from splicing to nonsense-mediated mRNA decay (NMD). It is assembled by the splicing machinery onto mRNA in a sequence-independent manner. A fundamental open question is whether the EJC is deposited onto all exon‒exon junctions or only on a subset of them. Several previous studies have made observations supportive of the latter, yet these have been limited by methodological constraints. RESULTS: In this study, we sought to overcome these limitations via the integration of two different approaches for transcriptome-wide mapping of EJCs. Our results revealed that nearly all, if not all, internal exons consistently harbor an EJC in Drosophila, demonstrating that EJC presence is an inherent consequence of the splicing reaction. Furthermore, our study underscores the limitations of eCLIP methods in fully elucidating the landscape of RBP binding sites. Our findings highlight how highly specific (low false positive) methodologies can lead to erroneous interpretations due to partial sensitivity (high false negatives). CONCLUSIONS: This study contributes to our understanding of EJC deposition and its association with pre-mRNA splicing. The universal presence of EJC on internal exons underscores its significance in ensuring proper mRNA processing. Additionally, our observations highlight the need to consider both specificity and sensitivity in RBP mapping methodologies.

Negative and ambisense RNA virus ribonucleocapsids: more than protective armor.

SUMMARYNegative and ambisense RNA viruses are the causative agents of important human diseases such as influenza, measles, Lassa fever, and Ebola hemorrhagic fever. The viral genome of these RNA viruses consists of one or more single-stranded RNA molecules that are encapsidated by viral nucleocapsid proteins to form a ribonucleoprotein complex (RNP). This RNP acts as protection, as a scaffold for RNA folding, and as the context for viral replication and transcription by a viral RNA polymerase. However, the roles of the viral nucleoproteins extend beyond these functions during the viral infection cycle. Recent advances in structural biology techniques and analysis methods have provided new insights into the formation, function, dynamics, and evolution of negative sense virus nucleocapsid proteins, as well as the role that they play in host innate immune responses against viral infection. In this review, we discuss the various roles of nucleocapsid proteins, both in the context of RNPs and in RNA-free states, as well as the open questions that remain.

Structure of the Spring Viraemia of Carp Virus Ribonucleoprotein Complex Reveals Its Assembly Mechanism and Application in Antiviral Drug Screening.

Spring viremia of carp virus (SVCV) is a highly pathogenic Vesiculovirus infecting the common carp, yet neither a vaccine nor effective therapies are available to treat spring viremia of carp (SVC). Like all negative-sense viruses, SVCV contains an RNA genome that is encapsidated by the nucleoprotein (N) in the form of a ribonucleoprotein (RNP) complex, which serves as the template for viral replication and transcription. Here, the three-dimensional (3D) structure of SVCV RNP was resolved through cryo-electron microscopy (cryo-EM) at a resolution of 3.7 Å. RNP assembly was stabilized by N and C loops; RNA was wrapped in the groove between the N and C lobes with 9 nt nucleotide per protomer. Combined with mutational analysis, our results elucidated the mechanism of RNP formation. The RNA binding groove of SVCV N was used as a target for drug virtual screening, and it was found suramin had a good antiviral effect. This study provided insights into RNP assembly, and anti-SVCV drug screening was performed on the basis of this structure, providing a theoretical basis and efficient drug screening method for the prevention and treatment of SVC. IMPORTANCE Aquaculture accounts for about 70% of global aquatic products, and viral diseases severely harm the development of aquaculture industry. Spring viremia of carp virus (SVCV) is the pathogen causing highly contagious spring viremia of carp (SVC) disease in cyprinids, especially common carp (Cyprinus carpio), yet neither a vaccine nor effective therapies are available to treat this disease. In this study, we have elucidated the mechanism of SVCV ribonucleoprotein complex (RNP) formation by resolving the 3D structure of SVCV RNP and screened antiviral drugs based on the structure. It is found that suramin could competitively bind to the RNA binding groove and has good antiviral effects both in vivo and in vitro. Our study provides a template for rational drug discovery efforts to treat and prevent SVCV infections.

Principles of mitoribosomal small subunit assembly in eukaryotes.

Mitochondrial ribosomes (mitoribosomes) synthesize proteins encoded within the mitochondrial genome that are assembled into oxidative phosphorylation complexes. Thus, mitoribosome biogenesis is essential for ATP production and cellular metabolism1. Here we used cryo-electron microscopy to determine nine structures of native yeast and human mitoribosomal small subunit assembly intermediates, illuminating the mechanistic basis for how GTPases are used to control early steps of decoding centre formation, how initial rRNA folding and processing events are mediated, and how mitoribosomal proteins have active roles during assembly. Furthermore, this series of intermediates from two species with divergent mitoribosomal architecture uncovers both conserved principles and species-specific adaptations that govern the maturation of mitoribosomal small subunits in eukaryotes. By revealing the dynamic interplay between assembly factors, mitoribosomal proteins and rRNA that are required to generate functional subunits, our structural analysis provides a vignette for how molecular complexity and diversity can evolve in large ribonucleoprotein assemblies.

An integrated ion-exchange membrane-based microfluidic device for irreversible dissociation and quantification of miRNA from ribonucleoproteins.

Ribonucleoproteins (RNPs), particularly microRNA-induced silencing complex (miRISC), have been associated with cancer-related gene regulation. Specific RNA-protein associations in miRISC complexes or those found in let-7 lin28A complexes can downregulate tumor-suppressing genes and can be directly linked to cancer. The high protein-RNA electrostatic binding affinity is a particular challenge for the quantification of the associated microRNAs (miRNAs). We report here the first microfluidic point-of-care assay that allows direct quantification of RNP-associated RNAs, which has the potential to greatly advance RNP profiling for liquid biopsy. Key to the technology is an integrated cation-anion exchange membrane (CEM/AEM) platform for rapid and irreversible dissociation (k = 0.0025 s-1) of the RNP (Cas9-miR-21) complex and quantification of its associated miR-21 in 40 minutes. The CEM-induced depletion front is used to concentrate the RNP at the depletion front such that the high electric field (>100 V cm-1) within the concentration boundary layer induces irreversible dissociation of the low KD (∼0.5 nM) complex, with ∼100% dissociation even though the association rate (kon = 6.1 s-1) is 1000 times higher. The high field also electrophoretically drives the dissociated RNA out of the concentrated zone without reassociation. A detection limit of 1.1 nM is achieved for Cy3 labelled miR-21.

A structural understanding of influenza virus genome replication.

Influenza virus contains a single-stranded negative-sense RNA genome. Replication of the genome is carried out by the viral RNA-dependent RNA polymerase in the context of the viral ribonucleoprotein (RNP) complex, through a positive-sense complementary RNA intermediate. Genome replication is tightly controlled through interactions with accessory viral and host factors. Propelled by developments in recombinant protein expression, and technical improvements in X-ray crystallography and cryo-electron microscopy, snapshots of the replication process have been captured. Here, we review how recent structural data shed light on the molecular mechanisms of influenza virus genome replication, in particular, encapsidation of nascent RNA, de novo RNP assembly, and regulation of replication initiation through interactions with host and viral cues.

Efficient Editing of the CXCR4 Locus Using Cas9 Ribonucleoprotein Complexes Stabilized with Polyglutamic Acid.

Gene editing using the CRISPR/Cas9 system provides new opportunities to treat human diseases. Approaches aimed at increasing the efficiency of genome editing are therefore important to develop. To increase the level of editing of the CXCR4 locus, which is a target for gene therapy of HIV infection, the Cas9 protein was modified by introducing additional NLS signals and ribonucleoprotein complexes of Cas9 and guide RNA were stabilized with poly-L-glutamic acid. The approach allowed a 1.8-fold increase in the level of CXCR4 knockout in the CEM/R5 T cell line and a 2-fold increase in the level of knock-in of the HIV-1 fusion peptide inhibitor MT-C34 in primary CD4+ T lymphocytes.

Sequence-specific remodeling of a topologically complex RNP substrate by Spb4.

DEAD-box ATPases are ubiquitous enzymes essential in all aspects of RNA biology. However, the limited in vitro catalytic activities described for these enzymes are at odds with their complex cellular roles, most notably in driving large-scale RNA remodeling steps during the assembly of ribonucleoproteins (RNPs). We describe cryo-EM structures of 60S ribosomal biogenesis intermediates that reveal how context-specific RNA unwinding by the DEAD-box ATPase Spb4 results in extensive, sequence-specific remodeling of rRNA secondary structure. Multiple cis and trans interactions stabilize Spb4 in a post-catalytic, high-energy intermediate that drives the organization of the three-way junction at the base of rRNA domain IV. This mechanism explains how limited strand separation by DEAD-box ATPases is leveraged to provide non-equilibrium directionality and ensure efficient and accurate RNP assembly.

Structures of a mobile intron retroelement poised to attack its structured DNA target.

Group II introns are ribozymes that catalyze their self-excision and function as retroelements that invade DNA. As retrotransposons, group II introns form ribonucleoprotein (RNP) complexes that roam the genome, integrating by reversal of forward splicing. Here we show that retrotransposition is achieved by a tertiary complex between a structurally elaborate ribozyme, its protein mobility factor, and a structured DNA substrate. We solved cryo-electron microscopy structures of an intact group IIC intron-maturase retroelement that was poised for integration into a DNA stem-loop motif. By visualizing the RNP before and after DNA targeting, we show that it is primed for attack and fits perfectly with its DNA target. This study reveals design principles of a prototypical retroelement and reinforces the hypothesis that group II introns are ancient elements of genetic diversification.

Influenza A virus use of BinCARD1 to facilitate the binding of viral NP to importin α7 is counteracted by TBK1-p62 axis-mediated autophagy.

As a major component of the viral ribonucleoprotein (vRNP) complex in influenza A virus (IAV), nucleoprotein (NP) interacts with isoforms of importin α family members, leading to the import of itself  and vRNP complex into the nucleus, a process pivotal in the replication cycle of IAV. In this study, we found that BinCARD1, an isoform of Bcl10-interacting protein with CARD (BinCARD), was leveraged by IAV for efficient viral replication. BinCARD1 promoted the nuclear import of the vRNP complex and newly synthesized NP and thus enhanced vRNP complex activity. Moreover, we found that BinCARD1 interacted with NP to promote NP binding to importin α7, an adaptor in the host nuclear import pathway. However, we also found that BinCARD1 promoted RIG-I-mediated innate immune signaling by mediating Lys63-linked polyubiquitination of TRAF3, and that TBK1 appeared to degrade BinCARD1. We showed that BinCARD1 was polyubiquitinated at residue K103 through a Lys63 linkage, which was recognized by the TBK1-p62 axis for autophagic degradation. Overall, our data demonstrate that IAV leverages BinCARD1 as an important host factor that promotes viral replication, and two mechanisms in the host defense system are triggered-innate immune signaling and autophagic degradation-to mitigate the promoting effect of BinCARD1 on the life cycle of IAV.

Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9.

Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ωRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo-electron microscopy structure of IscB-ωRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements.

GraPES: The Granule Protein Enrichment Server for prediction of biological condensate constituents.

Phase separation-based condensate formation is a novel working paradigm in biology, helping to rationalize many important cellular phenomena including the assembly of membraneless organelles. Uncovering the functional impact of cellular condensates requires a better knowledge of these condensates' constituents. Herein, we introduce the webserver GraPES (Granule Protein Enrichment Server), a user-friendly online interface containing the MaGS and MaGSeq predictors, which provide propensity scores for proteins' localization into cellular condensates. Our webpage contains models trained on human (Homo sapiens) and yeast (Saccharomyces cerevisiae) stress granule proteins. MaGS utilizes experimentally-based protein features for prediction, whereas MaGSeq is an entirely protein sequence-based implementation. GraPES is implemented in HTML/CSS and Javascript and is freely available for public use at https://grapes.msl.ubc.ca/. Documentation for using the provided webtools, descriptions of their methodology, and implementation notes can be found on the webpage.

Physicochemical and Functional Characterization of Differential CRISPR-Cas9 Ribonucleoprotein Complexes.

Advances in gene-editing technology enable efficient, targeted ex vivo engineering of different cell types, which offer a potential therapeutic platform for most challenging disease areas. CRISPR-Cas9 is a widely used gene-editing tool in therapeutic applications. The quality of gene-editing reagents (i.e., Cas9 nuclease, single guide (sg)RNA) is associated with the final cellular product quality as they can impact the gene-editing accuracy and efficiency. To assess the impact of the quality of Cas9 protein and sgRNA in the formation of a Cas9 ribonucleoprotein (RNP) complex, stability, and functional activities, we developed a size exclusion chromatography method that utilizes multiple detectors and an in vitro DNA cleavage assay using anion-exchange chromatography. Using these methods, we characterized the formation and stability of Cas9 RNP complexes associated with Cas9 and sgRNA characteristics as well as their functional activities. Multi-angle light scattering characterization showed different types and levels of aggregates in different source sgRNA materials, which contribute to form different Cas9 RNP complexes. The aggregations irreversibly dissociated at high temperatures. When the Cas9 RNP complexes derived from non-heated and heated sgRNAs were characterized, the data showed that specific RNP peaks were impacted. The Cas9 RNP complexes derived from the heated sgRNA retained their biological function and cleaved the double-strand target DNA at a higher rate. This work provides new tools to characterize the Cas9 RNP complex formation, stability, and functional activity and provides insights into sgRNA properties and handling procedures to better control the Cas9 RNP complex formation.

What are the distinguishing features and size requirements of biomolecular condensates and their implications for RNA-containing condensates?

Exciting recent work has highlighted that numerous cellular compartments lack encapsulating lipid bilayers (often called "membraneless organelles"), and that their structure and function are central to the regulation of key biological processes, including transcription, RNA splicing, translation, and more. These structures have been described as "biomolecular condensates" to underscore that biomolecules can be significantly concentrated in them. Many condensates, including RNA granules and processing bodies, are enriched in proteins and nucleic acids. Biomolecular condensates exhibit a range of material states from liquid- to gel-like, with the physical process of liquid-liquid phase separation implicated in driving or contributing to their formation. To date, in vitro studies of phase separation have provided mechanistic insights into the formation and function of condensates. However, the link between the often micron-sized in vitro condensates with nanometer-sized cellular correlates has not been well established. Consequently, questions have arisen as to whether cellular structures below the optical resolution limit can be considered biomolecular condensates. Similarly, the distinction between condensates and discrete dynamic hub complexes is debated. Here we discuss the key features that define biomolecular condensates to help understand behaviors of structures containing and generating RNA.