Isolation of Reconstructed Functional Ribonucleoprotein Complexes of Machupo Virus.

Arenaviruses initiate infection by delivering a transcriptionally competent ribonucleoprotein (RNP) complex into the cytosol of host cells. The arenavirus RNP consists of the large (L) RNA-dependent RNA polymerase (RdRP) bound to a nucleoprotein (NP)-encapsidated genomic RNA (viral RNA [vRNA]) template. During transcription and replication, L must transiently displace RNA-bound NP to allow for template access into the RdRP active site. Concomitant with RNA replication, new subunits of NP must be added to the nascent complementary RNAs (cRNA) as they emerge from the product exit channel of L. Interactions between L and NP thus play a central role in arenavirus gene expression. We developed an approach to purify recombinant functional RNPs from mammalian cells in culture using a synthetic vRNA and affinity-tagged L and NP. Negative-stain electron microscopy of purified RNPs revealed they adopt diverse and flexible structures, like RNPs of other Bunyavirales members. Monodispersed L-NP and trimeric ring-like NP complexes were also obtained in excess of flexible RNPs, suggesting that these heterodimeric structures self-assemble in the absence of suitable RNA templates. This work allows for further biochemical analysis of the interaction between arenavirus L and NP proteins and provides a framework for future high-resolution structural analyses of this replication-associated complex. IMPORTANCE Arenaviruses are rodent-borne pathogens that can cause severe disease in humans. All arenaviruses begin the infection cycle with delivery of the virus replication machinery into the cytoplasm of the host cell. This machinery consists of an RNA-dependent RNA polymerase-which copies the viral genome segments and synthesizes all four viral mRNAs-bound to the two nucleoprotein-encapsidated genomic RNAs. How this complex assembles remains a mystery. Our findings provide direct evidence for the formation of diverse intracellular arenavirus replication complexes using purification strategies for the polymerase, nucleoprotein, and genomic RNA of Machupo virus, which causes Bolivian hemorrhagic fever in humans. We demonstrate that the polymerase and nucleoprotein assemble into higher-order structures within cells, providing a model for the molecular events of arenavirus RNA synthesis. These findings provide a framework for probing the architectures and functions of the arenavirus replication machinery and thus advancing antiviral strategies targeting this essential complex.

Purification of RiboNucleoProtein Particles by MS2-MBP Affinity Chromatography.

RiboNucleoProtein particles (RNPs), which are composed of RNAs and proteins, play essential roles in many biological processes. The isolation of these molecular machines is a critical step to better understand their mechanisms of action. In this chapter, we describe the MS2-MBP affinity chromatography used to purify the protein content of the RNPs formed with an RNA of interest in a nuclear extract. Substrate RNAs are furnished with a tag consisting of three stem-loops that provide specific binding sites for the phage MS2 protein. Here, we successfully applied this method to isolate RNPs formed with subfragments of the long noncoding RNA ANRIL (Antisense Noncoding RNA in the INK4 Locus).

Efficient purification and assembly of ribonucleoprotein complex for interaction analysis by MST assay coupled with GaMD simulations.

Here, we describe a generic protocol for monitoring protein-RNA interaction using a cleavable GFP fusion of a recombinant RNA-binding protein. We detail each expression and purification step, including high salt and heparin column for contaminant RNA removal. After the assembly of RNA into the ribonucleoprotein complex, the MicroScale Thermophoresis assay enables the binding affinity to be obtained quickly with a small amount of sample. Further Gaussian accelerated molecular dynamics simulations allow us to analyze protein:RNA interactions in detail. For complete details on the use and execution of this protocol, please refer to Gao et al. (2020).

An RNA tagging approach for system-wide RNA-binding proteome profiling and dynamics investigation upon transcription inhibition.

RNA-protein interactions play key roles in epigenetic, transcriptional and posttranscriptional regulation. To reveal the regulatory mechanisms of these interactions, global investigation of RNA-binding proteins (RBPs) and monitor their changes under various physiological conditions are needed. Herein, we developed a psoralen probe (PP)-based method for RNA tagging and ribonucleic-protein complex (RNP) enrichment. Isolation of both coding and noncoding RNAs and mapping of 2986 RBPs including 782 unknown candidate RBPs from HeLa cells was achieved by PP enrichment, RNA-sequencing and mass spectrometry analysis. The dynamics study of RNPs by PP enrichment after the inhibition of RNA synthesis provides the first large-scale distribution profile of RBPs bound to RNAs with different decay rates. Furthermore, the remarkably greater decreases in the abundance of the RBPs obtained by PP-enrichment than by global proteome profiling suggest that PP enrichment after transcription inhibition offers a valuable way for large-scale evaluation of the candidate RBPs.

Viral crosslinking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes.

The initial interactions between incoming, pre-replicated virion RNA and host protein factors are important in infection and immunity. Yet currently there are no methods to study these crucial events. We established VIR-CLASP (VIRal Cross-Linking And Solid-phase Purification) to identify the primary viral RNA-host protein interactions. First, host cells are infected with 4-thiouridine (4SU)-labeled RNA viruses and irradiated with 365 nm light to crosslink 4SU-labeled viral genomes and interacting proteins from host or virus. The crosslinked RNA binding proteins (RBPs) are purified by solid-phase reversible immobilization (SPRI) beads with protein-denaturing buffers, and then identified by proteomics. With VIR-CLASP, only the incoming virion RNAs are labeled with 4SU, so crosslinking events specifically occur between proteins and pre-replicated virion RNA. Since solid-phase purification under protein-denaturing conditions, rather than sequence-specific nucleic acid purification, is used to pull-down total RNA and crosslinked RBPs, this method facilitates investigation of potentially all RNA viruses, regardless of RNA sequence. Preparation of 4SU-labeled virus takes ∼7 days and VIR-CLASP takes 1 day.

Differential Centrifugation to Enrich Bacterial Ribonucleoprotein Bodies (BR bodies) from Caulobacter crescentus.

Bacterial RNP bodies (BR bodies) contain the mRNA decay machinery, but the collection of associated RNAs and proteins are poorly defined. Here, we present a protocol for the rapid differential centrifugation-based enrichment of BR bodies from Caulobacter crescentus cells. As native BR bodies are highly labile and dissociate by degrading internal mRNAs, an active site mutant of RNase E, which blocks dissolution of BR bodies, allows BR-body stabilization during enrichment. For complete details on the use and execution of this protocol, please refer to Al-Husini et al. (2020).

Comparative analyses of alphaviral RNA:Protein complexes reveals conserved host-pathogen interactions.

The identification of host / pathogen interactions is essential to both understanding the molecular biology of infection and developing rational intervention strategies to overcome disease. Alphaviruses, such as Sindbis virus, Chikungunya virus, and Venezuelan Equine Encephalitis virus are medically relevant positive-sense RNA viruses. As such, they must interface with the host machinery to complete their infectious lifecycles. Nonetheless, exhaustive RNA:Protein interaction discovery approaches have not been reported for any alphavirus species. Thus, the breadth and evolutionary conservation of host interactions on alphaviral RNA function remains a critical gap in the field. Herein we describe the application of the Cross-Link Assisted mRNP Purification (CLAMP) strategy to identify conserved alphaviral interactions. Through comparative analyses, conserved alphaviral host / pathogen interactions were identified. Approximately 100 unique host proteins were identified as a result of these analyses. Ontological assessments reveal enriched Molecular Functions and Biological Processes relevant to alphaviral infection. Specifically, as anticipated, Poly(A) RNA Binding proteins are significantly enriched in virus specific CLAMP data sets. Moreover, host proteins involved in the regulation of mRNA stability, proteasome mediated degradation, and a number of 14-3-3 proteins were identified. Importantly, these data expand the understanding of alphaviral host / pathogen interactions by identifying conserved interactants.

Single and Combined Methods to Specifically or Bulk-Purify RNA-Protein Complexes.

The ribonome interconnects the proteome and the transcriptome. Specific biology is situated at this interface, which can be studied in bulk using omics approaches or specifically by targeting an individual protein or RNA species. In this review, we focus on both RNA- and ribonucleoprotein-(RNP) centric methods. These methods can be used to study the dynamics of the ribonome in response to a stimulus or to identify the proteins that interact with a specific RNA species. The purpose of this review is to provide and discuss an overview of strategies to cross-link RNA to proteins and the currently available RNA- and RNP-centric approaches to study RNPs. We elaborate on some major challenges common to most methods, involving RNP yield, purity and experimental cost. We identify the origin of these difficulties and propose to combine existing approaches to overcome these challenges. The solutions provided build on the recently developed organic phase separation protocols, such as Cross-Linked RNA eXtraction (XRNAX), orthogonal organic phase separation (OOPS) and Phenol-Toluol extraction (PTex).

Phase-separated bacterial ribonucleoprotein bodies organize mRNA decay.

In bacteria, mRNA decay is controlled by megadalton scale macromolecular assemblies called, "RNA degradosomes," composed of nucleases and other RNA decay associated proteins. Recent advances in bacterial cell biology have shown that RNA degradosomes can assemble into phase-separated structures, termed bacterial ribonucleoprotein bodies (BR-bodies), with many analogous properties to eukaryotic processing bodies and stress granules. This review will highlight the functional role that BR-bodies play in the mRNA decay process through its organization into a membraneless organelle in the bacterial cytoplasm. This review will also highlight the phylogenetic distribution of BR-bodies across bacterial species, which suggests that these phase-separated structures are broadly distributed across bacteria, and in evolutionarily related mitochondria and chloroplasts. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Export and Localization > RNA Localization RNA Turnover and Surveillance > Regulation of RNA Stability.

Identifying Trypanosome Protein-RNA Interactions Using RIP-Seq.

Trypanosomatids rely primarily on posttranscriptional mechanisms for the control of gene expression, with regulation of RNA processing, localization, degradation, and translation by RNA-binding proteins (RBPs). To determine the mechanisms by which RBPs control gene expression in trypanosomatids, transcriptome-wide identification of mRNA targets and mapping of the RNA-binding site is required. Here we present our most current RIP-Seq (RNA immunoprecipitation followed by high-throughput sequencing) protocol that we generally apply to elucidate RNA/protein interactions in Trypanosoma brucei. The technique provides valuable information about the workings of messenger ribonucleoprotein (mRNP) networks and trypanosome gene expression mechanisms.

Transcript specific mRNP capture from Drosophila egg-chambers for proteomic analysis.

mRNA binding proteins (RBPs) play a major role in post-transcriptional control of gene expression. To understand the complex regulatory processes regulating a specific mRNA during its life-time, a comprehensive view of the bound RBPs is essential. Here, we describe a method for transcript-specific isolation of endogenous ribonucleoprotein complexes (RNPs) from Drosophila egg-chambers. The method, which is based on in-solution hybridization of short biotinylated antisense DNA oligonucleotide probes to multiple segments of a transcript of interest allows unbiased identification of associated proteins by quantitative proteomics.

Fast and unbiased purification of RNA-protein complexes after UV cross-linking.

Post-transcriptional regulation of gene expression in cells is facilitated by formation of RNA-protein complexes (RNPs). While many methods to study eukaryotic (m)RNPs rely on purification of polyadenylated RNA, other important regulatory RNA classes or bacterial mRNA could not be investigated at the same depth. To overcome this limitation, we developed Phenol Toluol extraction (PTex), a novel and unbiased method for the purification of UV cross-linked RNPs in living cells. PTex is a fast (2-3 h) and simple protocol. The purification principle is solely based on physicochemical properties of cross-linked RNPs, enabling us to interrogate RNA-protein interactions system-wide and beyond poly(A) RNA from a variety of species and source material. Here, we are presenting an introduction of the underlying separation principles and give a detailed discussion of the individual steps as well as incorporation of PTex in high-throughput pipelines.

Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins.

CRISPR/Cas9 ribonucleoprotein (RNP) complexes are promising biological tools with diverse biomedical applications. However, to date there are no efficient methods that can produce these proteins at large scales and low cost. Here, we present a streamlined method for direct production of Cas9 RNPs from Escherichia coli by co-expression of Cas9 and the target-specific single-guided RNAs. Harnessing an ultrahigh-affinity CL7/Im7 purification system recently developed we achieve one-step purification of the self-assembling CRISPR/Cas RNPs, including the commonly used Cas9 and Cas12a, within half a day and with a ~fourfold higher yield than incumbent methods. The prepared Cas RNPs show remarkable stability in the absence of RNase inhibitors, as well as profound gene-editing efficiency in vitro and in vivo. Our method is convenient, cost-effective, and can be used to prepare other CRISPR/Cas RNPs.

Investigational antiviral therapies for the treatment of influenza.

INTRODUCTION: Influenza viral ribonucleoprotein complexes (vRNPs) play a key role in viral transcription and replication; hence, the recent development of novel anti-influenza drugs targeting vRNPs has garnered widespread interest. AREAS COVERED: We discuss the function of the constituents of vRNPs and summarize those vRNPs-targeted synthetic drugs that are in preclinical and early clinical development. EXPERT OPINION: vRNPs contain high-value drug targets; such targets include the subunits PA, PB1, PB2, and NP. Developing a new generation of antiviral therapies with strategies that utilize existing drugs, natural compounds originated from new resources and novel drug combinations may open up new therapeutic approaches to influenza.

Defining the RNA interactome by total RNA-associated protein purification.

The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)-associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA-associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA-protein complexes using silica beads. In a variant approach (PAR-TRAPP), RNAs were labelled with 4-thiouracil prior to 350 nm crosslinking. PAR-TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBPs. In comparison, TRAPP identified many more proteins not expected to bind RNA, and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNAs. Exploiting PAR-TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA-peptide conjugates, using iTRAPP, providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBPome characterization.

Purification of cross-linked RNA-protein complexes by phenol-toluol extraction.

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.

Automatic Counting of Intra-Cellular Ribonucleo-Protein Aggregates in Saccharomyces cerevisiae Using a Textural Approach.

In the context of microbiology, recent studies show the importance of ribonucleo-protein aggregates (RNPs) for the understanding of mechanisms involved in cell responses to specific environmental conditions. The assembly and disassembly of aggregates is a dynamic process, the characterization of the stage of their evolution can be performed by the evaluation of their number. The aim of this study is to propose a method to automatically determine the count of RNPs. We show that the determination of a precise count is an issue by itself and hence, we propose three textural approaches: a classical point of view using Haralick features, a frequency point of view with generalized Fourier descriptors, and a structural point of view with Zernike moment descriptors (ZMD). These parameters are then used as inputs for a supervised classification in order to determine the most relevant. An experiment using a specific Saccharomyces cerevisiae strain presenting a fusion between a protein found in RNPs (PAB1) and the green fluorescent protein was performed to benchmark this approach. The fluorescence was observed with two-photon fluorescence microscopy. Results show that the textural approach, by mixing ZMD with Haralick features, allows for the characterization of the number of RNPs.

Isoelectric Focusing on Non-Denaturing Flatbed Gels.

Isoelectric focusing (IEF) serves as a very useful procedure for cell protein separation and characterization. We have used this method to study antibody clonotype changes. Here we discuss the use of a sensitive native flatbed isoelectric focusing method to analyze specific antibody clonotype changes in a patient with systemic lupus erythematosus, who developed autoantibodies to the Ro 60 autoantigen under observation. Patient sera samples collected over several years were used for analysis using flatbed IEF. Following electrofocusing, the gel is analyzed by affinity immunoblotting utilizing Ro 60-coated nitrocellulose membrane to determine oligoclonality of the anti-Ro 60 containing sera.

Highly efficient in vitro translation of authentic affinity-purified messenger ribonucleoprotein complexes.

Cell-free systems are widely used to study mechanisms and regulation of translation, but the use of in vitro transcribed (IVT) mRNAs as translation substrates limits their efficiency and utility. Here, we present an approach for in vitro translation of messenger ribonucleoprotein (mRNP) complexes affinity purified in association with tagged mRNAs expressed in mammalian cells. We show that in vitro translation of purified mRNPs is much more efficient than that achieved using standard IVT mRNA substrates and is compatible with physiological ionic conditions. The high efficiency of affinity-purified mRNP in vitro translation is attributable to both copurified protein components and proper mRNA processing and modification. Further, we use translation inhibitors to show that translation of purified mRNPs consists of separable phases of run-off elongation by copurified ribosomes and de novo initiation by ribosomes present in the translation extracts. We expect that this in vitro system will enhance mechanistic studies of eukaryotic translation and translation-associated processes by allowing the use of endogenous mRNPs as translation substrates under physiological buffer conditions.

Isolation and Characterization of Endogenous RNPs from Brain Tissues.

Identification of physiological target RNAs and protein interactors bound to RNA-binding proteins is a key prerequisite to understand the underlying mechanisms of posttranscriptional expression control and RNA granule assembly. Here, we describe a multistep biochemical approach to isolate endogenous ribonucleoprotein particles from brain tissues by exploiting differential centrifugation and gradient fractionation followed by immunoprecipitation with monospecific, affinity-purified antibodies directed against selected RNA-binding proteins. This protocol results in highly enriched endogenous ribonucleoprotein particles that then can be analyzed by mass spectrometry (for proteins composition) and microarray or RNA sequencing technologies (for target mRNAs).