RNA-protein Complexes Research Articles

RNP-Based Control Systems for Genetic Circuits in Synthetic Biology Beyond CRISPR.

Ribonucleoproteins (RNPs) are RNA-protein complexes utilized natively in both prokaryotes and eukaryotes to regulate essential processes within the cell. Over the past few years, many of these native systems have been adapted to provide control over custom genetic targets. Engineered RNP-based control systems allow for fine-tune regulation of desired targets, by providing customizable nucleotide-nucleotide interactions. However, as there have been several engineered RNP systems developed recently, identifying an optimal system for various bioprocesses is challenging. Here, we review the most successful engineered RNP systems and their applications to survey the current state of the field. Additionally, we provide selection criteria to provide users a streamlined method for identifying an RNP control system most useful to their own work. Lastly, we discuss future applications of RNP control systems and how they can be utilized to address the current grand challenges of the synthetic biology community.

Pull-down of Biotinylated RNA and Associated Proteins.

Mapping networks of RNA-protein interactions in cells is essential for understanding the inner workings of many biological processes, including RNA processing, trafficking, and translation. Current in vivo methods for studying protein-RNA interactions rely mostly on purification of poly(A) transcripts, which represent only ~2-3% of total RNAs (Figure 1). Alternate robust methods for tagging RNA molecules with an RNA aptamer (e.g., MS2-, U1A- and biotin-RNA aptamer) and capturing the RNA-protein complex by the respective aptamer-specific partner are not extensively studied. Here, we describe a protocol (Figure 2) in which a biotin-RNA aptamer, referred to as the RNA mimic of biotin (RMB), was conjugated separately to two small RNA secondary structures that contribute to trafficking and translating HAC1 mRNA in the budding yeast Saccharomyces cerevisiae. The RMB-tagged RNA was expressed in yeast cells from a constitutive promoter. The biotinylated RNA bound to proteins was pulled down from the cell lysate by streptavidin agarose beads. RNA was detected by RT-PCR (Figure 3) and associated proteins by mass spectrometry (Figure 4). Our findings show that an RNA aptamer tag to RNA molecule is an effective method to explore the functional roles of RNA-protein networks in vivo.

Disruption of C. elegans embryonic P granules upon dlc-1(RNAi) is not associated with P granule component loss.

Dynein light chain (DLC-1) is a light chain component of the dynein motor complex, it functions as an allosteric regulator of multi-subunit protein complexes and promotes P granule integrity in the C. elegans embryo. P granules are RNA-protein complexes located in the C. elegans germline that are important for RNA regulation and fertility. To further study the role of DLC-1 during C. elegans embryogenesis we performed quantitative tandem mass tag mass spectrometry on embryos after dlc-1 knock down. The amount of core P granule components and nucleoporin proteins did not change after dlc-1(RNAi). These results show that DLC-1 does not help regulate P granule protein levels and support the model that DLC-1 facilitates phase separation of P granule components in vivo .

Assessing the in vitro Binding Affinity of Protein-RNA Interactions Using an RNA Pull-down Technique.

RNA is a vital component of the cell and is involved in a diverse range of cellular processes through a variety of functions. However, many of these functions cannot be performed without interactions with proteins. There are currently several techniques used to study protein-RNA interactions, such as electrophoretic mobility shift assay, fluorescence anisotropy, and filter binding. RNA-pulldown is a technique that uses biotinylated RNA probes to capture protein-RNA complexes of interest. First, the RNA probe and a recombinant protein are incubated to allow the in vitro interaction to occur. The fraction of bound protein is then captured by a biotin pull-down using streptavidin-agarose beads, followed by elution and immunoblotting for the recombinant protein with a His-tag-reactive probe. Overall, this method does not require specialized equipment outside what is typically found in a modern molecular laboratory and easily facilitates the maintenance of an RNase-free environment. This protocol was validated in: Nucleic Acids Res (2020), DOI: 10.1093/nar/gkaa029 Graphical abstract.

Analyzing the Composition and Organization of Ribonucleoprotein Complexes by APEX-Seq.

Diverse protein-RNA complexes assemble in cells, and their composition and localization regulate the fate of mRNAs. Here, we detail APEX-Seq, an experimental strategy to capture protein-RNA interactions and profile their sub-cellular organization by in vivo proximity labeling and high-throughput sequencing. APEX-Seq relies on direct proximity labeling of RNAs by the peroxidase enzyme APEX2, which can be targeted to specific sites in the cell or fused to proteins of interest. Direct RNA proximity labeling promises new insights into the dynamic behavior of RNA, addressing length scales beyond direct physical contact but too short for microscopy. APEX-Seq should be widely applicable to diverse biological questions and in many cell types, enabling comprehensive studies of the spatial transcriptome and its dynamics over time.

Identification and targeting of G-quadruplex structures in MALAT1 long non-coding RNA

RNA G-quadruplexes (rG4s) have functional roles in many cellular processes in diverse organisms. While a number of rG4 examples have been reported in coding messenger RNAs (mRNA), so far only limited works have studied rG4s in non-coding RNAs (ncRNAs), especially in long non-coding RNAs (lncRNAs) that are of emerging interest and significance in biology. Herein, we report that MALAT1 lncRNA contains conserved rG4 motifs, forming thermostable rG4 structures with parallel topology. We also show that rG4s in MALAT1 lncRNA can interact with NONO protein with high specificity and affinity in vitro and in nuclear cell lysate, and we provide cellular data to support that NONO protein recognizes MALAT1 lncRNA via rG4 motifs. Notably, we demonstrate that rG4s in MALAT1 lncRNA can be targeted by the rG4-specific small molecule, peptide, and L-aptamer, leading to the dissociation of MALAT1 rG4-NONO protein interaction. Altogether, this study uncovers new and important rG4s in MALAT1 lncRNAs, reveals their specific interactions with NONO protein, offers multiple strategies for targeting MALAT1 and its RNA–protein complex via its rG4 structure and illustrates the prevalence and significance of rG4s in ncRNAs.

Long non-coding RNA AC122108.1 promotes lung adenocarcinoma brain metastasis and progression through the Wnt/β-catenin pathway by directly binding to aldolase A.

BackgroundBrain metastasis (BM) is a major pathological subtype of lung adenocarcinoma (LAD), but the pathogenic mechanisms of BM remain unclear. The potential prognostic biomarkers and therapeutic targets for BM of LAD urgently need to be identified. AC122108.1 is a recently discovered new long non-coding ribonucleic acid (RNA).MethodsAC122108 was found to be overexpressed in a LAD BM cell model, and upregulated in 64.52% of LAD BM tissues. AC122108 is an independent factor of BM during LAD development; however, the molecular mechanisms and clinical significance of AC122108.1 in LAD have not yet been established. Additionally, in vitro and in vivo experiments showed that the direct binding of AC122108.1 with aldolase A (ALDOA) enhanced the proliferation, apoptosis, invasiveness, migration, and metastasis of LAD cells.ResultsThis RNA-protein complex decreased the stability of the β-catenin destruction complex, leading to the accumulation of β-catenin in the cytoplasm and ultimately its translocation into the nucleus to activate Wnt(wingless/integrated)/β-catenin signaling.ConclusionsOverall, AC122108.1 promotes LAD BM and its progression through the Wnt/β-catenin pathway by directly binding to ALDOA. This study provides insights into the regulatory mechanism of the LAD BM. AC122108.1 may serve as a potential therapeutic target and prognostic biomarker of LAD.

HIV-1 Packaging Visualised by In-Gel SHAPE.

HIV-1 packages two copies of its gRNA into virions via an interaction with the viral structural protein Gag. Both copies and their native RNA structure are essential for virion infectivity. The precise stepwise nature of the packaging process has not been resolved. This is largely due to a prior lack of structural techniques that follow RNA structural changes within an RNA–protein complex. Here, we apply the in-gel SHAPE (selective 2’OH acylation analysed by primer extension) technique to study the initiation of HIV-1 packaging, examining the interaction between the packaging signal RNA and the Gag polyprotein, and compare it with that of the NC domain of Gag alone. Our results imply interactions between Gag and monomeric packaging signal RNA in switching the RNA conformation into a dimerisation-competent structure, and show that the Gag–dimer complex then continues to stabilise. These data provide a novel insight into how HIV-1 regulates the translation and packaging of its genome.

A viral RNA hijacks host machinery using dynamic conformational changes of a tRNA-like structure

A tricky mimicry RNA viruses use dynamic, multifunctional folded elements to hijack host cellular machinery. Bonilla et al . used cryo–electron microscopy (cryo-EM) to explore an RNA element from Brome mosaic virus that tricks host cell tyrosine transfer RNA synthetase (TyrRS) into adding a tyrosine to the viral genome’s 3′ end. Visualizing this RNA both in isolation and bound to a cellular TyrRS revealed a bound structure unlike the canonical transfer RNA L-like shape and conformational rearrangements in the RNA upon binding to the TyrRS, which suggests a multistep process of enzyme recognition. This study highlights the power of cryo-EM to illustrate dynamic processes involving small structured RNAs and RNA-protein complexes. —DJ

CRISPR-based RNA-binding protein mapping in live cells

RNA-binding proteins (RBPs) are involved in all aspects of RNA metabolism, and RNA-RBP interactions are important for cell homeostasis and viral replication. The global RNA-binding proteome was recently reported; however, little is known about the proteins that bind to specific RNAs. In this study, we describe a novel CRISPR-based RNA interaction proteomics method in live cells. In brief, dCas13a with an HA tag was expressed in cells and bound to an RNA of interest with the help of gRNA. The RNA-protein complexes physically bound to dCas13a-HA were crosslinked using UV light and captured using anti-HA beads, after which the proteins were purified and identified using mass spectrometry. We optimized this system and subsequently applied it to U1 small nuclear RNA, which revealed 226 proteins.

Identifying Protein Interactomes of Target RNAs Using HyPR-MS.

RNA-protein interactions are integral to maintaining proper cellular function and homeostasis, and the disruption of key RNA-protein interactions is central to many disease states. HyPR-MS (hybridization purification of RNA-protein complexes followed by mass spectrometry) is a highly versatile and efficient technology which enables multiplexed discovery of specific RNA-protein interactomes. This chapter provides extensive guidance for successful application of HyPR-MS to the system and target RNA(s) of interest, as well as a detailed description of the fundamental HyPR-MS procedure, including: (1) experimental design of controls, capture oligonucleotides, and qPCR assays; (2) formaldehyde cross-linking of cell culture; (3) cell lysis and RNA solubilization; (4) isolation of target RNA(s); (5) RNA purification and RT-qPCR analysis; (6) protein preparation and mass spectrometric analysis; and (7) mass spectrometric data analysis.

Riboregulation in bacteria: From general principles to novel mechanisms of the trp attenuator and its sRNA and peptide products.

Gene expression strategies ensuring bacterial survival and competitiveness rely on cis- and trans-acting RNA-regulators (riboregulators). Among the cis-acting riboregulators are transcriptional and translational attenuators, and antisense RNAs (asRNAs). The trans-acting riboregulators are small RNAs (sRNAs) that bind proteins or base pairs with other RNAs. This classification is artificial since some regulatory RNAs act both in cis and in trans, or function in addition as small mRNAs. A prominent example is the archetypical, ribosome-dependent attenuator of tryptophan (Trp) biosynthesis genes. It responds by transcription attenuation to two signals, Trp availability and inhibition of translation, and gives rise to two trans-acting products, the attenuator sRNA rnTrpL and the leader peptide peTrpL. In Escherichia coli, rnTrpL links Trp availability to initiation of chromosome replication and in Sinorhizobium meliloti, it coordinates regulation of split tryptophan biosynthesis operons. Furthermore, in S. meliloti, peTrpL is involved in mRNA destabilization in response to antibiotic exposure. It forms two types of asRNA-containing, antibiotic-dependent ribonucleoprotein complexes (ARNPs), one of them changing the target specificity of rnTrpL. The posttranscriptional role of peTrpL indicates two emerging paradigms: (1) sRNA reprograming by small molecules and (2) direct involvement of antibiotics in regulatory RNPs. They broaden our view on RNA-based mechanisms and may inspire new approaches for studying, detecting, and using antibacterial compounds. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Structural mechanism of GTPase-powered ribosome-tRNA movement

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP–Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

Statistical potentials for RNA-protein interactions optimized by CMA-ES

Characterizing RNA-protein interactions remains an important endeavor, complicated by the difficulty in obtaining the relevant structures. Evaluating model structures via statistical potentials is in principle straight-forward and effective. However, given the relatively small size of the existing learning set of RNA-protein complexes optimization of such potentials continues to be problematic. Notably, interaction-based statistical potentials have problems in addressing large RNA-protein complexes. In this study, we adopted a novel strategy with covariance matrix adaptation (CMA-ES) to calculate statistical potentials, successfully identifying native docking poses.

ProNAB: database for binding affinities of protein-nucleic acid complexes and their mutants.

Protein–nucleic acid interactions are involved in various biological processes such as gene expression, replication, transcription, translation and packaging. The binding affinities of protein–DNA and protein–RNA complexes are important for elucidating the mechanism of protein–nucleic acid recognition. Although experimental data on binding affinity are reported abundantly in the literature, no well-curated database is currently available for protein–nucleic acid binding affinity. We have developed a database, ProNAB, which contains more than 20 000 experimental data for the binding affinities of protein–DNA and protein–RNA complexes. Each entry provides comprehensive information on sequence and structural features of a protein, nucleic acid and its complex, experimental conditions, thermodynamic parameters such as dissociation constant (Kd), binding free energy (ΔG) and change in binding free energy upon mutation (ΔΔG), and literature information. ProNAB is cross-linked with GenBank, UniProt, PDB, ProThermDB, PROSITE, DisProt and Pubmed. It provides a user-friendly web interface with options for search, display, sorting, visualization, download and upload the data. ProNAB is freely available at https://web.iitm.ac.in/bioinfo2/pronab/ and it has potential applications such as understanding the factors influencing the affinity, development of prediction tools, binding affinity change upon mutation and design complexes with the desired affinity.

Identification of RNA Base Pairs and Complete Assignment of Nucleobase Resonances by Proton-Detected Solid-State NMR Spectroscopy at 100 kHz MAS.

Knowledge of RNA structure, either in isolation or in complex, is fundamental to understand the mechanism of cellular processes. Solid‐state NMR (ssNMR) is applicable to high molecular‐weight complexes and does not require crystallization; thus, it is well‐suited to study RNA as part of large multicomponent assemblies. Recently, we solved the first structures of both RNA and an RNA‐protein complex by ssNMR using conventional 13C‐ and 15N‐detection. This approach is limited by the severe overlap of the RNA peaks together with the low sensitivity of multidimensional experiments. Here, we overcome the limitations in sensitivity and resolution by using 1H‐detection at fast MAS rates. We develop experiments that allow the identification of complete nucleobase spin‐systems together with their site‐specific base pair pattern using sub‐milligram quantities of one uniformly labelled RNA sample. These experiments provide rapid access to RNA secondary structure by ssNMR in protein‐RNA complexes of any size.

Protein-DNA complex structure modeling based on structural template

DNA-binding is an important feature of proteins, and protein-DNA interaction involves in many life processes. Various computational methods have been developed to predict protein-DNA complex structures due to the difficulty of experimentally obtaining protein-DNA complex structures. However, prediction of protein-DNA complex is still a challenging problem compared with prediction of protein-RNA complex, this may be due to the large conformational changes between bound and unbound structure in both protein and DNA. We extend PRIME 2.0 to PRIME 2.0.1 to model protein-DNA complex structures. By comparing sequence and structure alignment methods, we found that structure-based methods can find more templates than sequence-based methods. The results of all-to-all structure alignments showed that DNA structure plays an important role in prediction of protein-DNA complex structure. By exploring the relationship of sequence and structure, we found that in protein-DNA interaction, numerous structures with dissimilar sequences have similar 3D structures and perform the similar function.

RNA Granules in the Mitochondria and Their Organization under Mitochondrial Stresses.

The human mitochondrial genome (mtDNA) regulates its transcription products in specialised and distinct ways as compared to nuclear transcription. Thanks to its mtDNA mitochondria possess their own set of tRNAs, rRNAs and mRNAs that encode a subset of the protein subunits of the electron transport chain complexes. The RNA regulation within mitochondria is organised within specialised, membraneless, compartments of RNA-protein complexes, called the Mitochondrial RNA Granules (MRGs). MRGs were first identified to contain nascent mRNA, complexed with many proteins involved in RNA processing and maturation and ribosome assembly. Most recently, double-stranded RNA (dsRNA) species, a hybrid of the two complementary mRNA strands, were found to form granules in the matrix of mitochondria. These RNA granules are therefore components of the mitochondrial post-transcriptional pathway and as such play an essential role in mitochondrial gene expression. Mitochondrial dysfunctions in the form of, for example, RNA processing or RNA quality control defects, or inhibition of mitochondrial fission, can cause the loss or the aberrant accumulation of these RNA granules. These findings underline the important link between mitochondrial maintenance and the efficient expression of its genome.

Spatiotemporally-resolved mapping of RNA binding proteins via functional proximity labeling reveals a mitochondrial mRNA anchor promoting stress recovery

Proximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, we show that it is possible to map specific protein subclasses within distinct compartments of living cells. In particular, we develop a method to enrich subcompartment-specific RNA binding proteins (RBPs) by combining peroxidase-catalyzed PL with organic-aqueous phase separation of crosslinked protein-RNA complexes (“APEX-PS”). We use APEX-PS to generate datasets of nuclear, nucleolar, and outer mitochondrial membrane (OMM) RBPs, which can be mined for novel functions. For example, we find that the OMM RBP SYNJ2BP retains specific nuclear-encoded mitochondrial mRNAs at the OMM during translation stress, facilitating their local translation and import of protein products into the mitochondrion during stress recovery. Functional PL in general, and APEX-PS in particular, represent versatile approaches for the discovery of proteins with novel function in specific subcellular compartments.

Engineering Crystal Packing in RNA-Protein Complexes II: A Historical Perspective from the Structural Studies of the Spliceosome.

Cryo-electron microscopy has greatly advanced our understanding of how the spliceosome cycles through different conformational states to conduct the chemical reactions that remove introns from pre-mRNA transcripts. The Cryo-EM structures were built upon decades of crystallographic studies of various spliceosomal RNA-protein complexes. In this review we give an overview of the crystal structures solved in the Nagai group, utilizing many of the strategies to design crystal packing as described in the accompanying paper.