RNA Tagging Research Articles

A modular platform for bioluminescent RNA tracking

AbstractA complete understanding of RNA biology requires methods for tracking transcripts in vivo. Common strategies rely on fluorogenic probes that are limited in sensitivity, dynamic range, and depth of interrogation, owing to their need for excitation light and tissue autofluorescence. To overcome these challenges, we report a bioluminescent platform for serial imaging of RNAs. The RNA tags are engineered to recruit light-emitting luciferase fragments (termed RNA lanterns) upon transcription. Robust photon production is observed for RNA targets both in cells and in live animals. Importantly, only a single copy of the tag is necessary for sensitive detection, in sharp contrast to fluorescent platforms requiring multiple repeats. Overall, this work provides a foundational platform for visualizing RNA dynamics from the micro to the macro scale.

Live-Cell Imaging of mRNA Using a Pepper RNA Tag.

Live-cell imaging of mRNA enables tracking of mRNA localization and its dynamics in real time. This is fundamentally important in understanding how cells use RNA to regulate gene expression and orchestrate biological processes. Here, we describe a method of using an engineered RNA tag, called Pepper RNA tag, to visualizing mRNA in living cells. In this method, an mRNA of interest engineered to contain the Pepper RNA tag turns on the fluorescence signals of fluorogenic proteins, which enables tracking of mRNA in living cells.

Beyond RNA binding domains: determinants of protein-RNA binding.

RNA binding proteins (RBPs) are composed of RNA binding domains (RBDs) often linked via intrinsically disordered regions (IDRs). Structural and biochemical analysis have shown that disordered linkers contribute to RNA binding by orienting the adjacent RBDs and also characterized certain disordered repeats that directly contact the RNA. However, the relative contribution of IDRs and predicted RBDs to the in-vivo binding pattern is poorly explored. Here, we upscaled the RNA tagging method to map the transcriptome-wide binding of sixteen RBPs in budding yeast. We then performed extensive sequence mutations to distinguish binding determinants within predicted RBDs and the surrounding IDRs in eight of these. The majority of the predicted RBDs tested were not individually essential for mRNA binding, while multiple IDRs that lacked predicted RNA binding potential appeared essential for binding affinity or specificity. Our results provide new insights into the function of poorly studied RBPs and emphasize the complex and distributed encoding of RBP-RNA interaction in-vivo.

Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar KD.

Fluorogenic RNA aptamer tags with high affinity enable RNA purification and imaging. The G-quadruplex (G4) based Mango (M) series of aptamers were selected to bind a thiazole orange based (TO1-Biotin) ligand. Using a chemical biology and reselection approach, we have produced a MII.2 aptamer-ligand complex with a remarkable set of properties: Its unprecedented KD of 45 pM, formaldehyde resistance (8% v/v), temperature stability and ligand photo-recycling properties are all unusual to find simultaneously within a small RNA tag. Crystal structures demonstrate how MII.2, which differs from MII by a single A23U mutation, and modification of the TO1-Biotin ligand to TO1-6A-Biotin achieves these results. MII binds TO1-Biotin heterogeneously via a G4 surface that is surrounded by a stadium of five adenosines. Breaking this pseudo-rotational symmetry results in a highly cooperative and homogeneous ligand binding pocket: A22 of the G4 stadium stacks on the G4 binding surface while the TO1-6A-Biotin ligand completely fills the remaining three quadrants of the G4 ligand binding face. Similar optimization attempts with MIII.1, which already binds TO1-Biotin in a homogeneous manner, did not produce such marked improvements. We use the novel features of the MII.2 complex to demonstrate a powerful optically-based RNA purification system.

Comparative analysis of microbiome inhabiting oxygenated and deoxygenated habitats using V3 and V6 metabarcoding of 16S rRNA gene

We examine how oxygen levels and the choice of 16S ribosomal RNA (rRNA) tags impact marine bacterial communities using Next-Generation amplicon sequencing. Analyzing V3 and V6 regions, we assess microbial composition in both Oxygen minimum zones (OMZ) and non-OMZ (NOMZ) areas in the Arabian Sea (AS) and the Central Indian Ocean basin (CIOB) respectively. Operational taxonomic units (OTUs) at 97% similarity showed slightly higher richness and diversity with V6 compared to V3. Vertical diversity patterns were consistent across both regions. NOMZ showed greater richness and diversity than OMZ. AS and CIOB exhibited significant differences in bacterial community, diversity, and relative abundance at the order and family levels. Alteromonadaceae dominated the OMZ, while Pelagibacteraceae dominated the NOMZ. Synechococcaceae were found exclusively at 250 m in OMZ. Bacteria putatively involved in nitrification, denitrification, and sulfurylation were detected at both sites. Dissolved oxygen significantly influenced microbial diversity at both sites, while seasonal environmental parameters affected diversity consistently, with no observed temporal variation.

Revealing the hidden RBP-RNA interactions with RNA modification enzyme-based strategies.

RNA-binding proteins (RBPs) are powerful and versatile regulators in living creatures, playing fundamental roles in organismal development, metabolism, and various diseases by the regulation of gene expression at multiple levels. The requirements of deep research on RBP function have promoted the rapid development of RBP-RNA interplay detection methods. Recently, the detection method of fusing RNA modification enzymes (RME) with RBP of interest has become a hot topic. Here, we reviewed RNA modification enzymes in adenosine deaminases that act onRNA (ADAR), terminal nucleotidyl transferase (TENT), and activation-induced cytosine deaminase/ApoB mRNA editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family, regarding the biological function, biochemical activity, and substrate specificity originated from enzyme selves, their domains and partner proteins. In addition, we discussed the RME activity screening system, and the RME mutations with engineered enzyme activity. Furthermore, we provided a systematic overview of the basic principles, advantages, disadvantages, and applications of the RME-based and cross-linking and immunopurification (CLIP)-based RBP target profiling strategies, including targets of RNA-binding proteins identified by editing (TRIBE), RNA tagging, surveying targets by APOBEC-mediated profiling (STAMP), CLIP-seq, and their derivative technology. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Processing > RNA Editing and Modification.

Systematic expansion of Riboglow-FLIM by varying the RNA tag

Systematic expansion of Riboglow-FLIM by varying the RNA tag

Evaluating Riboglow-FLIM probes for RNA sensing.

We recently developed Riboglow-FLIM, where we genetically tag and track RNA molecules in live cells through measuring the fluorescence lifetime of a small molecule probe that binds the RNA tag. Here, we systematically and quantitatively evaluated key elements of Riboglow-FLIM that may serve as the foundation for Riboglow-FLIM applications and further tool development efforts. Our investigation focused on measuring changes in fluorescence lifetime of representative Riboglow-FLIM probes with different linkers and fluorophores in different environments. In vitro measurements revealed distinct lifetime differences among the probe variants as a result of different linker designs and fluorophore selections. To expand on the platform's versatility, probes in a wide variety of mammalian cell types were examined using fluorescence lifetime imaging microscopy (FLIM), and possible effects on cell physiology were evaluated by metabolomics. The results demonstrated that variations in lifetime were dependent on both probe and cell type. Interestingly, distinct differences in lifetime values were observed between cell lines, while no overall change in cell health was measured. These findings underscore the importance of probe selection and cellular environment when employing Riboglow-FLIM for RNA detection, serving as a foundation for future tool development and applications across diverse fields and biological systems.

RNA Analysis Using Immunoassay Detection Format.

Oligonucleotide probe tagging and reverse transcriptase polymerase-chain reaction (RT-PCR) are the most widely used techniques currently used for detecting and analyzing RNA. RNA detection using labeled oligonucleotide probe-based approaches is suitable for point-of-care (POC) applications but lacks assay sensitivity, whereas RT-PCR requires complex instrumentation. As an alternative, immunoassay detection formats coupled with isothermal RNA amplification techniques have been proposed for handheld assay development. In this chapter, we describe a robust technique comprising of: (a) target RNA tagging with a complementary oligonucleotide probe labeled with a hapten moiety to form a DNA/RNA duplex hybrid; (b) complexing the DNA/RNA duplex with a pre-coated antibody (Ab) directed at the hapten moiety; (c) sandwich complex formation with an Ab that selectively recognizes the DNA/RNA structural motif; and (d) detection of the sandwich complex using a secondary Ab enzyme conjugate targeting the anti-DNA/RNA Ab followed by standard enzyme-linked immunosorbent assay (ELISA) visualization.

Establishing Riboglow-FLIM to visualize noncoding RNAs inside live zebrafish embryos

The central role of RNAs in health and disease calls for robust tools to visualize RNAs in living systems through fluorescence microscopy. Live zebrafish embryos are a popular system to investigate multicellular complexity as disease models. However, RNA visualization approaches in whole organisms are notably underdeveloped. Here, we establish our RNA tagging and imaging platform Riboglow-FLIM for complex cellular imaging applications by systematically evaluating FLIM capabilities. We use adherent mammalian cells as models for RNA visualization. Additional complexity of analyzing RNAs in whole mammalian animals is achieved by injecting these cells into a zebrafish embryo system for cell-by-cell analysis in this model of multicellularity. We first evaluate all variable elements of Riboglow-FLIM quantitatively before assessing optimal use in whole animals. In this way, we demonstrate that a model noncoding RNA can be detected robustly and quantitatively inside live zebrafish embryos using a far-red Cy5-based variant of the Riboglow platform. We can clearly resolve cell-to-cell heterogeneity of different RNA populations by this methodology, promising applicability in diverse fields.

Targeted RNA condensation in living cells via genetically encodable triplet repeat tags.

Living systems contain various membraneless organelles that segregate proteins and RNAs via liquid-liquid phase separation. Inspired by nature, many protein-based synthetic compartments have been engineered in vitro and in living cells. Here, we introduce a genetically encoded CAG-repeat RNA tag to reprogram cellular condensate formation and recruit various non-phase-transition RNAs for cellular modulation. With the help of fluorogenic RNA aptamers, we have systematically studied the formation dynamics, spatial distributions, sizes and densities of these cellular RNA condensates. The cis- and trans-regulation functions of these CAG-repeat tags in cellular RNA localization, life time, RNA-protein interactions and gene expression have also been investigated. Considering the importance of RNA condensation in health and disease, we expect that these genetically encodable modular and self-assembled tags can be widely used for chemical biology and synthetic biology studies.

Interactions between terminal ribosomal RNA helices stabilize the E. coli large ribosomal subunit.

The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the Escherichia coli 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.

Visualizing orthogonal RNAs simultaneously in live mammalian cells by fluorescence lifetime imaging microscopy (FLIM)

Visualization of RNAs in live cells is critical to understand biology of RNA dynamics and function in the complex cellular environment. Detection of RNAs with a fluorescent marker frequently involves genetically fusing an RNA aptamer tag to the RNA of interest, which binds to small molecules that are added to live cells and have fluorescent properties. Engineering efforts aim to improve performance and add versatile features. Current efforts focus on adding multiplexing capabilities to tag and visualize multiple RNAs simultaneously in the same cell. Here, we present the fluorescence lifetime-based platform Riboglow-FLIM. Our system requires a smaller tag and has superior cell contrast when compared with intensity-based detection. Because our RNA tags are derived from a large bacterial riboswitch sequence family, the riboswitch variants add versatility for using multiple tags simultaneously. Indeed, we demonstrate visualization of two RNAs simultaneously with orthogonal lifetime-based tags.

Illuminating RNA through multiplexing in live mammalian cells.

Subcellular localization of RNAs play a central role in biology. Intense efforts are underway to develop tools for RNA fluorescence detection, but a common limitation is reliance on fluorescence intensity. Here, we present Riboglow-FLIM to illuminate localizations of RNAs in live mammalian cells. Riboglow-FLIM builds upon the Riboglow platform, where a fluorescent probe binds a genetically encoded RNA tag, leading to increasing fluorescence intensity and lifetime of the probe upon RNA binding. As a foundation for a fluorescence lifetime sensor, we demonstrate that lifetime detection leads to robust cellular contrast. We find that cellular contrast for FLIM measurements are superior in direct comparison with fluorescence intensity. Importantly, the RNA tag in Riboglow-FLIM is derived from a phylogenetically diverse family of RNA sequences. We then explore if variations in the RNA tag sequence lead to distinguishable lifetime signals. We demonstrate that lifetime changes observed for RNA tag variants were robust, establishing the RNA tag element of the Riboglow platform as a variable to achieve sensor orthogonality. As a proof-of-concept, we use a model system to demonstrate orthogonality of two RNA tags to visualize two different RNAs with distinct subcellular localizations simultaneously (the long non-coding RNA NORAD and an mRNA). Importantly, by using lifetime as a readout, we are visualizing two RNAs simultaneously with a single channel of detection. Lifetime microscopy image analysis was robust by diverse image processing workflows, including phasor approach and multiexponential reconvolution fitting. We are expanding the available Riboglow-FLIM RNA tags by exploring phylogenetic diversity and structure/function relationships of the RNA sequence that binds the probe ligand. Current efforts are underway to add additional capabilities, including expanding the platform to visualize RNAs in complex model systems like tumor spheroids and developing a split-Riboglow sensor to visualize mRNA splicing.

Harmonizing the growing fluorogenic RNA aptamer toolbox for RNA detection and imaging.

The field of fluorogenic RNA aptamers is a burgeoning research area that aims to address the lack of naturally fluorescent RNA molecules for RNA detection and imaging. These small RNA tags bind to their fluorogenic ligands resulting in significant fluorescent enhancement, leading to a molar brightness comparable to or exceeding that of fluorescent proteins. In the past decade, multiple light-up RNA aptamer systems have been isolated that bind to a broad range of ligands involving several distinct mechanisms of fluorogenicity. This review discusses the selection methods used to isolate fluorogenic RNA aptamers. More than seventy fluorogenic aptamer:ligand pairs are evaluated using objective parameters (e.g., molar brightness, binding affinity, fluorophore exchange capabilities and other details). General guidelines for choosing fluorescent RNA tools, with an emphasis on single-molecule detection and multi-colour imaging applications are provided. Lastly the importance of global standards for evaluating fluorogenic RNA aptamer systems is discussed.

A Novel Method to Isolate RNase MRP Using RNA Streptavidin Aptamer Tags.

Interactions between RNA-binding proteins and RNA molecules are at the center of multiple biological processes. Therefore, accurate characterization of the composition of ribonucleoprotein complexes (RNPs) is crucial. Ribonuclease (RNase) for mitochondrial RNA processing (MRP) and RNase P are highly similar RNPs that play distinct roles at the cellular level; as a consequence, the specific isolation of either of these complexes is essential to study their biochemical function. Since their protein components are nearly identical, purification of these endoribonucleases using protein-centric methods is not feasible. Here, we describe a procedure employing an optimized high-affinity streptavidin-binding RNA aptamer, termed S1m, to purify RNase MRP free of RNase P. This report details all steps from the RNA tagging to the characterization of the purified material. We show that using the S1m tag allows efficient isolation of active RNase MRP.

Fluorescent RNA Tags for In Situ RNA Imaging in Living Cells

AbstractRNA imaging paves the way to investigate RNA function and regulation from the aspect of RNA subcellular localization and expression level. In the past decades, various highly precise and genetic coding fluorescent RNA, which can bind and light up the fluorophore by their specific sequence/structure, has been developed for RNA imaging, including Broccoli, Mango, SRB‐2, RhoBAST, and Pepper et al. In this Review, we summarize the RNA imaging approaches and the progress made in the development of fluorescent RNA. Next, we emphasize the application of fluorescent RNA for in situ RNA imaging in living cells. Of particular note, besides the artificially selected fluorescent RNAs, a new kind of fluorophore that can bind and light up intrinsic RNA structure is described together for the first time. Finally, a summary of current strategies for designing fluorescent RNA and future perspectives are also presented. We hope this expanding topic will inspire the study of the architecture and functions of these fascinating molecular machinery in biological systems.

Genetically encodable tagging and sensing systems for fluorescent RNA imaging

Live cell imaging of RNAs is crucial to interrogate their fundamental roles in various biological processes. The highly spatiotemporal dynamic nature of RNA abundance and localization has presented great challenges for RNA imaging. Genetically encodable tagging and sensing (GETS) systems that can be continuously produced in living systems have afforded promising tools for imaging and sensing RNA dynamics in live cells. Here we review the recent advances of GETS systems that have been developed for RNA tagging and sensing in live cells. We first describe the various GETS systems using MS2-bacteriophage-MS2 coat protein, pumilio homology domain and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9/13 for RNA labeling and tracking. The progresses of GETS systems for fluorogenic labeling and/or sensing RNAs by engineering light-up RNA aptamers, CRISPR-Cas9 systems and RNA aptamer stabilized fluorogenic proteins are then elaborated. The challenges and future perspectives in this field are finally discussed. With the continuing development, GETS systems will afford powerful tools to elucidate RNA biology in living systems.

Abstract LB130: High-throughput RNA sequencing directly from cell lysates enables reproducible phenotypic profiling for assessing novel compounds and treatments in cancer research

Abstract New oncology therapies are driven by multiple approaches, with a heavy emphasis on small-molecule therapeutics to target cancer cells and provide a therapeutic benefit to cancer patients. These small-molecule compounds can be used as a stand-alone treatment approach or may be combined with other compounds to bring enhanced effects. Compound therapeutic discovery relies heavily on high-throughput screening assays for measurement of phenotypic responses following compound treatment of oncology cell cultures which can range from very targeted assays to wide-ranging unbiased approaches. Standard whole transcriptome RNA-seq is a preferred method for unbiased measurement of phenotypic responses but is often overlooked due to limited scalability and high sample screening costs. To address these challenges, we have developed a high-throughput gene expression (HT-GEx) assay that combines a variety of strategies to enable a low-cost and high-throughput alternative to standard RNA-seq. First, a simplified workflow removes upstream RNA isolation steps and tags transcripts directly from cell lysate. This is achieved by incorporating both a sample barcode and a unique molecular index (UMI) during the reverse transcription reaction. Next, we leverage a 3’ end counting approach to enable a reduction in sequencing depth coverage (i.e. sample cost) without compromising gene detection sensitivity. With these key advances, a high-plex and high-throughput screening assay is achievable at a reduced cost by removing the need to purify RNA, tagging transcripts early in the workflow to allow pooling of samples and reducing sequencing depth. We have combined these high-throughput and cost-saving strategies and present results which confirm HT-GEx offers results on par with standard RNA-seq. Gene detection sensitivity based on number of genes detected per millions of reads is highly similar, with gene detection sensitivity saturated at approximately 2 million reads. Additionally, the number of genes detected between replicates for RNA samples and lysate samples demonstrate strong linear correlation, implying highly reproducible results. Thus, high-throughput gene expression is an ideal method for phenotypic screening in oncology cell lines following stand-alone or combined compound treatments. Citation Format: Andrea O'Hara, Michael Stephens, Ilaria DeVito, Laure Turner, Christopher Mozdzierz, Haythem Latif, Ginger Zhou. High-throughput RNA sequencing directly from cell lysates enables reproducible phenotypic profiling for assessing novel compounds and treatments in cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB130.

Detecting RNA dynamics in live mammalian cells with fluorescence lifetime‐based sensors

RNA function is closely linked with subcellular localization and interaction with other biological molecules such as other RNAs and proteins. Acute cellular perturbations and disease mutations may alter RNA dynamics and hence function. Therefore, visualizing RNAs in live cells is critically important for a mechanistic understanding. However, robust fluorescence labels for specific RNA molecules to achieve quantitative detection in the complex cellular environment for diverse RNA species are under‐developed. Typically, fluorescent RNA tags are engineered where a genetically encoded short RNA binds a small molecule that conveys fluorescence properties. These efforts aim to generate large fluorescence intensity changes of the fluorophore upon binding the RNA. Here, we use fluorescence lifetime as an orthogonal approach for a fluorescence RNA sensor. We used the Riboglow platform, an RNA tagging platform that relies on binding of a small fluorescent molecule to a short RNA sequence. First, we compared probe fluorescence signal in live cells in the presence and absence of the RNA tag. We found that fluorescence lifetime provides substantially improved contrast compared with fluorescence intensity. Second, we assessed sensitivity of monitoring subcellular RNA localization dynamics live via fluorescence lifetime. Third, we are exploring modularity of the system by varying the RNA‐ and probe component to enable multi‐parameter RNA detection. Together, we find that fluorescence lifetime offers advantages to track RNAs and quantify localization versus fluorescence intensity. We demonstrated the versatility of this system in diverse cellular examples. Our system is the first platform that exploits fluorescence lifetime for live RNA tracking in cells. We plan to expand this imaging modality further to quantitatively assess RNA dynamics.