Single RNA Molecules Research Articles

A Single Cell but Many Different Transcripts: A Journey into the World of Long Non-Coding RNAs.

In late 2012 it was evidenced that most of the human genome is transcribed but only a small percentage of the transcripts are translated. This observation supported the importance of non-coding RNAs and it was confirmed in several organisms. The most abundant non-translated transcripts are long non-coding RNAs (lncRNAs). In contrast to protein-coding RNAs, they show a more cell-specific expression. To understand the function of lncRNAs, it is fundamental to investigate in which cells they are preferentially expressed and to detect their subcellular localization. Recent improvements of techniques that localize single RNA molecules in tissues like single-cell RNA sequencing and fluorescence amplification methods have given a considerable boost in the knowledge of the lncRNA functions. In recent years, single-cell transcription variability was associated with non-coding RNA expression, revealing this class of RNAs as important transcripts in the cell lineage specification. The purpose of this review is to collect updated information about lncRNA classification and new findings on their function derived from single-cell analysis. We also retained useful for all researchers to describe the methods available for single-cell analysis and the databases collecting single-cell and lncRNA data. Tables are included to schematize, describe, and compare exposed concepts.

Vesicular Stomatitis Virus Transcription Is Inhibited by TRIM69 in the Interferon-Induced Antiviral State.

Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs), many of which are responsible for the cellular antiviral state in which the replication of numerous viruses is blocked. How the majority of individual ISGs inhibit the replication of particular viruses is unknown. We conducted a loss-of-function screen to identify genes required for the activity of alpha interferon (IFN-α) against vesicular stomatitis virus, Indiana serotype (VSVIND), a prototype negative-strand RNA virus. Our screen revealed that TRIM69, a member of the tripartite motif (TRIM) family of proteins, is a VSVIND inhibitor. TRIM69 potently inhibited VSVIND replication through a previously undescribed transcriptional inhibition mechanism. Specifically, TRIM69 physically associates with the VSVIND phosphoprotein (P), requiring a specific peptide target sequence encoded therein. P is a cofactor for the viral polymerase and is required for viral RNA synthesis, as well as the assembly of replication compartments. By targeting P, TRIM69 inhibits pioneer transcription of the incoming virion-associated minus-strand RNA, thereby preventing the synthesis of viral mRNAs, and consequently impedes all downstream events in the VSVIND replication cycle. Unlike some TRIM proteins, TRIM69 does not inhibit viral replication by inducing degradation of target viral proteins. Rather, higher-order TRIM69 multimerization is required for its antiviral activity, suggesting that TRIM69 functions by sequestration or anatomical disruption of the viral machinery required for VSVIND RNA synthesis.IMPORTANCE Interferons are important antiviral cytokines that work by inducing hundreds of host genes whose products inhibit the replication of many viruses. While the antiviral activity of interferon has long been known, the identities and mechanisms of action of most interferon-induced antiviral proteins remain to be discovered. We identified gene products that are important for the antiviral activity of interferon against vesicular stomatitis virus (VSV), a model virus that whose genome consists of a single RNA molecule with negative-sense polarity. We found that a particular antiviral protein, TRIM69, functions by a previously undescribed molecular mechanism. Specifically, TRIM69 interacts with and inhibits the function of a particular phosphoprotein (P) component of the viral transcription machinery, preventing the synthesis of viral messenger RNAs.

Quantitative Studies of Muscleblind Proteins and Their Interaction With TCF4 RNA Foci Support Involvement in the Mechanism of Fuchs' Dystrophy.

PurposeFuchs' endothelial corneal dystrophy (FECD) is a major cause of vision loss and the most common nucleotide repeat disorder, affecting 4% of United States population greater than 40 years of age. Seventy percent of FECD cases are due to an intronic CTG expansion within the TCF4 gene, resulting in accumulation of CUG repeat RNA nuclear foci in corneal endothelium. Each endothelial cell has approximately two sense foci, and each focus is a single RNA molecule. This study aimed to obtain a better understanding of how rare repeat RNA species lead to disease.MethodsWe quantitatively examined muscleblind-like (MBNL) proteins and their interaction with foci in both patient-derived corneal endothelial cell lines and human corneal endothelial tissue.ResultsUsing fluorescent in situ hybridization and immunofluorescence, we found that depletion of both MBNL1 and MBNL2 reduces nuclear RNA foci formed by the repeat, suggesting that both are necessary for foci. Quantitative studies of RNA and protein copy number revealed MBNLs to be abundant in the total cellular pool in endothelial cell lines but are much lower in human corneal endothelial tissue. Studies using human tissue nuclear and cytoplasmic fractions indicate that most MBNL proteins are localized to the cytoplasm.ConclusionsThe low levels of MBNL1/2 in corneal tissue, in combination with the small fraction of protein in the nucleus, may make corneal endothelial cells especially susceptible to sequestration of MBNL1/2 by CUG repeat RNA. These observations may explain how a limited number of RNA molecules can cause widespread alteration of splicing and late-onset degenerative FECD.

High-fidelity amplified FISH for the detection and allelic discrimination of single mRNA molecules

Amplification of signals by the hybridization chain reaction (HCR) is a powerful approach for increasing signal strength in single-molecule fluorescence in situ hybridization, but probes tagged with an HCR initiator sequence are prone to producing false signals. Here we describe a system of interacting hairpin binary probes in which the HCR initiator sequence is conditionally sequestered. The binding of these probes to a perfectly complementary target unmasks the initiator, enabling the generation of an amplified signal. This probe system can distinguish single-nucleotide variations within single mRNA molecules and produces amplified signals in situ for both mutant and wild-type variants, each in a distinguishable color. This technology will augment studies of imbalanced allelic expression and will be useful for the detection of somatic mutations in cancer biopsies. By tiling these probes along the length of an mRNA target, enhanced signals can be obtained, thereby enabling the scanning of tissue sections for gene expression utilizing lower magnification microscopy, overcoming tissue autofluorescence, and allowing the detection of low-abundance biomarkers in flow cytometry.

Bioengineering of a single long noncoding RNA molecule that carries multiple small RNAs.

Noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs), regulate target gene expression and can be used as tools for understanding biological processes and identifying new therapeutic targets. Currently, ncRNA molecules for research and therapeutic use are limited to ncRNA mimics made by chemical synthesis. We have recently established a high-yield and cost-effective method of producing bioengineered or biologic ncRNA agents (BERAs) through bacterial fermentation, which is based on a stable tRNA/pre-miR-34a carrier (~ 180nt) that accommodates target small RNAs. Nevertheless, it remains a challenge to heterogeneously express longer ncRNAs (e.g., > 260nt), and it is unknown if single BERA may carry multiple small RNAs. To address this issue, we hypothesized that an additional human pre-miR-34a could be attached to the tRNA/pre-miR-34a scaffold to offer a new tRNA/pre-miR-34a/pre-miR-34a carrier (~ 296nt) for the accommodation of multiple small RNAs. We thus designed ten different combinatorial BERAs (CO-BERAs) that include different combinations of miRNAs, siRNAs, and antagomirs. Our data showed that all target CO-BERAs were successfully expressed in Escherichia coli at high levels, greater than 40% in total bacterial RNAs. Furthermore, recombinant CO-BERAs were purified to a high degree of homogeneity by fast protein liquid chromatography methods. In addition, CO-BERAs exhibited strong anti-proliferative activities against a variety of human non-small cell lung cancer cell lines. These results support the production of long ncRNA molecules carrying different warhead small RNAs for multi-targeting which may open avenues for developing new biologic RNAs as experimental, diagnostic, and therapeutic tools.

Multiplexed detection of RNA using MERFISH and branched DNA amplification

Multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows simultaneous imaging of numerous RNA species in their native cellular environment and hence spatially resolved single-cell transcriptomic measurements. However, the relatively modest brightness of signals from single RNA molecules can become limiting in a number of applications, such as increasing the imaging throughput, imaging shorter RNAs, and imaging samples with high degrees of background, such as some tissue samples. Here, we report a branched DNA (bDNA) amplification approach for MERFISH measurements. This approach produces a drastic signal increase in RNA FISH samples without increasing the fluorescent spot size for individual RNAs or increasing the variation in brightness from spot to spot, properties that are important for MERFISH imaging. Using this amplification approach in combination with MERFISH, we demonstrated RNA imaging and profiling with a near 100% detection efficiency. We further demonstrated that signal amplification improves MERFISH performance when fewer FISH probes are used for each RNA species, which should allow shorter RNAs to be imaged. We anticipate that the combination of bDNA amplification with MERFISH should facilitate many other applications and extend the range of biological questions that can be addressed by this technique in both cell culture and tissues.

Transcriptional Regulation of the Equol Biosynthesis Gene Cluster in Adlercreutzia equolifaciens DSM19450T.

Given the emerging evidence of equol’s benefit to human health, understanding its synthesis and regulation in equol-producing bacteria is of paramount importance. Adlercreutzia equolifaciens DSM19450T is a human intestinal bacterium—for which the whole genome sequence is publicly available—that produces equol from the daidzein isoflavone. In the present work, daidzein (between 50 to 200 μM) was completely metabolized by cultures of A. equolifaciens DSM19450T after 10 h of incubation. However, only about one third of the added isoflavone was transformed into dihydrodaidzein and then into equol. Transcriptional analysis of the ORFs and intergenic regions of the bacterium’s equol gene cluster was therefore undertaken using RT-PCR and RT-qPCR techniques with the aim of identifying the genetic elements of equol biosynthesis and its regulation mechanisms. Compared to controls cultured without daidzein, the expression of all 13 contiguous genes in the equol cluster was enhanced in the presence of the isoflavone. Depending on the gene and the amount of daidzein in the medium, overexpression varied from 0.5- to about 4-log10 units. Four expression patterns of transcription were identified involving genes within the cluster. The genes dzr, ddr and tdr, which code for daidzein reductase, dihydrodaidzein reductase and tetrahydrodaidzein reductase respectively, and which have been shown involved in equol biosynthesis, were among the most strongly expressed genes in the cluster. These expression patterns correlated with the location of four putative ρ-independent terminator sequences in the cluster. All the intergenic regions were amplified by RT-PCR, indicating the operon to be transcribed as a single RNA molecule. These findings provide new knowledge on the metabolic transformation of daidzein into equol by A. equolifaciens DSM19450T, which might help in efforts to increase the endogenous formation of this compound and/or its biotechnological production.

Probing RNA structure and interaction dynamics at the single molecule level

RNA structures and their dynamic fluctuations lie at the heart of understanding key biological process such as transcription, splicing, translation and RNA decay. While conventional bulk assays have proven to identify and characterize key pathway intermediates, the generally dynamic nature of RNA structures renders the information obtained from time and ensemble averaging techniques necessarily lacking in critical details. Here we detail Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS), a method that readily monitors structural fluctuations of single RNA molecules through the repetitive interaction of fluorescent probes with an unlabeled, surface-immobilized RNA target of virtually any length and in any biological context. In addition, we demonstrate the broad applicability of SiM-KARTS by kinetically fingerprinting the binding of cognate tRNA ligand to single immobilized T-box riboswitch molecules. SiM-KARTS represents a valuable tool for probing biologically relevant structure and interaction features of potentially many diverse RNA metabolic pathways.

Piezo1 Channels Contribute to Flow‐Induced Intracellular Calcium Transients in Mouse Cortical Collecting Duct (CCD)

Within the CCD, an acute increase in tubular fluid flow rate (TFFR) (i) subjects principal (PC) and intercalated (IC) cells therein to biomechanical forces including hydrostatic pressure/circumferential stretch (CS), fluid shear stress (FSS) and drag/torque on PC cilia, and (ii) leads to a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i) characterized by an immediate high amplitude increase to a peak value within ~10 s due to release of IP3‐sensitive internal Ca2+ stores coupled to extracellular Ca2+ entry at the basolateral membrane, followed by a decay to a plateau elevation in [Ca2+]i that exceeds baseline, sustained by luminal Ca2+ entry (Liu et al. AJP Renal Physiology 2003, 2005; 2007). The latter increase in [Ca2+]i is necessary for flow induced K+ secretion (FIKS) in the microperfused mammalian CCD. The identity of the basolateral Ca2+ entry path is unknown. We sought to examine whether Piezo channels, a family of mechanically‐activated Ca2+ permeable channels, contribute to this response. RNAscope detected single Piezo1 RNA molecules in mouse renal cortex. Immunofluorescence studies in mice expressing Piezo1‐tdTomato construct revealed robust basolateral and faint apical Piezo1 localization in both PC and IC. Flow‐induced [Ca2+]i transients, inferred from fluorescence intensity ratios (FIRs), were measured in microperfused mouse CCDs loaded with the Ca2+ indicator fura‐2 and then subject to low or high TFFRs, in the absence or presence of GsMTx4 (piezo‐blocker) or Yoda1 (piezo1‐activator). IC in CCDs from male and female mice preincubated with luminal or bath GsMTx4 (5 μM) failed to exhibit a flow‐induced increase in [Ca2+]i in response to an acute increase in TFFR. Addition of luminal or bath Yoda 1 (1 μM) to CCDs perfused at a low TFFR led to an increase in [Ca2+]i over 90 s in both PC and IC; addition of Yoda1 (10 μM) to the bath further increased [Ca2+]i. We speculate that mechanosensitive Piezo1 channels, expressed on the basolateral>apical membranes of PC and IC in the mouse CCD, contribute to the increase in [Ca2+]i triggered by an increase in TFFR.Support or Funding InformationNIDDK: R01 DK38470, R01 DK51391 and P30 DK079307This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of putative packaging signals in the RNA of foot-and-mouth disease virus (FMDV)

Viruses in the picornavirus family comprise a single molecule of positive sense RNA contained within a simple non-enveloped capsid. The mechanism for RNA packaging is not well understood. We have developed a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA, we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem-loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. To further test the function of these putative packaging signals (PPS), we have developed a trans-encapsidation assay using subgenomic replicons expressing GFP, helper virus and flow cytometry. The results of these studies provide evidence for the involvement of predicted RNA structures in picornavirus packaging and offer readily transferable methodologies for identifying packaging requirements in many other viruses.

Enhancers Predominantly Regulate Gene Expression <i>in vivo</i> Via Transcription Initiation

Gene transcription occurs via a cycle of linked events including initiation, promoter proximal pausing and elongation of RNA polymerase II (Pol II). A key question is how do transcriptional enhancers influence these events to control gene expression? Here we have used a new approach to quantify transcriptional initiation and pausing in vivo, while simultaneously identifying transcription start sites (TSSs) and pause-sites (TPSs) from single RNA molecules. When analyzed in parallel with nascent RNA-seq, these data show that differential gene expression is achieved predominantly via changes in transcription initiation rather than Pol II pausing. Using genetically engineered mouse models deleted for specific enhancers we show that these elements control gene expression via Pol II recruitment and/or initiation rather than via promoter proximal pause release. Together, our data show that enhancers, in general, control gene expression predominantly by Pol II recruitment and initiation rather than via pausing.

Nucleotide and structural label identification in single RNA molecules with quantum tunneling spectroscopy.

Although a number of advances have been made in RNA sequencing and structural characterization, the lack of a method for directly determining the sequence and structure of single RNA molecules has limited our ability to probe heterogeneity in gene expression at the level of single cells. Here we present a method for direct nucleotide identification and structural label mapping of single RNA molecules via Quantum Molecular Sequencing (QMSeq). The method combines non-perturbative quantum tunneling spectroscopy to probe the molecular orbitals of ribonucleotides, new experimental biophysical parameters that fingerprint these molecular orbitals, and a machine learning classification algorithm to distinguish between the ribonucleotides. The algorithm uses tunneling spectroscopy measurements on an unknown ribonucleotide to determine its chemical identity and the presence of local chemical modifications. Combining this with structure-dependent chemical labeling presents the possibility of mapping both the sequence and local structure of individual RNA molecules. By optimizing the base-calling algorithm, we show a high accuracy for both ribonucleotide discrimination (>99.8%) and chemical label identification (>98%) with a relatively modest molecular coverage (35 repeat measurements). This lays the groundwork for simultaneous sequencing and structural mapping of single unknown RNA molecules, and paves the way for probing the sequence-structure-function relationship within the transcriptome at an unprecedented level of detail.

Dynamic Imaging of Nascent RNA Reveals General Principles of Transcription Dynamics And Stochastic Splice Site Selection

The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex and nascent RNA sequencing further reveals widespread splice site selection within introns. We propose and validate a unified theoretical model to explain the general feature of transcription and pervasive stochastic splice site selection.

Lessons from (pre-)mRNA Imaging.

Cells are complex assemblies of molecules organized into organelles and membraneless compartments, each playing important roles in ensuring cellular homeostasis. The different steps of the gene expression pathway take place within these various cellular compartments, and studying gene regulation and RNA metabolism requires incorporating the spatial as well as temporal separation and progression of these processes. Microscopy has been a valuable tool to study RNA metabolism, as it allows the study of biomolecules in the context of intact individual cells, embryos or tissues, preserving cellular context often lost in experimental approaches that require the collection and lysis of cells in large numbers to obtain sufficient material for different types of assays. Indeed, from the first detection of RNAs and ribosomes in cells to today's ability to study the behaviour of single RNA molecules in living cells, or the expression profile and localization of hundreds of mRNA simultaneously in cells, constant effort in developing tools for microscopy has extensively contributed to our understanding of gene regulation. In this chapter, we will describe the role various microscopy approaches have played in shaping our current understanding of mRNA metabolism and outline how continuous development of new approaches might help in finding answers to outstanding questions or help to look at old dogmas through a new lens.

Live-Cell Imaging of mRNP-NPC Interactions in Budding Yeast.

Single-molecule resolution imaging has become an important tool in the study of cell biology. Aptamer-based approaches (e.g., MS2 and PP7) allow for detection of single RNA molecules in living cells and have been used to study various aspects of mRNA metabolism, including mRNP nuclear export. Here we outline an imaging protocol for the study of interactions between mRNPs and nuclear pore complexes (NPCs) in the yeast S. cerevisiae, including mRNPexport. We describe in detail the steps that allow for high-resolution live-cell mRNP imaging and measurement of mRNP interactions with NPCs using simultaneous two-color imaging. Our protocol discusses yeast strain construction, choice of marker proteins to label the nuclear pore complex, as well as imaging conditions that allow high signal-to-noise data acquisition. Moreover, we describe various aspects of postacquisition image analysis for single molecule tracking and image registration allowing for the characterization of mRNP-NPC interactions.

Visualization of Single mRNAs in Live Neurons.

Transcription and post-transcriptional regulations are critical in gene expression. To study the spatiotemporal regulation of RNA inside a cell, techniques for high-resolution imaging of RNA have been developed. In this chapter, we describe RNA fluorescent labeling methods using MS2 and PP7 systems to detect single RNA molecules in live neurons. We use hippocampal neurons cultured from knock-in mouse models in which β-actin or Arc mRNAs are tagged with MS2 or PP7 stem-loops. Adeno-associated virus (AAV) or lentiviral vectors are used to express MS2 or PP7 capsid proteins fused with GFP in those neurons. Then, GFP-labeled RNAs in live neurons can be detected by epifluorescence microscopy, and their moving pathways can be analyzed using single-particle tracking software. For these processes, we introduce protocols for neuron culture, transfection, imaging, and particle tracking methods.

Modular Architecture of Bacterial RNase P Ribozymes as a Structural Platform for RNA Nanostructure Design.

Ribonuclease P (RNase P) is a class of enzymes involved in the processing of precursor tRNAs to remove their 5'-leader sequences. Ribonuclease P enzymes are classified into two completely distinct classes, i.e. an RNA-based enzyme and a protein-only enzyme. The RNA-based enzyme functions as a ribozyme in which the catalytic machinery is supported by its RNA component consisting of a single RNA molecule. Bacterial RNase P RNAs are a classical class of ribozymes and their structures and catalytic mechanisms have been studied extensively. The bacterial RNase P ribozyme has a modular tertiary structure consisting of two large domains, each of which can self-fold without the partner domain. Such modular architecture, identification of which provided important insight into the function of this ribozyme, is attractive as a structural platform to design functional RNA nanostructures. The first section of this article briefly summarizes the diversity of RNase P mainly focusing on RNA-based enzymes. The second section describes the structures of bacterial RNase P ribozymes from the viewpoint of their application as modular tools in RNA nanostructure design. The last section summarizes the current state and next steps in modular engineering of RNase P RNAs, including possible design of RNase P ribozyme-based nanostructures.

Imaging the Life and Death of mRNAs in Single Cells.

RNA plays a central role in gene expression from its transcription in the nucleus through translation and degradation in the cytoplasm. Technological advances in fluorescent microscopy and labeling methodologies have made it possible to detect single molecules of RNA in both fixed and living cells. Here, we focus on the recent developments in RNA imaging that have allowed quantitatively measuring the lives of individual transcripts from birth to death and all the events in between in single cells and tissues. Direct observation of RNAs within their native cellular environment has revealed a complex layer of spatial and temporal regulation that has profoundly impacted our understanding of RNA biology.

Possible technical elements for the ultimate nucleotide sequencers

The DNA sequencing technology invented in 1970's has been a leading tool for genomics and its sequel ─ precision medicine. The needs for advancement of the DNA sequencing technology comes from increasing resolutions in cellular and molecular studies, not only DNA or cDNA sequences but also their covalent modifications, such as various kinds of DNA or RNA methylations. Sequencing single RNA molecules directly without conversion into cDNAs appears to be the last generation of nucleotide sequencers. Some cutting-edge technological elements are clearly expected for the ultimate design of nucleotide sequencers, which must include, but not limited to, nanopores, nanofabrication, surface- or tip-enhanced Raman spectrometry, single-molecule photonics and plasmonics, artificial-intelligence based data analysis tools. The complexity of biological systems is also calling for new syntheses, frameworks, paradigms and more concerted efforts and projects internationally. Here we discuss possible roles and capacities of such technical elements for the fourth-generation nucleotide sequencers in details.

Recognition Tunneling of Canonical and Modified RNA Nucleotides for Their Identification with the Aid of Machine Learning.

In the present study, we demonstrate a tunneling nanogap technique to identify individual RNA nucleotides, which can be used as a mechanism to read the nucleobases for direct sequencing of RNA in a solid-state nanopore. The tunneling nanogap is composed of two electrodes separated by a distance of <3 nm and functionalized with a recognition molecule. When a chemical entity is captured in the gap, it generates electron tunneling currents, a process we call recognition tunneling (RT). Using RT nanogaps created in a scanning tunneling microscope (STM), we acquired the electron tunneling signals for the canonical and two modified RNA nucleotides. To call the individual RNA nucleotides from the RT data, we adopted a machine learning algorithm, support vector machine (SVM), for the data analysis. Through the SVM, we were able to identify the individual RNA nucleotides and distinguish them from their DNA counterparts with reasonably high accuracy. Since each RNA nucleoside contains a hydroxyl group at the 2'-position of its sugar ring in an RNA strand, it allows for the formation of a tunneling junction at a larger nanogap compared to the DNA nucleoside in a DNA strand, which lacks the 2' hydroxyl group. It also proves advantageous for the manufacture of RT devices. This study is a proof-of-principle demonstration for the development of an RT nanopore device for directly sequencing single RNA molecules, including those bearing modifications.