Single-cell nascent RNA sequencing unveils coordinated global transcription.

Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers1,2. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations3. However, fundamental questions about the temporal regulation of transcription and enhancer-gene coordination remain unanswered, primarily because of the absence of a single-cell perspective on active transcription. In this study, we present scGRO-seq-a new single-cell nascent RNA sequencing assay that uses click chemistry-and unveil coordinated transcription throughout the genome. We demonstrate the episodic nature of transcription and the co-transcription of functionally related genes. scGRO-seq can estimate burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells and can leverage replication-dependent non-polyadenylated histone gene transcription to elucidate cell cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO-seq enables the identification of networks of enhancers and genes. Our results suggest that the bursting of transcription at super-enhancers precedes bursting from associated genes. By imparting insights into the dynamic nature of global transcription and the origin and propagation of transcription signals, we demonstrate the ability of scGRO-seq to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression.

fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer's disease using an automated quantitative analysis workflow.

In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue.

Expression and Clinical Significance of POLR1D in Colorectal Cancer.

PURPOSE: RNA polymerase I subunit D (POLR1D) is involved in the synthesis of ribosomal RNA precursors and small RNAs, but its mechanism in the development and progression of colorectal cancer (CRC) remains ambiguous. Thus, this research aimed to investigate POLR1D's expression and significance in human CRC patients and evaluate its association with clinicopathological characteristics. METHODS: Matched fresh-frozen cancerous and non-cancerous tissues were collected from 100 patients diagnosed with CRC. Immunohistochemical, Western blot, and quantitative real-time polymerase chain reaction analyses were adopted to validate the correlation between POLR1D expression and clinicopathological parameters in CRC tissues and adjacent normal tissues (ANTs). RESULTS: POLR1D expression in CRC tissues was significantly higher than in the ANTs. χ2 test and Spearman's correlative analysis showed that a high POLR1D expression is significantly associated with clinical stage, Dukes stage, tumor differentiation, depth of invasion, and metastasis (p < 0.05). It is not correlated with gender, age, and tumor location and size (p > 0.05). Kaplan-Meier survival curves show that the overall survival (OS) time for the low expression group is remarkably longer than for the high expression group (p < 0.0015). Univariate and multivariate analyses indicate that a high POLR1D expression is an independent prognostic factor for poor OS (p = 0.000). CONCLUSION: The findings of this study strongly indicate that POLR1D plays a pivotal role in the occurrence and progression of CRC. It might be an independent adverse prognostic factor for CRC patients and could serve as a potential therapeutic target for clinical diagnosis in CRC and anticancer drug development.

Decoding the grammar of transcriptional regulation from RNA polymerase measurements: models and their applications.

The genomic revolution has indubitably brought about a paradigm shift in the field of molecular biology, wherein we can sequence, write and re-write genomes. In spite of achieving such feats, we still lack a quantitative understanding of how cells integrate environmental and intra-cellular signals at the promoter and accordingly regulate the production of messenger RNAs. This current state of affairs is being redressed by recent experimental breakthroughs which enable the counting of RNA polymerase molecules (or the corresponding nascent RNAs) engaged in the process of transcribing a gene at the single-cell level. Theorists, in conjunction, have sought to unravel the grammar of transcriptional regulation by harnessing the various statistical properties of these measurements. In this review, we focus on the recent progress in developing falsifiable models of transcription that aim to connect the molecular mechanisms of transcription to single-cell polymerase measurements. We discuss studies where the application of such models to the experimental data have led to novel mechanistic insights into the process of transcriptional regulation. Such interplay between theory and experiments will likely contribute towards the exciting journey of unfurling the governing principles of transcriptional regulation ranging from bacteria to higher organisms.

Influenza virus RNA polymerase may be activated inside the virion.

Influenza A and B virions are packaged with their polymerases to catalyse RNA-dependent RNA polymerase activity. Since there is no evidence to rule in or out the permissiveness of influenza virions to triphosphate ribonucleotides, we functionally evaluated this. We found the means to stimulate influenza A and B RNA polymerase activity inside the virion, called natural endogenous RNA polymerase (NERP) activity. Stimulation of NERP activity increased up to 3 log10 viral RNA content, allowing the detection of influenza virus in otherwise undetectable clinical samples. NERP activation also improved our capacity to sequence misidentified regions of the influenza genome from clinical samples. By treating the samples with the ribavirin triphosphate we inhibited NERP activity, which confirms our hypothesis and highlights that this assay could be used to screen antiviral drugs. Altogether, our data show that NERP activity could be explored to increase molecular diagnostic sensitivity and/or to develop antiviral screening assays.

Rapid Detection of Avian Influenza A Virus (H7N9) by Lateral Flow Dipstick Recombinase Polymerase Amplification.

Avian influenza A (H7N9) virus has caused several epidemics and infection in both human and poultry. With mutation, the H7N9 virus gained its fifth endemic in China. Early diagnosis is crucial for the control of viral spread in poultry and prognosis of infected patients. In this study, we developed and evaluated a lateral flow dipstick recombinase polymerase amplification (LFD-RPA) assay for rapid detection of both hemagglutinin and neuraminidase gene of H7N9. Our H7-LFD-RPA and N9-LFD-RPA assay were able to detect 32 fg H7N9 nucleic acid which is more convenient and rapid than previous methods. Through detecting 50 influenza positive samples, cross-reaction was not found with other subtypes of influenza virus. The 100% analytical specificity and sufficient analytical sensitivity results agreed the real time RT-PCR assay. The results data demonstrated that our method performed well and could be applied to the detection of H7N9 virus. This LFD-RPA assay provides a candidate method for rapid point-of-care diagnosis of H7N9.

In Vitro Analysis of DNA-Protein Interactions in Gene Transcription Using DNAzyme-Based Electrochemical Assay.

The interaction between protein and DNA elements controls a variety of functions of genomes. The development of a convenient and cost-effective method for investigating the sequence specificity of DNA-binding proteins represents an important challenge. In response, we have introduced an electrochemical assay in this work for specific and sensitive analysis of interaction between protein and nucleic acid in nucleic extracts, based on the protein-induced distinctive motion behavior of DNA deoxyribozyme (DNAzyme) on an electrode surface. As a proof of principle, we have also presented assays for the rapid, sensitive, and selective detection of three transcription factors (NF-κB, SP6 RNA polymerase, and HNF-4α), as well as the analysis of binding affinity of the mutated protein-binding sequence, and even screening of the binding sequence of HNF-4α protein in vitro. This work may open new opportunity for in-depth profiling of the sequence specificity of DNA-binding proteins and study of nucleotide polymorphisms in known protein-binding sites.

Single Molecule Localization and Discrimination of DNA-Protein Complexes by Controlled Translocation Through Nanocapillaries.

Through the use of optical tweezers we performed controlled translocations of DNA-protein complexes through nanocapillaries. We used RNA polymerase (RNAP) with two binding sites on a 7.2 kbp DNA fragment and a dCas9 protein tailored to have five binding sites on λ-DNA (48.5 kbp). Measured localization of binding sites showed a shift from the expected positions on the DNA that we explained using both analytical fitting and a stochastic model. From the measured force versus stage curves we extracted the nonequilibrium work done during the translocation of a DNA-protein complex and used it to obtain an estimate of the effective charge of the complex. In combination with conductivity measurements, we provided a proof of concept for discrimination between different DNA-protein complexes simultaneous to the localization of their binding sites.

The PRP6-like splicing factor STA1 is involved in RNA-directed DNA methylation by facilitating the production of Pol V-dependent scaffold RNAs.

DNA methylation is a conserved epigenetic marker in plants and animals. In Arabidopsis, DNA methylation can be established through an RNA-directed DNA methylation (RdDM) pathway. By screening for suppressors of ros1, we identified STA1, a PRP6-like splicing factor, as a new RdDM regulator. Whole-genome bisulfite sequencing suggested that STA1 and the RdDM pathway share a large number of common targets in the Arabidopsis genome. Small RNA deep sequencing demonstrated that STA1 is predominantly involved in the accumulation of the siRNAs that depend on both Pol IV and Pol V. Moreover, the sta1 mutation partially reduces the levels of Pol V-dependent RNA transcripts. Immunolocalization assay indicated that STA1 signals are exclusively present in the Cajal body and overlap with AGO4 in most nuclei. STA1 signals are also partially overlap with NRPE1. Localization of STA1 to AGO4 and NRPE1 signals is probably related to the function of STA1 in the RdDM pathway. Based on these results, we propose that STA1 acts downstream of siRNA biogenesis and facilitates the production of Pol V-dependent RNA transcripts in the RdDM pathway.

Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation.

Studying the structure and dynamics of proteins in live cells is essential to understanding their physiological activities and mechanisms, and to validating in vitro characterization. Improvements in labeling and imaging technologies are starting to allow such in vivo studies; however, a number of technical challenges remain. Recently, we developed an electroporation-based protocol for internalization, which allows biomolecules labeled with organic fluorophores to be introduced at high efficiency into live E. coli (Crawford et al. in Biophys J 105 (11):2439-2450, 2013). Here, we address important challenges related to internalization of proteins, and optimize our method in terms of (1) electroporation buffer conditions; (2) removal of dye contaminants from stock protein samples; and (3) removal of non-internalized molecules from cell suspension after electroporation. We illustrate the usability of the optimized protocol by demonstrating high-efficiency internalization of a 10-kDa protein, the ω subunit of RNA polymerase. Provided that suggested control experiments are carried out, any fluorescently labeled protein of up to 60 kDa could be internalized using our method. Further, we probe the effect of electroporation voltage on internalization efficiency and cell viability and demonstrate that, whilst internalization increases with increased voltage, cell viability is compromised. However, due to the low number of damaged cells in our samples, the major fraction of loaded cells always corresponds to non-damaged cells. By taking care to include only viable cells into analysis, our method allows physiologically relevant studies to be performed, including in vivo measurements of protein diffusion, localization and intramolecular dynamics via single-molecule Förster resonance energy transfer.

Single-stranded loops as end-label polarity markers for double-stranded linear DNA templates in atomic force microscopy.

Visualization of DNA-protein interactions by atomic force microscopy (AFM) has deepened our understanding of molecular processes such as DNA transcription. Interpretation of systems where more than one protein acts on a single template, however, is complicated by protein molecules migrating along the DNA. Single-molecule AFM imaging experiments can reveal more information if the polarity of the template can be determined. A nucleic acid-based approach to end-labelling is desirable because it does not compromise the sample preparation procedures for biomolecular AFM. Here, we report a method involving oligonucleotide loop-primed synthesis for the end labelling of double-stranded DNA to discriminate the polarity of linear templates at the single-molecule level. Single-stranded oligonucleotide primers were designed to allow loop formation while retaining 3'-single-strand extensions to facilitate primer annealing to the template. Following a DNA polymerase extension, the labelled templates were shown to have the ability to form open promoter complexes on a model nested gene template using two Escherichia coli RNA polymerases in a convergent transcription arrangement. Analysis of the AFM images indicates that the added loops have no effect on the ability of the promoters to recruit RNA polymerase. This labelling strategy is proposed as a generic methodology for end-labelling linear DNA for studying DNA-protein interactions by AFM.

Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells.

Quantitative spatial distributions of ribosomes (S2-YFP) and RNA polymerase (RNAP; ß'-yGFP) in live Escherichia coli are measured by superresolution fluorescence microscopy. In moderate growth conditions, nucleoid-ribosome segregation is strong, and RNAP localizes to the nucleoid lobes. The mean copy numbers per cell are 4600 RNAPs and 55,000 ribosomes. Only 10-15% of the ribosomes lie within the densest part of the nucleoid lobes, and at most 4% of the RNAPs lie in the two ribosome-rich endcaps. The predominant observed diffusion coefficient of ribosomes is D(ribo) = 0.04 µm(2) s(-1), attributed to free mRNA being translated by one or more 70S ribosomes. We find no clear evidence of subdiffusion, as would arise from tethering of ribosomes to the DNA. The degree of DNA-ribosome segregation strongly suggests that in E. coli most translation occurs on free mRNA transcripts that have diffused into the ribosome-rich regions. Both RNAP and ribosome radial distributions extend to the cytoplasmic membrane, consistent with the transertion hypothesis. However, few if any RNAP copies lie near the membrane of the endcaps. This suggests that if transertion occurs, it exerts a direct radially expanding force on the nucleoid, but not a direct axially expanding force.

Extending the tools of single-molecule fluorescence imaging to problems in microbiology.

Single-molecule fluorescence microscopy enables non-invasive, high-sensitivity, high-resolution imaging, and this direct, quantitative method has recently been extended to understanding organization, dynamics and cooperativity of macromolecules in prokaryotes. In this issue of Molecular Microbiology, Bakshi et al. (2012) examine fluorescently labelled ribosomes and RNA polymerase (RNAP) in live Escherichia coli cells. By localizing individual molecules with 30 nm scale accuracy, they resolve the spatial distribution of RNAP (and thus of the E. coli nucleoid) and of the ribosomes, measure diffusion rates, and sensitively count protein copy numbers. This work represents an exciting achievement in terms of applying biophysical methods to live cells and quantitatively answering important questions in physiologically relevant conditions. In particular, the authors directly relate the positions, dynamics, and numbers of ribosomes and RNAP to transcription and translation in E. coli. The results indicate that, since the ribosomes and the nucleoid are well segregated, translation and transcription must be predominantly uncoupled. As well, the radial extension of ribosomes and RNAP to the cytoplasmic membrane is consistent with the hypothesis of transertion (simultaneous insertion of membrane proteins upon translation).

Optimized design and synthesis of a cell-permeable biarsenical cyanine probe for imaging tagged cytosolic bacterial proteins.

To optimize cellular delivery and specific labeling of tagged cytosolic proteins by biarsenical fluorescent probes built around a cyanine dye (Cy3) scaffold, we have systematically varied the polarity of the N-alkyl chain (i.e., 4-5 methylene groups appended by a sulfonate or methoxy ester moiety) and arsenic capping reagent (ethanedithiol versus benzenedithiol). Optimal live-cell labeling and visualization of tagged cytosolic proteins is reported using an ethanedithiol capping reagent with the uncharged methoxy ester functionalized N-alkyl chains. These measurements demonstrate the general utility of this new class of photostable and highly fluorescent biarsenical probes based on the cyanine dye scaffold for in vivo labeling of tagged cellular proteins for live cell imaging measurements of protein dynamics.

The dynamic protein partnership of RNA polymerase in Bacillus subtilis.

In prokaryotes, transcription results from the activity of a 400 kDa RNA polymerase (RNAP) protein complex composed of at least five subunits (2α, ß, ß', ω). To ensure adequate responses to changing environmental cues, RNAP activity is tightly controlled by means of interacting regulatory proteins. Here, we report the affinity-purification of the Bacillus subtilis RNAP complexes from cells in different growth states and stress conditions, and the quantitative assessment by mass spectrometry of the dynamic changes in the composition of the RNAP complex. The stoichiometry of RNA polymerase was determined by a comparison of two mass spectrometry-based quantification methods: a label-based and a label-free method. The validated label-free method was then used to quantify the proteins associated with RNAP. The levels of sigma factors bound to RNAP varied during growth and exposure to stress. Elongation factors, helicases such as HelD and PcrA, and novel unknown proteins were also associated with RNAP complexes. The content in 6S RNAs of purified RNAP complexes increased at the onset of the stationary phase. These quantitative variations in the protein and RNA composition of the RNAP complexes well correlate with the known physiology of B. subtilis cells under different conditions.

FRET (fluorescence resonance energy transfer) sheds light on transcription.

The complex organization of the transcription machinery has been revealed mainly by biochemical and crystallographic studies. X-ray structures describe RNA polymerases and transcription complexes on an atomic level, but fail to portray their dynamic nature. The use of fluorescence techniques has made it possible to add a new layer of information to our understanding of transcription by providing details about the structural rearrangement of mobile elements and the network of interactions within transcription complexes in solution and in real-time.

Hands on Native Mass Spectrometry Analysis of Multi-protein Complexes.

By maintaining intact multi-protein complexes in the gas-phase, native mass spectrometry provides their molecular weight with very good accuracy compared to other methods (typically native PAGE or SEC-MALS) (Marcoux and Robinson, Structure 21:1541-1550, 2013). Besides, heterogeneous samples, in terms of both oligomeric states and ligand-bound species can be fully characterized. Here we thoroughly describe the analysis of several oligomeric protein complexes ranging from a 16 = kDa dimer to a 801-kDa tetradecameric complex on different instrumental setups.

The Emerging Challenge of Diagnosing Drug-resistant Tubercular Uveitis: Experience of 110 Eyes from North India.

Background: Rapid and timely diagnosis of tubercular uveitis (TBU) is of paramount importance to save these eyes from blindness. The present study was, therefore, undertaken to carry out a comparative evaluation of Gene Xpert MTB/RIF (Xpert), MTBDRplus and Multiplex PCR (MPCR) for the diagnosis of TBU. These tests were performed on vitreous fluid of 110 patients with presumed TBU and 90 controls. rpoB gene sequencing confirmed Rifampicin resistance.Results: Xpert, MTBDRplus and MPCR were positive in 19(17.2%),38 (34.5%) and 79 (71.8 %) patients, respectively. All tests were negative in all controls. Rif resistance was detected in 3 by Xpert and 7 by MTBDRplus. MPCR followed by rpoB gene sequencing detected Rif resistance in 6 cases. One case of false Rif resistance was reported each by MTBDRplus and Xpert.Conclusion: MPCR followed by rpoB sequencing is a robust technique for the diagnosis of paucibacilliary condition like TBU and reliable detection of drug resistance.

Single-molecule analysis of 1D diffusion and transcription elongation of T7 RNA polymerase along individual stretched DNA molecules.

Using total internal reflection fluorescence microscopy, we directly visualize in real-time, the 1D Brownian motion and transcription elongation of T7 RNA polymerase along aligned DNA molecules bound to substrates by molecular combing. We fluorescently label T7 RNA polymerase with antibodies and use flow to convect them orthogonally to the DNA alignment direction, permitting observation and estimation of the protein diffusivity along the DNA at the single-molecule level. Our observations suggest that the 1D diffusion coefficient varies from molecule to molecule over the range 6.1 x 10(-11) cm2/s to 4.3 x 10(-9) cm2/s. We also observe binding and transcription by T7 RNA polymerases on single combed T7 DNA molecules with an apparent association rate of 1.6 microM(-1)s(-1). From the measured dependence of the rate of transcription on concentration of nucleotide triphosphate, we infer that the combed DNA molecules capable of interacting with proteins are under an average tension of 25 pN.

Triad pattern algorithm for predicting strong promoter candidates in bacterial genomes.

BACKGROUND: Bacterial promoters, which increase the efficiency of gene expression, differ from other promoters by several characteristics. This difference, not yet widely exploited in bioinformatics, looks promising for the development of relevant computational tools to search for strong promoters in bacterial genomes. RESULTS: We describe a new triad pattern algorithm that predicts strong promoter candidates in annotated bacterial genomes by matching specific patterns for the group I sigma70 factors of Escherichia coli RNA polymerase. It detects promoter-specific motifs by consecutively matching three patterns, consisting of an UP-element, required for interaction with the alpha subunit, and then optimally-separated patterns of -35 and -10 boxes, required for interaction with the sigma70 subunit of RNA polymerase. Analysis of 43 bacterial genomes revealed that the frequency of candidate sequences depends on the A+T content of the DNA under examination. The accuracy of in silico prediction was experimentally validated for the genome of a hyperthermophilic bacterium, Thermotoga maritima, by applying a cell-free expression assay using the predicted strong promoters. In this organism, the strong promoters govern genes for translation, energy metabolism, transport, cell movement, and other as-yet unidentified functions. CONCLUSION: The triad pattern algorithm developed for predicting strong bacterial promoters is well suited for analyzing bacterial genomes with an A+T content of less than 62%. This computational tool opens new prospects for investigating global gene expression, and individual strong promoters in bacteria of medical and/or economic significance.