Nascent ribosomal RNA act as surfactant that suppresses growth of fibrillar centers in nucleolus.

Liquid-liquid phase separation (LLPS) has been thought to be the biophysical principle governing the assembly of the multiphase structures of nucleoli, the site of ribosomal biogenesis. Condensates assembled through LLPS increase their sizes to minimize the surface energy as far as their components are available. However, multiple microphases, fibrillar centers (FCs), dispersed in a nucleolus are stable and their sizes do not grow unless the transcription of pre-ribosomal RNA (pre-rRNA) is inhibited. To understand the mechanism of the suppression of the FC growth, we here construct a minimal theoretical model by taking into account nascent pre-rRNAs tethered to FC surfaces by RNA polymerase I. The prediction of this theory was supported by our experiments that quantitatively measure the dependence of the size of FCs on the transcription level. This work sheds light on the role of nascent RNAs in controlling the size of nuclear bodies.

Molecular Dynamics Simulations Elucidate the Molecular Basis of Pre-mRNA Translocation by the Prp2 Spliceosomal Helicase.

The spliceosome machinery catalyzes precursor-messenger RNA (pre-mRNA) splicing by undergoing at each splicing cycle assembly, activation, catalysis, and disassembly processes, thanks to the concerted action of specific RNA-dependent ATPases/helicases. Prp2, a member of the DExH-box ATPase/helicase family, harnesses the energy of ATP hydrolysis to translocate a single pre-mRNA strand in the 5' to 3' direction, thus promoting spliceosome remodeling to its catalytic-competent state. Here, we established the functional coupling between ATPase and helicase activities of Prp2. Namely, extensive multi-µs molecular dynamics simulations allowed us to unlock how, after pre-mRNA selection, ATP binding, hydrolysis, and dissociation induce a functional typewriter-like rotation of the Prp2 C-terminal domain. This movement, endorsed by an iterative swing of interactions established between specific Prp2 residues with the nucleobases at 5'- and 3'-ends of pre-mRNA, promotes pre-mRNA translocation. Notably, some of these Prp2 residues are conserved in the DExH-box family, suggesting that the translocation mechanism elucidated here may be applicable to all DExH-box helicases.

Revealing nascent RNA processing dynamics with nano-COP.

During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3'-end cleavage, and polyadenylation. Here we describe nanopore analysis of co-transcriptional processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing, which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3' ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read-based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 d.

miRWoods: Enhanced precursor detection and stacked random forests for the sensitive detection of microRNAs.

MicroRNAs are conserved, endogenous small RNAs with critical post-transcriptional regulatory functions throughout eukaryota, including prominent roles in development and disease. Despite much effort, microRNA annotations still contain errors and are incomplete due especially to challenges related to identifying valid miRs that have small numbers of reads, to properly locating hairpin precursors and to balancing precision and recall. Here, we present miRWoods, which solves these challenges using a duplex-focused precursor detection method and stacked random forests with specialized layers to detect mature and precursor microRNAs, and has been tuned to optimize the harmonic mean of precision and recall. We trained and tuned our discovery pipeline on data sets from the well-annotated human genome, and evaluated its performance on data from mouse. Compared to existing approaches, miRWoods better identifies precursor spans, and can balance sensitivity and specificity for an overall greater prediction accuracy, recalling an average of 10% more annotated microRNAs, and correctly predicts substantially more microRNAs with only one read. We apply this method to the under-annotated genomes of Felis catus (domestic cat) and Bos taurus (cow). We identified hundreds of novel microRNAs in small RNA sequencing data sets from muscle and skin from cat, from 10 tissues from cow and also from human and mouse cells. Our novel predictions include a microRNA in an intron of tyrosine kinase 2 (TYK2) that is present in both cat and cow, as well as a family of mirtrons with two instances in the human genome. Our predictions support a more expanded miR-2284 family in the bovine genome, a larger mir-548 family in the human genome, and a larger let-7 family in the feline genome.

Accurate detection of intracellular microRNAs using functional Mo2C quantum dots nanoprobe.

A programmable molecular beacon (MB) with a good discrimination capability for mature and precursor microRNAs was loaded onto the surface of Mo2C quantum dots (QDs) for accurate detection of intracellular mature microRNAs.

An estimate of the total number of true human miRNAs.

While the number of human miRNA candidates continuously increases, only a few of them are completely characterized and experimentally validated. Toward determining the total number of true miRNAs, we employed a combined in silico high- and experimental low-throughput validation strategy. We collected 28 866 human small RNA sequencing data sets containing 363.7 billion sequencing reads and excluded falsely annotated and low quality data. Our high-throughput analysis identified 65% of 24 127 mature miRNA candidates as likely false-positives. Using northern blotting, we experimentally validated miRBase entries and novel miRNA candidates. By exogenous overexpression of 108 precursors that encode 205 mature miRNAs, we confirmed 68.5% of the miRBase entries with the confirmation rate going up to 94.4% for the high-confidence entries and 18.3% of the novel miRNA candidates. Analyzing endogenous miRNAs, we verified the expression of 8 miRNAs in 12 different human cell lines. In total, we extrapolated 2300 true human mature miRNAs, 1115 of which are currently annotated in miRBase V22. The experimentally validated miRNAs will contribute to revising targetomes hypothesized by utilizing falsely annotated miRNAs.

Adaboost-SVM-based probability algorithm for the prediction of all mature miRNA sites based on structured-sequence features.

The significant role of microRNAs (miRNAs) in various biological processes and diseases has been widely studied and reported in recent years. Several computational methods associated with mature miRNA identification suffer various limitations involving canonical biological features extraction, class imbalance, and classifier performance. The proposed classifier, miRFinder, is an accurate alternative for the identification of mature miRNAs. The structured-sequence features were proposed to precisely extract miRNA biological features, and three algorithms were selected to obtain the canonical features based on the classifier performance. Moreover, the center of mass near distance training based on K-means was provided to improve the class imbalance problem. In particular, the AdaBoost-SVM algorithm was used to construct the classifier. The classifier training process focuses on incorrectly classified samples, and the integrated results use the common decision strategies of the weak classifier with different weights. In addition, the all mature miRNA sites were predicted by different classifiers based on the features of different sites. Compared with other methods, the performance of the classifiers has a high degree of efficacy for the identification of mature miRNAs. MiRFinder is freely available at https://github.com/wangying0128/miRFinder .

The hierarchical structure of the perichromosomal layer comprises Ki67, ribosomal RNAs, and nucleolar proteins.

The perichromosomal layer (PCL) is a structure that surrounds mitotic chromosomes, found in both animal and plant cells. It comprises various proteins and RNAs, mainly derived from the nucleolus. Several functions for the PCL have been suggested; however, the mechanism of PCL organization during mitosis remains unclear. The localization of several nucleolar proteins to the PCL is reportedly dependent on pre-ribosomal RNAs and the marker of proliferation, Ki67, which is a major PCL-localized protein. Here we demonstrate that, although the removal of pre-ribosomal RNAs from the PCL causes PCL delocalization of several nucleolar proteins, it does not affect the localization of Ki67. Conversely, Ki67 depletion results in the dissociation of both pre-ribosomal RNAs and nucleolar proteins from the PCL, which indicates that Ki67 is required for the PCL accumulation of pre-ribosomal RNAs, to which several nucleolar proteins are associated. Given these findings, we propose a model for PCL organization that comprises three essential layers: the scaffolding protein Ki67, pre-ribosomal RNAs for linkage, and outer nucleolar proteins.

Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification.

MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. They are stable in body fluids and pose great potential to serve as biomarkers. Here, we present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on two-step RT-qPCR with SYBR-green detection chemistry called Two-tailed RT-qPCR. It takes advantage of novel, target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespectively of the position of the mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of seven logs and a sensitivity sufficient to detect down to ten target miRNA molecules. It is capable to capture the full isomiR repertoire, leading to accurate representation of the complete miRNA content in a sample. The reverse transcription step can be multiplexed and the miRNA profiles measured with Two-tailed RT-qPCR show excellent correlation with the industry standard TaqMan miRNA assays (r2 = 0.985). Moreover, Two-tailed RT-qPCR allows for rapid testing with a total analysis time of less than 2.5 hours.

Improved RT-PCR Assay to Quantitate the Pri-, Pre-, and Mature microRNAs with Higher Efficiency and Accuracy.

Understanding of the functional significance of microRNAs (miRNAs) requires efficient and accurate detection method. In this study, we developed an improved miRNAs quantification system based on quantitative real-time polymerase chain reaction (qRT-PCR). This method showed higher efficiency and accuracy to survey the expression of primary miRNAs (pri-miRNAs), precursor miRNAs (pre-miRNAs), and mature miRNAs. Instead of relative quantification method, we quantified the pri-miRNAs and pre-miRNAs with absolute qRT-PCR based on SYBR Green I fluorescence. This improvement corrected for the inaccuracy caused by the differences in amplicon length and PCR efficiency. We also used SYBR Green method to quantify mature miRNAs based on the stem-loop qRT-PCR method. We extended the pairing part of the stem-loop reverse transcript (RT) primer from 6 to 11 bp, which greatly increased the efficiency of reverse transcription PCR (RT-PCR). The performance of the improved RT primer was tested using synthetic mature miRNAs and tissue RNA samples. Results showed that the improved RT primer demonstrated dynamic range of seven orders of magnitude and sensitivity of detection of hundreds of copies of miRNA molecules.

Dead or alive: molecular assessment of microbial viability.

Nucleic acid-based analytical methods, ranging from species-targeted PCRs to metagenomics, have greatly expanded our understanding of microbiological diversity in natural samples. However, these methods provide only limited information on the activities and physiological states of microorganisms in samples. Even the most fundamental physiological state, viability, cannot be assessed cross-sectionally by standard DNA-targeted methods such as PCR. New PCR-based strategies, collectively called molecular viability analyses, have been developed that differentiate nucleic acids associated with viable cells from those associated with inactivated cells. In order to maximize the utility of these methods and to correctly interpret results, it is necessary to consider the physiological diversity of life and death in the microbial world. This article reviews molecular viability analysis in that context and discusses future opportunities for these strategies in genetic, metagenomic, and single-cell microbiology.

A view of pre-mRNA splicing from RNase R resistant RNAs.

During pre-mRNA splicing, exons in the primary transcript are precisely connected to generate an mRNA. Intron lariat RNAs are formed as by-products of this process. In addition, some exonic circular RNAs (circRNAs) may also result from exon skipping as by-products. Lariat RNAs and circRNAs are both RNase R resistant RNAs. RNase R is a strong 3' to 5' exoribonuclease, which efficiently degrades linear RNAs, such as mRNAs and rRNAs; therefore, the circular parts of lariat RNAs and the circRNAs can be segregated from eukaryotic total RNAs by their RNase R resistance. Thus, RNase R resistant RNAs could provide unexplored splicing information not available from mRNAs. Analyses of these RNAs identified repeating splicing phenomena, such as re-splicing of mature mRNAs and nested splicing. Moreover, circRNA might function as microRNA sponges. There is an enormous variety of endogenous circRNAs, which are generally synthesized in cells and tissues.

Quantification of two viral transcripts by real time PCR to investigate human herpesvirus type 6 active infection.

BACKGROUND: Human herpesvirus 6 (HHV-6) causes exanthema subitum and is associated with symptomatic reactivations in immunocompromised patients, particularly after hematopoietic stem cell transplantation. The detection of viral mRNA can help to make the difference between latent, chromosomally integrated and true replicating virus. It can also be a useful tool to investigate viral multiplication in different cell types. OBJECTIVES: To develop molecular tools for the detection and quantification HHV-6 transcripts that can be used in a clinical setting. STUDY-DESIGN: Two one-step reverse-transcriptase quantitative real-time PCR (RT-qPCR) were developed for the quantification of the immediate early U90 and the late U100 mRNAs. Viral mRNA loads were compared to viral DNA loads during infection in vitro and in blood samples collected from stem cell transplanted patients. RESULTS: Analytical performances of the two quantitative real-time PCR were good. In vitro, kinetics of both transcripts was well correlated with DNA levels. Sixty blood samples from patients with active HHV-6 infection were analyzed. Overall agreement of qualitative results for HHV-6 DNA, U90 RNA and U100 RNA was good. HHV-6 DNA loads were significantly higher than mRNA loads. In clinical samples, the amounts of U100 and U90 mRNAs were low and their detection was mainly associated to viral DNA loads upper than 1000 copies/ml of blood. CONCLUSION: The new assays are sensitive and reliable methods for the monitoring of viral transcription in vitro and in vivo. As their detection is associated to high DNA loads in vivo, they can be helpful tools for the diagnosis of active infection.

Fluorescent indicator displacement assay of ligands targeting 10 microRNA precursors.

Fluorescent indicator displacement (FID) assay is a rapid and convenient assay for identifying new ligands that bind to the target molecules. In our previous studies, we have shown that a series of 2,7-diaminoalkoxy xanthone and thioxanthone derivatives can be used as fluorescent indicators for detecting the interaction between RNA and a ligand. The xanthone and thioxanthone fluorochromes showed efficient fluorescence quenching upon binding to target RNA. Subsequent displacement of the bound-fluorochrome with a ligand that binds more strongly to the target RNA led to the recovery of the fluorescence by releasing the fluorochrome from RNA. Here we report a pilot screening of a chemical library that contains 9600 structurally diverse compounds for molecules that bind to a specific miRNA precursor using the FID assay.

Studies on the assembly characteristics of large subunit ribosomal proteins in S. cerevisae.

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.

Computational prediction of the localization of microRNAs within their pre-miRNA.

MicroRNAs (miRNAs) are short RNA species derived from hairpin-forming miRNA precursors (pre-miRNA) and acting as key posttranscriptional regulators. Most computational tools labeled as miRNA predictors are in fact pre-miRNA predictors and provide no information about the putative miRNA location within the pre-miRNA. Sequence and structural features that determine the location of the miRNA, and the extent to which these properties vary from species to species, are poorly understood. We have developed miRdup, a computational predictor for the identification of the most likely miRNA location within a given pre-miRNA or the validation of a candidate miRNA. MiRdup is based on a random forest classifier trained with experimentally validated miRNAs from miRbase, with features that characterize the miRNA-miRNA* duplex. Because we observed that miRNAs have sequence and structural properties that differ between species, mostly in terms of duplex stability, we trained various clade-specific miRdup models and obtained increased accuracy. MiRdup self-trains on the most recent version of miRbase and is easy to use. Combined with existing pre-miRNA predictors, it will be valuable for both de novo mapping of miRNAs and filtering of large sets of candidate miRNAs obtained from transcriptome sequencing projects. MiRdup is open source under the GPLv3 and available at http://www.cs.mcgill.ca/∼blanchem/mirdup/.

[Epstein-Barr virus-associated lymphoproliferations and lymphomas]. / Epstein-Barr-Virus-assoziierte Lymphoproliferationen und Lymphome.

Epstein-Barr virus (EBV) is a lymphotropic herpesvirus infecting > 95 % of the worldwide population. In case of an immunodeficiency of various causes, the virus may lead to the development of a wide spectrum of lymphoproliferations and lymphomas. This encompasses mononucleosis-like lymphoproliferations, hyperplasias of various B-cell subsets as well as aggressive non-Hodgkin lymphomas and classical Hodgkin lymphoma. These lesions occur frequently extranodal and present with a polymorphous histology with angioinvasion and necrosis. Clinical data combined with the immunohistological detection of CD30 expression in the activated infected cells and the demonstration of EBV-encoded proteins and RNA transcripts are helpful for achieving precise classification of these lesions.

Detection of potential TNA and RNA nucleoside precursors in a prebiotic mixture by pure shift diffusion-ordered NMR spectroscopy.

In the context of prebiotic chemistry, one of the characteristics of mixed nitrogenous-oxygenous chemistry is its propensity to give rise to highly complex reaction mixtures. There is therefore an urgent need to develop improved spectroscopic techniques if onerous chromatographic separations are to be avoided. One potential avenue is the combination of pure shift methodology, in which NMR spectra are measured with greatly improved resolution by suppressing multiplet structure, with diffusion-ordered spectroscopy, in which NMR signals from different species are distinguished through their different rates of diffusion. Such a combination has the added advantage of working with intact mixtures, allowing analyses to be carried out without perturbing mixtures in which chemical entities are part of a network of reactions in equilibrium. As part of a systems chemistry approach towards investigating the self-assembly of potentially prebiotic small molecules, we have analysed the complex mixture arising from mixing glycolaldehyde and cyanamide, in a first application of pure shift DOSY NMR to the characterisation of a partially unknown reaction composition. The work presented illustrates the potential of pure shift DOSY to be applied to chemistries that give rise to mixtures of compounds in which the NMR signal resolution is poor. The direct formation of potential RNA and TNA nucleoside precursors, amongst other adducts, was observed. These preliminary observations may have implications for the potentially prebiotic assembly chemistry of pyrimidine threonucleotides, and therefore of TNA, by using recently reported chemistries that yield the activated pyridimidine ribonucleotides.

Rapid antimicrobial susceptibility testing by sensitive detection of precursor rRNA using a novel electrochemical biosensing platform.

Precursor rRNA (pre-rRNA) is an intermediate stage in the formation of mature rRNA and is a useful marker for cellular metabolism and growth rate. We developed an electrochemical sensor assay for Escherichia coli pre-rRNA involving hybridization of capture and detector probes with tail sections that are spliced away during rRNA maturation. A ternary self-assembled monolayer (SAM) prepared on gold electrode surfaces by coassembly of thiolated capture probes with hexanedithiol and posttreatment with 6-mercapto-1-hexanol minimized the background signal and maximized the signal-to-noise ratio. Inclusion of internal calibration controls allowed accurate estimation of the pre-rRNA copy number per cell. As expected, the ratio of pre-rRNA to mature rRNA was low during stationary phase and high during log phase. Pre-rRNA levels were highly dynamic, ranging from 2 copies per cell during stationary phase to ~1,200 copies per cell within 60 min of inoculation into fresh growth medium. Specificity of the assay for pre-rRNA was validated using rifampin and chloramphenicol, which are known inhibitors of pre-rRNA synthesis and processing, respectively. The DNA gyrase inhibitor, ciprofloxacin, was found to act similarly to rifampin; a decline in pre-rRNA was detectable within 15 min in ciprofloxacin-susceptible bacteria. Assays for pre-rRNA provide insight into cellular metabolism and are promising predictors of antibiotic susceptibility.

Detection of pre-mRNA splicing in vitro by an RNA-templated fluorogenic reaction.

RNA splicing is an important target for basic research of disease mechanisms and for drug discovery. Here, we report a new method for analysis of the in vitro RNA splicing process that produces fluorescence using a reduction-triggered fluorescence (RETF) probe. The fluorescence signal is produced only when the two probes bind side-by-side with a specific RNA target. Precursor messenger RNA and mature messenger RNA originating from the chicken δ-crystallin (CDC) gene were successfully discriminated in solution using an RETF probe with the assistance of helper oligonucleotide strands. Also, we successfully applied RETF probes to the detection of emerging mature mRNA in an in vitro splicing process.