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.

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.

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.

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.

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.

Molecular architecture of the human pre-mRNA 3' processing complex.

Pre-mRNA 3' end formation is an essential step in eukaryotic gene expression. Over half of human genes produce alternatively polyadenylated mRNAs, suggesting that regulated polyadenylation is an important mechanism for posttranscriptional gene control. Although a number of mammalian mRNA 3' processing factors have been identified, the full protein composition of the 3' processing machinery has not been determined, and its structure is unknown. Here we report the purification and subsequent proteomic and structural characterization of human mRNA 3' processing complexes. Remarkably, the purified 3' processing complex contains approximately 85 proteins, including known and new core 3' processing factors and over 50 proteins that may mediate crosstalk with other processes. Electron microscopic analyses show that the core 3' processing complex has a distinct "kidney" shape and is approximately 250 A in length. Together, our data has revealed the complexity and molecular architecture of the pre-mRNA 3' processing complex.

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/.

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.

In vitro quantification of specific microRNA using molecular beacons.

MicroRNAs (miRNAs), a class of non-coding RNAs, have become a major focus of molecular biology research because of their diverse genomic origin and ability to regulate an array of cellular processes. Although the biological functions of miRNA are yet to be fully understood, tissue levels of specific miRNAs have been shown to correlate with pathological development of disease. Here, we demonstrate that molecular beacons can readily distinguish mature- and pre-miRNAs, and reliably quantify miRNA expression. We found that molecular beacons with DNA, RNA and combined locked nucleic acid (LNA)-DNA backbones can all detect miRNAs of low (<1 nM) concentrations in vitro, with RNA beacons having the highest detection sensitivity. Furthermore, we found that molecular beacons have the potential to distinguish miRNAs that have slight variations in their nucleotide sequence. These results suggest that the molecular beacon-based approach to assess miRNA expression and distinguish mature and precursor miRNA species is quite robust, and has the promise for assessing miRNA levels in biological samples.

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.

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.

Exon, intron and splice site locations in the spliceosomal B complex.

In recent years, electron microscopy (EM) has allowed the generation of three-dimensional structure maps of several spliceosomal complexes. However, owing to their limited resolution, little is known at present about the location of the pre-mRNA, the spliceosomal small nuclear ribonucleoprotein or the spliceosome's active site within these structures. In this work, we used EM to localise the intron and the 5' and 3' exons of a model pre-mRNA, as well as the U2-associated protein SF3b155, in pre-catalytic spliceosomes (i.e. B complexes) by labelling them with an antibody that bears colloidal gold. Our data reveal that the intron and both exons, together with SF3b155, are located in specific regions of the head domain of the B complex. These results represent an important first step towards identifying functional sites in the spliceosome. The gold-labelling method adopted here can be applied to other spliceosomal complexes and may thus contribute significantly to our overall understanding of the pre-mRNA splicing process.

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.

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.

Analysis of microRNA signatures using size-coded ligation-mediated PCR.

The expression pattern and regulatory functions of microRNAs (miRNAs) are intensively investigated in various tissues, cell types and disorders. Differential miRNA expression signatures have been revealed in healthy and unhealthy tissues using high-throughput profiling methods. For further analyses of miRNA signatures in biological samples, we describe here a simple and efficient method to detect multiple miRNAs simultaneously in total RNA. The size-coded ligation-mediated polymerase chain reaction (SL-PCR) method is based on size-coded DNA probe hybridization in solution, followed-by ligation, PCR amplification and gel fractionation. The new method shows quantitative and specific detection of miRNAs. We profiled miRNAs of the let-7 family in a number of organisms, tissues and cell types and the results correspond with their incidence in the genome and reported expression levels. Finally, SL-PCR detected let-7 expression changes in human embryonic stem cells as they differentiate to neuron and also in young and aged mice brain and bone marrow. We conclude that the method can efficiently reveal miRNA signatures in a range of biological samples.

Identification of human miRNA precursors that resemble box C/D snoRNAs.

There are two main classes of small nucleolar RNAs (snoRNAs): the box C/D snoRNAs and the box H/ACA snoRNAs that function as guide RNAs to direct sequence-specific modification of rRNA precursors and other nucleolar RNA targets. A previous computational and biochemical analysis revealed a possible evolutionary relationship between miRNA precursors and some box H/ACA snoRNAs. Here, we investigate a similar evolutionary relationship between a subset of miRNA precursors and box C/D snoRNAs. Computational analyses identified 84 intronic miRNAs that are encoded within either box C/D snoRNAs, or in precursors showing similarity to box C/D snoRNAs. Predictions of the folded structures of these box C/D snoRNA-like miRNA precursors resemble the structures of known box C/D snoRNAs, with the boxes C and D often in close proximity in the folded molecule. All five box C/D snoRNA-like miRNA precursors tested (miR-27b, miR-16-1, mir-28, miR-31 and let-7g) bind to fibrillarin, a specific protein component of functional box C/D snoRNP complexes. The data suggest that a subset of small regulatory RNAs may have evolved from box C/D snoRNAs.

[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.

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.

Colon cancer associated transcript-1: a novel RNA expressed in malignant and pre-malignant human tissues.

Early detection of colorectal cancer (CRC) is currently based on fecal occult blood testing (FOBT) and colonoscopy, both which can significantly reduce CRC-related mortality. However, FOBT has low-sensitivity and specificity, whereas colonoscopy is labor- and cost-intensive. Therefore, the discovery of novel biomarkers that can be used for improved CRC screening, diagnosis, staging and as targets for novel therapies is of utmost importance. To identify novel CRC biomarkers we utilized representational difference analysis (RDA) and characterized a colon cancer associated transcript (CCAT1), demonstrating consistently strong expression in adenocarcinoma of the colon, while being largely undetectable in normal human tissues (p < 000.1). CCAT1 levels in CRC are on average 235-fold higher than those found in normal mucosa. Importantly, CCAT1 is strongly expressed in tissues representing the early phase of tumorigenesis: in adenomatous polyps and in tumor-proximal colonic epithelium, as well as in later stages of the disease (liver metastasis, for example). In CRC-associated lymph nodes, CCAT1 overexpression is detectable in all H&E positive, and 40.0% of H&E and immunohistochemistry negative lymph nodes, suggesting very high sensitivity. CCAT1 is also overexpressed in 40.0% of peripheral blood samples of patients with CRC but not in healthy controls. CCAT1 is therefore a highly specific and readily detectable marker for CRC and tumor-associated tissues.

RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae.

We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3'-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3'-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3'-end cleavage and polyadenylation, that is, cotranscriptionally.