Heterogeneous RNA Research Articles

Visualization of RNA structure models within the Integrative Genomics Viewer.

Analyses of the interrelationships between RNA structure and function are increasingly important components of genomic studies. The SHAPE-MaP strategy enables accurate RNA structure probing and realistic structure modeling of kilobase-length noncoding RNAs and mRNAs. Existing tools for visualizing RNA structure models are not suitable for efficient analysis of long, structurally heterogeneous RNAs. In addition, structure models are often advantageously interpreted in the context of other experimental data and gene annotation information, for which few tools currently exist. We have developed a module within the widely used and well supported open-source Integrative Genomics Viewer (IGV) that allows visualization of SHAPE and other chemical probing data, including raw reactivities, data-driven structural entropies, and data-constrained base-pair secondary structure models, in context with linear genomic data tracks. We illustrate the usefulness of visualizing RNA structure in the IGV by exploring structure models for a large viral RNA genome, comparing bacterial mRNA structure in cells with its structure under cell- and protein-free conditions, and comparing a noncoding RNA structure modeled using SHAPE data with a base-pairing model inferred through sequence covariation analysis.

Single-Nucleotide Resolution Mapping of Hepatitis B Virus Promoters in Infected Human Livers and Hepatocellular Carcinoma.

ABSTRACTHepatitis B virus (HBV) is a major cause of liver diseases, including hepatocellular carcinoma (HCC), and more than 650,000 people die annually due to HBV-associated liver failure. Extensive studies of individual promoters have revealed that heterogeneous RNA 5′ ends contribute to the complexity of HBV transcriptome and proteome. Here, we provide a comprehensive map of HBV transcription start sites (TSSs) in human liver, HCC, and blood, as well as several experimental replication systems, at a single-nucleotide resolution. Using CAGE (cap analysis of gene expression) analysis of 16 HCC/nontumor liver pairs, we identify 17 robust TSSs, including a novel promoter for the X gene located in the middle of the gene body, which potentially produces a shorter X protein translated from the conserved second start codon, and two minor antisense transcripts that might represent viral noncoding RNAs. Interestingly, transcription profiles were similar in HCC and nontumor livers, although quantitative analysis revealed highly variable patterns of TSS usage among clinical samples, reflecting precise regulation of HBV transcription initiation at each promoter. Unlike the variety of TSSs found in liver and HCC, the vast majority of transcripts detected in HBV-positive blood samples are pregenomic RNA, most likely generated and released from liver. Our quantitative TSS mapping using the CAGE technology will allow better understanding of HBV transcriptional responses in further studies aimed at eradicating HBV in chronic carriers.IMPORTANCE Despite the availability of a safe and effective vaccine, HBV infection remains a global health problem, and current antiviral protocols are not able to eliminate the virus in chronic carriers. Previous studies of the regulation of HBV transcription have described four major promoters and two enhancers, but little is known about their activity in human livers and HCC. We deeply sequenced the HBV RNA 5′ ends in clinical human samples and experimental models by using a new, sensitive and quantitative method termed cap analysis of gene expression (CAGE). Our data provide the first comprehensive map of global TSS distribution over the entire HBV genome in the human liver, validating already known promoters and identifying novel locations. Better knowledge of HBV transcriptional activity in the clinical setting has critical implications in the evaluation of therapeutic approaches that target HBV replication.

RNA–DNA Chimeras in the Context of an RNA World Transition to an RNA/DNA World

AbstractThe RNA world hypothesis posits that DNA and proteins were later inventions of early life, or the chemistry that gave rise to life. Most scenarios put forth for the emergence of DNA assume a clean separation of RNA and DNA polymer, and a smooth transition between RNA and DNA. However, based on the reality of “clutter” and lack of sophisticated separation/discrimination mechanisms in a protobiological (and/or prebiological) world, heterogeneous RNA–DNA backbone containing chimeric sequences could have been common—and have not been fully considered in models transitioning from an RNA world to an RNA–DNA world. Herein we show that there is a significant decrease in Watson–Crick duplex stability of the heterogeneous backbone chimeric duplexes that would impede base‐pair mediated interactions (and functions). These results point to the difficulties for the transition from one homogeneous system (RNA) to another (RNA/DNA) in an RNA world with a heterogeneous mixture of ribo‐ and deoxyribonucleotides and sequences, while suggesting an alternative scenario of prebiological accumulation and co‐evolution of homogeneous systems (RNA and DNA).

Altered Glycosylation Patterns Increase Immunogenicity of a Subunit Hepatitis C Virus Vaccine, Inducing Neutralizing Antibodies Which Confer Protection in Mice.

A prophylactic vaccine with high efficacy and low cost is urgently needed for global control of HCV infection. Induction of broadly neutralizing antibodies against most HCV genotypes has been challenging due to the antigenic diversity of the HCV genome. Here, we refined a high-yield subunit HCV vaccine that elicited broadly neutralizing antibody responses in preclinical trials. We found that soluble HCV E2 protein (sE2) produced in insect cells is distinctly glycosylated and is more immunogenic than sE2 produced in mammalian cells, suggesting that glycosylation patterns should be taken into consideration in efforts to generate antibody-based recombinant vaccines against HCV. We further showed that sE2 vaccination confers protection against HCV infection in a genetically humanized mouse model. Thus, our work identified a promising broadly protective HCV vaccine candidate that should be considered for further preclinical and clinical development.

RNA structure through multidimensional chemical mapping.

The discoveries of myriad non-coding RNA molecules, each transiting through multiple flexible states in cells or virions, present major challenges for structure determination. Advances in high-throughput chemical mapping give new routes for characterizing entire transcriptomes in vivo, but the resulting one-dimensional data generally remain too information-poor to allow accurate de novo structure determination. Multidimensional chemical mapping (MCM) methods seek to address this challenge. Mutate-and-map (M2), RNA interaction groups by mutational profiling (RING-MaP and MaP-2D analysis) and multiplexed •OH cleavage analysis (MOHCA) measure how the chemical reactivities of every nucleotide in an RNA molecule change in response to modifications at every other nucleotide. A growing body of in vitro blind tests and compensatory mutation/rescue experiments indicate that MCM methods give consistently accurate secondary structures and global tertiary structures for ribozymes, ribosomal domains and ligand-bound riboswitch aptamers up to 200 nucleotides in length. Importantly, MCM analyses provide detailed information on structurally heterogeneous RNA states, such as ligand-free riboswitches that are functionally important but difficult to resolve with other approaches. The sequencing requirements of currently available MCM protocols scale at least quadratically with RNA length, precluding general application to transcriptomes or viral genomes at present. We propose a modify-cross-link-map (MXM) expansion to overcome this and other current limitations to resolving the in vivo 'RNA structurome'.

The Generation of Turnip Crinkle Virus-Like Particles in Plants by the Transient Expression of Wild-Type and Modified Forms of Its Coat Protein.

Turnip crinkle virus (TCV), a member of the genus carmovirus of the Tombusviridae family, has a genome consisting of a single positive-sense RNA molecule that is encapsidated in an icosahedral particle composed of 180 copies of a single type of coat protein. We have employed the CPMV-HT transient expression system to investigate the formation of TCV-like particles following the expression of the wild-type coat protein or modified forms of it that contain either deletions and/or additions. Transient expression of the coat protein in plants results in the formation of capsid structures that morphologically resemble TCV virions (T = 3 structure) but encapsidate heterogeneous cellular RNAs, rather than the specific TCV coat protein messenger RNA. Expression of an amino-terminal deleted form of the coat protein resulted in the formation of smaller T = 1 structures that are free of RNA. The possibility of utilizing TCV as a carrier for the presentation of foreign proteins on the particle surface was also explored by fusing the sequence of GFP to the C-terminus of the coat protein. The expression of coat protein-GFP hybrids permitted the formation of VLPs but the yield of particles is diminished compared to the yield obtained with unmodified coat protein. Our results confirm the importance of the N-terminus of the coat protein for the encapsidation of RNA and show that the coat protein's exterior P domain plays a key role in particle formation.

Expression of polyQ aggregates in Malpighian tubules leads to degeneration in Drosophila melanogaster

BackgroundPolyglutamine (polyQ) disorders are caused by expanded CAG (Glutamine) repeats in neurons in the brain. The expanded repeats are also expressed in the non-neuronal cells, however, their contribution to disease pathogenesis is not very well studied. In the present study, we have expressed a stretch of 127 Glutamine repeats in Malpighian tubules (MTs) of Drosophila melanogaster as these tissues do not undergo ecdysone induced histolysis during larval to pupal transition at metamorphosis. ResultsProgressive degeneration, which is the hallmark of neurodegeneration is also observed in MTs. The mutant protein forms inclusion bodies in the nucleus resulting in expansion of the nucleus and affect chromatin organization which appear loose and open, eventually resulting in DNA fragmentation and blebbing. A virtual absence of tubule lumen was observed followed by functional abnormalities. As development progressed, severe abnormalities affecting pupal epithelial morphogenesis processes were observed resulting in complete lethality. Distribution of heterogeneous RNA binding protein (hnRNP), HRB87F, Wnt/wingless and JNK signaling and expression of Relish was also found to be affected. Expression of resistance genes following polyQ expression was up regulated. ConclusionThe present study gives an insight into the effects of polyQ aggregates in non-neuronal tissues.

Native purification and labeling of RNA for single molecule fluorescence studies.

The recent discovery that non-coding RNAs are considerably more abundant and serve a much wider range of critical cellular functions than recognized over previous decades of research into molecular biology has sparked a renewed interest in the study of structure-function relationships of RNA. To perform their functions in the cell, RNAs must dominantly adopt their native conformations, avoiding deep, non-productive kinetic traps that may exist along a frustrated (rugged) folding free energy landscape. Intracellularly, RNAs are synthesized by RNA polymerase and fold co-transcriptionally starting from the 5' end, sometimes with the aid of protein chaperones. By contrast, in the laboratory RNAs are commonly generated by in vitro transcription or chemical synthesis, followed by purification in a manner that includes the use of high concentrations of urea, heat and UV light (for detection), resulting in the denaturation and subsequent refolding of the entire RNA. Recent studies into the nature of heterogeneous RNA populations resulting from this process have underscored the need for non-denaturing (native) purification methods that maintain the co-transcriptional fold of an RNA. Here, we present protocols for the native purification of an RNA after its in vitro transcription and for fluorophore and biotin labeling methods designed to preserve its native conformation for use in single molecule fluorescence resonance energy transfer (smFRET) inquiries into its structure and function. Finally, we present methods for taking smFRET data and for analyzing them, as well as a description of plausible overall preparation schemes for the plethora of non-coding RNAs.

RNA EDITING IN Calotropis procera MITOCHONDRIAL NADHDEHYDROGENASE SUBUNIT 3 GENE

Nad3 (NADH-dehydrogenase subunit 3) gene from genomic (accession no. KP171516) and cDNA (accession no. KP171517) was identified in desert plant Calotropis procera using RNA seq and DNA seq data. A number of cytosines are altered to be recognized as uridines in transcripts of the nad3 locus in mitochondria. The nucleotide modifications were found at 11 different nucleotide positions (nucleotide no. 44, 62, 80, 209, 215,230, 247, 266, 275, 317 and 349) within the nad3 coding region. Heterogeneous RNA editing in C. procera nad3 RNA was not detected in this study. These alterations in the mRNA sequence change codon identities to specify 11 amino acids. The alteration in nucleotides leads to codons alteration specifying different amino acids, the common being proline to leuciene (P-L). Other changes were serine to leucine (S-L), serine to phenylalanine (S-F), proline to serine and arginine to tryptophan (R-W). These alterations are common in mitochondrial nad3 gene of most plant species with few differences according to the properties of the amino acids involved.

Widespread RNA metabolism impairment in sporadic inclusion body myositis TDP43-proteinopathy

TDP43 protein mislocalization is a hallmark of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal dementia, and mutations in the gene encoding TDP43 cause both disorders, further highlighting its role in disease pathogenesis. TDP43 is a heterogenous ribonucleoprotein, therefore suggesting that alterations in RNA metabolism play a role in these disorders, although direct evidence in patients is lacking. Sporadic inclusion body myositis (sIBM) is the most common acquired myopathy occurring in adults aged older than 50 years and abnormal cytoplasmic accumulations of TDP43 have been consistently described in sIBM myofibers. Here, we exploit high quality RNA from frozen sIBM muscle biopsies for transcriptomic studies on TDP43-proteinopathy patient tissue. Surprisingly, we found widespread sIBM-specific changes in the RNA metabolism pathways themselves. Consistent with this finding, we describe novel RNA binding proteins to mislocalize in the cytoplasm of sIBM myofibers and splicing changes in MAPT, a gene previously shown to play a role in sIBM. Our data indicate widespread alterations of RNA metabolism are a novel aspect of disease pathogenesis in sIBM. These findings also document an association, in TDP43-proteinopathy patients, between heterogenous ribonucleoprotein pathology and RNA metabolism alterations and carry importance for neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal dementia.

Heterologous expression of artificial miRNAs from rice dwarf virus in transgenic rice

In contrast to hairpin RNAs, in which heterogeneous small RNAs are processed from double-stranded RNA to have potential off-target effects on endogenous other genes, artificial miRNAs (amiRNAs) have advantages of exquisite specificity and non-transitivity to thus target individual genes and groups of endogenous genes. Earlier studies showed that amiRNA engineering based on osa-miRNA528 precursor could efficiently trigger endogenous gene silencing and modulate agronomic traits in rice. However, both the expression efficiency of heterologous amiRNAs based on osa-miRNA528 precursor and the correlation of copy number with the relative expression level of amiRNAs remain unknown. In the present study, five amiRNAs (S9-1174, S9-1192, S11-864, S11-868 and S11-869) targeting different sites of S9 and S11 negative strands in rice dwarf virus (RDV) genome were constructed using endogenous osa-miRNA528 precursor as backbone. After identification by Northern blot, two amiRNAs (S9-1174 and S9-1192) targeting S9 negative strand in RDV genome were highly expressed, whereas in three tested amiRNAs targeting S11 negative strand in RDV genome, only two amiRNAs (S11-868 and S11-869) were processed efficiently. T0 generation transgenic rice containing amiRNAs (S9-1174, S9-1192, S11-868 and S11-869) exhibited different expression ratios of amiRNAs, accounting for 90.0, 90.0, 66.7 and 77.8 %, respectively. In addition, combination analysis with the relative amiRNA expression levels and its copy number revealed that the relative expression levels of amiRNAs had no relation to the copy number of T-DNA insert in transgenic rice.

Correlating transcriptional networks to breast cancer survival: a large-scale coexpression analysis

Weighted gene coexpression network analysis (WGCNA) is a powerful 'guilt-by-association'-based method to extract coexpressed groups of genes from large heterogeneous messenger RNA expression data sets. We have utilized WGCNA to identify 11 coregulated gene clusters across 2342 breast cancer samples from 13 microarray-based gene expression studies. A number of these transcriptional modules were found to be correlated to clinicopathological variables (e.g. tumor grade), survival endpoints for breast cancer as a whole (disease-free survival, distant disease-free survival and overall survival) and also its molecular subtypes (luminal A, luminal B, HER2+ and basal-like). Examples of findings arising from this work include the identification of a cluster of proliferation-related genes that when upregulated correlated to increased tumor grade and were associated with poor survival in general. The prognostic potential of novel genes, for example, ubiquitin-conjugating enzyme E2S (UBE2S) within this group was confirmed in an independent data set. In addition, gene clusters were also associated with survival for breast cancer molecular subtypes including a cluster of genes that was found to correlate with prognosis exclusively for basal-like breast cancer. The upregulation of several single genes within this coexpression cluster, for example, the potassium channel, subfamily K, member 5 (KCNK5) was associated with poor outcome for the basal-like molecular subtype. We have developed an online database to allow user-friendly access to the coexpression patterns and the survival analysis outputs uncovered in this study (available at http://glados.ucd.ie/Coexpression/).

RNA species whose transcription is totally silent in pre-MBT stage are not mRNA but rRNA and possible involvement of weak bases (ammonium salts and/or amines) in the transcriptional silence of rRNA genes during the pre-MBT stage in <i>Xenopus</i> early embryos

In Xenopus laevis embryogenesis, fertilized eggs undergo 12 cycles of synchronous divisions and reach the stage called midblastula transition (MBT). It has long been believed that during the first 12 cycles of cleavage (pre-MBT stage), transcriptional activity of the zygotic nuclei is totally absent. However, heterogeneous mRNA-like RNA is synthesized in pre-MBT stage embryos, and exogenously-injected bacterial CAT genes with SV40 promoter are expressed from the cleavage stage. Nevertheless, the synthesis of rRNA as detected by rRNA-specific2’-O-methylation does not take place in pre-MBT embryos and starts only from the latter half of the MBT stage, corroborating the fact that formation of definitive nucleoli as well as the transcription of microinjected rRNA genes starts only at and after MBT stage. Thus, while mRNA-like RNA synthesis occurs from pre-MBT stage, synthesis of rRNA is controlled in the way that transcription of rRNA genes is totally silent during pre-MBT stage and is initiated only at the latter half of MBT stage. Once initiated, the rate of the synthesis of rRNA is constant throughout later stages on a per-cell basis. We searched substances which are responsible for the transcriptional silence of rRNA genes during the pre-MBT stage. Weak bases such as ammonium ion and amines selectively inhibited rRNA synthesis at the transcriptional level in post-MBT stage embryo cells. Since we found that the level of ammonia extracted from embryos is much higher in pre-MBT embryos than in post-MBT embryos, we suggest that weak bases like ammonium ion could be responsible for the transcriptional silence of rRNA genes by slightly increasing intracellular pH during the pre-MBT.

Modified FRAP Methodology with Millisecond Time Resolution for the Investigation of Heterogeneous RNA Polymerase II Diffusion Dynamics

We have developed a laser scanning fluorescence recovery after photobleaching (FRAP) methodology capable of investigating protein diffusion and binding dynamics over several decades in time, from the millisecond through minute timescale. A several micron diameter bleach spot is established within 800 us, followed by fluorescence monitoring by a line scan through the bleach region. This method of FRAP permits the recovery to be captured with 1.6 ms time resolution, enabling observation of short time diffusion and long time binding kinetics. The rapid time resolution, precisely defined bleach geometry, and well-characterized bleach depth enable this method to be made highly quantitative through the application of a diffusion-anomalous subdiffusion model. This modeling can provide insight into the local environment through which biological molecules move. We have applied this novel implementation to investigate a fluorescently labeled subunit of RNA Polymerase II (Rpb3-GFP) in the polytene nuclei of Drosophila salivary glands. This unique chromosomal architecture creates segregated nucleoplasmic and chromatin regions. This has allowed for discriminating observations of protein dynamics while diffusing in the presence or absence of DNA obstructions and binding sites, permitting observation of three distinct regimes of protein diffusion - nucleoplasmic (absence of binding/obstruction), intrachromatin (binding/obstruction), and active transcription regions. We have observed that Rpb3-GFP diffuses anomalously in the nucleoplasm, in contrast to our studies of GFP expressed in polytene cells, which moves by Brownian diffusion through the nucleoplasm. Given the comparable size between the proteins and absence of possible chromatin binding sites, this indicates that Rpb3-GFP is engaged with other RNAP II subunits, giving rise to a distribution of diffusion coefficients, manifest as anomalous subdiffusion. We also see highly anomalous behavior for Rbp3-GFP moving through chromatin regions coupled with reduced diffusion coefficients.

Long interspersed nuclear elements (LINEs) show tissue-specific, mosaic genome and methylation-unrestricted, widespread expression of noncoding RNAs in somatic tissues of the rat

We report strong somatic and germ line expression of LINE RNAs in eight different tissues of rat by using a novel ~2.8 kb genomic PstI-LINE DNA (P1-LINE) isolated from the rat brain. P1-LINE is present in a 93 kb LINE-SINE-cluster in sub-telomeric region of chromosome 12 (12p12) and as multiple truncated copies interspersed in all rat chromosomes. P1-LINEs occur as inverted repeats at multiple genomic loci in tissue-specific and mosaic patterns. P1-LINE RNAs are strongly expressed in brain, liver, lungs, heart, kidney, testes, spleen and thymus into large to small heterogeneous RNAs (~5.0 to 0.2 kb) in tissue-specific and dynamic patterns in individual rats. P1-LINE DNA is strongly methylated at CpG-dinucleotides in most genomic copies in all the tissues and weakly hypomethylated in few copies in some tissues. Small (700–75 nt) P1-LINE RNAs expressed in all tissues may be possible precursors for small regulatory RNAs (PIWI-interacting/piRNAs) bioinformatically derived from P1-LINE. The strong and dynamic expression of LINE RNAs from multiple chromosomal loci and the putative piRNAs in somatic tissues of rat under normal physiological conditions may define functional chromosomal domains marked by LINE RNAs as long noncoding RNAs (lncRNAs) unrestricted by DNA methylation. The tissue-specific, dynamic RNA expression and mosaic genomic distribution of LINEs representing a steady-state genomic flux of retrotransposon RNAs suggest for biological role of LINE RNAs as long ncRNAs and small piRNAs in mammalian tissues independent of their cellular fate for translation, reverse-transcription and retrotransposition. This may provide evolutionary advantages to LINEs and mammalian genomes.

A Magnetic Bead-Integrated Chip for the Large Scale Manufacture of Normalized esiRNAs

The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified in regulating the migration capability of Hela cells. We believe that this approach provides a more robust and cost-effective choice for manufacturing esiRNAs than current approaches, and therefore these heterogeneous RNA strands may have utility in most intensive and extensive applications.

Epigenetic regulation of glucocorticoid receptor expression in aorta from mice with hyperhomocysteinemia

Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease but its underlying molecular pathology is not understood. Homocysteine is metabolically linked to the epigenetic process of DNA methylation. Tissue-specific changes in DNA methylation have been observed in HHcy but little is known about vascular tissue. The objective of this study was to determine if changes in the epigenetic regulation of glucocorticoid receptor (GR) expression (encoded by Nr3c1) in aorta are associated with HHcy. C57BL/6 mice heterozygous for disruption of the cystathionine-β-synthase gene (Cbs +/-) and controls (Cbs +/+) were fed a control or high methionine/low folate (HH) diet to induce HHcy. Cbs +/- and Cbs +/+ fed the HH diet had higher plasma total homocysteine levels (19.9 ± 3.2 and 7.0 ± 0.9 μM, respectively) than Cbs +/+ mice fed the control diet (2.7 ± 0.2 μM), and this was accompanied by lower Nr3c1 mRNA and lower GR protein in aorta. The Nr3c1 gene contains multiple first exons producing heterogeneous RNA transcripts expressed in a tissue-specific manner. We identified expression of two transcripts in aorta. Bisulfite pyrosequencing found increased methylation of the promoter regions for these transcripts at sites corresponding to Sp1 and Nrf1 binding sites. Chromatin immunoprecipitation found lower binding of Nrf1 to the Nr3c1 promoter but higher expression of Nrf1 protein in aorta from mice with HHcy. These findings show methylation and silencing of vascular Nr3c1 expression and suggest a role for epigenetic regulation of gene expression in HHcy.

Thermodynamics of Ligand Binding to a Heterogeneous RNA Population in the Malachite Green Aptamer

The malachite green aptamer binds two closely related ligands, malachite green (MG) and tetramethylrosamine (TMR), with nearly equal affinity. The MG ligand consists of three phenyl rings emanating from a central carbon, while TMR has two of the three rings connected by an ether linkage. The binding pockets for MG and TMR in the aptamer, known from high-resolution structures, differ only in the conformation of a few nucleotides. Herein, we applied isothermal titration calorimetry (ITC) to compare the thermodynamics of binding of MG and TMR to the aptamer. Binding heat capacities were obtained from ITC titrations over the temperature range of 15-60 °C. Two temperature regimes were found for MG binding: one from 15 to 45 °C where MG bound with a large negative heat capacity and an apparent stoichiometry (n) of ~0.4 and another from 50 to 60 °C where MG bound with a positive heat capacity and an n of ~1.1. The binding of TMR, on the other hand, revealed only one temperature regime for binding, with a more modest negative heat capacity and an n of ~1.2. The large difference in heat capacity between the two ligands suggests that significantly more conformational rearrangement occurs upon the binding of MG than that of TMR, which is consistent with differences in solvent accessible surface area calculated for available ligand-bound structures. Lastly, we note that the binding stoichiometry of MG was improved not only by an increase in the temperature but also by a decrease in the concentration of Mg(2+) or an increase in the time between ITC injections. These studies suggest that binding of a dynamical ligand to a functional RNA requires the RNA itself to have significant dynamics.

Characterization of microRNAs expression profiling in one group of Chinese urothelial cell carcinoma identified by Solexa sequencing

ObjectiveThe goal of this study was to extend the known repertoire of microRNAs (miRNAs) expressed in urothelial bladder cancer (BCa) of the Chinese population and further understand the molecular events of miRNAs underlying urothelial bladder tumorigenesis at the global genome level. Materials and methodsWe separated well-characterized epithelial tumor cells from 20 moderately differentiated or poorly differentiated BCa specimens by laser capture microdissection (LCM) and pooled these cells of interest prior to RNA analysis. Ten normal bladder epithelia (NBE) samples were pooled as the control. After preparation of small RNAs library, the 2 samples were sequenced simultaneously by the next generation high through-put Solexa sequencing technology. ResultsWe employed the next generation high through-put Solexa sequencing technology to clone and identify miRNAs in BCa and NBE, and generated 11,146,610, and 10,263,845 high quality sequence reads, respectively. According to the analysis of size distribution, 22 nt class was the most abundant group of small RNAs in the BCa. Likewise, the 20 and 22 nt sequences were significantly greater than shorter or longer sequences, and accounted for 59.55% of the total sequence number of NBE library. The whole-genome-scale data mining suggested that BCa and NBE libraries both contained multiple and heterogeneous small RNA species. On further analysis, the sequencing data revealed that different miRNAs showed clearly in-house differential expression levels in BCa and NBE and 74 miRNAs aberrantly expressed between BCa and NBE at the global genome level. We also predicted 13 novel miRNAs in both BCa and NBE libraries. ConclusionsOur results suggest that BCa miRNAs include a large proportion of conserved miRNAs and a set of non-conserved miRNAs with low expression levels. These known and newly identified miRNAs at the population level significantly enhance our knowledge of BCa miRNAs expression profiling and provide insights into miRNAs oncogenesis and oncotherapy in BCa. Further studies are necessary to elucidate the roles of miRNAs in urothelial bladder tumorigenesis and determine the potential of miRNAs as diagnostic and prognostic tools or therapeutic targets for BCa in the future.

Single cell transcriptomics of neighboring hyphae of Aspergillus niger

Single cell profiling was performed to assess differences in RNA accumulation in neighboring hyphae of the fungus Aspergillus niger. A protocol was developed to isolate and amplify RNA from single hyphae or parts thereof. Microarray analysis resulted in a present call for 4 to 7% of the A. niger genes, of which 12% showed heterogeneous RNA levels. These genes belonged to a wide range of gene categories.