Science.gov

Sample records for small rna analysis

  1. Computational analysis of small RNA cloning data.

    PubMed

    Berninger, Philipp; Gaidatzis, Dimos; van Nimwegen, Erik; Zavolan, Mihaela

    2008-01-01

    Cloning and sequencing is the method of choice for small regulatory RNA identification. Using deep sequencing technologies one can now obtain up to a billion nucleotides--and tens of millions of small RNAs--from a single library. Careful computational analyses of such libraries enabled the discovery of miRNAs, rasiRNAs, piRNAs, and 21U RNAs. Given the large number of sequences that can be obtained from each individual sample, deep sequencing may soon become an alternative to oligonucleotide microarray technology for mRNA expression profiling. In this report we present the methods that we developed for the annotation and expression profiling of small RNAs obtained through large-scale sequencing. These include a fast algorithm for finding nearly perfect matches of small RNAs in sequence databases, a web-accessible software system for the annotation of small RNA libraries, and a Bayesian method for comparing small RNA expression across samples.

  2. DSAP: deep-sequencing small RNA analysis pipeline.

    PubMed

    Huang, Po-Jung; Liu, Yi-Chung; Lee, Chi-Ching; Lin, Wei-Chen; Gan, Richie Ruei-Chi; Lyu, Ping-Chiang; Tang, Petrus

    2010-07-01

    DSAP is an automated multiple-task web service designed to provide a total solution to analyzing deep-sequencing small RNA datasets generated by next-generation sequencing technology. DSAP uses a tab-delimited file as an input format, which holds the unique sequence reads (tags) and their corresponding number of copies generated by the Solexa sequencing platform. The input data will go through four analysis steps in DSAP: (i) cleanup: removal of adaptors and poly-A/T/C/G/N nucleotides; (ii) clustering: grouping of cleaned sequence tags into unique sequence clusters; (iii) non-coding RNA (ncRNA) matching: sequence homology mapping against a transcribed sequence library from the ncRNA database Rfam (http://rfam.sanger.ac.uk/); and (iv) known miRNA matching: detection of known miRNAs in miRBase (http://www.mirbase.org/) based on sequence homology. The expression levels corresponding to matched ncRNAs and miRNAs are summarized in multi-color clickable bar charts linked to external databases. DSAP is also capable of displaying miRNA expression levels from different jobs using a log(2)-scaled color matrix. Furthermore, a cross-species comparative function is also provided to show the distribution of identified miRNAs in different species as deposited in miRBase. DSAP is available at http://dsap.cgu.edu.tw.

  3. Analysis of microRNA transcriptome by deep sequencing of small RNA libraries of peripheral blood

    PubMed Central

    2010-01-01

    Background MicroRNAs are a class of small non-coding RNAs that regulate mRNA expression at the post - transcriptional level and thereby many fundamental biological processes. A number of methods, such as multiplex polymerase chain reaction, microarrays have been developed for profiling levels of known miRNAs. These methods lack the ability to identify novel miRNAs and accurately determine expression at a range of concentrations. Deep or massively parallel sequencing methods are providing suitable platforms for genome wide transcriptome analysis and have the ability to identify novel transcripts. Results The results of analysis of small RNA sequences obtained by Solexa technology of normal peripheral blood mononuclear cells, tumor cell lines K562 and HL60 are presented. In general K562 cells displayed overall low level of miRNA population and also low levels of DICER. Some of the highly expressed miRNAs in the leukocytes include several members of the let-7 family, miR-21, 103, 185, 191 and 320a. Comparison of the miRNA profiles of normal versus K562 or HL60 cells revealed a specific set of differentially expressed molecules. Correlation of the miRNA with that of mRNA expression profiles, obtained by microarray, revealed a set of target genes showing inverse correlation with miRNA levels. Relative expression levels of individual miRNAs belonging to a cluster were found to be highly variable. Our computational pipeline also predicted a number of novel miRNAs. Some of the predictions were validated by Real-time RT-PCR and or RNase protection assay. Organization of some of the novel miRNAs in human genome suggests that these may also be part of existing clusters or form new clusters. Conclusions We conclude that about 904 miRNAs are expressed in human leukocytes. Out of these 370 are novel miRNAs. We have identified miRNAs that are differentially regulated in normal PBMC with respect to cancer cells, K562 and HL60. Our results suggest that post - transcriptional

  4. Comprehensive analysis of human small RNA sequencing data provides insights into expression profiles and miRNA editing

    PubMed Central

    Gong, Jing; Wu, Yuliang; Zhang, Xiantong; Liao, Yifang; Sibanda, Vusumuzi Leroy; Liu, Wei; Guo, An-Yuan

    2014-01-01

    MicroRNAs (miRNAs) play key regulatory roles in various biological processes and diseases. A comprehensive analysis of large scale small RNA sequencing data (smRNA-seq) will be very helpful to explore tissue or disease specific miRNA markers and uncover miRNA variants. Here, we systematically analyzed 410 human smRNA-seq datasets, which samples are from 24 tissue/disease/cell lines. We tested the mapping strategies and found that it was necessary to make multiple-round mappings with different mismatch parameters. miRNA expression profiles revealed that on average ∼70% of known miRNAs were expressed at low level or not expressed (RPM < 1) in a sample and only ∼9% of known miRNAs were relatively highly expressed (RPM > 100). About 30% known miRNAs were not expressed in all of our used samples. The miRNA expression profiles were compiled into an online database (HMED, http://bioinfo.life.hust.edu.cn/smallRNA/). Dozens of tissue/disease specific miRNAs, disease/control dysregulated miRNAs and miRNAs with arm switching events were discovered. Further, we identified some highly confident editing sites including 24 A-to-I sites and 23 C-to-U sites. About half of them were widespread miRNA editing sites in different tissues. We characterized that the 2 types of editing sites have different features with regard to location, editing level and frequency. Our analyses for expression profiles, specific miRNA markers, arm switching, and editing sites, may provide valuable information for further studies of miRNA function and biomarker finding. PMID:25692236

  5. "Well-determined" regions in RNA secondary structure prediction: analysis of small subunit ribosomal RNA.

    PubMed Central

    Zuker, M; Jacobson, A B

    1995-01-01

    Recent structural analyses of genomic RNAs from RNA coliphages suggest that both well-determined base paired helices and well-determined structural domains that are identified by "energy dot plot" analysis using the RNA folding package mfold, are likely to be predicted correctly. To test these observations with another group of large RNAs, we have analyzed 15 ribosomal RNAs. Published secondary structure models that were derived by comparative sequence analysis were used to evaluate the predicted structures. Both the optimal predicted fold and the predicted "energy dot plot" of each sequence were examined. Each prediction was obtained from a single computer run on an entire ribosomal RNA sequence. All predicted base pairs in optimal foldings were examined for agreement with proven base pairs in the comparative models. Our analyses show that the overall correspondence between the predicted and comparative models varied for different RNAs and ranges from a low of 27% to high of 70%, with a mean value of 49%. The correspondence improves to a mean value of 81% when the analysis is limited to well-determined helices. In addition to well-determined helices, large well-determined structural domains can be observed in "energy dot plots" of some 16S ribosomal RNAs. The predicted domains correspond closely with structural domains that are found by the comparative method in the same RNAs. Our analyses also show that measuring the agreement between predicted and comparative secondary structure models underestimates the reliability of structural prediction by mfold. PMID:7544463

  6. iMir: An integrated pipeline for high-throughput analysis of small non-coding RNA data obtained by smallRNA-Seq

    PubMed Central

    2013-01-01

    Background Qualitative and quantitative analysis of small non-coding RNAs by next generation sequencing (smallRNA-Seq) represents a novel technology increasingly used to investigate with high sensitivity and specificity RNA population comprising microRNAs and other regulatory small transcripts. Analysis of smallRNA-Seq data to gather biologically relevant information, i.e. detection and differential expression analysis of known and novel non-coding RNAs, target prediction, etc., requires implementation of multiple statistical and bioinformatics tools from different sources, each focusing on a specific step of the analysis pipeline. As a consequence, the analytical workflow is slowed down by the need for continuous interventions by the operator, a critical factor when large numbers of datasets need to be analyzed at once. Results We designed a novel modular pipeline (iMir) for comprehensive analysis of smallRNA-Seq data, comprising specific tools for adapter trimming, quality filtering, differential expression analysis, biological target prediction and other useful options by integrating multiple open source modules and resources in an automated workflow. As statistics is crucial in deep-sequencing data analysis, we devised and integrated in iMir tools based on different statistical approaches to allow the operator to analyze data rigorously. The pipeline created here proved to be efficient and time-saving than currently available methods and, in addition, flexible enough to allow the user to select the preferred combination of analytical steps. We present here the results obtained by applying this pipeline to analyze simultaneously 6 smallRNA-Seq datasets from either exponentially growing or growth-arrested human breast cancer MCF-7 cells, that led to the rapid and accurate identification, quantitation and differential expression analysis of ~450 miRNAs, including several novel miRNAs and isomiRs, as well as identification of the putative mRNA targets of

  7. Analysis and application of viroid-specific small RNAs generated by viroid-inducing RNA silencing.

    PubMed

    Adkar-Purushothama, Charith Raj; Zhang, Zhixiang; Li, Shifang; Sano, Teruo

    2015-01-01

    Viroids are noncoding RNA pathogens inducing severe to mild disease symptoms on agriculturally important crop plants. Viroid replication is entirely dependent on host transcription machinery, and their replication/accumulation in the infected cells can activate RNA silencing-a host defense mechanism that targets the viroid itself. RNA silencing produces in the cell large amounts of viroid-specific small RNAs of 21-24-nucleotides by cleaving (or "dicing") entire molecules of viroid RNA. However, viroid replication is resistant to the effects of RNA silencing and disrupts the normal regulation of host gene expression, finally resulting in the development of disease symptoms on infected plant. The molecular mechanisms of biological processes involving RNA silencing and underlying various aspects of viroid-host interaction, such as symptom expression, are of special interests to both basic and applied areas of viroid research. Here we present a method to create infectious viroid cDNA clones and RNA transcripts, the starting material for such analyses, using Hop stunt viroid as an example. Next we describe methods for the preparation and analysis of viroid-specific small RNAs by deep sequencing using tomato plants infected with Potato spindle tuber viroid as an example. Finally we introduce bioinformatics tools and methods necessary to process, analyze, and characterize these viroid-specific small RNAs. These bioinformatic methods provide a powerful new tool for the detection and discovery of both known and new viroid species. PMID:25287502

  8. Deep Sequencing Analysis of Nucleolar Small RNAs: RNA Isolation and Library Preparation.

    PubMed

    Bai, Baoyan; Laiho, Marikki

    2016-01-01

    The nucleolus is a subcellular compartment with a key essential function in ribosome biogenesis. The nucleolus is rich in noncoding RNAs, mostly the ribosomal RNAs and small nucleolar RNAs. Surprisingly, also several miRNAs have been detected in the nucleolus, raising the question as to whether other small RNA species are present and functional in the nucleolus. We have developed a strategy for stepwise enrichment of nucleolar small RNAs from the total nucleolar RNA extracts and subsequent construction of nucleolar small RNA libraries which are suitable for deep sequencing. Our method successfully isolates the small RNA population from total RNAs and monitors the RNA quality in each step to ensure that small RNAs recovered represent the actual small RNA population in the nucleolus and not degradation products from larger RNAs. We have further applied this approach to characterize the distribution of small RNAs in different cellular compartments. PMID:27576723

  9. Comparative analysis of secondary structure of insect mitochondrial small subunit ribosomal RNA using maximum weighted matching.

    PubMed

    Page, R D

    2000-10-15

    Comparative analysis is the preferred method of inferring RNA secondary structure, but its use requires considerable expertise and manual effort. As the importance of secondary structure for accurate sequence alignment and phylogenetic analysis becomes increasingly realised, the need for secondary structure models for diverse taxonomic groups becomes more pressing. The number of available structures bears little relation to the relative diversity or importance of the different taxonomic groups. Insects, for example, comprise the largest group of animals and yet are very poorly represented in secondary structure databases. This paper explores the utility of maximum weighted matching (MWM) to help automate the process of comparative analysis by inferring secondary structure for insect mitochondrial small subunit (12S) rRNA sequences. By combining information on correlated changes in substitutions and helix dot plots, MWM can rapidly generate plausible models of secondary structure. These models can be further refined using standard comparative techniques. This paper presents a secondary structure model for insect 12S rRNA based on an alignment of 225 insect sequences and an alignment for 16 exemplar insect sequences. This alignment is used as a template for a web server that automatically generates secondary structures for insect sequences.

  10. Tracking Cryptosporidium parvum by sequence analysis of small double-stranded RNA.

    PubMed Central

    Xiao, L.; Limor, J.; Bern, C.; Lal, A. A.

    2001-01-01

    We sequenced a 173-nucleotide fragment of the small double-stranded viruslike RNA of Cryptosporidium parvum isolates from 23 calves and 38 humans. Sequence diversity was detected at 17 sites. Isolates from the same outbreak had identical double-stranded RNA sequences, suggesting that this technique may be useful for tracking Cryptosporidium infection sources. PMID:11266306

  11. CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains.

    PubMed

    Wright, Patrick R; Georg, Jens; Mann, Martin; Sorescu, Dragos A; Richter, Andreas S; Lott, Steffen; Kleinkauf, Robert; Hess, Wolfgang R; Backofen, Rolf

    2014-07-01

    CopraRNA (Comparative prediction algorithm for small RNA targets) is the most recent asset to the Freiburg RNA Tools webserver. It incorporates and extends the functionality of the existing tool IntaRNA (Interacting RNAs) in order to predict targets, interaction domains and consequently the regulatory networks of bacterial small RNA molecules. The CopraRNA prediction results are accompanied by extensive postprocessing methods such as functional enrichment analysis and visualization of interacting regions. Here, we introduce the functionality of the CopraRNA and IntaRNA webservers and give detailed explanations on their postprocessing functionalities. Both tools are freely accessible at http://rna.informatik.uni-freiburg.de. PMID:24838564

  12. CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains

    PubMed Central

    Wright, Patrick R.; Georg, Jens; Mann, Martin; Sorescu, Dragos A.; Richter, Andreas S.; Lott, Steffen; Kleinkauf, Robert; Hess, Wolfgang R.; Backofen, Rolf

    2014-01-01

    CopraRNA (Comparative prediction algorithm for small RNA targets) is the most recent asset to the Freiburg RNA Tools webserver. It incorporates and extends the functionality of the existing tool IntaRNA (Interacting RNAs) in order to predict targets, interaction domains and consequently the regulatory networks of bacterial small RNA molecules. The CopraRNA prediction results are accompanied by extensive postprocessing methods such as functional enrichment analysis and visualization of interacting regions. Here, we introduce the functionality of the CopraRNA and IntaRNA webservers and give detailed explanations on their postprocessing functionalities. Both tools are freely accessible at http://rna.informatik.uni-freiburg.de. PMID:24838564

  13. miRMOD: a tool for identification and analysis of 5' and 3' miRNA modifications in Next Generation Sequencing small RNA data.

    PubMed

    Kaushik, Abhinav; Saraf, Shradha; Mukherjee, Sunil K; Gupta, Dinesh

    2015-01-01

    In the past decade, the microRNAs (miRNAs) have emerged to be important regulators of gene expression across various species. Several studies have confirmed different types of post-transcriptional modifications at terminal ends of miRNAs. The reports indicate that miRNA modifications are conserved and functionally significant as it may affect miRNA stability and ability to bind mRNA targets, hence affecting target gene repression. Next Generation Sequencing (NGS) of the small RNA (sRNA) provides an efficient and reliable method to explore miRNA modifications. The need for dedicated software, especially for users with little knowledge of computers, to determine and analyze miRNA modifications in sRNA NGS data, motivated us to develop miRMOD. miRMOD is a user-friendly, Microsoft Windows and Graphical User Interface (GUI) based tool for identification and analysis of 5' and 3' miRNA modifications (non-templated nucleotide additions and trimming) in sRNA NGS data. In addition to identification of miRNA modifications, the tool also predicts and compares the targets of query and modified miRNAs. In order to compare binding affinities for the same target, miRMOD utilizes minimum free energies of the miRNA:target and modified-miRNA:target interactions. Comparisons of the binding energies may guide experimental exploration of miRNA post-transcriptional modifications. The tool is available as a stand-alone package to overcome large data transfer problems commonly faced in web-based high-throughput (HT) sequencing data analysis tools. miRMOD package is freely available at http://bioinfo.icgeb.res.in/miRMOD. PMID:26623179

  14. Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa.

    PubMed

    Morin, Ryan D; Aksay, Gozde; Dolgosheina, Elena; Ebhardt, H Alexander; Magrini, Vincent; Mardis, Elaine R; Sahinalp, S Cenk; Unrau, Peter J

    2008-04-01

    The diversity of microRNAs and small-interfering RNAs has been extensively explored within angiosperms by focusing on a few key organisms such as Oryza sativa and Arabidopsis thaliana. A deeper division of the plants is defined by the radiation of the angiosperms and gymnosperms, with the latter comprising the commercially important conifers. The conifers are expected to provide important information regarding the evolution of highly conserved small regulatory RNAs. Deep sequencing provides the means to characterize and quantitatively profile small RNAs in understudied organisms such as these. Pyrosequencing of small RNAs from O. sativa revealed, as expected, approximately 21- and approximately 24-nt RNAs. The former contained known microRNAs, and the latter largely comprised intergenic-derived sequences likely representing heterochromatin siRNAs. In contrast, sequences from Pinus contorta were dominated by 21-nt small RNAs. Using a novel sequence-based clustering algorithm, we identified sequences belonging to 18 highly conserved microRNA families in P. contorta as well as numerous clusters of conserved small RNAs of unknown function. Using multiple methods, including expressed sequence folding and machine learning algorithms, we found a further 53 candidate novel microRNA families, 51 appearing specific to the P. contorta library. In addition, alignment of small RNA sequences to the O. sativa genome revealed six perfectly conserved classes of small RNA that included chloroplast transcripts and specific types of genomic repeats. The conservation of microRNAs and other small RNAs between the conifers and the angiosperms indicates that important RNA silencing processes were highly developed in the earliest spermatophytes. Genomic mapping of all sequences to the O. sativa genome can be viewed at http://microrna.bcgsc.ca/cgi-bin/gbrowse/rice_build_3/.

  15. Identification and phylogenetic analysis of morphologically similar naked amoebae using small subunit ribosomal RNA.

    PubMed

    Sims, Gary P; Aitken, Robert; Rogerson, Andrew

    2002-01-01

    Fan-shaped, naked amoebae are commonly encountered in samples from freshwater and marine habitats suggesting that they are an important component of the microbial food web. However, there are considerable problems in both detecting these amoebae and identifying them, given their morphological similarity. In this study we used restriction analysis and partial sequence analysis of the small-subunit 18S ribosomal RNA gene to examine the phylogenetic relationships between nine "fan-shaped" Vannella and Platyamoeba species. The molecular phylogeny showed that the marine Vannella and Platyamoeba isolates are closely related, whereas the freshwater isolates are disparate. Thus, the current reliance on the fine structure of the cell coat (glycocalyx) used to separate these genera is not justified. The study also highlights sequence elements that might be targeted by fluorescent probes for the direct detection of these amoebae in field samples. The molecular data were also used to aid the identification of three unknown fan-shaped isolates. All three unknowns resembled Vannella or Platyamoeba. However, one of the strains (a small < 10 microm, benthic, fan-shaped amoeba) probably represents a new genus.

  16. Functional analysis of the sea urchin U7 small nuclear RNA

    SciTech Connect

    Gilmartin, G.M.; Schaufele, F.; Schaffner, G.; Birnstiel, M.L.

    1988-03-01

    U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. The authors analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The fist domain encompasses the 5'-terminal sequence, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing. Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome fo the historne mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.

  17. A systems biology approach for miRNA-mRNA expression patterns analysis in non-small cell lung cancer.

    PubMed

    Najafi, Ali; Tavallaei, Mahmood; Hosseini, Sayed Mostafa

    2016-01-01

    Non-small cell lung cancers (NSCLCs) is a prevalent and heterogeneous subtype of lung cancer accounting for 85 percent of patients. MicroRNAs (miRNAs), a class of small endogenous non-coding RNAs, incorporate into regulation of gene expression post-transcriptionally. Therefore, deregulation of miRNAs' expression has provided further layers of complexity to the molecular etiology and pathogenesis of different diseases and malignancies. Although, until now considerable number of studies has been carried out to illuminate this complexity in NSCLC, they have remained less effective in their goal due to lack of a holistic and integrative systems biology approach which considers all natural elaborations of miRNAs' function. It is able to reliably nominate most affected signaling pathways and therapeutic target genes by deregulated miRNAs during a particular pathological condition. Herein, we utilized a holistic systems biology approach, based on appropriate re-analyses of microarray datasets followed by reliable data filtering, to analyze integrative and combinatorial deregulated miRNA-mRNA interaction network in NSCLC, aiming to ascertain miRNA-dysregulated signaling pathway and potential therapeutic miRNAs and mRNAs which represent a lion' share during various aspects of NSCLC's pathogenesis. Our systems biology approach introduced and nominated 1) important deregulated miRNAs in NSCLCs compared with normal tissue 2) significant and confident deregulated mRNAs which were anti-correlatively targeted by deregulated miRNA in NSCLCs and 3) dysregulated signaling pathways in association with deregulated miRNA-mRNAs interactions in NSCLCs. These results introduce possible mechanism of function of deregulated miRNAs and mRNAs in NSCLC that could be used as potential therapeutic targets.

  18. Conformational readout of RNA by small ligands

    PubMed Central

    Kligun, Efrat; Mandel-Gutfreund, Yael

    2013-01-01

    RNA molecules have highly versatile structures that can fold into myriad conformations, providing many potential pockets for binding small molecules. The increasing number of available RNA structures, in complex with proteins, small ligands and in free form, enables the design of new therapeutically useful RNA-binding ligands. Here we studied RNA ligand complexes from 10 RNA groups extracted from the protein data bank (PDB), including adaptive and non-adaptive complexes. We analyzed the chemical, physical, structural and conformational properties of binding pockets around the ligand. Comparing the properties of ligand-binding pockets to the properties of computed pockets extracted from all available RNA structures and RNA-protein interfaces, revealed that ligand-binding pockets, mainly the adaptive pockets, are characterized by unique properties, specifically enriched in rare conformations of the nucleobase and the sugar pucker. Further, we demonstrate that nucleotides possessing the rare conformations are preferentially involved in direct interactions with the ligand. Overall, based on our comprehensive analysis of RNA-ligand complexes, we suggest that the unique conformations adopted by RNA nucleotides play an important role in RNA recognition by small ligands. We term the recognition of a binding site by a ligand via the unique RNA conformations “RNA conformational readout.” We propose that “conformational readout” is a general way by which RNA binding pockets are recognized and selected from an ensemble of different RNA states. PMID:23618839

  19. Biases in small RNA deep sequencing data

    PubMed Central

    Raabe, Carsten A.; Tang, Thean-Hock; Brosius, Juergen; Rozhdestvensky, Timofey S.

    2014-01-01

    High-throughput RNA sequencing (RNA-seq) is considered a powerful tool for novel gene discovery and fine-tuned transcriptional profiling. The digital nature of RNA-seq is also believed to simplify meta-analysis and to reduce background noise associated with hybridization-based approaches. The development of multiplex sequencing enables efficient and economic parallel analysis of gene expression. In addition, RNA-seq is of particular value when low RNA expression or modest changes between samples are monitored. However, recent data uncovered severe bias in the sequencing of small non-protein coding RNA (small RNA-seq or sRNA-seq), such that the expression levels of some RNAs appeared to be artificially enhanced and others diminished or even undetectable. The use of different adapters and barcodes during ligation as well as complex RNA structures and modifications drastically influence cDNA synthesis efficacies and exemplify sources of bias in deep sequencing. In addition, variable specific RNA G/C-content is associated with unequal polymerase chain reaction amplification efficiencies. Given the central importance of RNA-seq to molecular biology and personalized medicine, we review recent findings that challenge small non-protein coding RNA-seq data and suggest approaches and precautions to overcome or minimize bias. PMID:24198247

  20. Comparative Small RNA Analysis of Pollen Development in Autotetraploid and Diploid Rice.

    PubMed

    Li, Xiang; Shahid, Muhammad Qasim; Wu, Jinwen; Wang, Lan; Liu, Xiangdong; Lu, Yonggen

    2016-01-01

    MicroRNAs (miRNAs) play key roles in plant reproduction. However, knowledge on microRNAome analysis in autotetraploid rice is rather limited. Here, high-throughput sequencing technology was employed to analyze miRNAomes during pollen development in diploid and polyploid rice. A total of 172 differentially expressed miRNAs (DEM) were detected in autotetraploid rice compared to its diploid counterpart, and 57 miRNAs were specifically expressed in autotetraploid rice. Of the 172 DEM, 115 and 61 miRNAs exhibited up- and down-regulation, respectively. Gene Ontology analysis on the targets of up-regulated DEM showed that they were enriched in transport and membrane in pre-meiotic interphase, reproduction in meiosis, and nucleotide binding in single microspore stage. osa-miR5788 and osa-miR1432-5p_R+1 were up-regulated in meiosis and their targets revealed interaction with the meiosis-related genes, suggesting that they may involve in the genes regulation associated with the chromosome behavior. Abundant 24 nt siRNAs associated with transposable elements were found in autotetraploid rice during pollen development; however, they significantly declined in diploid rice, suggesting that 24 nt siRNAs may play a role in pollen development. These findings provide a foundation for understanding the effect of polyploidy on small RNA expression patterns during pollen development that cause pollen sterility in autotetraploid rice. PMID:27077850

  1. Comparative Small RNA Analysis of Pollen Development in Autotetraploid and Diploid Rice

    PubMed Central

    Li, Xiang; Shahid, Muhammad Qasim; Wu, Jinwen; Wang, Lan; Liu, Xiangdong; Lu, Yonggen

    2016-01-01

    MicroRNAs (miRNAs) play key roles in plant reproduction. However, knowledge on microRNAome analysis in autotetraploid rice is rather limited. Here, high-throughput sequencing technology was employed to analyze miRNAomes during pollen development in diploid and polyploid rice. A total of 172 differentially expressed miRNAs (DEM) were detected in autotetraploid rice compared to its diploid counterpart, and 57 miRNAs were specifically expressed in autotetraploid rice. Of the 172 DEM, 115 and 61 miRNAs exhibited up- and down-regulation, respectively. Gene Ontology analysis on the targets of up-regulated DEM showed that they were enriched in transport and membrane in pre-meiotic interphase, reproduction in meiosis, and nucleotide binding in single microspore stage. osa-miR5788 and osa-miR1432-5p_R+1 were up-regulated in meiosis and their targets revealed interaction with the meiosis-related genes, suggesting that they may involve in the genes regulation associated with the chromosome behavior. Abundant 24 nt siRNAs associated with transposable elements were found in autotetraploid rice during pollen development; however, they significantly declined in diploid rice, suggesting that 24 nt siRNAs may play a role in pollen development. These findings provide a foundation for understanding the effect of polyploidy on small RNA expression patterns during pollen development that cause pollen sterility in autotetraploid rice. PMID:27077850

  2. plantDARIO: web based quantitative and qualitative analysis of small RNA-seq data in plants.

    PubMed

    Patra, Deblina; Fasold, Mario; Langenberger, David; Steger, Gerhard; Grosse, Ivo; Stadler, Peter F

    2014-01-01

    High-throughput sequencing techniques have made it possible to assay an organism's entire repertoire of small non-coding RNAs (ncRNAs) in an efficient and cost-effective manner. The moderate size of small RNA-seq datasets makes it feasible to provide free web services to the research community that provide many basic features of a small RNA-seq analysis, including quality control, read normalization, ncRNA quantification, and the prediction of putative novel ncRNAs. DARIO is one such system that so far has been focussed on animals. Here we introduce an extension of this system to plant short non-coding RNAs (sncRNAs). It includes major modifications to cope with plant-specific sncRNA processing. The current version of plantDARIO covers analyses of mapping files, small RNA-seq quality control, expression analyses of annotated sncRNAs, including the prediction of novel miRNAs and snoRNAs from unknown expressed loci and expression analyses of user-defined loci. At present Arabidopsis thaliana, Beta vulgaris, and Solanum lycopersicum are covered. The web tool links to a plant specific visualization browser to display the read distribution of the analyzed sample. The easy-to-use platform of plantDARIO quantifies RNA expression of annotated sncRNAs from different sncRNA databases together with new sncRNAs, annotated by our group. The plantDARIO website can be accessed at http://plantdario.bioinf.uni-leipzig.de/. PMID:25566282

  3. plantDARIO: web based quantitative and qualitative analysis of small RNA-seq data in plants

    PubMed Central

    Patra, Deblina; Fasold, Mario; Langenberger, David; Steger, Gerhard; Grosse, Ivo; Stadler, Peter F.

    2014-01-01

    High-throughput sequencing techniques have made it possible to assay an organism's entire repertoire of small non-coding RNAs (ncRNAs) in an efficient and cost-effective manner. The moderate size of small RNA-seq datasets makes it feasible to provide free web services to the research community that provide many basic features of a small RNA-seq analysis, including quality control, read normalization, ncRNA quantification, and the prediction of putative novel ncRNAs. DARIO is one such system that so far has been focussed on animals. Here we introduce an extension of this system to plant short non-coding RNAs (sncRNAs). It includes major modifications to cope with plant-specific sncRNA processing. The current version of plantDARIO covers analyses of mapping files, small RNA-seq quality control, expression analyses of annotated sncRNAs, including the prediction of novel miRNAs and snoRNAs from unknown expressed loci and expression analyses of user-defined loci. At present Arabidopsis thaliana, Beta vulgaris, and Solanum lycopersicum are covered. The web tool links to a plant specific visualization browser to display the read distribution of the analyzed sample. The easy-to-use platform of plantDARIO quantifies RNA expression of annotated sncRNAs from different sncRNA databases together with new sncRNAs, annotated by our group. The plantDARIO website can be accessed at http://plantdario.bioinf.uni-leipzig.de/. PMID:25566282

  4. De novo transcriptome and small RNA analysis of two Chinese willow cultivars reveals stress response genes in Salix matsudana.

    PubMed

    Rao, Guodong; Sui, Jinkai; Zeng, Yanfei; He, Caiyun; Duan, Aiguo; Zhang, Jianguo

    2014-01-01

    Salix matsudana Koidz. is a deciduous, rapidly growing, and drought resistant tree and is one of the most widely distributed and commonly cultivated willow species in China. Currently little transcriptomic and small RNAomic data are available to reveal the genes involve in the stress resistant in S. matsudana. Here, we report the RNA-seq analysis results of both transcriptome and small RNAome data using Illumina deep sequencing of shoot tips from two willow variants(Salix. matsudana and Salix matsudana Koidz. cultivar 'Tortuosa'). De novo gene assembly was used to generate the consensus transcriptome and small RNAome, which contained 106,403 unique transcripts with an average length of 944 bp and a total length of 100.45 MB, and 166 known miRNAs representing 35 miRNA families. Comparison of transcriptomes and small RNAomes combined with quantitative real-time PCR from the two Salix libraries revealed a total of 292 different expressed genes(DEGs) and 36 different expressed miRNAs (DEMs). Among the DEGs and DEMs, 196 genes and 24 miRNAs were up regulated, 96 genes and 12 miRNA were down regulated in S. matsudana. Functional analysis of DEGs and miRNA targets showed that many genes were involved in stress resistance in S. matsudana. Our global gene expression profiling presents a comprehensive view of the transcriptome and small RNAome which provide valuable information and sequence resources for uncovering the stress response genes in S. matsudana. Moreover the transcriptome and small RNAome data provide a basis for future study of genetic resistance in Salix.

  5. De Novo Transcriptome and Small RNA Analysis of Two Chinese Willow Cultivars Reveals Stress Response Genes in Salix matsudana

    PubMed Central

    Zeng, Yanfei; He, Caiyun; Duan, Aiguo; Zhang, Jianguo

    2014-01-01

    Salix matsudana Koidz. is a deciduous, rapidly growing, and drought resistant tree and is one of the most widely distributed and commonly cultivated willow species in China. Currently little transcriptomic and small RNAomic data are available to reveal the genes involve in the stress resistant in S. matsudana. Here, we report the RNA-seq analysis results of both transcriptome and small RNAome data using Illumina deep sequencing of shoot tips from two willow variants(Salix. matsudana and Salix matsudana Koidz. cultivar ‘Tortuosa’). De novo gene assembly was used to generate the consensus transcriptome and small RNAome, which contained 106,403 unique transcripts with an average length of 944 bp and a total length of 100.45 MB, and 166 known miRNAs representing 35 miRNA families. Comparison of transcriptomes and small RNAomes combined with quantitative real-time PCR from the two Salix libraries revealed a total of 292 different expressed genes(DEGs) and 36 different expressed miRNAs (DEMs). Among the DEGs and DEMs, 196 genes and 24 miRNAs were up regulated, 96 genes and 12 miRNA were down regulated in S. matsudana. Functional analysis of DEGs and miRNA targets showed that many genes were involved in stress resistance in S. matsudana. Our global gene expression profiling presents a comprehensive view of the transcriptome and small RNAome which provide valuable information and sequence resources for uncovering the stress response genes in S. matsudana. Moreover the transcriptome and small RNAome data provide a basis for future study of genetic resistance in Salix. PMID:25275458

  6. Molecular analysis of lungworms from European bison (Bison bonasus) on the basis of small subunit ribosomal RNA gene (SSU).

    PubMed

    Pyziel, Anna M

    2014-03-01

    Dictyocaulosis (Nematoda: Trichostrongyloidea) is a widespread parasitosis of the European bison (Bison bonasus) inhabiting Bialowieza Primeval Forest. Bearing in mind the current coexistence of bison with wild cervids, and with domestic ruminants in the 19th and 20th century, the need arose for molecular identification of lungworm species. Molecular analysis was done on adult lungworms that were obtained from the respiratory track of four free-roaming bison euthanized as a part of the population health control program. As the result of the study four identical small subunit-ribosomal RNA gene sequences from the lungworms were obtained and deposited in GenBank as sequence, 1708 bp long (GenBank KC771250). Comparative analysis of the SSU rRNA sequences revealed the European bison to be a host for the bovine lungworm Dictyocaulus viviparus.

  7. Genome-wide Annotation, Identification, and Global Transcriptomic Analysis of Regulatory or Small RNA Gene Expression in Staphylococcus aureus

    PubMed Central

    Weiss, Andy; Broach, William H.; Wiemels, Richard E.; Mogen, Austin B.; Rice, Kelly C.

    2016-01-01

    ABSTRACT In Staphylococcus aureus, hundreds of small regulatory or small RNAs (sRNAs) have been identified, yet this class of molecule remains poorly understood and severely understudied. sRNA genes are typically absent from genome annotation files, and as a consequence, their existence is often overlooked, particularly in global transcriptomic studies. To facilitate improved detection and analysis of sRNAs in S. aureus, we generated updated GenBank files for three commonly used S. aureus strains (MRSA252, NCTC 8325, and USA300), in which we added annotations for >260 previously identified sRNAs. These files, the first to include genome-wide annotation of sRNAs in S. aureus, were then used as a foundation to identify novel sRNAs in the community-associated methicillin-resistant strain USA300. This analysis led to the discovery of 39 previously unidentified sRNAs. Investigating the genomic loci of the newly identified sRNAs revealed a surprising degree of inconsistency in genome annotation in S. aureus, which may be hindering the analysis and functional exploration of these elements. Finally, using our newly created annotation files as a reference, we perform a global analysis of sRNA gene expression in S. aureus and demonstrate that the newly identified tsr25 is the most highly upregulated sRNA in human serum. This study provides an invaluable resource to the S. aureus research community in the form of our newly generated annotation files, while at the same time presenting the first examination of differential sRNA expression in pathophysiologically relevant conditions. PMID:26861020

  8. Revised small subunit rRNA analysis provides further evidence that Foraminifera are related to Cercozoa.

    PubMed

    Berney, Cédric; Pawlowski, Jan

    2003-01-01

    There is accumulating evidence that the general shape of the ribosomal DNA-based phylogeny of Eukaryotes is strongly biased by the long-branch attraction phenomenon, leading to an artifactual basal clustering of groups that are probably highly derived. Among these groups, Foraminifera are of particular interest, because their deep phylogenetic position in ribosomal trees contrasts with their Cambrian appearance in the fossil record. A recent actin-based phylogeny of Eukaryotes has proposed that Foraminifera might be closely related to Cercozoa and, thus, branch among the so-called crown of Eukaryotes. Here, we reanalyze the small-subunit ribosomal RNA gene (SSU rDNA) phylogeny by removing all long-branching lineages that could artifactually attract foraminiferan sequences to the base of the tree. Our analyses reveal that Foraminifera branch together with the marine testate filosean Gromia oviformis as a sister group to Cercozoa, in agreement with actin phylogeny. Our study confirms the utility of SSU rDNA as a phylogenetic marker of megaevolutionary history, provided that the artifacts due to the heterogeneity of substitution rates in ribosomal genes are circumvented.

  9. The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs

    PubMed Central

    Hu, Hongtao; Rashotte, Aaron M.; Singh, Narendra K.; Weaver, David B.; Goertzen, Leslie R.; Singh, Shree R.; Locy, Robert D.

    2015-01-01

    MicroRNAs (miRNAs) and secondary small interfering RNAs (principally phased siRNAs or trans-acting siRNAs) are two distinct subfamilies of small RNAs (sRNAs) that are emerging as key regulators of posttranscriptional gene expression in plants. Both miRNAs and secondary-siRNAs (sec-siRNAs) are processed from longer RNA precursors by DICER-LIKE proteins (DCLs). Gossypium arboreum L., also known as tree cotton or Asian cotton, is a diploid, possibly ancestral relative of tetraploid Gossypium hirsutum L., the predominant type of commercially grown cotton worldwide known as upland cotton. To understand the biological significance of these gene regulators in G. arboreum, a bioinformatics analysis was performed on G. arboreum small RNAs produced from G. arboreum leaf, flower, and boll tissues. Consequently, 263 miRNAs derived from 353 precursors, including 155 conserved miRNAs (cs-miRNAs) and 108 novel lineage-specific miRNAs (ls-miRNAs). Along with miRNAs, 2,033 miRNA variants (isomiRNAs) were identified as well. Those isomiRNAs with variation at the 3’-miRNA end were expressed at the highest levels, compared to other types of variants. In addition, 755 pha-siRNAs derived 319 pha-siRNA gene transcripts (PGTs) were identified, and the potential pha-siRNA initiators were predicted. Also, 2,251 non-phased siRNAs were found as well, of which 1,088 appeared to be produced by so-called cis- or trans-cleavage of the PGTs observed at positions differing from pha-siRNAs. Of those sRNAs, 148 miRNAs/isomiRNAs and 274 phased/non-phased siRNAs were differentially expressed in one or more pairs of tissues examined. Target analysis revealed that target genes for both miRNAs and pha-siRNAs are involved a broad range of metabolic and enzymatic activities. We demonstrate that secondary siRNA production could result from initial cleavage of precursors by both miRNAs or isomiRNAs, and that subsequently produced phased and unphased siRNAs could result that also serve as triggers of a

  10. Gene Expression and MicroRNA Expression Analysis in Small Arteries of Spontaneously Hypertensive Rats. Evidence for ER Stress.

    PubMed

    Palao, Teresa; Swärd, Karl; Jongejan, Aldo; Moerland, Perry D; de Vos, Judith; van Weert, Angela; Arribas, Silvia M; Groma, Gergely; vanBavel, Ed; Bakker, Erik N T P

    2015-01-01

    Small arteries are known to develop functional and structural alterations in hypertension. However, the mechanisms of this remodeling are not fully understood. We hypothesized that altered gene expression is associated with the development of hypertension in mesenteric arteries of spontaneously hypertensive rats (SHR). Three sublines of SHR and normotensive Wistar Kyoto rats (WKY) were studied at 6 weeks and 5 months of age. MiRNA and mRNA microarray experiments were performed and analyzed with bioinformatical tools, including Ingenuity Pathway Analysis (IPA). Principal component analysis showed a clear separation in both miRNA and mRNA expression levels between both ages studied, demonstrating strong age-related changes in expression. At the miRNA level, IPA identified differences between SHR and WKY related to metabolic diseases, cellular growth, and proliferation. The mRNAs differentially expressed between SHR and WKY were related to metabolism, cellular movement and proliferation. The most strongly upregulated gene (9.2-fold) was thrombospondin 4 (Thbs4), a protein involved in the endoplasmic reticulum (ER) stress response that activates transcription factor 6α (ATF6α). ATF6α downstream targets were also differentially expressed in SHR vs. WKY. Differential expression of THBS4, the cleaved form of ATF6α, and two of its targets were further confirmed at the protein level by western blot. In summary, these data revealed a number of genes (n = 202) and miRNAs (n = 3) in mesenteric arteries of SHR that had not been related to hypertension previously. The most prominent of these, Thbs4, is related to vascular ER stress that is associated with hypertension.

  11. Characterization of the rDNA unit and sequence analysis of the small subunit rRNA and 5.8S rRNA genes from Tritrichomonas foetus.

    PubMed

    Chakrabarti, D; Dame, J B; Gutell, R R; Yowell, C A

    1992-05-01

    The ribosomal RNA gene unit of the protozoan parasite Tritrichomonas foetus has been cloned and analyzed. Southern blot analysis of the genomic DNA showed that the ribosomal RNA gene unit is organized as a tandem head to tail repeat with a unit length of 6 kb. By Northern analysis a primary transcript of 5.8 kb was detected. Copy number analysis showed the presence of 12 copies of the ribosomal RNA gene unit. The lengths of the small subunit ribosomal RNA and 5.8S ribosomal RNA are 1571 bp and 159 bp, respectively, as determined by sequence analysis. The T. foetus small subunit ribosomal RNA sequence is one of the shortest eukaryotic small subunit rRNA sequences, similar in length to those from 2 other amitochondrial protists. Although shorter than the majority of the eukaryotic small subunit ribosomal RNAs, this sequence maintains the primary and secondary structure common to all eukaryotic small subunit ribosomal RNA structures, while truncating sequences found within the eukaryotic variable regions. The length of the large subunit ribosomal RNA was measured at 2.5 kb.

  12. Analysis of small RNA production patterns among the two potato spindle tuber viroid variants in tomato plants.

    PubMed

    Adkar-Purushothama, Charith Raj; Perreault, Jean-Pierre; Sano, Teruo

    2015-12-01

    In order to analyze the production of small RNA (sRNA) by viroids upon infecting the plants, the tomato plants (Solanum lycopersicum cultivar Rutgers) were inoculated with the variants of Potato spindle tuber viroid (PSTVd). After 21-days of postinoculation, total RNA was extracted and subjected for deep-sequencing using Illumina HiSeq platform. The primers were trimmed and only 21- to 24-nt long sRNAs were filtered after quality check of the raw data. The filtered sRNA population was then mapped against both the genomic (+) and antigenomic (-) strands of the respective PSTVd variants using standard pattern-matching algorithm. The profiling of viroid derived sRNA (vd-sRNA) revealed that the viroids are susceptible to host RNA silencing mechanism. High-throughput sequence data linked to this project have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE69225.

  13. Analysis of the small RNA spf in the plant pathogen Pseudomonas syringae pv. tomato strain DC3000

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteria contain small non-coding RNAs (ncRNAs) that are typically responsible for altering transcription, translation, or mRNA stability. ncRNAs are important because they often regulate virulence factors and susceptibility to various stresses. Here, the regulation of a recently described ncRNA of ...

  14. Structure of the small ribosomal subunit RNA of the pulmonate snail, Limicolaria kambeul, and phylogenetic analysis of the Metazoa.

    PubMed

    Winnepennickx, B; Backeljau, T; van de Peer, Y; De Wachter, R

    1992-09-01

    The complete nucleotide sequence of the small ribosomal subunit RNA of the gastropod, Limicolaria kambeul, was determined and used to infer a secondary structure model. In order to clarify the phylogenetic position of the Mollusca among the Metazoa, an evolutionary tree was constructed by neighbor-joining, starting from an alignment of small ribosomal subunit RNA sequences. The Mollusca appear to be a monophyletic group, related to Arthropoda and Chordata in an unresolved trichotomy. PMID:1505675

  15. Comparative RNA-sequencing analysis of myocardial and circulating small RNAs in human heart failure and their utility as biomarkers.

    PubMed

    Akat, Kemal Marc; Moore-McGriff, D'Vesharronne; Morozov, Pavel; Brown, Miguel; Gogakos, Tasos; Correa Da Rosa, Joel; Mihailovic, Aleksandra; Sauer, Markus; Ji, Ruiping; Ramarathnam, Aarthi; Totary-Jain, Hana; Williams, Zev; Tuschl, Thomas; Schulze, P Christian

    2014-07-29

    Heart failure (HF) is associated with high morbidity and mortality and its incidence is increasing worldwide. MicroRNAs (miRNAs) are potential markers and targets for diagnostic and therapeutic applications, respectively. We determined myocardial and circulating miRNA abundance and its changes in patients with stable and end-stage HF before and at different time points after mechanical unloading by a left ventricular assist device (LVAD) by small RNA sequencing. miRNA changes in failing heart tissues partially resembled that of fetal myocardium. Consistent with prototypical miRNA-target-mRNA interactions, target mRNA levels were negatively correlated with changes in abundance for highly expressed miRNAs in HF and fetal hearts. The circulating small RNA profile was dominated by miRNAs, and fragments of tRNAs and small cytoplasmic RNAs. Heart- and muscle-specific circulating miRNAs (myomirs) increased up to 140-fold in advanced HF, which coincided with a similar increase in cardiac troponin I (cTnI) protein, the established marker for heart injury. These extracellular changes nearly completely reversed 3 mo following initiation of LVAD support. In stable HF, circulating miRNAs showed less than fivefold differences compared with normal, and myomir and cTnI levels were only captured near the detection limit. These findings provide the underpinning for miRNA-based therapies and emphasize the usefulness of circulating miRNAs as biomarkers for heart injury performing similar to established diagnostic protein biomarkers.

  16. PHYLOGENETIC ANALYSIS OF CRYPTOSPORIDIUM PARASITES BASED ON THE SMALL SUBUNIT RIBOSOMAL RNA GENE LOCUS

    EPA Science Inventory

    ABSTRACT
    Biologic data support the presence of multiple species in the genus Cryptosporidium, but
    a recent analysis of the available genetic data has suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxono...

  17. Analysis of U3 snoRNA and small subunit processome components in the parasitic protist Entamoeba histolytica.

    PubMed

    Srivastava, Ankita; Ahamad, Jamaluddin; Ray, Ashwini Kumar; Kaur, Devinder; Bhattacharya, Alok; Bhattacharya, Sudha

    2014-02-01

    In the early branching parasitic protist Entamoeba histolytica, pre-rRNA synthesis continues when cells are subjected to growth stress, but processing slows down and unprocessed pre-rRNA accumulates. To gain insight into the regulatory mechanisms leading to accumulation, it is necessary to define the pre-rRNA processing machinery in E. histolytica. We searched the E. histolytica genome sequence for homologs of the SSU processome, which contains the U3snoRNA, and 72 proteins in yeast. We could identify 57 of the proteins with high confidence. Of the rest, 6 were absent in human, and 4 were non-essential in yeast. The remaining 5 were absent in other parasite genomes as well. Analysis of U3snoRNA showed that the E. histolytica U3snoRNA adopted the same conserved secondary structure as seen in yeast and human. The predicted structure was verified by chemical modification followed by primer extension (SHAPE). Further we showed that the predicted interactions of Eh_U3snoRNA boxes A and A' with pre-18S rRNA were highly conserved both in position and sequence. The predicted interactions of 5'-hinge and 3'-hinge sequences of Eh_U3 snoRNA with the 5'-ETS sequences were conserved in position but not in sequence. Transcription of selected genes of SSU processome was tested by northern analysis, and transcripts of predicted sizes were obtained. During serum starvation, when unprocessed pre-RNA accumulated, the transcript levels of some of these genes declined. This is the first report on pre-rRNA processing machinery in E. histolytica, and shows that the components are well conserved with respect to yeast and human.

  18. Systematic analysis of plant mitochondrial and chloroplast small RNAs suggests organelle-specific mRNA stabilization mechanisms

    PubMed Central

    Ruwe, Hannes; Wang, Gongwei; Gusewski, Sandra; Schmitz-Linneweber, Christian

    2016-01-01

    Land plant organellar genomes encode a small number of genes, many of which are essential for respiration and photosynthesis. Organellar gene expression is characterized by a multitude of RNA processing events that lead to stable, translatable transcripts. RNA binding proteins (RBPs), have been shown to generate and protect transcript termini and eventually induce the accumulation of short RNA footprints. We applied knowledge of such RBP-derived footprints to develop software (sRNA miner) that enables identification of RBP footprints, or other clusters of small RNAs, in organelles. We used this tool to determine mitochondrial and chloroplast cosRNAs (clustered organellar sRNAs) in Arabidopsis. We found that in mitochondria, cosRNAs coincide with transcript 3′-ends, but are largely absent from 5′-ends. In chloroplasts this bias is absent, suggesting a different mode of 5′ processing, possibly owing to different sets of RNases. Furthermore, we identified a large number of cosRNAs that represent silenced insertions of mitochondrial DNA in the nuclear genome of Arabidopsis. Steady-state RNA analyses demonstrate that cosRNAs display differential accumulation during development. Finally, we demonstrate that the chloroplast RBP PPR10 associates in vivo with its cognate cosRNA. A hypothetical role of cosRNAs as competitors of mRNAs for PPR proteins is discussed. PMID:27235415

  19. Systematic analysis of plant mitochondrial and chloroplast small RNAs suggests organelle-specific mRNA stabilization mechanisms.

    PubMed

    Ruwe, Hannes; Wang, Gongwei; Gusewski, Sandra; Schmitz-Linneweber, Christian

    2016-09-01

    Land plant organellar genomes encode a small number of genes, many of which are essential for respiration and photosynthesis. Organellar gene expression is characterized by a multitude of RNA processing events that lead to stable, translatable transcripts. RNA binding proteins (RBPs), have been shown to generate and protect transcript termini and eventually induce the accumulation of short RNA footprints. We applied knowledge of such RBP-derived footprints to develop software (sRNA miner) that enables identification of RBP footprints, or other clusters of small RNAs, in organelles. We used this tool to determine mitochondrial and chloroplast cosRNAs (clustered organellar sRNAs) in Arabidopsis. We found that in mitochondria, cosRNAs coincide with transcript 3'-ends, but are largely absent from 5'-ends. In chloroplasts this bias is absent, suggesting a different mode of 5' processing, possibly owing to different sets of RNases. Furthermore, we identified a large number of cosRNAs that represent silenced insertions of mitochondrial DNA in the nuclear genome of Arabidopsis. Steady-state RNA analyses demonstrate that cosRNAs display differential accumulation during development. Finally, we demonstrate that the chloroplast RBP PPR10 associates in vivo with its cognate cosRNA. A hypothetical role of cosRNAs as competitors of mRNAs for PPR proteins is discussed. PMID:27235415

  20. SeqBuster, a bioinformatic tool for the processing and analysis of small RNAs datasets, reveals ubiquitous miRNA modifications in human embryonic cells.

    PubMed

    Pantano, Lorena; Estivill, Xavier; Martí, Eulàlia

    2010-03-01

    High-throughput sequencing technologies enable direct approaches to catalog and analyze snapshots of the total small RNA content of living cells. Characterization of high-throughput sequencing data requires bioinformatic tools offering a wide perspective of the small RNA transcriptome. Here we present SeqBuster, a highly versatile and reliable web-based toolkit to process and analyze large-scale small RNA datasets. The high flexibility of this tool is illustrated by the multiple choices offered in the pre-analysis for mapping purposes and in the different analysis modules for data manipulation. To overcome the storage capacity limitations of the web-based tool, SeqBuster offers a stand-alone version that permits the annotation against any custom database. SeqBuster integrates multiple analyses modules in a unique platform and constitutes the first bioinformatic tool offering a deep characterization of miRNA variants (isomiRs). The application of SeqBuster to small-RNA datasets of human embryonic stem cells revealed that most miRNAs present different types of isomiRs, some of them being associated to stem cell differentiation. The exhaustive description of the isomiRs provided by SeqBuster could help to identify miRNA-variants that are relevant in physiological and pathological processes. SeqBuster is available at http://estivill_lab.crg.es/seqbuster.

  1. SeqBuster, a bioinformatic tool for the processing and analysis of small RNAs datasets, reveals ubiquitous miRNA modifications in human embryonic cells.

    PubMed

    Pantano, Lorena; Estivill, Xavier; Martí, Eulàlia

    2010-03-01

    High-throughput sequencing technologies enable direct approaches to catalog and analyze snapshots of the total small RNA content of living cells. Characterization of high-throughput sequencing data requires bioinformatic tools offering a wide perspective of the small RNA transcriptome. Here we present SeqBuster, a highly versatile and reliable web-based toolkit to process and analyze large-scale small RNA datasets. The high flexibility of this tool is illustrated by the multiple choices offered in the pre-analysis for mapping purposes and in the different analysis modules for data manipulation. To overcome the storage capacity limitations of the web-based tool, SeqBuster offers a stand-alone version that permits the annotation against any custom database. SeqBuster integrates multiple analyses modules in a unique platform and constitutes the first bioinformatic tool offering a deep characterization of miRNA variants (isomiRs). The application of SeqBuster to small-RNA datasets of human embryonic stem cells revealed that most miRNAs present different types of isomiRs, some of them being associated to stem cell differentiation. The exhaustive description of the isomiRs provided by SeqBuster could help to identify miRNA-variants that are relevant in physiological and pathological processes. SeqBuster is available at http://estivill_lab.crg.es/seqbuster. PMID:20008100

  2. A rapid and sensitive non-radioactive method applicable for genome-wide analysis of Saccharomyces cerevisiae genes involved in small RNA biology.

    PubMed

    Wu, Jingyan; Huang, Hsiao-Yun; Hopper, Anita K

    2013-04-01

    Conventional isolation and detection methods for small RNAs from yeast cells have been designed for a limited number of samples. In order to be able to conduct a genome-wide assessment of how each gene product impacts upon small RNAs, we developed a rapid method for analysing small RNAs from Saccharomyces cerevisiae wild-type (wt) and mutants cells in the deletion and temperature-sensitive (ts) collections. Our method implements three optimized techniques: a procedure for growing small yeast cultures in 96-deepwell plates, a fast procedure for small RNA isolation from the plates, and a sensitive non-radioactive northern method for RNA detection. The RNA isolation procedure requires only 4 h for processing 96 samples, is highly reproducible and yields RNA of good quality and quantity. The non-radioactive northern method employs digoxigenin (DIG)-labelled DNA probes and chemiluminescence. It detects femtomole levels of small RNAs within 1 min exposure time. We minimized the processing time for large-scale analysis and optimized the stripping and reprobing procedures for analyses of multiple RNAs from a single membrane. The method described is rapid, sensitive, safe and cost-effective for genome-wide screens of novel genes involved in the biogenesis, subcellular trafficking and stability of small RNAs. Moreover, it will be useful to educational laboratory class venues and to research institutions with limited access to radioisotopes or robots.

  3. A small RNA targets pokeweed antiviral protein transcript.

    PubMed

    Klenov, Alexander; Neller, Kira C M; Burns, Lydia A; Krivdova, Gabriela; Hudak, Katalin A

    2016-03-01

    Ribosome-inactivating proteins (RIPs) are a class of plant defense proteins with N-glycosidase activity (EC 3.2.2.22). Pokeweed antiviral protein (PAP) is a Type I RIP isolated from the pokeweed plant, Phytolacca americana, thought to confer broad-spectrum virus resistance in this plant. Through a combination of standard molecular techniques and RNA sequencing analysis, we report here that a small RNA binds and cleaves the open reading frame of PAP mRNA. Additionally, sRNA targeting of PAP is dependent on jasmonic acid (JA), a plant hormone important for defense against pathogen infection and herbivory. Levels of small RNA increased with JA treatment, as did levels of PAP mRNA and protein, suggesting that the small RNA functions to moderate the expression of PAP in response to this hormone. The association between JA and PAP expression, mediated by sRNA299, situates PAP within a signaling pathway initiated by biotic stress. The consensus sequence of sRNA299 was obtained through bioinformatic analysis of pokeweed small RNA sequencing. To our knowledge, this is the first account of a sRNA targeting a RIP gene.

  4. Genome-wide analysis of small nucleolar RNAs of Leishmania major reveals a rich repertoire of RNAs involved in modification and processing of rRNA

    PubMed Central

    Eliaz, Dror; Doniger, Tirza; Tkacz, Itai Dov; Biswas, Viplov Kumar; Gupta, Sachin Kumar; Kolev, Nikolay G; Unger, Ron; Ullu, Elisabetta; Tschudi, Christian; Michaeli, Shulamit

    2015-01-01

    Trypanosomatids are protozoan parasites and the causative agent of infamous infectious diseases. These organisms regulate their gene expression mainly at the post-transcriptional level and possess characteristic RNA processing mechanisms. In this study, we analyzed the complete repertoire of Leishmania major small nucleolar (snoRNA) RNAs by performing RNA-seq analysis on RNAs that were affinity-purified using the C/D snoRNA core protein, SNU13, and the H/ACA core protein, NHP2. This study revealed a large collection of C/D and H/ACA snoRNAs, organized in gene clusters generally containing both snoRNA types. Abundant snoRNAs were identified and predicted to guide trypanosome-specific rRNA cleavages. The repertoire of snoRNAs was compared to that of the closely related Trypanosoma brucei, and 80% of both C/D and H/ACA molecules were found to have functional homologues. The comparative analyses elucidated how snoRNAs evolved to generate molecules with analogous functions in both species. Interestingly, H/ACA RNAs have great flexibility in their ability to guide modifications, and several of the RNA species can guide more than one modification, compensating for the presence of single hairpin H/ACA snoRNA in these organisms. Placing the predicted modifications on the rRNA secondary structure revealed hypermodification regions mostly in domains which are modified in other eukaryotes, in addition to trypanosome-specific modifications. PMID:25970223

  5. Analysis of Serum microRNA Expression Profiles and Comparison with Small Intestinal microRNA Expression Profiles in Weaned Piglets.

    PubMed

    Tao, Xin; Xu, Ziwei; Men, Xiaoming

    2016-01-01

    Weaning stress induces tissue injuries and impairs health and growth in piglets, especially during the first week post-weaning. MicroRNAs (miRNAs) play vital roles in regulating stresses and diseases. Our previous study found multiple differentially expressed miRNAs in small intestine of piglets at four days post-weaning. To better understand the roles of miRNAs during weaning stress, we analyzed the serum miRNA expressional profile in weaned piglets (at four days post-weaning) and in suckling piglets (control) of the same age using miRNA microarray technology. We detected a total of 300 expressed miRNAs, 179 miRNAs of which were differentially expressed between the two groups. The miRNA microarray results were validated by RT-qPCR. The biological functions of these differentially expressed miRNAs were predicted by GO terms and KEGG pathway annotations. We identified 10 highly expressed miRNAs in weaned piglets including miR-31, miR-205, and miR-21 (upregulated) and miR-144, miR-30c-5p, miR-363, miR-194a, miR-186, miR-150, and miR-194b-5p (downregulated). Additionally, miR-194b-5p expression was significantly downregulated in serum and small intestine of weaned piglets. Our results suggest that weaning stress affects serum miRNA profiles in piglets. And serum miR-194b-5p levels can reflect its expressional changes in small intestine of piglets by weaning stress. PMID:27632531

  6. Analysis of Serum microRNA Expression Profiles and Comparison with Small Intestinal microRNA Expression Profiles in Weaned Piglets

    PubMed Central

    Tao, Xin; Xu, Ziwei; Men, Xiaoming

    2016-01-01

    Weaning stress induces tissue injuries and impairs health and growth in piglets, especially during the first week post-weaning. MicroRNAs (miRNAs) play vital roles in regulating stresses and diseases. Our previous study found multiple differentially expressed miRNAs in small intestine of piglets at four days post-weaning. To better understand the roles of miRNAs during weaning stress, we analyzed the serum miRNA expressional profile in weaned piglets (at four days post-weaning) and in suckling piglets (control) of the same age using miRNA microarray technology. We detected a total of 300 expressed miRNAs, 179 miRNAs of which were differentially expressed between the two groups. The miRNA microarray results were validated by RT-qPCR. The biological functions of these differentially expressed miRNAs were predicted by GO terms and KEGG pathway annotations. We identified 10 highly expressed miRNAs in weaned piglets including miR-31, miR-205, and miR-21 (upregulated) and miR-144, miR-30c-5p, miR-363, miR-194a, miR-186, miR-150, and miR-194b-5p (downregulated). Additionally, miR-194b-5p expression was significantly downregulated in serum and small intestine of weaned piglets. Our results suggest that weaning stress affects serum miRNA profiles in piglets. And serum miR-194b-5p levels can reflect its expressional changes in small intestine of piglets by weaning stress. PMID:27632531

  7. Argonaute: The executor of small RNA function.

    PubMed

    Azlan, Azali; Dzaki, Najat; Azzam, Ghows

    2016-08-20

    The discovery of small non-coding RNAs - microRNA (miRNA), short interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) - represents one of the most exciting frontiers in biology specifically on the mechanism of gene regulation. In order to execute their functions, these small RNAs require physical interactions with their protein partners, the Argonaute (AGO) family proteins. Over the years, numerous studies have made tremendous progress on understanding the roles of AGO in gene silencing in various organisms. In this review, we summarize recent progress of AGO-mediated gene silencing and other cellular processes in which AGO proteins have been implicated with a particular focus on progress made in flies, humans and other model organisms as compliment. PMID:27569398

  8. Conservation of small RNA pathways in platypus.

    PubMed

    Murchison, Elizabeth P; Kheradpour, Pouya; Sachidanandam, Ravi; Smith, Carly; Hodges, Emily; Xuan, Zhenyu; Kellis, Manolis; Grützner, Frank; Stark, Alexander; Hannon, Gregory J

    2008-06-01

    Small RNA pathways play evolutionarily conserved roles in gene regulation and defense from parasitic nucleic acids. The character and expression patterns of small RNAs show conservation throughout animal lineages, but specific animal clades also show variations on these recurring themes, including species-specific small RNAs. The monotremes, with only platypus and four species of echidna as extant members, represent the basal branch of the mammalian lineage. Here, we examine the small RNA pathways of monotremes by deep sequencing of six platypus and echidna tissues. We find that highly conserved microRNA species display their signature tissue-specific expression patterns. In addition, we find a large rapidly evolving cluster of microRNAs on platypus chromosome X1, which is unique to monotremes. Platypus and echidna testes contain a robust Piwi-interacting (piRNA) system, which appears to be participating in ongoing transposon defense. PMID:18463306

  9. The potential of circulating extracellular small RNAs (smexRNA) in veterinary diagnostics—Identifying biomarker signatures by multivariate data analysis

    PubMed Central

    Melanie, Spornraft; Benedikt, Kirchner; Pfaffl, Michael W.; Irmgard, Riedmaier

    2015-01-01

    Worldwide growth and performance-enhancing substances are used in cattle husbandry to increase productivity. In certain countries however e.g., in the EU, these practices are forbidden to prevent the consumers from potential health risks of substance residues in food. To maximize economic profit, ‘black sheep‘ among farmers might circumvent the detection methods used in routine controls, which highlights the need for an innovative and reliable detection method. Transcriptomics is a promising new approach in the discovery of veterinary medicine biomarkers and also a missing puzzle piece, as up to date, metabolomics and proteomics are paramount. Due to increased stability and easy sampling, circulating extracellular small RNAs (smexRNAs) in bovine plasma were small RNA-sequenced and their potential to serve as biomarker candidates was evaluated using multivariate data analysis tools. After running the data evaluation pipeline, the proportion of miRNAs (microRNAs) and piRNAs (PIWI-interacting small non-coding RNAs) on the total sequenced reads was calculated. Additionally, top 10 signatures were compared which revealed that the readcount data sets were highly affected by the most abundant miRNA and piRNA profiles. To evaluate the discriminative power of multivariate data analyses to identify animals after veterinary drug application on the basis of smexRNAs, OPLS-DA was performed. In summary, the quality of miRNA models using all mapped reads for both treatment groups (animals treated with steroid hormones or the β-agonist clenbuterol) is predominant to those generated with combined data sets or piRNAs alone. Using multivariate projection methodologies like OPLS-DA have proven the best potential to generate discriminative miRNA models, supported by small RNA-Seq data. Based on the presented comparative OPLS-DA, miRNAs are the favorable smexRNA biomarker candidates in the research field of veterinary drug abuse. PMID:27077039

  10. The potential of circulating extracellular small RNAs (smexRNA) in veterinary diagnostics-Identifying biomarker signatures by multivariate data analysis.

    PubMed

    Melanie, Spornraft; Benedikt, Kirchner; Pfaffl, Michael W; Irmgard, Riedmaier

    2015-09-01

    Worldwide growth and performance-enhancing substances are used in cattle husbandry to increase productivity. In certain countries however e.g., in the EU, these practices are forbidden to prevent the consumers from potential health risks of substance residues in food. To maximize economic profit, 'black sheep' among farmers might circumvent the detection methods used in routine controls, which highlights the need for an innovative and reliable detection method. Transcriptomics is a promising new approach in the discovery of veterinary medicine biomarkers and also a missing puzzle piece, as up to date, metabolomics and proteomics are paramount. Due to increased stability and easy sampling, circulating extracellular small RNAs (smexRNAs) in bovine plasma were small RNA-sequenced and their potential to serve as biomarker candidates was evaluated using multivariate data analysis tools. After running the data evaluation pipeline, the proportion of miRNAs (microRNAs) and piRNAs (PIWI-interacting small non-coding RNAs) on the total sequenced reads was calculated. Additionally, top 10 signatures were compared which revealed that the readcount data sets were highly affected by the most abundant miRNA and piRNA profiles. To evaluate the discriminative power of multivariate data analyses to identify animals after veterinary drug application on the basis of smexRNAs, OPLS-DA was performed. In summary, the quality of miRNA models using all mapped reads for both treatment groups (animals treated with steroid hormones or the β-agonist clenbuterol) is predominant to those generated with combined data sets or piRNAs alone. Using multivariate projection methodologies like OPLS-DA have proven the best potential to generate discriminative miRNA models, supported by small RNA-Seq data. Based on the presented comparative OPLS-DA, miRNAs are the favorable smexRNA biomarker candidates in the research field of veterinary drug abuse.

  11. Flow Analysis of Amino Acids by Using a Newly Developed Aminoacyl-tRNA Synthetase-Immobilized, Small Reactor Column-Based Assay.

    PubMed

    Kugimiya, Akimitsu; Konishi, Hidenori; Fukada, Rie

    2016-03-01

    Abnormal concentrations of amino acids in blood and urine can be indicative of several diseases, including cancer and diabetes. Therefore, analyses that examine amino acid concentrations are useful for the diagnosis of such diseases. In this study, we developed an enzyme-immobilized, small reactor column for flow analysis of amino acid concentrations. For the recognition of asparagine and lysine, asparaginyl-tRNA synthetase and lysyl-tRNA synthase were immobilized onto microparticles, respectively, and coupled with coloration reagents for spectrophotometric detection. This assay has some advantages in the analytical field, such as the ability to detect small amounts of analyte, allowing for the use of a small reaction volume, and ensuring a rapid and efficient reaction rate. This approach provided selective quantitation of up to 480 μM of asparagine and lysine in 200 mM Tris-HCl buffer (pH 8.0).

  12. Flow Analysis of Amino Acids by Using a Newly Developed Aminoacyl-tRNA Synthetase-Immobilized, Small Reactor Column-Based Assay.

    PubMed

    Kugimiya, Akimitsu; Konishi, Hidenori; Fukada, Rie

    2016-03-01

    Abnormal concentrations of amino acids in blood and urine can be indicative of several diseases, including cancer and diabetes. Therefore, analyses that examine amino acid concentrations are useful for the diagnosis of such diseases. In this study, we developed an enzyme-immobilized, small reactor column for flow analysis of amino acid concentrations. For the recognition of asparagine and lysine, asparaginyl-tRNA synthetase and lysyl-tRNA synthase were immobilized onto microparticles, respectively, and coupled with coloration reagents for spectrophotometric detection. This assay has some advantages in the analytical field, such as the ability to detect small amounts of analyte, allowing for the use of a small reaction volume, and ensuring a rapid and efficient reaction rate. This approach provided selective quantitation of up to 480 μM of asparagine and lysine in 200 mM Tris-HCl buffer (pH 8.0). PMID:26554858

  13. Small RNA combination therapy for lung cancer

    PubMed Central

    Xue, Wen; Dahlman, James E.; Tammela, Tuomas; Khan, Omar F.; Sood, Sabina; Dave, Apeksha; Cai, Wenxin; Chirino, Leilani M.; Yang, Gillian R.; Bronson, Roderick; Crowley, Denise G.; Sahay, Gaurav; Schroeder, Avi; Langer, Robert; Anderson, Daniel G.; Jacks, Tyler

    2014-01-01

    MicroRNAs (miRNAs) and siRNAs have enormous potential as cancer therapeutics, but their effective delivery to most solid tumors has been difficult. Here, we show that a new lung-targeting nanoparticle is capable of delivering miRNA mimics and siRNAs to lung adenocarcinoma cells in vitro and to tumors in a genetically engineered mouse model of lung cancer based on activation of oncogenic Kirsten rat sarcoma viral oncogene homolog (Kras) and loss of p53 function. Therapeutic delivery of miR-34a, a p53-regulated tumor suppressor miRNA, restored miR-34a levels in lung tumors, specifically down-regulated miR-34a target genes, and slowed tumor growth. The delivery of siRNAs targeting Kras reduced Kras gene expression and MAPK signaling, increased apoptosis, and inhibited tumor growth. The combination of miR-34a and siRNA targeting Kras improved therapeutic responses over those observed with either small RNA alone, leading to tumor regression. Furthermore, nanoparticle-mediated small RNA delivery plus conventional, cisplatin-based chemotherapy prolonged survival in this model compared with chemotherapy alone. These findings demonstrate that RNA combination therapy is possible in an autochthonous model of lung cancer and provide preclinical support for the use of small RNA therapies in patients who have cancer. PMID:25114235

  14. Analysis of Epstein-Barr virus-encoded small RNA 1 expression in benign lymphoepithelial salivary gland lesions.

    PubMed

    DiGiuseppe, J A; Wu, T C; Corio, R L

    1994-06-01

    Salivary gland enlargement resulting from benign lymphoepithelial lesions may be seen in patients with Sjögren's syndrome (SS) and in the early stages of human immunodeficiency virus (HIV) infection. Although the pathogenesis of these lesions is thought to differ in SS and HIV infection, Epstein-Barr virus (EBV) has been suggested as a pathogenetic agent in both cases. We have assessed the presence of latent EBV infection in a series of 15 lymphoepithelial salivary gland lesions using RNA-RNA in situ hybridization with digoxigenin-labeled riboprobes complementary to the abundantly-expressed EBV-encoded small RNA 1 (EBER1). Two of four benign lymphoepithelial lesions (BLEL) from patients seropositive for HIV expressed EBER1 in lymphocyte nuclei in a fashion similar to that described previously in HIV-associated persistent generalized lymphadenopathy (PGL). By contrast, EBER1 was not expressed in any of four BLEL from patients with SS, or seven lymphoepithelial cysts (LEC) and BLEL from patients with neither HIV infection nor SS. These data suggest that latent EBV infection does not play a major pathogenetic role in the lymphoepithelial salivary gland lesions associated with SS or those seen in patients without systemic disease. In the case of HIV-associated salivary gland lesions, the data are consistent with earlier proposals that these BLEL result from the involvement of intraparotid lymph nodes by PGL.

  15. Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect

    PubMed Central

    Veldhoen, Sandra; Laufer, Sandra D.; Trampe, Alexander; Restle, Tobias

    2006-01-01

    Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGα, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGα-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC50 value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGα/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGα/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGα/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGα-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed. PMID:17135188

  16. Genome-Wide Small RNA Analysis of Soybean Reveals Auxin-Responsive microRNAs that are Differentially Expressed in Response to Salt Stress in Root Apex

    PubMed Central

    Sun, Zhengxi; Wang, Youning; Mou, Fupeng; Tian, Yinping; Chen, Liang; Zhang, Senlei; Jiang, Qiong; Li, Xia

    2016-01-01

    Root growth and the architecture of the root system in Arabidopsis are largely determined by root meristematic activity. Legume roots show strong developmental plasticity in response to both abiotic and biotic stimuli, including symbiotic rhizobia. However, a global analysis of gene regulation in the root meristem of soybean plants is lacking. In this study, we performed a global analysis of the small RNA transcriptome of root tips from soybean seedlings grown under normal and salt stress conditions. In total, 71 miRNA candidates, including known and novel variants of 59 miRNA families, were identified. We found 66 salt-responsive miRNAs in the soybean root meristem; among them, 22 are novel miRNAs. Interestingly, we found auxin-responsive cis-elements in the promoters of many salt-responsive miRNAs, implying that these miRNAs may be regulated by auxin and auxin signaling plays a key role in regulating the plasticity of the miRNAome and root development in soybean. A functional analysis of miR399, a salt-responsive miRNA in the root meristem, indicates the crucial role of this miRNA in modulating soybean root developmental plasticity. Our data provide novel insight into the miRNAome-mediated regulatory mechanism in soybean root growth under salt stress. PMID:26834773

  17. Polymers in Small-Interfering RNA Delivery

    PubMed Central

    Singha, Kaushik; Namgung, Ran

    2011-01-01

    This review will cover the current strategies that are being adopted to efficiently deliver small interfering RNA using nonviral vectors, including the use of polymers such as polyethylenimine, poly(lactic-co-glycolic acid), polypeptides, chitosan, cyclodextrin, dendrimers, and polymers-containing different nanoparticles. The article will provide a brief and concise account of underlying principle of these polymeric vectors and their structural and functional modifications which were intended to serve different purposes to affect efficient therapeutic outcome of small-interfering RNA delivery. The modifications of these polymeric vectors will be discussed with reference to stimuli-responsiveness, target specific delivery, and incorporation of nanoconstructs such as carbon nanotubes, gold nanoparticles, and silica nanoparticles. The emergence of small-interfering RNA as the potential therapeutic agent and its mode of action will also be mentioned in a nutshell. PMID:21749290

  18. Functional analysis of microRNA and transcription factor synergistic regulatory network based on identifying regulatory motifs in non-small cell lung cancer

    PubMed Central

    2013-01-01

    Background Lung cancer, especially non-small cell lung cancer, is a leading cause of malignant tumor death worldwide. Understanding the mechanisms employed by the main regulators, such as microRNAs (miRNAs) and transcription factors (TFs), still remains elusive. The patterns of their cooperation and biological functions in the synergistic regulatory network have rarely been studied. Results Here, we describe the first miRNA-TF synergistic regulation network in human lung cancer. We identified important regulators (MYC, NFKB1, miR-590, and miR-570) and significant miRNA-TF synergistic regulatory motifs by random simulations. The two most significant motifs were the co-regulation of miRNAs and TFs, and TF-mediated cascade regulation. We also developed an algorithm to uncover the biological functions of the human lung cancer miRNA-TF synergistic regulatory network (regulation of apoptosis, cellular protein metabolic process, and cell cycle), and the specific functions of each miRNA-TF synergistic subnetwork. We found that the miR-17 family exerted important effects in the regulation of non-small cell lung cancer, such as in proliferation and cell cycle regulation by targeting the retinoblastoma protein (RB1) and forming a feed forward loop with the E2F1 TF. We proposed a model for the miR-17 family, E2F1, and RB1 to demonstrate their potential roles in the occurrence and development of non-small cell lung cancer. Conclusions This work will provide a framework for constructing miRNA-TF synergistic regulatory networks, function analysis in diseases, and identification of the main regulators and regulatory motifs, which will be useful for understanding the putative regulatory motifs involving miRNAs and TFs, and for predicting new targets for cancer studies. PMID:24200043

  19. The unusually long small subunit ribosomal RNA of Phreatamoeba balamuthi.

    PubMed Central

    Hinkle, G; Leipe, D D; Nerad, T A; Sogin, M L

    1994-01-01

    The small subunit ribosomal RNA (rRNA) of the anaerobic amoeba Phreatamoeba balamuthi is the longest 16S-like rRNA sequenced to date. Secondary structure analysis suggests that the additional sequence is incorporated in canonical eukaryotic expansion regions and is not due to the presence of introns. Reverse transcriptase sequencing of total RNA extracts confirmed that two uncommonly long expansion regions are present in native P. balamuthi 16S-like rRNA. Primary sequence comparison and similar secondary structure indicate a 61 base stem and loop repeat within an expansion region; a mechanism whereby the repeat may have been incorporated is presented. P. balamuthi provides further evidence that 16S-like rRNA length does not correlate with phylogenetic position. PMID:8127686

  20. Small RNA pathways in Schmidtea mediterranea.

    PubMed

    Resch, Alissa M; Palakodeti, Dasaradhi

    2012-01-01

    Planarians are bilaterally symmetrical fresh water organisms capable of regenerating body parts from small fragments following bodily injury. Planarians possess a specialized population of pluripotent cells called neoblasts, which are responsible for their unique regenerative ability. The study of planarian stem cell biology and regeneration has traditionally focused on the transcription factors and proteins that regulate signal transduction pathways. New evidence shows that small RNA molecules are important players in stem cell function and regeneration, yet little is known about the exact nature of their regulatory roles during the regenerative process. In this review, we discuss biogenesis of microRNAs and piwiRNAs and their functional role in key developmental pathways in vertebrates and invertebrates with an emphasis on recent studies on planarian small RNA pathways.

  1. miRNA, siRNA, piRNA and argonautes: news in small matters.

    PubMed

    Riedmann, Lucia T; Schwentner, Raphaela

    2010-01-01

    Since the discovery of the first microRNA (miRNA) family member lin-4 in Caenorhabditis elegans by Lee et al. and RNA interference (RNAi) by Andrew Fire and his colleagues in the 1990s, the new field of regulatory non-coding RNAs has enormously gained momentum and importance. Small regulatory RNAs comprise small interfering RNAs (siRNAs), miRNAs and Piwi-associated small RNAs (piRNAs). Generated from double-stranded RNAs (dsRNAs), siRNAs trigger sequence-specific mRNA decay also known as RNA interference (RNAi). miRNAs in association with Argonaute (AGO ) and GW182 proteins, forming the RNA-induced silencing complex (RISC), mediate fine tuning of gene expression and are involved in various biological key processes. An estimate of 500-1,000 miRNA genes exist in vertebrates and plants and about 100 in invertebrates. Each miRNA is predicted to target hundreds of mRNAs thus influencing key regulatory mechanisms of the cell. Consequently, deregulated miRNA expression has been suggested to contribute to the initiation and progression of human cancer and other diseases. piRNAs associated with Piwi proteins protect the animal germline from mobile genetic elements, thereby acting as a small RNA-based immune system. PMID:20200493

  2. Small non-coding RNA deregulation in endometrial carcinogenesis.

    PubMed

    Ravo, Maria; Cordella, Angela; Rinaldi, Antonio; Bruno, Giuseppina; Alexandrova, Elena; Saggese, Pasquale; Nassa, Giovanni; Giurato, Giorgio; Tarallo, Roberta; Marchese, Giovanna; Rizzo, Francesca; Stellato, Claudia; Biancardi, Rossella; Troisi, Jacopo; Di Spiezio Sardo, Attilio; Zullo, Fulvio; Weisz, Alessandro; Guida, Maurizio

    2015-03-10

    Small non-coding RNAs (sncRNAs) represent a heterogeneous group of <200nt-long transcripts comprising microRNAs, PIWI-interacting RNAs (piRNAs) and small-nucleolar-RNAs (snoRNAs) involved in physiological and pathological processes such as carcinogenesis and tumor progression. Aberrant sncRNA expression in cancer has been associated with specific clinical phenotypes, grading, staging, metastases development and resistance to therapy.Aim of the present work is to study the role of sncRNAs in endometrial carcinogenesis. Changes in sncRNA expression were identified by high-throughput genomic analysis of paired normal, hyperplastic and cancerous endometrial tissues obtained by endometrial biopsies (n = 10). Using smallRNA sequencing and microarrays we identified significant differences in sncRNA expression pattern between normal, hyperplastic and neoplastic endometrium. This led to the definition of a sncRNA signature (129 microRNAs, 2 of which not previously described, 10 piRNAs and 3 snoRNAs) of neoplastic transformation. Functional bioinformatics analysis identified as downstream targets multiple signaling pathways potentially involved in the hyperplastic and neoplastic tissue responses, including Wnt/β-catenin, and ERK/MAPK and TGF-β-Signaling.Considering the regulatory role of sncRNAs, this newly identified sncRNA signature is likely to reflect the events leading to endometrial cancer, which can be exploited to dissect the carcinogenic process including novel biomarkers for early and non-invasive diagnosis of these tumors. PMID:25686835

  3. Small non-coding RNA deregulation in endometrial carcinogenesis

    PubMed Central

    Ravo, Maria; Cordella, Angela; Rinaldi, Antonio; Bruno, Giuseppina; Alexandrova, Elena; Saggese, Pasquale; Nassa, Giovanni; Giurato, Giorgio; Tarallo, Roberta; Marchese, Giovanna; Rizzo, Francesca; Stellato, Claudia; Biancardi, Rossella; Troisi, Jacopo; Di Spiezio Sardo, Attilio; Zullo, Fulvio; Weisz, Alessandro; Guida, Maurizio

    2015-01-01

    Small non-coding RNAs (sncRNAs) represent a heterogeneous group of <200nt-long transcripts comprising microRNAs, PIWI-interacting RNAs (piRNAs) and small-nucleolar-RNAs (snoRNAs) involved in physiological and pathological processes such as carcinogenesis and tumor progression. Aberrant sncRNA expression in cancer has been associated with specific clinical phenotypes, grading, staging, metastases development and resistance to therapy. Aim of the present work is to study the role of sncRNAs in endometrial carcinogenesis. Changes in sncRNA expression were identified by high-throughput genomic analysis of paired normal, hyperplastic and cancerous endometrial tissues obtained by endometrial biopsies (n = 10). Using smallRNA sequencing and microarrays we identified significant differences in sncRNA expression pattern between normal, hyperplastic and neoplastic endometrium. This led to the definition of a sncRNA signature (129 microRNAs, 2 of which not previously described, 10 piRNAs and 3 snoRNAs) of neoplastic transformation. Functional bioinformatics analysis identified as downstream targets multiple signaling pathways potentially involved in the hyperplastic and neoplastic tissue responses, including Wnt/β-catenin, and ERK/MAPK and TGF-β-Signaling. Considering the regulatory role of sncRNAs, this newly identified sncRNA signature is likely to reflect the events leading to endometrial cancer, which can be exploited to dissect the carcinogenic process including novel biomarkers for early and non-invasive diagnosis of these tumors. PMID:25686835

  4. quenched-smFISH: Counting small RNA in Pathogenic Bacteria

    NASA Astrophysics Data System (ADS)

    Shepherd, Douglas; Li, Nan; Micheva-Viteva, Sofiya; Munsky, Brian; Hong-Geller, Elizabeth; Werner, James

    2014-03-01

    Here, we present a modification to single-molecule fluorescence in situ hybridization, quenched smFISH (q-smFISH), that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. Exploiting an automated, multi-color wide-field microscope and GPU-accelerated data analysis package, we analyzed the statistics of sRNA expression in thousands of individual Yersinia pseudotuberculosis and Yersinia pestis bacteria before and during a simulated infection. Before infection, we find only a small fraction of either bacteria express the small RNAs YSR35 or YSP8. The copy numbers of these RNA are increased during simulated infection, suggesting a role in pathogenesis. The ability to directly quantify expression level changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in bacterial regulatory networks.

  5. Quantitative and qualitative analysis of small RNAs in human endothelial cells and exosomes provides insights into localized RNA processing, degradation and sorting

    PubMed Central

    van Balkom, Bas W. M.; Eisele, Almut S.; Pegtel, D. Michiel; Bervoets, Sander; Verhaar, Marianne C.

    2015-01-01

    Exosomes are small vesicles that mediate cell–cell communication. They contain proteins, lipids and RNA, and evidence is accumulating that these molecules are specifically sorted for release via exosomes. We recently showed that endothelial-cell-produced exosomes promote angiogenesis in vivo in a small RNA-dependent manner. Recent deep sequencing studies in exosomes from lymphocytic origin revealed a broad spectrum of small RNAs. However, selective depletion or incorporation of small RNA species into endothelial exosomes has not been studied extensively. With next generation sequencing, we identified all known non-coding RNA classes, including microRNAs (miRNAs), small nucleolar RNAs, yRNAs, vault RNAs, 5p and 3p fragments of miRNAs and miRNA-like fragments. In addition, we mapped many fragments of messenger RNAs (mRNAs) and mitochondrial RNAs (mtRNAs). The distribution of small RNAs in exosomes revealed a considerable overlap with the distribution in the producing cells. However, we identified a remarkable enrichment of yRNA fragments and mRNA degradation products in exosomes consistent with yRNAs having a role in degradation of structured and misfolded RNAs in close proximity to endosomes. We propose that endothelial endosomes selectively sequester cytoplasmic RNA-degrading machineries taking part in gene regulation. The release of these regulatory RNAs via exosomes may have implications for endothelial cell–cell communication. PMID:26027894

  6. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    SciTech Connect

    Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

    1987-04-01

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone lambdaHB''-1 from a phage lambdagt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone lambdaHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone lambdaHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the lambdaHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambdaHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.

  7. Mapping the small RNA content of simian immunodeficiency virions (SIV).

    PubMed

    Brameier, Markus; Ibing, Wiebke; Höfer, Katharina; Montag, Judith; Stahl-Hennig, Christiane; Motzkus, Dirk

    2013-01-01

    Recent evidence indicates that regulatory small non-coding RNAs are not only components of eukaryotic cells and vesicles, but also reside within a number of different viruses including retroviral particles. Using ultra-deep sequencing we have comprehensively analyzed the content of simian immunodeficiency virions (SIV), which were compared to mock-control preparations. Our analysis revealed that more than 428,000 sequence reads matched the SIV mac239 genome sequence. Among these we could identify 12 virus-derived small RNAs (vsRNAs) that were highly abundant. Beside known retrovirus-enriched small RNAs, like 7SL-RNA, tRNA(Lys3) and tRNA(Lys) isoacceptors, we also identified defined fragments derived from small ILF3/NF90-associated RNA snaR-A14, that were enriched more than 50 fold in SIV. We also found evidence that small nucleolar RNAs U2 and U12 were underrepresented in the SIV preparation, indicating that the relative number or the content of co-isolated exosomes was changed upon infection. Our comprehensive atlas of SIV-incorporated small RNAs provides a refined picture of the composition of retrovirions, which gives novel insights into viral packaging. PMID:24086438

  8. Small RNA pathway genes identified by patterns of phylogenetic conservation and divergence

    PubMed Central

    Tabach, Yuval; Billi, Allison C.; Hayes, Gabriel D.; Newman, Martin A.; Zuk, Or; Gabel, Harrison; Kamath, Ravi; Yacoby, Keren; Chapman, Brad; Garcia, Susana M.; Borowsky, Mark; Kim, John K.; Ruvkun, Gary

    2013-01-01

    Genetic and biochemical analyses of RNA interference (RNAi) and microRNA (miRNA) pathways have revealed proteins such as Argonaute/PIWI and Dicer that process and present small RNAs to their targets. Well validated small RNA pathway cofactors, such as the Argonaute/PIWI proteins show distinctive patterns of conservation or divergence in particular animal, plant, fungal, and protist species. We compared 86 divergent eukaryotic genome sequences to discern sets of proteins that show similar phylogenetic profiles with known small RNA cofactors. A large set of additional candidate small RNA cofactors have emerged from functional genomic screens for defects in miRNA- or siRNA-mediated repression in C. elegans and D. melanogaster1,2 and from proteomic analyses of proteins co-purifying with validated small RNA pathway proteins3,4. The phylogenetic profiles of many of these candidate small RNA pathway proteins are similar to those of known small RNA cofactor proteins. We used a Bayesian approach to integrate the phylogenetic profile analysis with predictions from diverse transcriptional coregulation and proteome interaction datasets to assign a probability for each protein for a role in a small RNA pathway. Testing high-confidence candidates from this analysis for defects in RNAi silencing, we found that about half of the predicted small RNA cofactors are required for RNAi silencing. Many of the newly identified small RNA pathway proteins are orthologues of proteins implicated in RNA splicing. In support of a deep connection between the mechanism of RNA splicing and small RNA-mediated gene silencing, the presence of the Argonaute proteins and other small RNA components in the many species analysed strongly correlates with the number of introns in that species. PMID:23364702

  9. Analysis of the small RNA P16/RgsA in the plant pathogen Pseudomonas syringae pathovar tomato strain DC3000

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteria contain small non-coding RNAs (ncRNAs) that are responsible for altering transcription, translation, or mRNA stability. ncRNAs are important because they regulate virulence factors and susceptibility to various stresses. Here, the regulation of a recently described ncRNA of P. syringae DC30...

  10. Small RNA cloning and sequencing strategy affects host and viral microRNA expression signatures.

    PubMed

    Stik, Grégoire; Muylkens, Benoît; Coupeau, Damien; Laurent, Sylvie; Dambrine, Ginette; Messmer, Mélanie; Chane-Woon-Ming, Béatrice; Pfeffer, Sébastien; Rasschaert, Denis

    2014-07-10

    The establishment of the microRNA (miRNA) expression signatures is the basic element to investigate the role played by these regulatory molecules in the biology of an organism. Marek's disease virus 1 (MDV-1) is an avian herpesvirus that naturally infects chicken and induces T cells lymphomas. During latency, MDV-1, like other herpesviruses, expresses a limited subset of transcripts. These include three miRNA clusters. Several studies identified the expression of virus and host encoded miRNAs from MDV-1 infected cell cultures and chickens. But a high discrepancy was observed when miRNA cloning frequencies obtained from different cloning and sequencing protocols were compared. Thus, we analyzed the effect of small RNA library preparation and sequencing on the miRNA frequencies obtained from the same RNA samples collected during MDV-1 infection of chicken at different steps of the oncoviral pathogenesis. Qualitative and quantitative variations were found in the data, depending on the strategy used. One of the mature miRNA derived from the latency-associated-transcript (LAT), mdv1-miR-M7-5p, showed the highest variation. Its cloning frequency was 50% of the viral miRNA counts when a small scale sequencing approach was used. Its frequency was 100 times less abundant when determined through the deep sequencing approach. Northern blot analysis showed a better correlation with the miRNA frequencies found by the small scale sequencing approach. By analyzing the cellular miRNA repertoire, we also found a gap between the two sequencing approaches. Collectively, our study indicates that next-generation sequencing data considered alone are limited for assessing the absolute copy number of transcripts. Thus, the quantification of small RNA should be addressed by compiling data obtained by using different techniques such as microarrays, qRT-PCR and NB analysis in support of high throughput sequencing data. These observations should be considered when miRNA variations are studied

  11. Small Regulatory RNA and Legionella pneumophila

    PubMed Central

    Faucher, Sébastien P.; Shuman, Howard A.

    2011-01-01

    Legionella pneumophila is a gram-negative bacterial species that is ubiquitous in almost any aqueous environment. It is the agent of Legionnaires’ disease, an acute and often under-reported form of pneumonia. In mammals, L. pneumophila replicates inside macrophages within a modified vacuole. Many protein regulators have been identified that control virulence-related properties, including RpoS, LetA/LetS, and PmrA/PmrB. In the past few years, the importance of regulation of virulence factors by small regulatory RNA (sRNAs) has been increasingly appreciated. This is also the case in L. pneumophila where three sRNAs (RsmY, RsmZ, and 6S RNA) were recently shown to be important determinants of virulence regulation and 79 actively transcribed sRNAs were identified. In this review we describe current knowledge about sRNAs and their regulatory properties and how this relates to the known regulatory systems of L. pneumophila. We also provide a model for sRNA-mediated control of gene expression that serves as a framework for understanding the regulation of virulence-related properties of L. pneumophila. PMID:21833335

  12. Transcriptome and small RNA deep sequencing reveals deregulation of miRNA biogenesis in human glioma.

    PubMed

    Moore, Lynette M; Kivinen, Virpi; Liu, Yuexin; Annala, Matti; Cogdell, David; Liu, Xiuping; Liu, Chang-Gong; Sawaya, Raymond; Yli-Harja, Olli; Shmulevich, Ilya; Fuller, Gregory N; Zhang, Wei; Nykter, Matti

    2013-02-01

    Altered expression of oncogenic and tumour-suppressing microRNAs (miRNAs) is widely associated with tumourigenesis. However, the regulatory mechanisms underlying these alterations are poorly understood. We sought to shed light on the deregulation of miRNA biogenesis promoting the aberrant miRNA expression profiles identified in these tumours. Using sequencing technology to perform both whole-transcriptome and small RNA sequencing of glioma patient samples, we examined precursor and mature miRNAs to directly evaluate the miRNA maturation process, and examined expression profiles for genes involved in the major steps of miRNA biogenesis. We found that ratios of mature to precursor forms of a large number of miRNAs increased with the progression from normal brain to low-grade and then to high-grade gliomas. The expression levels of genes involved in each of the three major steps of miRNA biogenesis (nuclear processing, nucleo-cytoplasmic transport, and cytoplasmic processing) were systematically altered in glioma tissues. Survival analysis of an independent data set demonstrated that the alteration of genes involved in miRNA maturation correlates with survival in glioma patients. Direct quantification of miRNA maturation with deep sequencing demonstrated that deregulation of the miRNA biogenesis pathway is a hallmark for glioma genesis and progression.

  13. Transcriptome and Small RNA Deep Sequencing Reveals Deregulation of miRNA Biogenesis in Human Glioma

    PubMed Central

    Moore, Lynette M.; Kivinen, Virpi; Liu, Yuexin; Annala, Matti; Cogdell, David; Liu, Xiuping; Liu, Chang-Gong; Sawaya, Raymond; Yli-Harja, Olli; Shmulevich, Ilya; Fuller, Gregory N.; Zhang, Wei; Nykter, Matti

    2013-01-01

    Altered expression of oncogenic and tumor-suppressing microRNAs (miRNAs) is widely associated with tumorigenesis. However, the regulatory mechanisms underlying these alterations are poorly understood. We sought to shed light on the deregulation of miRNA biogenesis promoting the aberrant miRNA expression profiles identified in these tumors. Using sequencing technology to perform both whole-transcriptome and small RNA sequencing of glioma patient samples, we examined precursor and mature miRNAs to directly evaluate the miRNA maturation process, and interrogated expression profiles for genes involved in the major steps of miRNA biogenesis. We found that ratios of mature to precursor forms of a large number of miRNAs increased with the progression from normal brain to low-grade and then to high-grade gliomas. The expression levels of genes involved in each of the three major steps of miRNA biogenesis (nuclear processing, nucleo-cytoplasmic transport, and cytoplasmic processing) were systematically altered in glioma tissues. Survival analysis of an independent data set demonstrated that the alteration of genes involved in miRNA maturation correlates with survival in glioma patients. Direct quantification of miRNA maturation with deep sequencing demonstrated that deregulation of the miRNA biogenesis pathway is a hallmark for glioma genesis and progression. PMID:23007860

  14. High-quality RNA extraction from small cardamom tissues rich in polysaccharides and polyphenols.

    PubMed

    Nadiya, Fasiludeen; Anjali, Narayanannair; Gangaprasad, Appukuttannair; Sabu, Kalluvettankuzhy Krishnannair

    2015-09-15

    Due to the presence of a diverse array of metabolites, no standard method of RNA isolation is available for plants. We noted that polysaccharide and polyphenol contents of cardamom tissues critically hinder the RNA extraction procedure. Hence, we attempted several methods for obtaining intact mRNA and small RNA from various cardamom tissues. It was found that protocols involving a combination of commercial kits and conventional CTAB (cetyl trimethylammonium bromide) methods yielded RNA with good purity, higher yield, and good integrity. The total RNA isolated through this approach was found to be amenable for transcriptome and small RNA analysis through next-generation sequencing platforms. PMID:26048648

  15. High-quality RNA extraction from small cardamom tissues rich in polysaccharides and polyphenols.

    PubMed

    Nadiya, Fasiludeen; Anjali, Narayanannair; Gangaprasad, Appukuttannair; Sabu, Kalluvettankuzhy Krishnannair

    2015-09-15

    Due to the presence of a diverse array of metabolites, no standard method of RNA isolation is available for plants. We noted that polysaccharide and polyphenol contents of cardamom tissues critically hinder the RNA extraction procedure. Hence, we attempted several methods for obtaining intact mRNA and small RNA from various cardamom tissues. It was found that protocols involving a combination of commercial kits and conventional CTAB (cetyl trimethylammonium bromide) methods yielded RNA with good purity, higher yield, and good integrity. The total RNA isolated through this approach was found to be amenable for transcriptome and small RNA analysis through next-generation sequencing platforms.

  16. Evolution of small guide RNA genes in hyperthermophilic archaea.

    PubMed

    Randau, Lennart

    2015-04-01

    Profiling the RNA production in hyperthermophilic archaea revealed an abundance of small RNA-guided processes near the upper temperature limit of life. Archaea utilize the base-pairing ability of RNA guide sequences to target ribosomal RNAs, transfer RNAs, messenger RNAs, and viral genomes. Cellular processes that are guided by small RNAs include the modification of RNA molecules, trans-splicing, gene regulation, and RNA and DNA degradation. Here, a brief overview of our knowledge on small guide RNA genes in archaeal genomes is provided and examples of their putative roles in genome evolution are described.

  17. Compilation of small ribosomal subunit RNA structures.

    PubMed Central

    Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

    1993-01-01

    The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

  18. Small RNA profile of the cumulus-oocyte complex and early embryos in the pig.

    PubMed

    Yang, Cai-Xia; Du, Zhi-Qiang; Wright, Elane C; Rothschild, Max F; Prather, Randall S; Ross, Jason W

    2012-11-01

    Small RNA represent several unique noncoding RNA classes that have important function in the development of germ cells and early embryonic development. Deep sequencing was performed on small RNA from cumulus cells (recovered from germinal vesicle [GV] and metaphase II-arrested [MII] oocytes), GV and MII oocytes, in vitro fertilization-derived embryos at 60 h postfertilization (4- to 8-cell stage), and Day 6 blastocysts. Additionally, a heterologous miRNA microarray method was also used to identify miRNA expressed in the oocyte during in vitro maturation. Similar to the results of expression analysis of other species, these data demonstrate dynamic expression regulation of multiple classes of noncoding RNA during oocyte maturation and development to the blastocyst stage. Mapping small RNA to the pig genome indicates dynamic distribution of small RNA organization across the genome. Additionally, a cluster of miRNA and Piwi-interacting RNA (piRNA) was discovered on chromosome 6. Many of the small RNA mapped to annotated repetitive elements in the pig genome, of which the SINE/tRNA-Glu and LINE/L1 elements represented a large proportion. Two piRNA (piR84651 and piR16993) and seven miRNA (MIR574, MIR24, LET7E, MIR23B, MIR30D, MIR320, and MIR30C) were further characterized using quantitative RT-PCR. Secretory carrier membrane protein 4 (SCAMP4) was predicted to be subject to posttranscriptional gene regulation mediated by small RNA, by annotating small RNA reads mapped to exonic regions in the pig genome. Consistent with the prediction results, SCAMP4 was further confirmed to be differentially expressed at both transcriptional and translational levels. These data establish a small RNA expression profile of the pig cumulus-oocyte complex and early embryos and demonstrate their potential capacity to be utilized for predictions of functional posttranscriptional regulatory events.

  19. Small catalytic RNA: Structure, function and application

    SciTech Connect

    Monforte, J.A.

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the paperclip'' and hammerhead'' RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a hammerhead,'' to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 12{plus minus}1 nucleotides are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  20. RNA-Seq analysis of non-small cell lung cancer in female never-smokers reveals candidate cancer-associated long non-coding RNAs.

    PubMed

    Li, Jun; Bi, Lintao; Shi, Zhangzhen; Sun, Yanxia; Lin, Yumei; Shao, Hui; Zhu, Zhenxing

    2016-06-01

    We aimed to elucidate the potential mechanisms of long non-coding RNAs (lncRNAs) in the progression of non-small cell lung cancer (NSCLC). The microarray datasets of GSE37764, including 3 primary NSCLC tumors and 3 matched normal tissues isolated from 6 Korean female never-smokers, were downloaded from Gene Expression Omnibus database. The differentially expressed lncRNAs and mRNA in NSCLC samples were identified using NOISeq package. Co-expression network of differentially expressed lncRNAs and mRNA was established. Gene Ontology (GO) and pathway enrichment analysis were respectively performed. Finally, lncRNAs related to NSCLC were predicted by blasting the differentially expressed lncRNAs with all predicted lncRNAs related to NSCLC. A total of 182 and 539 differentially expressed lncRNAs and mRNA (109 up- and 73 down-regulated lncRNAs; 307 up- and 232 down-regulated mRNA) were respectively identified. Among them, 4 up-regulated lncRNAs, like lnc-geranylgeranyl diphosphate synthase 1 (GGPS1), lnc-zinc finger protein 793 (ZNF793) and lnc-serine/threonine kinase 4 (STK4), and 4 down-regulated lncRNAs including lnc-LOC284440 and lnc-peptidylprolyl isomerase E-like pseudogene (PPIEL), and lnc-zinc finger protein 461 (ZNF461) were predicted related to NSCLC. lncSSPS1, lnc-ZNF793 and lnc-STK4 were co-expressed with linker for activation of T cells (LAT) and Lck interacting transmembrane adaptor 1 (LIME1). Lnc-LOC284440, lnc-PPIEL and lnc-ZNF461 were co-expressed with Src-like-adaptor 2 (SLA2) and defensin beta 4A (DEFB4A). Our study indicates that immune response may be a crucial mechanism involved in NSCLC progression. Lnc-GGPS1, lnc-ZNF793, lnc-STK4, lnc-LOC284440, lnc-PPIEL, and lnc-ZNF461 may be involved in immune response for promoting NSCLC progression via co-expressing with LAT, LIME1, SLA2 and DEFB4A.

  1. Mapping the Small RNA Content of Simian Immunodeficiency Virions (SIV)

    PubMed Central

    Brameier, Markus; Ibing, Wiebke; Höfer, Katharina; Montag, Judith; Stahl-Hennig, Christiane; Motzkus, Dirk

    2013-01-01

    Recent evidence indicates that regulatory small non-coding RNAs are not only components of eukaryotic cells and vesicles, but also reside within a number of different viruses including retroviral particles. Using ultra-deep sequencing we have comprehensively analyzed the content of simian immunodeficiency virions (SIV), which were compared to mock-control preparations. Our analysis revealed that more than 428,000 sequence reads matched the SIV mac239 genome sequence. Among these we could identify 12 virus-derived small RNAs (vsRNAs) that were highly abundant. Beside known retrovirus-enriched small RNAs, like 7SL-RNA, tRNALys3 and tRNALys isoacceptors, we also identified defined fragments derived from small ILF3/NF90-associated RNA snaR-A14, that were enriched more than 50 fold in SIV. We also found evidence that small nucleolar RNAs U2 and U12 were underrepresented in the SIV preparation, indicating that the relative number or the content of co-isolated exosomes was changed upon infection. Our comprehensive atlas of SIV-incorporated small RNAs provides a refined picture of the composition of retrovirions, which gives novel insights into viral packaging. PMID:24086438

  2. Contribution of Small RNA Pathway Components in Plant Immunity

    PubMed Central

    Seo, Jang-Kyun; Wu, Jianguo; Lii, Yifan; Li, Yi; Jin, Hailing

    2013-01-01

    Small RNAs regulate a multitude of cellular processes, including development, stress responses, metabolism, and maintenance of genome integrity, in a sequence-specific manner. Accumulating evidence reveals that host endogenous small RNAs and small RNA pathway components play important roles in plant immune responses against various pathogens, including bacteria, fungi, oomycetes, and viruses. Small-RNA-mediated defense responses are regulated through diverse pathways and the components of these pathways, including Dicer-like proteins, RNA-dependent RNA polymerases, Argonaute proteins, and RNA polymerase IV and V, exhibit functional specificities as well as redundancy. In this review, we summarize the recent insights revealed mainly through the examination of two model plants, Arabidopsis and rice, with a primary focus on our emerging understanding of how these small RNA pathway components contribute to plant immunity. PMID:23489060

  3. Small catalytic RNA: Structure, function and application

    SciTech Connect

    Monforte, J.A.

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the ``paperclip`` and ``hammerhead`` RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a ``hammerhead,`` to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 12{plus_minus}1 nucleotides are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus_minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  4. Tat-dependent production of an HIV-1 TAR-encoded miRNA-like small RNA

    PubMed Central

    Harwig, Alex; Jongejan, Aldo; van Kampen, Antoine H. C.; Berkhout, Ben; Das, Atze T.

    2016-01-01

    Evidence is accumulating that retroviruses can produce microRNAs (miRNAs). To prevent cleavage of their RNA genome, retroviruses have to use an alternative RNA source as miRNA precursor. The transacting responsive (TAR) hairpin structure in HIV-1 RNA has been suggested as source for miRNAs, but how these small RNAs are produced without impeding virus replication remained unclear. We used deep sequencing analysis of AGO2-bound HIV-1 RNAs to demonstrate that the 3′ side of the TAR hairpin is processed into a miRNA-like small RNA. This ∼21 nt RNA product is able to repress the expression of mRNAs bearing a complementary target sequence. Analysis of the small RNAs produced by wild-type and mutant HIV-1 variants revealed that non-processive transcription from the HIV-1 LTR promoter results in the production of short TAR RNAs that serve as precursor. These TAR RNAs are cleaved by Dicer and processing is stimulated by the viral Tat protein. This biogenesis pathway differs from the canonical miRNA pathway and allows HIV-1 to produce the TAR-encoded miRNA-like molecule without cleavage of the RNA genome. PMID:26984525

  5. Tat-dependent production of an HIV-1 TAR-encoded miRNA-like small RNA.

    PubMed

    Harwig, Alex; Jongejan, Aldo; van Kampen, Antoine H C; Berkhout, Ben; Das, Atze T

    2016-05-19

    Evidence is accumulating that retroviruses can produce microRNAs (miRNAs). To prevent cleavage of their RNA genome, retroviruses have to use an alternative RNA source as miRNA precursor. The transacting responsive (TAR) hairpin structure in HIV-1 RNA has been suggested as source for miRNAs, but how these small RNAs are produced without impeding virus replication remained unclear. We used deep sequencing analysis of AGO2-bound HIV-1 RNAs to demonstrate that the 3' side of the TAR hairpin is processed into a miRNA-like small RNA. This ∼21 nt RNA product is able to repress the expression of mRNAs bearing a complementary target sequence. Analysis of the small RNAs produced by wild-type and mutant HIV-1 variants revealed that non-processive transcription from the HIV-1 LTR promoter results in the production of short TAR RNAs that serve as precursor. These TAR RNAs are cleaved by Dicer and processing is stimulated by the viral Tat protein. This biogenesis pathway differs from the canonical miRNA pathway and allows HIV-1 to produce the TAR-encoded miRNA-like molecule without cleavage of the RNA genome. PMID:26984525

  6. miRge - A Multiplexed Method of Processing Small RNA-Seq Data to Determine MicroRNA Entropy

    PubMed Central

    Myers, Jason R.; Gupta, Simone; Weng, Lien-Chun; Ashton, John M.; Cornish, Toby C.; Pandey, Akhilesh; Halushka, Marc K.

    2015-01-01

    Small RNA RNA-seq for microRNAs (miRNAs) is a rapidly developing field where opportunities still exist to create better bioinformatics tools to process these large datasets and generate new, useful analyses. We built miRge to be a fast, smart small RNA-seq solution to process samples in a highly multiplexed fashion. miRge employs a Bayesian alignment approach, whereby reads are sequentially aligned against customized mature miRNA, hairpin miRNA, noncoding RNA and mRNA sequence libraries. miRNAs are summarized at the level of raw reads in addition to reads per million (RPM). Reads for all other RNA species (tRNA, rRNA, snoRNA, mRNA) are provided, which is useful for identifying potential contaminants and optimizing small RNA purification strategies. miRge was designed to optimally identify miRNA isomiRs and employs an entropy based statistical measurement to identify differential production of isomiRs. This allowed us to identify decreasing entropy in isomiRs as stem cells mature into retinal pigment epithelial cells. Conversely, we show that pancreatic tumor miRNAs have similar entropy to matched normal pancreatic tissues. In a head-to-head comparison with other miRNA analysis tools (miRExpress 2.0, sRNAbench, omiRAs, miRDeep2, Chimira, UEA small RNA Workbench), miRge was faster (4 to 32-fold) and was among the top-two methods in maximally aligning miRNAs reads per sample. Moreover, miRge has no inherent limits to its multiplexing. miRge was capable of simultaneously analyzing 100 small RNA-Seq samples in 52 minutes, providing an integrated analysis of miRNA expression across all samples. As miRge was designed for analysis of single as well as multiple samples, miRge is an ideal tool for high and low-throughput users. miRge is freely available at http://atlas.pathology.jhu.edu/baras/miRge.html. PMID:26571139

  7. Ribozymes and Riboswitches: Modulation of RNA Function by Small Molecules†

    PubMed Central

    Zhang, Jinwei; Lau, Matthew W.; Ferré-D'Amaré, Adrian R.

    2010-01-01

    Diverse small molecules interact with catalytic RNAs (ribozymes) as substrates and cofactors, and their intracellular concentrations are sensed by gene-regulatory mRNA domains (riboswitches) that modulate transcription, splicing, translation, or RNA stability. Although recognition mechanisms vary from RNA to RNA, structural analyses reveal recurring strategies that arise from the intrinsic properties of RNA such as base pairing and stacking with conjugated heterocycles, and cation-dependent recognition of anionic functional groups. These studies also suggest that, to a first approximation, the magnitude of ligand-induced reorganization of an RNA is inversely proportional to the complexity of the riboswitch or ribozyme. How these small molecule binding-induced changes in RNA lead to alteration in gene expression is less well understood. While different riboswitches have been proposed to be under either kinetic or thermodynamic control, the biochemical and structural mechanisms that give rise to regulatory consequences downstream of small molecule recognition by RNAs mostly remain to be elucidated. PMID:20931966

  8. The evolving world of small RNAs from RNA viruses.

    PubMed

    Li, Mei-Ling; Weng, Kuo-Feng; Shih, Shin-Ru; Brewer, Gary

    2016-09-01

    RNA virus infection in plants and invertebrates can produce virus-derived small RNAs. These RNAs share features with host endogenous small interfering RNAs (siRNAs). They can potentially mediate RNA interference (RNAi) and related RNA silencing pathways, resulting in specific antiviral defense. Although most RNA silencing components such as Dicer, Ago2, and RISC are conserved among eukaryotic hosts, whether RNA virus infection in mammals can generate functional small RNAs that act in antiviral defense remains under discussion. Here, we review recent studies on the molecular and biochemical features of viral siRNAs and other virus-derived small RNAs from infected plants, arthropods, nematodes, and vertebrates and discuss the genetic pathways for their biogenesis and their roles in antiviral activity. WIREs RNA 2016, 7:575-588. doi: 10.1002/wrna.1351 For further resources related to this article, please visit the WIREs website. PMID:27046163

  9. The evolving world of small RNAs from RNA viruses.

    PubMed

    Li, Mei-Ling; Weng, Kuo-Feng; Shih, Shin-Ru; Brewer, Gary

    2016-09-01

    RNA virus infection in plants and invertebrates can produce virus-derived small RNAs. These RNAs share features with host endogenous small interfering RNAs (siRNAs). They can potentially mediate RNA interference (RNAi) and related RNA silencing pathways, resulting in specific antiviral defense. Although most RNA silencing components such as Dicer, Ago2, and RISC are conserved among eukaryotic hosts, whether RNA virus infection in mammals can generate functional small RNAs that act in antiviral defense remains under discussion. Here, we review recent studies on the molecular and biochemical features of viral siRNAs and other virus-derived small RNAs from infected plants, arthropods, nematodes, and vertebrates and discuss the genetic pathways for their biogenesis and their roles in antiviral activity. WIREs RNA 2016, 7:575-588. doi: 10.1002/wrna.1351 For further resources related to this article, please visit the WIREs website.

  10. Genome assembly of bell pepper endornavirus from small RNA.

    PubMed

    Sela, Noa; Luria, Neta; Dombrovsky, Aviv

    2012-07-01

    The family Endornaviridae infects diverse hosts, including plants, fungi, and oomycetes. Here we report for the first time the assembly of bell pepper endornavirus by next-generation sequencing of viral small RNA. Such a population of small RNA indicates the activation of the viral immunity silencing machinery by this cryptic virus, which probably encodes a novel silencing suppressor.

  11. Statistical Analysis of RNA Backbone

    PubMed Central

    Hershkovitz, Eli; Sapiro, Guillermo; Tannenbaum, Allen; Williams, Loren Dean

    2009-01-01

    Local conformation is an important determinant of RNA catalysis and binding. The analysis of RNA conformation is particularly difficult due to the large number of degrees of freedom (torsion angles) per residue. Proteins, by comparison, have many fewer degrees of freedom per residue. In this work, we use and extend classical tools from statistics and signal processing to search for clusters in RNA conformational space. Results are reported both for scalar analysis, where each torsion angle is separately studied, and for vectorial analysis, where several angles are simultaneously clustered. Adapting techniques from vector quantization and clustering to the RNA structure, we find torsion angle clusters and RNA conformational motifs. We validate the technique using well-known conformational motifs, showing that the simultaneous study of the total torsion angle space leads to results consistent with known motifs reported in the literature and also to the finding of new ones. PMID:17048391

  12. Comparative analysis of virus-specific small RNA profiles of three biologically distinct strains of Potato virus Y in infected potato (Solanum tuberosum) cv. Russet Burbank.

    PubMed

    Naveed, Khalid; Mitter, Neena; Harper, Artemus; Dhingra, Amit; Pappu, Hanu R

    2014-10-13

    Deep sequencing technology has enabled the analysis of small RNA profiles of virus-infected plants and could provide insights into virus-host interactions. Potato virus Y is an economically important viral pathogen of potato worldwide. In this study, we investigated the nature and relative levels of virus-derived small interfering RNAs (vsiRNAs) in potato cv. Russet Burbank infected with three biologically distinct and economically important strains of PVY, the ordinary strain (PVY-O), tobacco veinal-necrotic strain (PVY-N) and tuber necrotic strain (PVY-NTN). The analysis showed an overall abundance of vsiRNAs of 20-24nt in PVY-infected plants. Considerable differences were present in the distribution of vsiRNAs as well as total small RNAs. The 21nt class was the most prevalent in PVY-infected plants irrespective of the virus strain, whereas in healthy potato plants, the 24nt class was the most dominant. vsiRNAs were derived from every position in the PVY genome, though certain hotspots were identified for each of the PVY strains. Among the three strains used, the population of vsiRNAs of different size classes was relatively different with PVY-NTN accumulating the highest level of vsiRNAs, while PVY-N infected plants had the least population of vsiRNAs. Unique vsiRNAs mapping to PVY genome in PVY-infected plants amounted to 3.13, 1.93 and 1.70% for NTN, N and O, respectively. There was a bias in the generation of vsiRNAs from the plus strand of the genome in comparison to the negative strand. The highest number of total vsiRNAs was from the cytoplasmic inclusion protein gene (CI) in PVY-O and PVY-NTN strains, whereas from PVY-N, the NIb gene produced maximum total vsiRNAs. These findings indicate that the three PVY strains interact differently in the same host genetic background and provided insights into virus-host interactions in an important food crop. PMID:25036885

  13. Comparative analysis of virus-specific small RNA profiles of three biologically distinct strains of Potato virus Y in infected potato (Solanum tuberosum) cv. Russet Burbank.

    PubMed

    Naveed, Khalid; Mitter, Neena; Harper, Artemus; Dhingra, Amit; Pappu, Hanu R

    2014-10-13

    Deep sequencing technology has enabled the analysis of small RNA profiles of virus-infected plants and could provide insights into virus-host interactions. Potato virus Y is an economically important viral pathogen of potato worldwide. In this study, we investigated the nature and relative levels of virus-derived small interfering RNAs (vsiRNAs) in potato cv. Russet Burbank infected with three biologically distinct and economically important strains of PVY, the ordinary strain (PVY-O), tobacco veinal-necrotic strain (PVY-N) and tuber necrotic strain (PVY-NTN). The analysis showed an overall abundance of vsiRNAs of 20-24nt in PVY-infected plants. Considerable differences were present in the distribution of vsiRNAs as well as total small RNAs. The 21nt class was the most prevalent in PVY-infected plants irrespective of the virus strain, whereas in healthy potato plants, the 24nt class was the most dominant. vsiRNAs were derived from every position in the PVY genome, though certain hotspots were identified for each of the PVY strains. Among the three strains used, the population of vsiRNAs of different size classes was relatively different with PVY-NTN accumulating the highest level of vsiRNAs, while PVY-N infected plants had the least population of vsiRNAs. Unique vsiRNAs mapping to PVY genome in PVY-infected plants amounted to 3.13, 1.93 and 1.70% for NTN, N and O, respectively. There was a bias in the generation of vsiRNAs from the plus strand of the genome in comparison to the negative strand. The highest number of total vsiRNAs was from the cytoplasmic inclusion protein gene (CI) in PVY-O and PVY-NTN strains, whereas from PVY-N, the NIb gene produced maximum total vsiRNAs. These findings indicate that the three PVY strains interact differently in the same host genetic background and provided insights into virus-host interactions in an important food crop.

  14. Analysis RNA-seq and Noncoding RNA.

    PubMed

    Arrigoni, Alberto; Ranzani, Valeria; Rossetti, Grazisa; Panzeri, Ilaria; Abrignani, Sergio; Bonnal, Raoul J P; Pagani, Massimiliano

    2016-01-01

    RNA-Seq is an approach to transcriptome profiling that uses deep-sequencing technologies to detect and accurately quantify RNA molecules originating from a genome at a given moment in time. In recent years, the advent of RNA-Seq has facilitated genome-wide expression profiling, including the identification of novel and rare transcripts like noncoding RNAs and novel alternative splicing isoforms.Here, we describe the analytical steps required for the identification and characterization of noncoding RNAs starting from RNA-Seq raw samples, with a particular emphasis on long noncoding RNAs (lncRNAs). PMID:27659980

  15. Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases.

    PubMed

    Takahashi, Yuki; Nishikawa, Makiya; Kobayashi, Naoki; Takakura, Yoshinobu

    2005-07-20

    Silencing of oncogenes or other genes contributing to tumor malignancy or progression by RNA interference (RNAi) offers a promising approach to treating tumor patients. To achieve RNAi-based tumor therapy, a small interfering RNA (siRNA) or siRNA-expressing vector needs to be delivered to tumor cells, but little information about its in vivo delivery has been reported. In this study, we examined whether the expression of the target gene in tumor cells can be suppressed by the delivery of RNAi effectors to primary and metastatic tumor cells. To quantitatively evaluate the RNAi effects in tumor cells, mouse melanoma B16-BL6 cells were stably transfected with both firefly (a model target gene) and sea pansy (an internal standard gene) luciferase genes to obtain B16-BL6/dual Luc cells. The target gene expression in subcutaneous primary tumors of B16-BL6/dual Luc cells was significantly suppressed by direct injection of the RNAi effectors followed by electroporation. The expression in metastatic hepatic tumors was also significantly reduced by an intravenous injection of either RNAi effector by the hydrodynamics-based procedure. These results indicate that the both RNAi effectors have a potential to silence target gene in tumor cells in vivo when successfully delivered to tumor cells. PMID:15936841

  16. DNApi: A De Novo Adapter Prediction Algorithm for Small RNA Sequencing Data

    PubMed Central

    Tsuji, Junko; Weng, Zhiping

    2016-01-01

    With the rapid accumulation of publicly available small RNA sequencing datasets, third-party meta-analysis across many datasets is becoming increasingly powerful. Although removing the 3´ adapter is an essential step for small RNA sequencing analysis, the adapter sequence information is not always available in the metadata. The information can be also erroneous even when it is available. In this study, we developed DNApi, a lightweight Python software package that predicts the 3´ adapter sequence de novo and provides the user with cleansed small RNA sequences ready for down stream analysis. Tested on 539 publicly available small RNA libraries accompanied with 3´ adapter sequences in their metadata, DNApi shows near-perfect accuracy (98.5%) with fast runtime (~2.85 seconds per library) and efficient memory usage (~43 MB on average). In addition to 3´ adapter prediction, it is also important to classify whether the input small RNA libraries were already processed, i.e. the 3´ adapters were removed. DNApi perfectly judged that given another batch of datasets, 192 publicly available processed libraries were “ready-to-map” small RNA sequence. DNApi is compatible with Python 2 and 3, and is available at https://github.com/jnktsj/DNApi. The 731 small RNA libraries used for DNApi evaluation were from human tissues and were carefully and manually collected. This study also provides readers with the curated datasets that can be integrated into their studies. PMID:27736901

  17. Small RNA Transcriptome of the Oral Microbiome during Periodontitis Progression.

    PubMed

    Duran-Pinedo, Ana E; Yost, Susan; Frias-Lopez, Jorge

    2015-10-01

    The oral microbiome is one of the most complex microbial communities in the human body, and due to circumstances not completely understood, the healthy microbial community becomes dysbiotic, giving rise to periodontitis, a polymicrobial inflammatory disease. We previously reported the results of community-wide gene expression changes in the oral microbiome during periodontitis progression and identified signatures associated with increasing severity of the disease. Small noncoding RNAs (sRNAs) are key players in posttranscriptional regulation, especially in fast-changing environments such as the oral cavity. Here, we expanded our analysis to the study of the sRNA metatranscriptome during periodontitis progression on the same samples for which mRNA expression changes were analyzed. We observed differential expression of 12,097 sRNAs, identifying a total of 20 Rfam sRNA families as being overrepresented in progression and 23 at baseline. Gene ontology activities regulated by the differentially expressed (DE) sRNAs included amino acid metabolism, ethanolamine catabolism, signal recognition particle-dependent cotranslational protein targeting to membrane, intron splicing, carbohydrate metabolism, control of plasmid copy number, and response to stress. In integrating patterns of expression of protein coding transcripts and sRNAs, we found that functional activities of genes that correlated positively with profiles of expression of DE sRNAs were involved in pathogenesis, proteolysis, ferrous iron transport, and oligopeptide transport. These findings represent the first integrated sequencing analysis of the community-wide sRNA transcriptome of the oral microbiome during periodontitis progression and show that sRNAs are key regulatory elements of the dysbiotic process leading to disease.

  18. Small RNA Transcriptome of the Oral Microbiome during Periodontitis Progression.

    PubMed

    Duran-Pinedo, Ana E; Yost, Susan; Frias-Lopez, Jorge

    2015-10-01

    The oral microbiome is one of the most complex microbial communities in the human body, and due to circumstances not completely understood, the healthy microbial community becomes dysbiotic, giving rise to periodontitis, a polymicrobial inflammatory disease. We previously reported the results of community-wide gene expression changes in the oral microbiome during periodontitis progression and identified signatures associated with increasing severity of the disease. Small noncoding RNAs (sRNAs) are key players in posttranscriptional regulation, especially in fast-changing environments such as the oral cavity. Here, we expanded our analysis to the study of the sRNA metatranscriptome during periodontitis progression on the same samples for which mRNA expression changes were analyzed. We observed differential expression of 12,097 sRNAs, identifying a total of 20 Rfam sRNA families as being overrepresented in progression and 23 at baseline. Gene ontology activities regulated by the differentially expressed (DE) sRNAs included amino acid metabolism, ethanolamine catabolism, signal recognition particle-dependent cotranslational protein targeting to membrane, intron splicing, carbohydrate metabolism, control of plasmid copy number, and response to stress. In integrating patterns of expression of protein coding transcripts and sRNAs, we found that functional activities of genes that correlated positively with profiles of expression of DE sRNAs were involved in pathogenesis, proteolysis, ferrous iron transport, and oligopeptide transport. These findings represent the first integrated sequencing analysis of the community-wide sRNA transcriptome of the oral microbiome during periodontitis progression and show that sRNAs are key regulatory elements of the dysbiotic process leading to disease. PMID:26187962

  19. Small RNA Transcriptome of the Oral Microbiome during Periodontitis Progression

    PubMed Central

    Duran-Pinedo, Ana E.; Yost, Susan

    2015-01-01

    The oral microbiome is one of the most complex microbial communities in the human body, and due to circumstances not completely understood, the healthy microbial community becomes dysbiotic, giving rise to periodontitis, a polymicrobial inflammatory disease. We previously reported the results of community-wide gene expression changes in the oral microbiome during periodontitis progression and identified signatures associated with increasing severity of the disease. Small noncoding RNAs (sRNAs) are key players in posttranscriptional regulation, especially in fast-changing environments such as the oral cavity. Here, we expanded our analysis to the study of the sRNA metatranscriptome during periodontitis progression on the same samples for which mRNA expression changes were analyzed. We observed differential expression of 12,097 sRNAs, identifying a total of 20 Rfam sRNA families as being overrepresented in progression and 23 at baseline. Gene ontology activities regulated by the differentially expressed (DE) sRNAs included amino acid metabolism, ethanolamine catabolism, signal recognition particle-dependent cotranslational protein targeting to membrane, intron splicing, carbohydrate metabolism, control of plasmid copy number, and response to stress. In integrating patterns of expression of protein coding transcripts and sRNAs, we found that functional activities of genes that correlated positively with profiles of expression of DE sRNAs were involved in pathogenesis, proteolysis, ferrous iron transport, and oligopeptide transport. These findings represent the first integrated sequencing analysis of the community-wide sRNA transcriptome of the oral microbiome during periodontitis progression and show that sRNAs are key regulatory elements of the dysbiotic process leading to disease. PMID:26187962

  20. Abstract shape analysis of RNA.

    PubMed

    Janssen, Stefan; Giegerich, Robert

    2014-01-01

    Abstract shape analysis abstract shape analysis is a method to learn more about the complete Boltzmann ensemble of the secondary structures of a single RNA molecule. Abstract shapes classify competing secondary structures into classes that are defined by their arrangement of helices. It allows us to compute, in addition to the structure of minimal free energy, a set of structures that represents relevant and interesting structural alternatives. Furthermore, it allows to compute probabilities of all structures within a shape class. This allows to ensure that our representative subset covers the complete Boltzmann ensemble, except for a portion of negligible probability. This chapter explains the main functions of abstract shape analysis, as implemented in the tool RNA shapes. RNA shapes It reports on some other types of analysis that are based on the abstract shapes idea and shows how you can solve novel problems by creating your own shape abstractions.

  1. Small RNA mediated regulation of seed germination

    PubMed Central

    Das, Shabari Sarkar; Karmakar, Prakash; Nandi, Asis Kumar; Sanan-Mishra, Neeti

    2015-01-01

    Mature seeds of most of the higher plants harbor dormant embryos and go through the complex process of germination under favorable environmental conditions. The germination process involves dynamic physiological, cellular and metabolic events that are controlled by the interplay of several gene products and different phytohormones. The small non-coding RNAs comprise key regulatory modules in the process of seed dormancy and germination. Recent studies have implicated the small RNAs in plant growth in correlation with various plant physiological processes including hormone signaling and stress response. In this review we provide a brief overview of the regulation of seed germination or dormancy while emphasizing on the current understanding of the role of small RNAs in this regard. We have also highlighted specific examples of stress responsive small RNAs in seed germination and discussed their future potential. PMID:26528301

  2. Conifers have a unique small RNA silencing signature

    PubMed Central

    Dolgosheina, Elena V.; Morin, Ryan D.; Aksay, Gozde; Sahinalp, S. Cenk; Magrini, Vincent; Mardis, Elaine R.; Mattsson, Jim; Unrau, Peter J.

    2008-01-01

    Plants produce small RNAs to negatively regulate genes, viral nucleic acids, and repetitive elements at either the transcriptional or post-transcriptional level in a process that is referred to as RNA silencing. While RNA silencing has been extensively studied across the different phyla of the animal kingdom (e.g., mouse, fly, worm), similar studies in the plant kingdom have focused primarily on angiosperms, thus limiting evolutionary studies of RNA silencing in plants. Here we report on an unexpected phylogenetic difference in the size distribution of small RNAs among the vascular plants. By extracting total RNA from freshly growing shoot tissue, we conducted a survey of small RNAs in 24 vascular plant species. We find that conifers, which radiated from the other seed-bearing plants ∼260 million years ago, fail to produce significant amounts of 24-nucleotide (nt) RNAs that are known to guide DNA methylation and heterochromatin formation in angiosperms. Instead, they synthesize a diverse population of small RNAs that are exactly 21-nt long. This finding was confirmed by high-throughput sequencing of the small RNA sequences from a conifer, Pinus contorta. A conifer EST search revealed the presence of a novel Dicer-like (DCL) family, which may be responsible for the observed change in small RNA expression. No evidence for DCL3, an enzyme that matures 24-nt RNAs in angiosperms, was found. We hypothesize that the diverse class of 21-nt RNAs found in conifers may help to maintain organization of their unusually large genomes. PMID:18566193

  3. ATP requirements and small interfering RNA structure in the RNA interference pathway.

    PubMed

    Nykänen, A; Haley, B; Zamore, P D

    2001-11-01

    We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs. A 5' phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.

  4. Small Interfering RNA Transfection Across a Phospholipid Membrane

    NASA Astrophysics Data System (ADS)

    Ngo, Van; Choubey, Amit; Kalia, Rajiv; Nakano, Aiichiro; Vashishta, Priya

    2012-02-01

    Small interfering RNA (siRNA) molecules play a pivotal role in silencing gene expression via the RNA interference mechanism. We have performed steered MD simulations to study the transfection of a bare siRNA and siRNA/Oleic Acid (OA) complex across the dipalmitoylphosphatidycholine (DPPC) bilayer at T = 323 K. Bare siRNA induces the formation of frustrated lipid gel domains, whereas in the presence of siRNA/OA complex the membrane is found to be in the liquid-ordered phase. In both cases the stress profiles across the membrane indicate that the membrane is under tension near the head groups and highly compressed at the water-hydrophobic interface. During transfection, the membrane is deformed and the lateral stress is significantly lowered for the bare siRNA and siRNA/OA complex. The bare siRNA transfects through a lipid-nanopore of hydrophilic head-groups and hydrophobic carbon chains, whereas the siRNA/OA complex transfects through a lipid-nanopore of hydrophilic head groups.

  5. Mining diverse small RNA species in the deep transcriptome.

    PubMed

    Vickers, Kasey C; Roteta, Leslie A; Hucheson-Dilks, Holli; Han, Leng; Guo, Yan

    2015-01-01

    Transcriptomes of many species are proving to be exquisitely diverse, and many investigators are now using high-throughput sequencing to quantify non-protein-coding RNAs, namely small RNAs (sRNA). Unfortunately, most studies are focused solely on microRNA changes, and many investigators are not analyzing the full compendium of sRNA species present in their large datasets. We provide here a rationale to include all types of sRNAs in sRNA sequencing analyses, which will aid in the discovery of their biological functions and physiological relevance.

  6. Modeling Small Noncanonical RNA Motifs with the Rosetta FARFAR Server.

    PubMed

    Yesselman, Joseph D; Das, Rhiju

    2016-01-01

    Noncanonical RNA motifs help define the vast complexity of RNA structure and function, and in many cases, these loops and junctions are on the order of only ten nucleotides in size. Unfortunately, despite their small size, there is no reliable method to determine the ensemble of lowest energy structures of junctions and loops at atomic accuracy. This chapter outlines straightforward protocols using a webserver for Rosetta Fragment Assembly of RNA with Full Atom Refinement (FARFAR) ( http://rosie.rosettacommons.org/rna_denovo/submit ) to model the 3D structure of small noncanonical RNA motifs for use in visualizing motifs and for further refinement or filtering with experimental data such as NMR chemical shifts. PMID:27665600

  7. A small RNA response at DNA ends in Drosophila.

    PubMed

    Michalik, Katharina M; Böttcher, Romy; Förstemann, Klaus

    2012-10-01

    Small RNAs have been implicated in numerous cellular processes, including effects on chromatin structure and the repression of transposons. We describe the generation of a small RNA response at DNA ends in Drosophila that is analogous to the recently reported double-strand break (DSB)-induced RNAs or Dicer- and Drosha-dependent small RNAs in Arabidopsis and vertebrates. Active transcription in the vicinity of the break amplifies this small RNA response, demonstrating that the normal messenger RNA contributes to the endogenous small interfering RNAs precursor. The double-stranded RNA precursor forms with an antisense transcript that initiates at the DNA break. Breaks are thus sites of transcription initiation, a novel aspect of the cellular DSB response. This response is specific to a double-strand break since nicked DNA structures do not trigger small RNA production. The small RNAs are generated independently of the exact end structure (blunt, 3'- or 5'-overhang), can repress homologous sequences in trans and may therefore--in addition to putative roles in repair--exert a quality control function by clearing potentially truncated messages from genes in the vicinity of the break.

  8. Computer-assisted annotation of murine Sertoli cell small RNA transcriptome.

    PubMed

    Ortogero, Nicole; Hennig, Grant W; Langille, Chad; Ro, Seungil; McCarrey, John R; Yan, Wei

    2013-01-01

    Mammalian genomes encode a large number of small noncoding RNAs (sncRNAs) that play regulatory roles during development and adulthood by affecting gene expression. Several sncRNA species, including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), endogenous small interfering RNAs (endo-siRNAs), and small nucleolar RNAs (snoRNAs), are abundantly expressed in the testis and required for normal testicular development and spermatogenesis. To evaluate global changes in sncRNA expression, the next-generation sequencing (NGS)-based sncRNA transcriptomic analysis has become routine, because it allows rapid determination of the small RNA transcriptome of a particular testicular cell type. However, annotation of small RNA NGS reads can be challenging due to the volume of reads obtained, which is usually in the millions. Therefore, we developed a computer-assisted sncRNA annotation protocol that could identify not only known sncRNAs but also previously uncharacterized ones. Using this protocol, we annotated NGS reads of a Sertoli cell sncRNA library, and we report to our knowledge the first comprehensive annotation of the sncRNA transcriptome of immature murine Sertoli cells. Moreover, the computer-assisted sncRNA annotation pipeline that we report is applicable for annotating NGS reads derived from other cell types and/or sequencing platforms.

  9. Functional Nanostructures for Effective Delivery of Small Interfering RNA Therapeutics

    PubMed Central

    Hong, Cheol Am; Nam, Yoon Sung

    2014-01-01

    Small interfering RNA (siRNA) has proved to be a powerful tool for target-specific gene silencing via RNA interference (RNAi). Its ability to control targeted gene expression gives new hope to gene therapy as a treatment for cancers and genetic diseases. However, siRNA shows poor pharmacological properties, such as low serum stability, off-targeting, and innate immune responses, which present a significant challenge for clinical applications. In addition, siRNA cannot cross the cell membrane for RNAi activity because of its anionic property and stiff structure. Therefore, the development of a safe, stable, and efficient system for the delivery of siRNA therapeutics into the cytoplasm of targeted cells is crucial. Several nanoparticle platforms for siRNA delivery have been developed to overcome the major hurdles facing the therapeutic uses of siRNA. This review covers a broad spectrum of non-viral siRNA delivery systems developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and discusses their characteristics and opportunities for clinical applications of therapeutic siRNA. PMID:25285170

  10. Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool.

    PubMed

    Giallombardo, Marco; Chacártegui Borrás, Jorge; Castiglia, Marta; Van Der Steen, Nele; Mertens, Inge; Pauwels, Patrick; Peeters, Marc; Rolfo, Christian

    2016-01-01

    The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2(-) (ΔΔct) (13). Control values were used as baseline and results are shown in logarithmic scale. PMID:27285610

  11. Design of a small molecule against an oncogenic noncoding RNA.

    PubMed

    Velagapudi, Sai Pradeep; Cameron, Michael D; Haga, Christopher L; Rosenberg, Laura H; Lafitte, Marie; Duckett, Derek R; Phinney, Donald G; Disney, Matthew D

    2016-05-24

    The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of disease-causing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif-small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-to-drug paradigm.

  12. A small RNA activates CFA synthase by isoform-specific mRNA stabilization

    PubMed Central

    Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

    2013-01-01

    Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability. PMID:24141880

  13. Functionalization of an Antisense Small RNA

    PubMed Central

    Rodrigo, Guillermo; Prakash, Satya; Cordero, Teresa; Kushwaha, Manish; Jaramillo, Alfonso

    2016-01-01

    In order to explore the possibility of adding new functions to preexisting genes, we considered a framework of riboregulation. We created a new riboregulator consisting of the reverse complement of a known riboregulator. Using computational design, we engineered a cis-repressing 5′ untranslated region that can be activated by this new riboregulator. As a result, both RNAs can orthogonally trans-activate translation of their cognate, independent targets. The two riboregulators can also repress each other by antisense interaction, although not symmetrically. Our work highlights that antisense small RNAs can work as regulatory agents beyond the antisense paradigm and that, hence, they could be interfaced with other circuits used in synthetic biology. PMID:26756967

  14. Crystallographic Analysis of Small Ribozymes and Riboswitches

    PubMed Central

    Lippa, Geoffrey M.; Liberman, Joseph A.; Jenkins, Jermaine L.; Krucinska, Jolanta; Salim, Mohammad; Wedekind, Joseph E.

    2016-01-01

    Ribozymes and riboswitches are RNA motifs that accelerate biological reactions and regulate gene expression in response to metabolite recognition, respectively. These RNA molecules gain functionality via complex folding that cannot be predicted a priori, and thus requires high-resolution three-dimensional structure determination to locate key functional attributes. Herein, we present an overview of the methods used to determine small RNA structures with an emphasis on RNA preparation, crystallization, and structure refinement. We draw upon examples from our own research in the analysis of the leadzyme ribozyme, the hairpin ribozyme, a class I preQ1 riboswitch, and variants of a larger class II preQ1 riboswitch. The methods presented provide a guide for comparable investigations of noncoding RNA molecules including a 48-solution, “first choice” RNA crystal screen compiled from our prior successes with commercially available screens. PMID:22315069

  15. Empirical insights into the stochasticity of small RNA sequencing.

    PubMed

    Qin, Li-Xuan; Tuschl, Thomas; Singer, Samuel

    2016-01-01

    The choice of stochasticity distribution for modeling the noise distribution is a fundamental assumption for the analysis of sequencing data and consequently is critical for the accurate assessment of biological heterogeneity and differential expression. The stochasticity of RNA sequencing has been assumed to follow Poisson distributions. We collected microRNA sequencing data and observed that its stochasticity is better approximated by gamma distributions, likely because of the stochastic nature of exponential PCR amplification. We validated our findings with two independent datasets, one for microRNA sequencing and another for RNA sequencing. Motivated by the gamma distributed stochasticity, we provided a simple method for the analysis of RNA sequencing data and showed its superiority to three existing methods for differential expression analysis using three data examples of technical replicate data and biological replicate data. PMID:27052356

  16. Empirical insights into the stochasticity of small RNA sequencing

    NASA Astrophysics Data System (ADS)

    Qin, Li-Xuan; Tuschl, Thomas; Singer, Samuel

    2016-04-01

    The choice of stochasticity distribution for modeling the noise distribution is a fundamental assumption for the analysis of sequencing data and consequently is critical for the accurate assessment of biological heterogeneity and differential expression. The stochasticity of RNA sequencing has been assumed to follow Poisson distributions. We collected microRNA sequencing data and observed that its stochasticity is better approximated by gamma distributions, likely because of the stochastic nature of exponential PCR amplification. We validated our findings with two independent datasets, one for microRNA sequencing and another for RNA sequencing. Motivated by the gamma distributed stochasticity, we provided a simple method for the analysis of RNA sequencing data and showed its superiority to three existing methods for differential expression analysis using three data examples of technical replicate data and biological replicate data.

  17. Final report for ER65039, The Role of Small RNA in Biomass Deposition

    SciTech Connect

    Hudson, Matthew E.

    2015-03-12

    Our objective in this project was to discover the role of sRNA in regulating both biomass biosynthesis and perenniality in the Andropogoneae feedstock grasses. Our central hypothesis was that there is a time-and space specific sRNA network playing a crucial role in regulating processes associated with cell wall biosynthesis, flowering time control, overwintering/juvenility, and nutrient sequestration in the feedstock grasses. To address this, we performed a large scale biological project consisting of the growth of material, generation of Illumina libraries, sequencing and analysis for small RNA, mRNA and Degradome / cmRNA. Our subsidiary objectives included analysis of the biology of small RNAs and the cell wall composition of Miscanthus. These objectives have all been completed, one publication is in print, one is submitted and several more are in progress.

  18. Functional annotations in bacterial genomes based on small RNA signatures.

    PubMed

    Sridhar, Jayavel; Rafi, Ziauddin Ahamed

    2008-04-04

    One of the key challenges in computational genomics is annotating coding genes and identification of regulatory RNAs in complete genomes. An attempt is made in this study which uses the regulatory RNA locations and their conserved flanking genes identified within the genomic backbone of template genome to search for similar RNA locations in query genomes. The search is based on recently reported coexistence of small RNAs and their conserved flanking genes in related genomes. Based on our study, 54 additional sRNA locations and functions of 96 uncharacterized genes are predicted in two draft genomes viz., Serratia marcesens Db1 and Yersinia enterocolitica 8081. Although most of the identified additional small RNA regions and their corresponding flanking genes are homologous in nature, the proposed anchoring technique could successfully identify four non-homologous small RNA regions in Y. enterocolitica genome also. The KEGG Orthology (KO) based automated functional predictions confirms the predicted functions of 65 flanking genes having defined KO numbers, out of the total 96 predictions made by this method. This coexistence based method shows more sensitivity than controlled vocabularies in locating orthologous gene pairs even in the absence of defined Orthology numbers. All functional predictions made by this study in Y. enterocolitica 8081 were confirmed by the recently published complete genome sequence and annotations. This study also reports the possible regions of gene rearrangements in these two genomes and further characterization of such RNA regions could shed more light on their possible role in genome evolution.

  19. Functional annotations in bacterial genomes based on small RNA signatures

    PubMed Central

    Sridhar, Jayavel; Rafi, Ziauddin Ahamed

    2008-01-01

    One of the key challenges in computational genomics is annotating coding genes and identification of regulatory RNAs in complete genomes. An attempt is made in this study which uses the regulatory RNA locations and their conserved flanking genes identified within the genomic backbone of template genome to search for similar RNA locations in query genomes. The search is based on recently reported coexistence of small RNAs and their conserved flanking genes in related genomes. Based on our study, 54 additional sRNA locations and functions of 96 uncharacterized genes are predicted in two draft genomes viz., Serratia marcesens Db1 and Yersinia enterocolitica 8081. Although most of the identified additional small RNA regions and their corresponding flanking genes are homologous in nature, the proposed anchoring technique could successfully identify four non-homologous small RNA regions in Y. enterocolitica genome also. The KEGG Orthology (KO) based automated functional predictions confirms the predicted functions of 65 flanking genes having defined KO numbers, out of the total 96 predictions made by this method. This coexistence based method shows more sensitivity than controlled vocabularies in locating orthologous gene pairs even in the absence of defined Orthology numbers. All functional predictions made by this study in Y. enterocolitica 8081 were confirmed by the recently published complete genome sequence and annotations. This study also reports the possible regions of gene rearrangements in these two genomes and further characterization of such RNA regions could shed more light on their possible role in genome evolution. PMID:18478081

  20. Delivery of small interfering RNA (siRNA) using the sleeping beauty transposon.

    PubMed

    Fletcher, Bradley S

    2010-11-01

    RNA interference (RNAi) is an evolutionarily conserved process that silences gene expression through double-stranded RNA species in a sequence-specific manner. Small interfering RNAs (siRNAs) can promote sequence-specific degradation and/or translational repression of target RNA by activation of the RNA-induced silencing complex (RISC). Traditionally, silencing in mammalian cells had been achieved by transfection of synthetically derived siRNA duplexes, resulting in transient gene suppression of the target sequence. As the technology was advanced, inhibitory short-hairpin-shaped RNAs (shRNAs) could be produced by transcription from RNA polymerase-III (pol-III)-driven promoters, such as H1, U6, or cytomegalovirus (CMV)-enhanced pol III promoters. Following transcription, the shRNAs are processed by the enzyme Dicer into active siRNA. This approach allows for the continuous production of siRNA within cells using a DNA template and offers increased options for delivery of the pol-III-driven transcriptional units. A number of different viral vectors, as well as plasmid DNAs, have been utilized to deliver shRNA to mammalian cells. Here, the Tc1/mariner DNA transposon Sleeping Beauty (SB) is used as a tool to deliver shRNA-encoding transcriptional units. The SB transposon system uses a "cut-and-paste" mechanism to insert the transposon into random TA dinucleotides within the target genome. The shRNAs are then processed and used for gene knockdown. PMID:21041394

  1. Colored petri net modeling of small interfering RNA-mediated messenger RNA degradation

    PubMed Central

    Nickaeen, Niloofar; Moein, Shiva; Heidary, Zarifeh; Ghaisari, Jafar

    2016-01-01

    Background: Mathematical modeling of biological systems is an attractive way for studying complex biological systems and their behaviors. Petri Nets, due to their ability to model systems with various levels of qualitative information, have been wildly used in modeling biological systems in which enough qualitative data may not be at disposal. These nets have been used to answer questions regarding the dynamics of different cell behaviors including the translation process. In one stage of the translation process, the RNA sequence may be degraded. In the process of degradation of RNA sequence, small-noncoding RNA molecules known as small interfering RNA (siRNA) match the target RNA sequence. As a result of this matching, the target RNA sequence is destroyed. Materials and Methods: In this context, the process of matching and destruction is modeled using Colored Petri Nets (CPNs). The model is constructed using CPNs which allow tokens to have a value or type on them. Thus, CPN is a suitable tool to model string structures in which each element of the string has a different type. Using CPNs, long RNA, and siRNA strings are modeled with a finite set of colors. The model is simulated via CPN Tools. Results: A CPN model of the matching between RNA and siRNA strings is constructed in CPN Tools environment. Conclusion: In previous studies, a network of stoichiometric equations was modeled. However, in this particular study, we modeled the mechanism behind the silencing process. Modeling this kind of mechanisms provides us with a tool to examine the effects of different factors such as mutation or drugs on the process. PMID:27376039

  2. Comprehensive experimental fitness landscape and evolutionary network for small RNA.

    PubMed

    Jiménez, José I; Xulvi-Brunet, Ramon; Campbell, Gregory W; Turk-MacLeod, Rebecca; Chen, Irene A

    2013-09-10

    The origin of life is believed to have progressed through an RNA world, in which RNA acted as both genetic material and functional molecules. The structure of the evolutionary fitness landscape of RNA would determine natural selection for the first functional sequences. Fitness landscapes are the subject of much speculation, but their structure is essentially unknown. Here we describe a comprehensive map of a fitness landscape, exploring nearly all of sequence space, for short RNAs surviving selection in vitro. With the exception of a small evolutionary network, we find that fitness peaks are largely isolated from one another, highlighting the importance of historical contingency and indicating that natural selection would be constrained to local exploration in the RNA world. PMID:23980164

  3. Comprehensive experimental fitness landscape and evolutionary network for small RNA.

    PubMed

    Jiménez, José I; Xulvi-Brunet, Ramon; Campbell, Gregory W; Turk-MacLeod, Rebecca; Chen, Irene A

    2013-09-10

    The origin of life is believed to have progressed through an RNA world, in which RNA acted as both genetic material and functional molecules. The structure of the evolutionary fitness landscape of RNA would determine natural selection for the first functional sequences. Fitness landscapes are the subject of much speculation, but their structure is essentially unknown. Here we describe a comprehensive map of a fitness landscape, exploring nearly all of sequence space, for short RNAs surviving selection in vitro. With the exception of a small evolutionary network, we find that fitness peaks are largely isolated from one another, highlighting the importance of historical contingency and indicating that natural selection would be constrained to local exploration in the RNA world.

  4. Mapping Long Noncoding RNA Chromatin Occupancy Using Capture Hybridization Analysis of RNA Targets (CHART).

    PubMed

    Vance, Keith W

    2017-01-01

    Capture Hybridization Analysis of RNA Targets (CHART) has recently been developed to map the genome-wide binding profile of chromatin-associated RNAs. This protocol uses a small number of 22-28 nucleotide biotinylated antisense oligonucleotides, complementary to regions of the target RNA that are accessible for hybridization, to purify RNAs from a cross-linked chromatin extract. RNA-chromatin complexes are next immobilized on beads, washed, and specifically eluted using RNase H. Associated genomic DNA is then sequenced using high-throughput sequencing technologies and mapped to the genome to identify RNA-chromatin associations on a large scale. CHART-based strategies can be applied to determine the nature and extent of long noncoding RNA (long ncRNA) association with chromatin genome-wide and identify direct long ncRNA transcriptional targets. PMID:27662869

  5. Exploring MicroRNA-Like Small RNAs in the Filamentous Fungus Fusarium oxysporum

    PubMed Central

    Jiang, Qiyan; Sun, Xianjun; Wang, Yong; Zhang, Hui; Hu, Zheng

    2014-01-01

    RNA silencing such as quelling and meiotic silencing by unpaired DNA (MSUD) and several other classes of special small RNAs have been discovered in filamentous fungi recently. More than four different mechanisms of microRNA-like RNAs (milRNAs) production have been illustrated in the model fungus Neurospora crassa including a dicer-independent pathway. To date, very little work focusing on small RNAs in fungi has been reported and no universal or particular characteristic of milRNAs were defined clearly. In this study, small RNA and degradome libraries were constructed and subsequently deep sequenced for investigating milRNAs and their potential cleavage targets on the genome level in the filamentous fungus F. oxysporum f. sp. lycopersici. As a result, there is no intersection of conserved miRNAs found by BLASTing against the miRBase. Further analysis showed that the small RNA population of F. oxysporum shared many common features with the small RNAs from N. crassa and other fungi. According to the known standards of miRNA prediction in plants and animals, milRNA candidates from 8 families (comprising 19 members) were screened out and identified. However, none of them could trigger target cleavage based on the degradome data. Moreover, most major signals of cleavage in transcripts could not match appropriate complementary small RNAs, suggesting that other predominant modes for milRNA-mediated gene regulation could exist in F. oxysporum. In addition, the PAREsnip program was utilized for comprehensive analysis and 3 families of small RNAs leading to transcript cleavage were experimentally validated. Altogether, our findings provided valuable information and important hints for better understanding the functions of the small RNAs and milRNAs in the fungal kingdom. PMID:25141304

  6. Selection and Characterization of Small Molecules That Bind the HIV-1 Frameshift Site RNA

    PubMed Central

    Marcheschi, Ryan J.; Mouzakis, Kathryn D.; Butcher, Samuel E.

    2009-01-01

    HIV-1 requires a −1 translational frameshift to properly synthesize the viral enzymes required for replication. The frameshift mechanism is dependent upon two RNA elements, a seven-nucleotide slippery sequence (UUUUUUA) and a downstream RNA structure. Frameshifting occurs with a frequency of ~5%, and increasing or decreasing this frequency may result in a decrease in viral replication. Here, we report the results of a high-throughput screen designed to find small molecules that bind to the HIV-1 frameshift site RNA. Out of 34,500 compounds screened, 202 were identified as positive hits. We show that one of these compounds, doxorubicin, binds the HIV-1 RNA with low micromolar affinity (Kd = 2.8 μM). This binding was confirmed and localized to the RNA using NMR. Further analysis revealed that this compound increased the RNA stability by approximately 5 °C and decreased translational frameshifting by 28% (±14%), as measured in vitro. PMID:19673541

  7. Small RNA profiles from virus-infected fresh market vegetables.

    PubMed

    Frizzi, Alessandra; Zhang, Yuanji; Kao, John; Hagen, Charles; Huang, Shihshieh

    2014-12-10

    Functional small RNAs, such as short interfering RNAs (siRNAs) and microRNAs (miRNAs), exist in freshly consumed fruits and vegetables. These siRNAs can be derived either from endogenous sequences or from viruses that infect them. Symptomatic tomatoes, watermelons, zucchini, and onions were purchased from grocery stores and investigated by small RNA sequencing. By aligning the obtained small RNA sequences to sequences of known viruses, four different viruses were identified as infecting these fruits and vegetables. Many of these virally derived small RNAs along with endogenous small RNAs were found to be highly complementary to human genes. However, the established history of safe consumption of these vegetables suggests that this sequence homology has little biological relevance. By extension, these results provide evidence for the safe use by humans and animals of genetically engineered crops using RNA-based suppression technologies, especially vegetable crops with virus resistance conferred by expression of siRNAs or miRNAs derived from viral sequences. PMID:25389086

  8. PSRna: Prediction of small RNA secondary structures based on reverse complementary folding method.

    PubMed

    Li, Jin; Xu, Chengzhen; Wang, Lei; Liang, Hong; Feng, Weixing; Cai, Zhongxi; Wang, Ying; Cong, Wang; Liu, Yunlong

    2016-08-01

    Prediction of RNA secondary structures is an important problem in computational biology and bioinformatics, since RNA secondary structures are fundamental for functional analysis of RNA molecules. However, small RNA secondary structures are scarce and few algorithms have been specifically designed for predicting the secondary structures of small RNAs. Here we propose an algorithm named "PSRna" for predicting small-RNA secondary structures using reverse complementary folding and characteristic hairpin loops of small RNAs. Unlike traditional algorithms that usually generate multi-branch loops and 5[Formula: see text] end self-folding, PSRna first estimated the maximum number of base pairs of RNA secondary structures based on the dynamic programming algorithm and a path matrix is constructed at the same time. Second, the backtracking paths are extracted from the path matrix based on backtracking algorithm, and each backtracking path represents a secondary structure. To improve accuracy, the predicted RNA secondary structures are filtered based on their free energy, where only the secondary structure with the minimum free energy was identified as the candidate secondary structure. Our experiments on real data show that the proposed algorithm is superior to two popular methods, RNAfold and RNAstructure, in terms of sensitivity, specificity and Matthews correlation coefficient (MCC). PMID:27045556

  9. PSRna: Prediction of small RNA secondary structures based on reverse complementary folding method.

    PubMed

    Li, Jin; Xu, Chengzhen; Wang, Lei; Liang, Hong; Feng, Weixing; Cai, Zhongxi; Wang, Ying; Cong, Wang; Liu, Yunlong

    2016-08-01

    Prediction of RNA secondary structures is an important problem in computational biology and bioinformatics, since RNA secondary structures are fundamental for functional analysis of RNA molecules. However, small RNA secondary structures are scarce and few algorithms have been specifically designed for predicting the secondary structures of small RNAs. Here we propose an algorithm named "PSRna" for predicting small-RNA secondary structures using reverse complementary folding and characteristic hairpin loops of small RNAs. Unlike traditional algorithms that usually generate multi-branch loops and 5[Formula: see text] end self-folding, PSRna first estimated the maximum number of base pairs of RNA secondary structures based on the dynamic programming algorithm and a path matrix is constructed at the same time. Second, the backtracking paths are extracted from the path matrix based on backtracking algorithm, and each backtracking path represents a secondary structure. To improve accuracy, the predicted RNA secondary structures are filtered based on their free energy, where only the secondary structure with the minimum free energy was identified as the candidate secondary structure. Our experiments on real data show that the proposed algorithm is superior to two popular methods, RNAfold and RNAstructure, in terms of sensitivity, specificity and Matthews correlation coefficient (MCC).

  10. Ageing and the Small, Non-Coding RNA World

    PubMed Central

    Kato, Masaomi; Slack, Frank J.

    2012-01-01

    MicroRNAs, a class of small, non-coding RNAs, are now widely known for their importance in many aspects of biology. These small regulatory RNAs have critical functions in diverse biological events, including development and disease. Recent findings show that microRNAs are essential for lifespan determination in the model organisms, C. elegans and Drosophila, suggesting that microRNAs are also involved in the complex process of ageing. Further, short RNA fragments derived from longer parental RNAs, such as transfer RNA cleavage fragments, have now emerged as a novel class of regulatory RNAs that inhibit translation in response to stress. In addition, the RNA editing pathway is likely to act in the double-stranded RNA-mediated silencing machinery to suppress unfavorable RNA interference activity in the ageing process. These multiple, redundant layers in gene regulatory networks may make it possible to both stably and flexibly regulate genetic pathways in ensuring robustness of developmental and ageing processes. PMID:22504407

  11. Development of Small RNA Delivery Systems for Lung Cancer Therapy

    PubMed Central

    Fujita, Yu; Kuwano, Kazuyoshi; Ochiya, Takahiro

    2015-01-01

    RNA interference (RNAi) has emerged as a powerful tool for studying target identification and holds promise for the development of therapeutic gene silencing. Recent advances in RNAi delivery and target selection provide remarkable opportunities for translational medical research. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA (mRNA) degradation. Two types of small RNA molecules, small interfering RNAs (siRNAs) and microRNAs (miRNAs), have a central function in RNAi technology. The success of RNAi-based therapeutic delivery may be dependent upon uncovering a delivery route, sophisticated delivery carriers, and nucleic acid modifications. Lung cancer is still the leading cause of cancer death worldwide, for which novel therapeutic strategies are critically needed. Recently, we have reported a novel platform (PnkRNA™ and nkRNA®) to promote naked RNAi approaches through inhalation without delivery vehicles in lung cancer xenograft models. We suggest that a new class of RNAi therapeutic agent and local drug delivery system could also offer a promising RNAi-based strategy for clinical applications in cancer therapy. In this article, we show recent strategies for an RNAi delivery system and suggest the possible clinical usefulness of RNAi-based therapeutics for lung cancer treatment. PMID:25756380

  12. Small RNA transcriptomes of mangroves evolve adaptively in extreme environments

    PubMed Central

    Wen, Ming; Lin, Xingqin; Xie, Munan; Wang, Yushuai; Shen, Xu; Liufu, Zhongqi; Wu, Chung-I; Shi, Suhua; Tang, Tian

    2016-01-01

    MicroRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are key players in plant stress responses. Here, we present the sRNA transcriptomes of mangroves Bruguiera gymnorrhiza and Kandelia candel. Comparative computational analyses and target predictions revealed that mangroves exhibit distinct sRNA regulatory networks that differ from those of glycophytes. A total of 32 known and three novel miRNA families were identified. Conserved and mangrove-specific miRNA targets were predicted; the latter were widely involved in stress responses. The known miRNAs showed differential expression between the mangroves and glycophytes, reminiscent of the adaptive stress-responsive changes in Arabidopsis. B. gymnorrhiza possessed highly abundant but less conserved TAS3 trans-acting siRNAs (tasiRNAs) in addition to tasiR-ARFs, with expanded potential targets. Our results indicate that the evolutionary alteration of sRNA expression levels and the rewiring of sRNA-regulatory networks are important mechanisms underlying stress adaptation. We also identified sRNAs that are involved in salt and/or drought tolerance and nutrient homeostasis as possible contributors to mangrove success in stressful environments. PMID:27278626

  13. Small RNA transcriptomes of mangroves evolve adaptively in extreme environments.

    PubMed

    Wen, Ming; Lin, Xingqin; Xie, Munan; Wang, Yushuai; Shen, Xu; Liufu, Zhongqi; Wu, Chung-I; Shi, Suhua; Tang, Tian

    2016-01-01

    MicroRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are key players in plant stress responses. Here, we present the sRNA transcriptomes of mangroves Bruguiera gymnorrhiza and Kandelia candel. Comparative computational analyses and target predictions revealed that mangroves exhibit distinct sRNA regulatory networks that differ from those of glycophytes. A total of 32 known and three novel miRNA families were identified. Conserved and mangrove-specific miRNA targets were predicted; the latter were widely involved in stress responses. The known miRNAs showed differential expression between the mangroves and glycophytes, reminiscent of the adaptive stress-responsive changes in Arabidopsis. B. gymnorrhiza possessed highly abundant but less conserved TAS3 trans-acting siRNAs (tasiRNAs) in addition to tasiR-ARFs, with expanded potential targets. Our results indicate that the evolutionary alteration of sRNA expression levels and the rewiring of sRNA-regulatory networks are important mechanisms underlying stress adaptation. We also identified sRNAs that are involved in salt and/or drought tolerance and nutrient homeostasis as possible contributors to mangrove success in stressful environments. PMID:27278626

  14. Using Small RNA Deep Sequencing Data to Detect Human Viruses.

    PubMed

    Wang, Fang; Sun, Yu; Ruan, Jishou; Chen, Rui; Chen, Xin; Chen, Chengjie; Kreuze, Jan F; Fei, ZhangJun; Zhu, Xiao; Gao, Shan

    2016-01-01

    Small RNA sequencing (sRNA-seq) can be used to detect viruses in infected hosts without the necessity to have any prior knowledge or specialized sample preparation. The sRNA-seq method was initially used for viral detection and identification in plants and then in invertebrates and fungi. However, it is still controversial to use sRNA-seq in the detection of mammalian or human viruses. In this study, we used 931 sRNA-seq runs of data from the NCBI SRA database to detect and identify viruses in human cells or tissues, particularly from some clinical samples. Six viruses including HPV-18, HBV, HCV, HIV-1, SMRV, and EBV were detected from 36 runs of data. Four viruses were consistent with the annotations from the previous studies. HIV-1 was found in clinical samples without the HIV-positive reports, and SMRV was found in Diffuse Large B-Cell Lymphoma cells for the first time. In conclusion, these results suggest the sRNA-seq can be used to detect viruses in mammals and humans. PMID:27066498

  15. Using Small RNA Deep Sequencing Data to Detect Human Viruses

    PubMed Central

    Wang, Fang; Sun, Yu; Ruan, Jishou; Chen, Rui; Chen, Xin; Chen, Chengjie; Kreuze, Jan F.; Fei, ZhangJun; Zhu, Xiao

    2016-01-01

    Small RNA sequencing (sRNA-seq) can be used to detect viruses in infected hosts without the necessity to have any prior knowledge or specialized sample preparation. The sRNA-seq method was initially used for viral detection and identification in plants and then in invertebrates and fungi. However, it is still controversial to use sRNA-seq in the detection of mammalian or human viruses. In this study, we used 931 sRNA-seq runs of data from the NCBI SRA database to detect and identify viruses in human cells or tissues, particularly from some clinical samples. Six viruses including HPV-18, HBV, HCV, HIV-1, SMRV, and EBV were detected from 36 runs of data. Four viruses were consistent with the annotations from the previous studies. HIV-1 was found in clinical samples without the HIV-positive reports, and SMRV was found in Diffuse Large B-Cell Lymphoma cells for the first time. In conclusion, these results suggest the sRNA-seq can be used to detect viruses in mammals and humans. PMID:27066498

  16. Global Mapping of Small RNA-Target Interactions in Bacteria.

    PubMed

    Melamed, Sahar; Peer, Asaf; Faigenbaum-Romm, Raya; Gatt, Yair E; Reiss, Niv; Bar, Amir; Altuvia, Yael; Argaman, Liron; Margalit, Hanah

    2016-09-01

    Small RNAs (sRNAs) associated with the RNA chaperon protein Hfq are key posttranscriptional regulators of gene expression in bacteria. Deciphering the sRNA-target interactome is an essential step toward understanding the roles of sRNAs in the cellular networks. We developed a broadly applicable methodology termed RIL-seq (RNA interaction by ligation and sequencing), which integrates experimental and computational tools for in vivo transcriptome-wide identification of interactions involving Hfq-associated sRNAs. By applying this methodology to Escherichia coli we discovered an extensive network of interactions involving RNA pairs showing sequence complementarity. We expand the ensemble of targets for known sRNAs, uncover additional Hfq-bound sRNAs encoded in various genomic regions along with their trans encoded targets, and provide insights into binding and possible cycling of RNAs on Hfq. Comparison of the sRNA interactome under various conditions has revealed changes in the sRNA repertoire as well as substantial re-wiring of the network between conditions. PMID:27588604

  17. Equilibrium self-assembly of small RNA viruses

    NASA Astrophysics Data System (ADS)

    Bruinsma, R. F.; Comas-Garcia, M.; Garmann, R. F.; Grosberg, A. Y.

    2016-03-01

    We propose a description for the quasiequilibrium self-assembly of small, single-stranded (ss) RNA viruses whose capsid proteins (CPs) have flexible, positively charged, disordered tails that associate with the negatively charged RNA genome molecules. We describe the assembly of such viruses as the interplay between two coupled phase-transition-like events: the formation of the protein shell (the capsid) by CPs and the condensation of a large ss viral RNA molecule. Electrostatic repulsion between the CPs competes with attractive hydrophobic interactions and attractive interaction between neutralized RNA segments mediated by the tail groups. An assembly diagram is derived in terms of the strength of attractive interactions between CPs and between CPs and the RNA molecules. It is compared with the results of recent studies of viral assembly. We demonstrate that the conventional theory of self-assembly, which does describe the assembly of empty capsids, is in general not applicable to the self-assembly of RNA-encapsidating virions.

  18. Equilibrium self-assembly of small RNA viruses.

    PubMed

    Bruinsma, R F; Comas-Garcia, M; Garmann, R F; Grosberg, A Y

    2016-03-01

    We propose a description for the quasiequilibrium self-assembly of small, single-stranded (ss) RNA viruses whose capsid proteins (CPs) have flexible, positively charged, disordered tails that associate with the negatively charged RNA genome molecules. We describe the assembly of such viruses as the interplay between two coupled phase-transition-like events: the formation of the protein shell (the capsid) by CPs and the condensation of a large ss viral RNA molecule. Electrostatic repulsion between the CPs competes with attractive hydrophobic interactions and attractive interaction between neutralized RNA segments mediated by the tail groups. An assembly diagram is derived in terms of the strength of attractive interactions between CPs and between CPs and the RNA molecules. It is compared with the results of recent studies of viral assembly. We demonstrate that the conventional theory of self-assembly, which does describe the assembly of empty capsids, is in general not applicable to the self-assembly of RNA-encapsidating virions.

  19. Small RNA transcriptomes of mangroves evolve adaptively in extreme environments.

    PubMed

    Wen, Ming; Lin, Xingqin; Xie, Munan; Wang, Yushuai; Shen, Xu; Liufu, Zhongqi; Wu, Chung-I; Shi, Suhua; Tang, Tian

    2016-01-01

    MicroRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are key players in plant stress responses. Here, we present the sRNA transcriptomes of mangroves Bruguiera gymnorrhiza and Kandelia candel. Comparative computational analyses and target predictions revealed that mangroves exhibit distinct sRNA regulatory networks that differ from those of glycophytes. A total of 32 known and three novel miRNA families were identified. Conserved and mangrove-specific miRNA targets were predicted; the latter were widely involved in stress responses. The known miRNAs showed differential expression between the mangroves and glycophytes, reminiscent of the adaptive stress-responsive changes in Arabidopsis. B. gymnorrhiza possessed highly abundant but less conserved TAS3 trans-acting siRNAs (tasiRNAs) in addition to tasiR-ARFs, with expanded potential targets. Our results indicate that the evolutionary alteration of sRNA expression levels and the rewiring of sRNA-regulatory networks are important mechanisms underlying stress adaptation. We also identified sRNAs that are involved in salt and/or drought tolerance and nutrient homeostasis as possible contributors to mangrove success in stressful environments.

  20. Small interfering RNA delivery through positively charged polymer nanoparticles

    NASA Astrophysics Data System (ADS)

    Dragoni, Luca; Ferrari, Raffaele; Lupi, Monica; Cesana, Alberto; Falcetta, Francesca; Ubezio, Paolo; D'Incalci, Maurizio; Morbidelli, Massimo; Moscatelli, Davide

    2016-03-01

    Small interfering RNA (siRNA) is receiving increasing attention with regard to the treatment of many genetic diseases, both acquired and hereditary, such as cancer and diabetes. Being a high molecular weight (MW) polyanion, siRNA is not able to cross a cell membrane, and in addition it is unstable in physiological conditions. Accordingly, a biocompatible nanocarrier able to deliver siRNA into cells is needed. In this work, we synthesized biocompatible positively charged nanoparticles (NPs) following a two-step process that involves ring opening polymerization (ROP) and emulsion free radical polymerization (EFRP). Firstly, we proved the possibility of fine tuning the NPs’ characteristics (e.g. size and surface charge) by changing the synthetic process parameters. Then the capability in loading and delivering undamaged siRNA into a cancer cell cytoplasm has been shown. This latter process occurs through the biodegradation of the polymer constituting the NPs, whose kinetics can be tuned by adjusting the polymer’s MW. Finally, the ability of NPs to carry siRNA inside the cells in order to inhibit their target gene has been demonstrated using green flourescent protein positive cells.

  1. Structure-function analysis of the trypanosomatid spliced leader RNA.

    PubMed Central

    Goncharov, I; Xu, Y X; Zimmer, Y; Sherman, K; Michaeli, S

    1998-01-01

    In trypanosomes, all mRNAs possess a spliced leader (SL) at their 5' end. SL is added to pre-mRNA via trans -splicing from a small RNA, the SL RNA. To examine structure-function aspects of the trypanosomatid SL RNA, an in vivo system was developed in the monogenetic trypanosomatid Leptomonas collosoma to analyze the function of chimeric and site-directed SL RNA mutants in trans -splicing. Stable cell lines expressing chimeric and mutated SL RNA from the authentic SL RNA regulatory unit were obtained. The chimeric RNA was expressed and assembled into an SL RNP particle, but could not serve as a substrate in splicing. Mutations in loop II and III of L.collosoma SL RNA formed the Y structure intermediate. In addition, a double SL RNA mutant in loop II, and positions 7 and 8 of the intron, also formed the Y structure intermediate, suggesting that these intron positions, although proposed to participate in the interaction of SL RNA with U5, may not be crucial for the first step of the trans -splicing reaction. A mutation in the exon located in loop I was not utilized in splicing, suggesting the importance of exon sequences for trans -splicing in trypanosomes. However, a double SL RNA mutant in loop II and exon position 31 was utilized in both steps of splicing; the mutant thus provides a model molecule for further analysis of positions essential for the function of the SL RNA. PMID:9547281

  2. Silent no more: Endogenous small RNA pathways promote gene expression.

    PubMed

    Wedeles, Christopher J; Wu, Monica Z; Claycomb, Julie M

    2014-01-01

    Endogenous small RNA pathways related to RNA interference (RNAi) play a well-documented role in protecting host genomes from the invasion of foreign nucleic acids. In C. elegans, the PIWI type Argonaute, PRG-1, through an association with 21U-RNAs, mediates a genome surveillance process by constantly scanning the genome for potentially deleterious invading elements. Upon recognition of foreign nucleic acids, PRG-1 initiates a cascade of cytoplasmic and nuclear events that results in heritable epigenetic silencing of these transcripts and their coding genomic loci. If the PRG-1/21U-RNA genome surveillance pathway has the capacity to target most of the C. elegans transcriptome, what mechanisms exist to protect endogenous transcripts from being silenced by this pathway? In this commentary, we discuss three recent publications that implicate the CSR-1 small RNA pathway in the heritable activation of germline transcripts, propose a model as to why not all epialleles behave similarly, and touch on the practical implications of these findings.

  3. Small Cofactors May Assist Protein Emergence from RNA World: Clues from RNA-Protein Complexes

    PubMed Central

    Shen, Liang; Ji, Hong-Fang

    2011-01-01

    It is now widely accepted that at an early stage in the evolution of life an RNA world arose, in which RNAs both served as the genetic material and catalyzed diverse biochemical reactions. Then, proteins have gradually replaced RNAs because of their superior catalytic properties in catalysis over time. Therefore, it is important to investigate how primitive functional proteins emerged from RNA world, which can shed light on the evolutionary pathway of life from RNA world to the modern world. In this work, we proposed that the emergence of most primitive functional proteins are assisted by the early primitive nucleotide cofactors, while only a minority are induced directly by RNAs based on the analysis of RNA-protein complexes. Furthermore, the present findings have significant implication for exploring the composition of primitive RNA, i.e., adenine base as principal building blocks. PMID:21789260

  4. Ultraviolet light-induced inhibition of small nuclear RNA synthesis.

    PubMed

    Eliceiri, B P; Choudhury, K; Scott, Q O; Eliceiri, G L

    1989-03-01

    Two apparently distinct types of inhibition of the synthesis of U1, U2, U3, U4, and U5 small nuclear RNA, induced by ultraviolet (UV) radiation, have been described before: immediate and delayed. Our present observation can be summarized as follows: a) neither the immediate nor the delayed inhibition appear to be mediated by the formation of cyclobutane pyrimidine dimers, since they were not prevented by photoreactivating light, in ICR 2A frog cells; b) the inhibition of U1 RNA synthesis, monitored in HeLA cells within the first few minutes after irradiation, extrapolated to a substantial suppression at time zero of postirradiation cell incubation, providing further support for the proposal that the immediate inhibition is a reaction separate from the delayed UV light-induced inhibition of U1 RNA synthesis; c) the transition from the pattern of the immediate inhibition to that of the delayed inhibition (disappearance of the UV-resistant fraction of U1 RNA synthesis and increased rate of inhibition) occurred gradually, without an apparent threshold, within the first 2 hr of incubation after irradiation; and d) the incident UV dose that resulted in a 37% level of residual U1 RNA synthesis (D37) during the delayed inhibition was about 7 J/m2, with an apparent UV dose threshold, and was about 60 J/m2 for the immediate inhibition. PMID:2925798

  5. Global small RNA chaperone Hfq and regulatory small RNAs are important virulence regulators in Erwinia amylovora.

    PubMed

    Zeng, Quan; McNally, R Ryan; Sundin, George W

    2013-04-01

    Hfq is a global small RNA (sRNA) chaperone that interacts with Hfq-regulated sRNAs and functions in the posttranscriptional regulation of gene expression. In this work, we identified Hfq to be a virulence regulator in the Gram-negative fire blight pathogen Erwinia amylovora. Deletion of hfq in E. amylovora Ea1189 significantly reduced bacterial virulence in both immature pear fruits and apple shoots. Analysis of virulence determinants in strain Ea1189Δhfq showed that Hfq exerts pleiotropic regulation of amylovoran exopolysaccharide production, biofilm formation, motility, and the type III secretion system (T3SS). Further characterization of biofilm regulation by Hfq demonstrated that Hfq limits bacterial attachment to solid surfaces while promoting biofilm maturation. Characterization of T3SS regulation by Hfq revealed that Hfq positively regulates the translocation and secretion of the major type III effector DspE and negatively controls the secretion of the putative translocator HrpK and the type III effector Eop1. Lastly, 10 Hfq-regulated sRNAs were identified using a computational method, and two of these sRNAs, RprA and RyhA, were found to be required for the full virulence of E. amylovora.

  6. Silencing-associated and meiosis-specific small RNA pathways in Paramecium tetraurelia

    PubMed Central

    Lepère, Gersende; Nowacki, Mariusz; Serrano, Vincent; Gout, Jean-François; Guglielmi, Gérard; Duharcourt, Sandra; Meyer, Eric

    2009-01-01

    Distinct small RNA pathways are involved in the two types of homology-dependent effects described in Paramecium tetraurelia, as shown by a functional analysis of Dicer and Dicer-like genes and by the sequencing of small RNAs. The siRNAs that mediate post-transcriptional gene silencing when cells are fed with double-stranded RNA (dsRNA) were found to comprise two subclasses. DCR1-dependent cleavage of the inducing dsRNA generates ∼23-nt primary siRNAs from both strands, while a different subclass of ∼24-nt RNAs, characterized by a short untemplated poly-A tail, is strictly antisense to the targeted mRNA, suggestive of secondary siRNAs that depend on an RNA-dependent RNA polymerase. An entirely distinct pathway is responsible for homology-dependent regulation of developmental genome rearrangements after sexual reproduction. During early meiosis, the DCL2 and DCL3 genes are required for the production of a highly complex population of ∼25-nt scnRNAs from all types of germline sequences, including both strands of exons, introns, intergenic regions, transposons and Internal Eliminated Sequences. A prominent 5′-UNG signature, and a minor fraction showing the complementary signature at positions 21–23, indicate that scnRNAs are cleaved from dsRNA precursors as duplexes with 2-nt 3′ overhangs at both ends, followed by preferential stabilization of the 5′-UNG strand. PMID:19103667

  7. Silencing-associated and meiosis-specific small RNA pathways in Paramecium tetraurelia.

    PubMed

    Lepère, Gersende; Nowacki, Mariusz; Serrano, Vincent; Gout, Jean-François; Guglielmi, Gérard; Duharcourt, Sandra; Meyer, Eric

    2009-02-01

    Distinct small RNA pathways are involved in the two types of homology-dependent effects described in Paramecium tetraurelia, as shown by a functional analysis of Dicer and Dicer-like genes and by the sequencing of small RNAs. The siRNAs that mediate post-transcriptional gene silencing when cells are fed with double-stranded RNA (dsRNA) were found to comprise two subclasses. DCR1-dependent cleavage of the inducing dsRNA generates approximately 23-nt primary siRNAs from both strands, while a different subclass of approximately 24-nt RNAs, characterized by a short untemplated poly-A tail, is strictly antisense to the targeted mRNA, suggestive of secondary siRNAs that depend on an RNA-dependent RNA polymerase. An entirely distinct pathway is responsible for homology-dependent regulation of developmental genome rearrangements after sexual reproduction. During early meiosis, the DCL2 and DCL3 genes are required for the production of a highly complex population of approximately 25-nt scnRNAs from all types of germline sequences, including both strands of exons, introns, intergenic regions, transposons and Internal Eliminated Sequences. A prominent 5'-UNG signature, and a minor fraction showing the complementary signature at positions 21-23, indicate that scnRNAs are cleaved from dsRNA precursors as duplexes with 2-nt 3' overhangs at both ends, followed by preferential stabilization of the 5'-UNG strand. PMID:19103667

  8. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Caers, A; De Rijk, P; De Wachter, R

    1998-01-01

    About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/ PMID:9399829

  9. Examining small molecule: HIV RNA interactions using arrayed imaging reflectometry

    NASA Astrophysics Data System (ADS)

    Chaimayo, Wanaruk; Miller, Benjamin L.

    2014-03-01

    Human Immunodeficiency Virus (HIV) has been the subject of intense research for more than three decades as it causes an uncurable disease: Acquired Immunodeficiency Syndrome, AIDS. In the pursuit of a medical treatment, RNAtargeted small molecules are emerging as promising targets. In order to understand the binding kinetics of small molecules and HIV RNA, association (ka) and dissociation (kd) kinetic constants must be obtained, ideally for a large number of sequences to assess selectivity. We have developed Aqueous Array Imaged Reflectometry (Aq-AIR) to address this challenge. Using a simple light interference phenomenon, Aq-AIR provides real-time high-throughput multiplex capabilities to detect binding of targets to surface-immobilized probes in a label-free microarray format. The second generation of Aq-AIR consisting of high-sensitivity CCD camera and 12-μL flow cell was fabricated. The system performance was assessed by real-time detection of MBNL1-(CUG)10 and neomycin B - HIV RNA bindings. The results establish this second-generation Aq-AIR to be able to examine small molecules binding to RNA sequences specific to HIV.

  10. Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli.

    PubMed

    Massé, Eric; Escorcia, Freddy E; Gottesman, Susan

    2003-10-01

    RyhB is a small antisense regulatory RNA that is repressed by the Fur repressor and negatively regulates at least six mRNAs encoding Fe-binding or Fe-storage proteins in Escherichia coli. When Fe is limiting, RyhB levels rise, and target mRNAs are rapidly degraded. RyhB is very stable when measured after treatment of cells with the transcription inhibitor rifampicin, but is unstable when overall mRNA transcription continues. We propose that RyhB turnover is coupled to and dependent on pairing with the target mRNAs. Degradation of both mRNA targets and RyhB is dependent on RNase E and is slowed in degradosome mutants. RyhB requires the RNA chaperone Hfq. In the absence of Hfq, RyhB is unstable, even when general transcription is inhibited; degradation is dependent upon RNase E. Hfq and RNase E bind similar sites on the RNA; pairing may allow loss of Hfq and access by RNase E. Two other Hfq-dependent small RNAs, DsrA and OxyS, are also stable when overall transcription is off, and unstable when it is not, suggesting that they, too, are degraded when their target mRNAs are available for pairing. Thus, this large class of regulatory RNAs share an unexpected intrinsic mechanism for shutting off their action.

  11. Assessment of microRNA differential expression and detection in multiplexed small RNA sequencing data.

    PubMed

    Campbell, Joshua D; Liu, Gang; Luo, Lingqi; Xiao, Ji; Gerrein, Joseph; Juan-Guardela, Brenda; Tedrow, John; Alekseyev, Yuriy O; Yang, Ivana V; Correll, Mick; Geraci, Mark; Quackenbush, John; Sciurba, Frank; Schwartz, David A; Kaminski, Naftali; Johnson, W Evan; Monti, Stefano; Spira, Avrum; Beane, Jennifer; Lenburg, Marc E

    2015-02-01

    Small RNA sequencing can be used to gain an unprecedented amount of detail into the microRNA transcriptome. The relatively high cost and low throughput of sequencing bases technologies can potentially be offset by the use of multiplexing. However, multiplexing involves a trade-off between increased number of sequenced samples and reduced number of reads per sample (i.e., lower depth of coverage). To assess the effect of different sequencing depths owing to multiplexing on microRNA differential expression and detection, we sequenced the small RNA of lung tissue samples collected in a clinical setting by multiplexing one, three, six, nine, or 12 samples per lane using the Illumina HiSeq 2000. As expected, the numbers of reads obtained per sample decreased as the number of samples in a multiplex increased. Furthermore, after normalization, replicate samples included in distinct multiplexes were highly correlated (R > 0.97). When detecting differential microRNA expression between groups of samples, microRNAs with average expression >1 reads per million (RPM) had reproducible fold change estimates (signal to noise) independent of the degree of multiplexing. The number of microRNAs detected was strongly correlated with the log2 number of reads aligning to microRNA loci (R = 0.96). However, most additional microRNAs detected in samples with greater sequencing depth were in the range of expression which had lower fold change reproducibility. These findings elucidate the trade-off between increasing the number of samples in a multiplex with decreasing sequencing depth and will aid in the design of large-scale clinical studies exploring microRNA expression and its role in disease.

  12. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    PubMed

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency. PMID:26567315

  13. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    PubMed

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency.

  14. ARGONAUTE PIWI domain and microRNA duplex structure regulate small RNA sorting in Arabidopsis

    PubMed Central

    Zhang, Xiaoming; Niu, DongDong; Carbonell, Alberto; Wang, Airong; Lee, Angel; Tun, Vinnary; Wang, Zonghua; Carrington, James C.; Chang, Chia-en A.; Jin, Hailing

    2014-01-01

    Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, the 5′-terminal nucleotide is important for sRNA sorting into different AGOs. Here, we show that miRNA duplex structure also contributes to miRNA sorting. Base-pairing at the 15th nucleotide of a miRNA duplex is important for miRNA sorting in both Arabidopsis AGO1 and AGO2. AGO2 favors miRNA duplexes with no middle mismatches, whereas AGO1 tolerates, or prefers, duplexes with central mismatches. AGO structure modeling and mutational analyses reveal that the QF-V motif within the conserved PIWI domain contributes to recognition of base-pairing at the 15th nucleotide of a duplex, while the DDDE catalytic core of AtAGO2 is important for recognition of the central nucleotides. Finally, we rescued the adaxialized phenotype of ago1-12, which is largely due to miR165 loss-of-function, by changing miR165 duplex structure which we predict redirects it to AGO2. PMID:25406978

  15. Molecular phylogeny of Stentor (Ciliophora: Heterotrichea) based on small subunit ribosomal RNA sequences.

    PubMed

    Gong, Ying-Chun; Yu, Yu-He; Zhu, Fei-Yun; Feng, Wei-Song

    2007-01-01

    To determine the phylogenetic position of Stentor within the Class Heterotrichea, the complete small subunit rRNA genes of three Stentor species, namely Stentor polymorphus, Stentor coeruleus, and Stentor roeseli, were sequenced and used to construct phylogenetic trees using the maximum parsimony, neighbor joining, and Bayesian analysis. With all phylogenetic methods, the genus Stentor was monophyletic, with S. roeseli branching basally. PMID:17300519

  16. An Integrative Analysis of microRNA and mRNA Profiling in CML Stem Cells.

    PubMed

    Nassar, Farah J; El Eit, Rabab; Nasr, Rihab

    2016-01-01

    Integrative analysis of microRNA (miRNA) and messenger RNA (mRNA) in Chronic Myeloid leukemia (CML) stem cells is an important technique to study the involvement of miRNA and their targets in CML stem cells self-renewal, maintenance, and therapeutic resistance. Here, we describe a simplified integrative analysis using Ingenuity Pathway Analysis software after performing proper RNA extraction, miRNA and mRNA microarray and data analysis. PMID:27581151

  17. Analysis of the small interfering RNA profiles of randomly inserted pTRM-TRI6 Fusarium graminearum mutants and their DON related phenotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Deoxynivalenol (DON) production by Fusarium graminearum requires activation of the trichothecene pathway in which TRI5 catalyzes the first step of trichothecene synthesis and TRI6 is a transcription factor activates the pathway. RNA interference (RNAi) has emerged as a useful fungal genetics tool f...

  18. Translocation of Small Interfering RNA and Cholesterol Molecules in Biomembranes

    NASA Astrophysics Data System (ADS)

    Kalia, Rajiv

    2013-03-01

    This presentation will focus on all-atom molecular dynamics (MD) simulation studies of (1) structural and mechanical barriers to translocation of small interfering RNA (siRNA) across a phospholipid bilayer, and (2) flip-flop dynamics of cholesterol (CHOL) molecules across a phospholipid bilayer. In the first case, we find that the siRNA induces a liquid-to-gel phase transformation. In the gel phase we find large compressive lateral stresses in the hydrocarbon chains of lipid molecules, which present a considerable barrier to siRNA passage across the bilayer. In the second case, we study spontaneous CHOL inter-leaflet transport (flip-flop), the effect of this process on mechanical stresses across the bilayer, and the role of CHOL in inducing molecular order in bilayer leaflets. The simulation was run for 15 microseconds and we found 24 CHOL flip-flop events over that duration. On average, a CHOL molecule migrates across the lipid bilayer in about 73 ns after a flip-flop event is triggered. We have calculated diffusion maps and determined free energy surfaces and flip-flop mechanisms for CHOL molecules. Work supported by NSF-OCI-0749360 and NSF-IOS-125317.

  19. Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts.

    PubMed

    Lueders, Tillmann; Friedrich, Michael W

    2003-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.

  20. Diverse Evolutionary Trajectories for Small RNA Biogenesis Genes in the Oomycete Genus Phytophthora

    PubMed Central

    Bollmann, Stephanie R.; Fang, Yufeng; Press, Caroline M.; Tyler, Brett M.; Grünwald, Niklaus J.

    2016-01-01

    Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. The genomes of several oomycetes including Phytophthora species such as the soybean pathogen P. sojae, have been sequenced, allowing evolutionary analysis of small RNA-processing enzymes. This study examined the evolutionary origins of the oomycete small RNA-related genes Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) through broad phylogenetic analyses of the key domains. Two Dicer gene homologs, DCL1 and DCL2, and one RDR homolog were cloned and analyzed from P. sojae. Gene expression analysis revealed only minor changes in transcript levels among different life stages. Oomycete DCL1 homologs clustered with animal and plant Dicer homologs in evolutionary trees, whereas oomycete DCL2 homologs clustered basally to the tree along with Drosha homologs. Phylogenetic analysis of the RDR homologs confirmed a previous study that suggested the last common eukaryote ancestor possessed three RDR homologs, which were selectively retained or lost in later lineages. Our analysis clarifies the position of some Unikont and Chromalveolate RDR lineages within the tree, including oomycete homologs. Finally, we analyzed alterations in the domain structure of oomycete Dicer and RDR homologs, specifically focusing on the proposed domain transfer of the DEAD-box helicase domain from Dicer to RDR. Implications of the oomycete domain structure are discussed, and possible roles of the two oomycete Dicer homologs are proposed. PMID:27014308

  1. Diverse Evolutionary Trajectories for Small RNA Biogenesis Genes in the Oomycete Genus Phytophthora.

    PubMed

    Bollmann, Stephanie R; Fang, Yufeng; Press, Caroline M; Tyler, Brett M; Grünwald, Niklaus J

    2016-01-01

    Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. The genomes of several oomycetes including Phytophthora species such as the soybean pathogen P. sojae, have been sequenced, allowing evolutionary analysis of small RNA-processing enzymes. This study examined the evolutionary origins of the oomycete small RNA-related genes Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) through broad phylogenetic analyses of the key domains. Two Dicer gene homologs, DCL1 and DCL2, and one RDR homolog were cloned and analyzed from P. sojae. Gene expression analysis revealed only minor changes in transcript levels among different life stages. Oomycete DCL1 homologs clustered with animal and plant Dicer homologs in evolutionary trees, whereas oomycete DCL2 homologs clustered basally to the tree along with Drosha homologs. Phylogenetic analysis of the RDR homologs confirmed a previous study that suggested the last common eukaryote ancestor possessed three RDR homologs, which were selectively retained or lost in later lineages. Our analysis clarifies the position of some Unikont and Chromalveolate RDR lineages within the tree, including oomycete homologs. Finally, we analyzed alterations in the domain structure of oomycete Dicer and RDR homologs, specifically focusing on the proposed domain transfer of the DEAD-box helicase domain from Dicer to RDR. Implications of the oomycete domain structure are discussed, and possible roles of the two oomycete Dicer homologs are proposed. PMID:27014308

  2. Nicotiana Small RNA Sequences Support a Host Genome Origin of Cucumber Mosaic Virus Satellite RNA

    PubMed Central

    Smith, Neil A.; Schumann, Ulrike; Fang, Yuan-Yuan; Dennis, Elizabeth S.; Zhang, Ren; Guo, Hui-Shan; Wang, Ming-Bo

    2015-01-01

    Satellite RNAs (satRNAs) are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS) transgene fused with a Cucumber mosaic virus (CMV) Y satellite RNA (Y-Sat) sequence (35S-GUS:Sat) was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM) to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs. PMID:25568943

  3. Small RNA viruses of insects: expression in plants and RNA silencing.

    PubMed

    Gordon, Karl H J; Waterhouse, Peter M

    2006-01-01

    Interest in insect small RNA viruses (SRVs) has grown slowly but steadily. A number of new viruses have been analyzed at the sequence level, adding to our knowledge of their diversity at the level of both individual virus species and families. In particular, a number of possible new virus families have emerged. This research has largely been driven by interest in their potential for pest control, as well as in their importance as the causal agents of disease in beneficial arthropods. At the same time, research into known viruses has made valuable contributions to our understanding of an emerging new field of central importance to molecular biology-the existence of RNA-based gene silencing, developmental control, and adaptive immune systems in eukaryotes. Subject to RNA-based adaptive immune responses in their hosts, viruses have evolved a variety of genes encoding proteins capable of suppressing the immune response. Such genes were first identified in plant viruses, but the first examples known from animal viruses were identified in insect RNA viruses. This chapter will address the diversity of insect SRVs, and attempts to harness their simplicity in the engineering of transgenic plants expressing viruses for resistance to insect pests. We also describe RNA interference and antiviral pathways identified in plants and animals, how they have led viruses to evolve genes capable of suppressing such adaptive immunity, and the problems presented by these pathways for the strategy of expressing viruses in transgenic plants. Approaches for countering these problems are also discussed.

  4. Mechanisms of immune system activation in mammalians by small interfering RNA (siRNA).

    PubMed

    Mansoori, Behzad; Mohammadi, Ali; Shir Jang, Solmaz; Baradaran, Behzad

    2016-11-01

    RNA interference (RNAi) guided by small interfering RNAs (siRNA), because of its potential to target and silence the expression of specific genes is utilized as an effective tool in a variety of biological applications. RNAi guided by siRNAs is a powerful tool to attain gene silencing in mammalian cells. One of the features which make siRNA as an amazing biological tool is extremely specific knockdown of target genes by degradation of analogous mRNAs. However, various non-specific effects limit the use of RNAi including the activation of innate immunity and inhibition of inadvertent target genes. One of the most common non-specific effects is inducing the innate immune system including cytoplasmic and endosomal activation of innate immune system, potentially offending the single in mammals. This activation is mainly interceded by immune cells, regularly through a Toll-like receptor (TLR) pathway. The siRNA sequence association of these pathways changes with the sort and position of the TLR involved. In contrast, non-immune cell activation can also arise generally siRNAs which enter into cytoplasm interacting with cytoplasmic RNA sensors such as retinoic acid-inducible gene I. Here, we explain the off-target effects of siRNAs that activate innate immune system and methods to alleviate them, to help enable impressive application of this exciting technology, Also we bold the aspect of molecular strategies permitting the design of therapeutic siRNAs with minute off-target effects.

  5. High throughput sequencing of small RNA component of leaves and inflorescence revealed conserved and novel miRNAs as well as phasiRNA loci in chickpea.

    PubMed

    Srivastava, Sangeeta; Zheng, Yun; Kudapa, Himabindu; Jagadeeswaran, Guru; Hivrale, Vandana; Varshney, Rajeev K; Sunkar, Ramanjulu

    2015-06-01

    Among legumes, chickpea (Cicer arietinum L.) is the second most important crop after soybean. MicroRNAs (miRNAs) play important roles by regulating target gene expression important for plant development and tolerance to stress conditions. Additionally, recently discovered phased siRNAs (phasiRNAs), a new class of small RNAs, are abundantly produced in legumes. Nevertheless, little is known about these regulatory molecules in chickpea. The small RNA population was sequenced from leaves and flowers of chickpea to identify conserved and novel miRNAs as well as phasiRNAs/phasiRNA loci. Bioinformatics analysis revealed 157 miRNA loci for the 96 highly conserved and known miRNA homologs belonging to 38 miRNA families in chickpea. Furthermore, 20 novel miRNAs belonging to 17 miRNA families were identified. Sequence analysis revealed approximately 60 phasiRNA loci. Potential target genes likely to be regulated by these miRNAs were predicted and some were confirmed by modified 5' RACE assay. Predicted targets are mostly transcription factors that might be important for developmental processes, and others include superoxide dismutases, plantacyanin, laccases and F-box proteins that could participate in stress responses and protein degradation. Overall, this study provides an inventory of miRNA-target gene interactions for chickpea, useful for the comparative analysis of small RNAs among legumes.

  6. Ancient and novel small RNA pathways compensate for the loss of piRNAs in multiple independent nematode lineages.

    PubMed

    Sarkies, Peter; Selkirk, Murray E; Jones, John T; Blok, Vivian; Boothby, Thomas; Goldstein, Bob; Hanelt, Ben; Ardila-Garcia, Alex; Fast, Naomi M; Schiffer, Phillip M; Kraus, Christopher; Taylor, Mark J; Koutsovoulos, Georgios; Blaxter, Mark L; Miska, Eric A

    2015-02-01

    Small RNA pathways act at the front line of defence against transposable elements across the Eukaryota. In animals, Piwi interacting small RNAs (piRNAs) are a crucial arm of this defence. However, the evolutionary relationships among piRNAs and other small RNA pathways targeting transposable elements are poorly resolved. To address this question we sequenced small RNAs from multiple, diverse nematode species, producing the first phylum-wide analysis of how small RNA pathways evolve. Surprisingly, despite their prominence in Caenorhabditis elegans and closely related nematodes, piRNAs are absent in all other nematode lineages. We found that there are at least two evolutionarily distinct mechanisms that compensate for the absence of piRNAs, both involving RNA-dependent RNA polymerases (RdRPs). Whilst one pathway is unique to nematodes, the second involves Dicer-dependent RNA-directed DNA methylation, hitherto unknown in animals, and bears striking similarity to transposon-control mechanisms in fungi and plants. Our results highlight the rapid, context-dependent evolution of small RNA pathways and suggest piRNAs in animals may have replaced an ancient eukaryotic RNA-dependent RNA polymerase pathway to control transposable elements.

  7. Small RNA profiling of Xenopus embryos reveals novel miRNAs and a new class of small RNAs derived from intronic transposable elements

    PubMed Central

    Harding, Joanne L.; Horswell, Stuart; Heliot, Claire; Armisen, Javier; Zimmerman, Lyle B.; Luscombe, Nicholas M.; Miska, Eric A.; Hill, Caroline S.

    2014-01-01

    Small RNA control of gene expression is critical for developmental processes in vertebrate embryos. To determine the dynamics of small RNA expression and to uncover novel small RNAs in the early vertebrate embryo, we performed high-throughput sequencing of all small RNAs in Xenopus tropicalis embryos at three developmental time points and in dissected halves of gastrula embryos. This analysis allowed us to identify novel microRNAs and we show that microRNA expression is highly dynamic and spatially localized in early embryos. In addition, we have developed a microRNA prediction pipeline and demonstrate that it has the power to predict new miRNAs that are experimentally detectable in frogs, mice, and humans. By combining the small RNA sequencing with mRNA profiling at the different developmental stages, we identify a new class of small noncoding RNAs that we name siteRNAs, which align in clusters to introns of protein-coding genes. We show that siteRNAs are derived from remnants of transposable elements present in the introns. We find that genes containing clusters of siteRNAs are transcriptionally repressed as compared with all genes. Furthermore, we show that this is true for individual genes containing siteRNA clusters, and that these genes are enriched in specific repressive histone modifications. Our data thus suggest a new mechanism of siteRNA-mediated gene silencing in vertebrates, and provide an example of how mobile elements can affect gene regulation. PMID:24065776

  8. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Van den Broeck, I; De Rijk, P; De Wachter, R

    1994-01-01

    The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library. PMID:7524022

  9. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Jansen, J; De Rijk, P; De Wachter, R

    1997-01-01

    The Antwerp database on small ribosomal subunit RNA now offers more than 6000 nucleotide sequences (August 1996). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. For ease of use, the complete database is made available to the scientific community via World Wide Web at URL http://rrna.uia.ac.be/ssu/ . PMID:9016516

  10. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Nicolaï, S; De Rijk, P; De Wachter, R

    1996-01-01

    The Antwerp database on small ribosomal subunit RNA offers over 4300 nucleotide sequences (August 1995). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. The complete database is made available to the scientific community through anonymous ftp and World Wide Web(WWW). PMID:8594609

  11. The small RNA SraG participates in PNPase homeostasis.

    PubMed

    Fontaine, Fanette; Gasiorowski, Elise; Gracia, Celine; Ballouche, Mathieu; Caillet, Joel; Marchais, Antonin; Hajnsdorf, Eliane

    2016-10-01

    The rpsO-pnp operon encodes ribosomal protein S15 and polynucleotide phosphorylase, a major 3'-5' exoribonuclease involved in mRNA decay in Escherichia coli The gene for the SraG small RNA is located between the coding regions of the rpsO and pnp genes, and it is transcribed in the opposite direction relative to the two genes. No function has been assigned to SraG. Multiple levels of post-transcriptional regulation have been demonstrated for the rpsO-pnp operon. Here we show that SraG is a new factor affecting pnp expression. SraG overexpression results in a reduction of pnp expression and a destabilization of pnp mRNA; in contrast, inhibition of SraG transcription results in a higher level of the pnp transcript. Furthermore, in vitro experiments indicate that SraG inhibits translation initiation of pnp Together, these observations demonstrate that SraG participates in the post-transcriptional control of pnp by a direct antisense interaction between SraG and PNPase RNAs. Our data reveal a new level of regulation in the expression of this major exoribonuclease.

  12. Polyploidy and small RNA regulation of cotton fiber development.

    PubMed

    Guan, Xueying; Song, Qingxin; Chen, Z Jeffrey

    2014-08-01

    Cotton is not only the most important source of renewal textile fibers, but also an excellent model for studying cell fate determination and polyploidy effects on gene expression and evolution of domestication traits. The combination of A and D-progenitor genomes into allotetraploid cotton induces intergenomic interactions and epigenetic effects, leading to the unequal expression of homoeologous genes. Small RNAs regulate the expression of transcription and signaling factors related to cellular growth, development and adaptation. An example is miRNA-mediated preferential degradation of homoeologous mRNAs encoding MYB-domain transcription factors that are required for the initiation of leaf trichomes in Arabidopsis and of seed fibers in cotton. This example of coevolution between small RNAs and their homoeologous targets could shape morphological traits such as fibers during the selection and domestication of polyploid crops.

  13. iSRAP – a one-touch research tool for rapid profiling of small RNA-seq data

    PubMed Central

    Quek, Camelia; Jung, Chol-hee; Bellingham, Shayne A.; Lonie, Andrew; Hill, Andrew F.

    2015-01-01

    Small non-coding RNAs have been significantly recognized as the key modulators in many biological processes, and are emerging as promising biomarkers for several diseases. These RNA species are transcribed in cells and can be packaged in extracellular vesicles, which are small vesicles released from many biotypes, and are involved in intercellular communication. Currently, the advent of next-generation sequencing (NGS) technology for high-throughput profiling has further advanced the biological insights of non-coding RNA on a genome-wide scale and has become the preferred approach for the discovery and quantification of non-coding RNA species. Despite the routine practice of NGS, the processing of large data sets poses difficulty for analysis before conducting downstream experiments. Often, the current analysis tools are designed for specific RNA species, such as microRNA, and are limited in flexibility for modifying parameters for optimization. An analysis tool that allows for maximum control of different software is essential for drawing concrete conclusions for differentially expressed transcripts. Here, we developed a one-touch integrated small RNA analysis pipeline (iSRAP) research tool that is composed of widely used tools for rapid profiling of small RNAs. The performance test of iSRAP using publicly and in-house available data sets shows its ability of comprehensive profiling of small RNAs of various classes, and analysis of differentially expressed small RNAs. iSRAP offers comprehensive analysis of small RNA sequencing data that leverage informed decisions on the downstream analyses of small RNA studies, including extracellular vesicles such as exosomes. PMID:26561006

  14. RNA-Seq Experiment and Data Analysis.

    PubMed

    Liang, Hanquan; Zeng, Erliang

    2016-01-01

    With the ability to obtain tens of millions of reads, high-throughput messenger RNA sequencing (RNA-Seq) data offers the possibility of estimating abundance of isoforms and finding novel transcripts. In this chapter, we describe a protocol to construct an RNA-Seq library for sequencing on Illumina NGS platforms, and a computational pipeline to perform RNA-Seq data analysis. The protocols described in this chapter can be applied to the analysis of differential gene expression in control versus 17β-estradiol treatment of in vivo or in vitro systems. PMID:26585130

  15. RNA-Seq Experiment and Data Analysis.

    PubMed

    Liang, Hanquan; Zeng, Erliang

    2016-01-01

    With the ability to obtain tens of millions of reads, high-throughput messenger RNA sequencing (RNA-Seq) data offers the possibility of estimating abundance of isoforms and finding novel transcripts. In this chapter, we describe a protocol to construct an RNA-Seq library for sequencing on Illumina NGS platforms, and a computational pipeline to perform RNA-Seq data analysis. The protocols described in this chapter can be applied to the analysis of differential gene expression in control versus 17β-estradiol treatment of in vivo or in vitro systems.

  16. Characterization and Small RNA Content of Extracellular Vesicles in Follicular Fluid of Developing Bovine Antral Follicles

    PubMed Central

    Navakanitworakul, Raphatphorn; Hung, Wei-Ting; Gunewardena, Sumedha; Davis, John S.; Chotigeat, Wilaiwan; Christenson, Lane K.

    2016-01-01

    Exosomes and microvesicles (i.e., extracellular vesicles: EVs) have been identified within ovarian follicular fluid and recent evidence suggests that EVs are able to elicit profound effects on ovarian cell function. While existence of miRNA within EVs has been reported, whether EV size and concentration as well as their cargos (i.e., proteins and RNA) change during antral follicle growth remains unknown. Extracellular vesicles isolated from follicular fluid of small, medium and large bovine follicles were similar in size, while concentration of EVs decreased progressively as follicle size increased. Electron microscopy indicated a highly purified population of the lipid bilayer enclosed vesicles that were enriched in exosome biomarkers including CD81 and Alix. Small RNA sequencing identified a large number of known and novel miRNAs that changed in the EVs of different size follicles. Ingenuity Pathway Analysis (IPA) indicated that miRNA abundant in small follicle EV preparations were associated with cell proliferation pathways, while those miRNA abundant in large follicle preparations were related to inflammatory response pathways. These studies are the first to demonstrate that EVs change in their levels and makeup during antral follicle development and point to the potential for a unique vesicle-mediated cell-to-cell communication network within the ovarian follicle. PMID:27158133

  17. Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants

    PubMed Central

    Kontra, Levente; Tavazza, Mario; Lucioli, Alessandra; Tavazza, Raffaela; Moxon, Simon; Medzihradszky, Anna; Burgyán, József

    2016-01-01

    RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination. PMID:27711201

  18. Small RNA pathways and diversity in model legumes: lessons from genomics

    PubMed Central

    Bustos-Sanmamed, Pilar; Bazin, Jérémie; Hartmann, Caroline; Crespi, Martin; Lelandais-Brière, Christine

    2013-01-01

    Small non-coding RNAs (smRNA) participate in the regulation of development, cell differentiation, adaptation to environmental constraints and defense responses in plants. They negatively regulate gene expression by degrading specific mRNA targets, repressing their translation or modifying chromatin conformation through homologous interaction with target loci. MicroRNAs (miRNA) and short-interfering RNAs (siRNA) are generated from long double stranded RNA (dsRNA) that are cleaved into 20–24-nucleotide dsRNAs by RNase III proteins called DICERs (DCL). One strand of the duplex is then loaded onto effective complexes containing different ARGONAUTE (AGO) proteins. In this review, we explored smRNA diversity in model legumes and compiled available data from miRBAse, the miRNA database, and from 22 reports of smRNA deep sequencing or miRNA identification genome-wide in three legumes: Medicago truncatula, soybean (Glycine max) and Lotus japonicus. In addition to conserved miRNAs present in other plant species, 229, 179, and 35 novel miRNA families were identified respectively in these 3 legumes, among which several seems legume-specific. New potential functions of several miRNAs in the legume-specific nodulation process are discussed. Furthermore, a new category of siRNA, the phased siRNAs, which seems to mainly regulate disease-resistance genes, was recently discovered in legumes. Despite that the genome sequence of model legumes are not yet fully completed, further analysis was performed by database mining of gene families and protein characteristics of DCLs and AGOs in these genomes. Although most components of the smRNA pathways are conserved, identifiable homologs of key smRNA players from non-legumes, like AGO10 or DCL4, could not yet be detected in M. truncatula available genomic and expressed sequence (EST) databases. In contrast to Arabidopsis, an important gene diversification was observed in the three legume models (for DCL2, AGO4, AGO2, and AGO10) or

  19. Small RNA pathways and diversity in model legumes: lessons from genomics.

    PubMed

    Bustos-Sanmamed, Pilar; Bazin, Jérémie; Hartmann, Caroline; Crespi, Martin; Lelandais-Brière, Christine

    2013-01-01

    Small non-coding RNAs (smRNA) participate in the regulation of development, cell differentiation, adaptation to environmental constraints and defense responses in plants. They negatively regulate gene expression by degrading specific mRNA targets, repressing their translation or modifying chromatin conformation through homologous interaction with target loci. MicroRNAs (miRNA) and short-interfering RNAs (siRNA) are generated from long double stranded RNA (dsRNA) that are cleaved into 20-24-nucleotide dsRNAs by RNase III proteins called DICERs (DCL). One strand of the duplex is then loaded onto effective complexes containing different ARGONAUTE (AGO) proteins. In this review, we explored smRNA diversity in model legumes and compiled available data from miRBAse, the miRNA database, and from 22 reports of smRNA deep sequencing or miRNA identification genome-wide in three legumes: Medicago truncatula, soybean (Glycine max) and Lotus japonicus. In addition to conserved miRNAs present in other plant species, 229, 179, and 35 novel miRNA families were identified respectively in these 3 legumes, among which several seems legume-specific. New potential functions of several miRNAs in the legume-specific nodulation process are discussed. Furthermore, a new category of siRNA, the phased siRNAs, which seems to mainly regulate disease-resistance genes, was recently discovered in legumes. Despite that the genome sequence of model legumes are not yet fully completed, further analysis was performed by database mining of gene families and protein characteristics of DCLs and AGOs in these genomes. Although most components of the smRNA pathways are conserved, identifiable homologs of key smRNA players from non-legumes, like AGO10 or DCL4, could not yet be detected in M. truncatula available genomic and expressed sequence (EST) databases. In contrast to Arabidopsis, an important gene diversification was observed in the three legume models (for DCL2, AGO4, AGO2, and AGO10) or

  20. Antitumor and Antimetastatic Effect of Small Immunostimulatory RNA against B16 Melanoma in Mice

    PubMed Central

    Kabilova, Tatyana O.; Sen’kova, Aleksandra V.; Nikolin, Valeriy P.; Popova, Nelly A.; Zenkova, Marina A.; Vlassov, Valentin V.; Chernolovskaya, Elena L.

    2016-01-01

    Small interfering RNAs, depending on their structure, delivery system and sequence, can stimulate innate and adaptive immunity. The aim of this study was to investigate the antitumor and antimetastatic effects of immunostimulatory 19-bp dsRNA with 3’- trinucleotide overhangs (isRNA) on melanoma B16 in C57Bl/6 mice. Recently developed novel cationic liposomes 2X3-DOPE were used for the in vivo delivery of isRNA. Administration of isRNA/2X3-DOPE complexes significantly inhibits melanoma tumor growth and metastasis. Histopathological analysis of spleen cross sections showed hyperplasia of the lymphoid white pulp and formation of large germinal centers after isRNA/2X3-DOPE administration, indicating activation of the immune system. The treatment of melanoma-bearing mice with isRNA/2X3-DOPE decreases the destructive changes in the liver parenchyma. Thus, the developed isRNA displays pronounced immunostimulatory, antitumor and antimetastatic properties against melanoma B16 and may be considered a potential agent in the immunotherapy of melanoma. PMID:26981617

  1. Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes.

    PubMed

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2011-10-28

    The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications. PMID:22001930

  2. Accessibility and conservation: general features of bacterial small RNA-mRNA interactions?

    PubMed

    Richter, Andreas S; Backofen, Rolf

    2012-07-01

    Bacterial small RNAs (sRNAs) are a class of structural RNAs that often regulate mRNA targets via post-transcriptional base pair interactions. We determined features that discriminate functional from non-functional interactions and assessed the influence of these features on genome-wide target predictions. For this purpose, we compiled a set of 71 experimentally verified sRNA-target pairs from Escherichia coli and Salmonella enterica. Furthermore, we collected full-length 5' untranslated regions by using genome-wide experimentally verified transcription start sites. Only interaction sites in sRNAs, but not in targets, show significant sequence conservation. In addition to this observation, we found that the base pairing between sRNAs and their targets is not conserved in general across more distantly related species. A closer inspection of RybB and RyhB sRNAs and their targets revealed that the base pairing complementarity is only conserved in a small subset of the targets. In contrast to conservation, accessibility of functional interaction sites is significantly higher in both sRNAs and targets in comparison to non-functional sites. Based on the above observations, we successfully used the following constraints to improve the specificity of genome-wide target predictions: the region of interaction initiation must be located in (1) highly accessible regions in both interaction partners and (2) unstructured conserved sRNA regions derived from reliability profiles of multiple sRNA alignments. Aligned sequences of homologous sRNAs, functional and non-functional targets, and a supplementary document with supplementary tables, figures and references are available at http://www. bioinf.uni-freiburg.de/Supplements/srna-interact-feat. PMID:22767260

  3. sRNAtoolbox: an integrated collection of small RNA research tools

    PubMed Central

    Rueda, Antonio; Barturen, Guillermo; Lebrón, Ricardo; Gómez-Martín, Cristina; Alganza, Ángel; Oliver, José L.; Hackenberg, Michael

    2015-01-01

    Small RNA research is a rapidly growing field. Apart from microRNAs, which are important regulators of gene expression, other types of functional small RNA molecules have been reported in animals and plants. MicroRNAs are important in host-microbe interactions and parasite microRNAs might modulate the innate immunity of the host. Furthermore, small RNAs can be detected in bodily fluids making them attractive non-invasive biomarker candidates. Given the general broad interest in small RNAs, and in particular microRNAs, a large number of bioinformatics aided analysis types are needed by the scientific community. To facilitate integrated sRNA research, we developed sRNAtoolbox, a set of independent but interconnected tools for expression profiling from high-throughput sequencing data, consensus differential expression, target gene prediction, visual exploration in a genome context as a function of read length, gene list analysis and blast search of unmapped reads. All tools can be used independently or for the exploration and downstream analysis of sRNAbench results. Workflows like the prediction of consensus target genes of parasite microRNAs in the host followed by the detection of enriched pathways can be easily established. The web-interface interconnecting all these tools is available at http://bioinfo5.ugr.es/srnatoolbox PMID:26019179

  4. Profiling small RNA reveals multimodal substructural signals in a Boltzmann ensemble

    PubMed Central

    Rogers, Emily; Heitsch, Christine E.

    2014-01-01

    As the biomedical impact of small RNAs grows, so does the need to understand competing structural alternatives for regions of functional interest. Suboptimal structure analysis provides significantly more RNA base pairing information than a single minimum free energy prediction. Yet computational enhancements like Boltzmann sampling have not been fully adopted by experimentalists since identifying meaningful patterns in this data can be challenging. Profiling is a novel approach to mining RNA suboptimal structure data which makes the power of ensemble-based analysis accessible in a stable and reliable way. Balancing abstraction and specificity, profiling identifies significant combinations of base pairs which dominate low-energy RNA secondary structures. By design, critical similarities and differences are highlighted, yielding crucial information for molecular biologists. The code is freely available via http://gtfold.sourceforge.net/profiling.html. PMID:25392423

  5. Small RNA sequencing identifies miRNA roles in ovule and fibre development.

    PubMed

    Xie, Fuliang; Jones, Don C; Wang, Qinglian; Sun, Runrun; Zhang, Baohong

    2015-04-01

    MicroRNAs (miRNAs) have been found to be differentially expressed during cotton fibre development. However, which specific miRNAs and how they are involved in fibre development is unclear. Here, using deep sequencing, 65 conserved miRNA families were identified and 32 families were differentially expressed between leaf and ovule. At least 40 miRNAs were either leaf or ovule specific, whereas 62 miRNAs were shared in both leaf and ovule. qRT-PCR confirmed these miRNAs were differentially expressed during fibre early development. A total of 820 genes were potentially targeted by the identified miRNAs, whose functions are involved in a series of biological processes including fibre development, metabolism and signal transduction. Many predicted miRNA-target pairs were subsequently validated by degradome sequencing analysis. GO and KEGG analyses showed that the identified miRNAs and their targets were classified to 1027 GO terms including 568 biological processes, 324 molecular functions and 135 cellular components and were enriched to 78 KEGG pathways. At least seven unique miRNAs participate in trichome regulatory interaction network. Eleven trans-acting siRNA (tasiRNA) candidate genes were also identified in cotton. One has never been found in other plant species and two of them were derived from MYB and ARF, both of which play important roles in cotton fibre development. Sixteen genes were predicted to be tasiRNA targets, including sucrose synthase and MYB2. Together, this study discovered new miRNAs in cotton and offered evidences that miRNAs play important roles in cotton ovule/fibre development. The identification of tasiRNA genes and their targets broadens our understanding of the complicated regulatory mechanism of miRNAs in cotton.

  6. Comparative RNA-seq-Based Transcriptome Analysis of the Virulence Characteristics of Methicillin-Resistant and -Susceptible Staphylococcus pseudintermedius Strains Isolated from Small Animals

    PubMed Central

    Couto, Natacha; Belas, Adriana; Oliveira, Manuela; Almeida, Paulo; Clemente, Carla

    2015-01-01

    Staphylococcus pseudintermedius is often associated with pyoderma, which can turn into a life-threatening disease. The dissemination of highly resistant isolates has occurred in the last 10 years and has challenged antimicrobial treatment of these infections considerably. We have compared the carriage of virulence genes and biofilm formation between methicillin-resistant and methicillin-susceptible S. pseudintermedius (MRSP and MSSP, respectively) isolates and their in vitro gene expression profiles by transcriptome sequencing (RNA-seq). Isolates were relatively unevenly distributed among the four agr groups, and agr type III predominated in MRSP. Five virulence genes were detected in all isolates. Only the spsO gene was significantly associated with MSSP isolates (P = 0.04). All isolates produced biofilm in brain heart infusion broth (BHIB)–4% NaCl. MSSP isolates produced more biofilm on BHIB and BHIB–1% glucose media than MRSP isolates (P = 0.03 and P = 0.02, respectively). Virulence genes encoding surface proteins and toxins (spsA, spsB, spsD, spsK, spsL, spsN, nucC, coa, and luk-I) and also prophage genes (encoding phage capsid protein, phage infection protein, two phage portal proteins, and a phage-like protein) were highly expressed in the MRSP isolate (compared with the MSSP isolate), suggesting they may play a role in the rapid and widespread dissemination of MRSP. This study indicates that MRSP may upregulate surface proteins, which may increase the adherence of MRSP isolates (especially sequence type 71 [ST71]) to corneocytes. MSSP isolates may have an increased ability to form biofilm under acidic circumstances, through upregulation of the entire arc operon. Complete understanding of S. pseudintermedius pathogenesis and host-pathogen signal interaction during infections is critical for the treatment and prevention of S. pseudintermedius infections. PMID:26621622

  7. Toward reprogramming bacteria with small molecules and RNA.

    PubMed

    Gallivan, Justin P

    2007-12-01

    A major goal of synthetic biology is to reprogram bacteria to carry out complex tasks, such as synthesizing and delivering drugs, and seeking and destroying environmental pollutants. Advances in molecular biology and bacterial genetics have made it straightforward to modify, insert, or delete genes in many bacterial strains, and advances in gene synthesis have opened the door to replacing entire genomes. However, rewriting the underlying genetic code is only part of the challenge of reprogramming cellular behavior. A remaining challenge is to control how and when the modified genes are expressed. Several recent studies have highlighted how synthetic riboswitches, which are RNA sequences that undergo a ligand-induced conformational change to alter gene expression, can be used to reprogram how bacteria respond to small molecules. PMID:17967431

  8. Computational analysis of bacterial RNA-Seq data

    PubMed Central

    McClure, Ryan; Balasubramanian, Divya; Sun, Yan; Bobrovskyy, Maksym; Sumby, Paul; Genco, Caroline A.; Vanderpool, Carin K.; Tjaden, Brian

    2013-01-01

    Recent advances in high-throughput RNA sequencing (RNA-seq) have enabled tremendous leaps forward in our understanding of bacterial transcriptomes. However, computational methods for analysis of bacterial transcriptome data have not kept pace with the large and growing data sets generated by RNA-seq technology. Here, we present new algorithms, specific to bacterial gene structures and transcriptomes, for analysis of RNA-seq data. The algorithms are implemented in an open source software system called Rockhopper that supports various stages of bacterial RNA-seq data analysis, including aligning sequencing reads to a genome, constructing transcriptome maps, quantifying transcript abundance, testing for differential gene expression, determining operon structures and visualizing results. We demonstrate the performance of Rockhopper using 2.1 billion sequenced reads from 75 RNA-seq experiments conducted with Escherichia coli, Neisseria gonorrhoeae, Salmonella enterica, Streptococcus pyogenes and Xenorhabdus nematophila. We find that the transcriptome maps generated by our algorithms are highly accurate when compared with focused experimental data from E. coli and N. gonorrhoeae, and we validate our system’s ability to identify novel small RNAs, operons and transcription start sites. Our results suggest that Rockhopper can be used for efficient and accurate analysis of bacterial RNA-seq data, and that it can aid with elucidation of bacterial transcriptomes. PMID:23716638

  9. Computational analysis of bacterial RNA-Seq data.

    PubMed

    McClure, Ryan; Balasubramanian, Divya; Sun, Yan; Bobrovskyy, Maksym; Sumby, Paul; Genco, Caroline A; Vanderpool, Carin K; Tjaden, Brian

    2013-08-01

    Recent advances in high-throughput RNA sequencing (RNA-seq) have enabled tremendous leaps forward in our understanding of bacterial transcriptomes. However, computational methods for analysis of bacterial transcriptome data have not kept pace with the large and growing data sets generated by RNA-seq technology. Here, we present new algorithms, specific to bacterial gene structures and transcriptomes, for analysis of RNA-seq data. The algorithms are implemented in an open source software system called Rockhopper that supports various stages of bacterial RNA-seq data analysis, including aligning sequencing reads to a genome, constructing transcriptome maps, quantifying transcript abundance, testing for differential gene expression, determining operon structures and visualizing results. We demonstrate the performance of Rockhopper using 2.1 billion sequenced reads from 75 RNA-seq experiments conducted with Escherichia coli, Neisseria gonorrhoeae, Salmonella enterica, Streptococcus pyogenes and Xenorhabdus nematophila. We find that the transcriptome maps generated by our algorithms are highly accurate when compared with focused experimental data from E. coli and N. gonorrhoeae, and we validate our system's ability to identify novel small RNAs, operons and transcription start sites. Our results suggest that Rockhopper can be used for efficient and accurate analysis of bacterial RNA-seq data, and that it can aid with elucidation of bacterial transcriptomes.

  10. Rapid RNA analysis of individual Caenorhabditis elegans.

    PubMed

    Ly, Kien; Reid, Suzanne J; Snell, Russell G

    2015-01-01

    Traditional RNA extraction methods rely on the use of hazardous chemicals such as phenol, chloroform, guanidinium thiocyanate to disrupt cells and inactivate RNAse simultaneously. RNA isolation from Caenorhabditis elegans presents another challenge due to its tough cuticle, therefore several repeated freeze-thaw cycles may be needed to disrupt the cuticle before the cell contents are released. In addition, a large number of animals are required for successful RNA isolation. To overcome these issues, we have developed a simple and efficient method using proteinase K and a brief heat treatment to release RNA of quality suitable for quantitative PCR analysis.The benefits of the method are: •Faster and safer compared to conventional RNA extraction methods•Released RNA can be used directly for cDNA synthesis without purification•As little as a single worm is sufficient.

  11. Deep Sequencing Analysis of Nucleolar Small RNAs: Bioinformatics.

    PubMed

    Bai, Baoyan; Laiho, Marikki

    2016-01-01

    Small RNAs (size 20-30 nt) of various types have been actively investigated in recent years, and their subcellular compartmentalization and relative concentrations are likely to be of importance to their cellular and physiological functions. Comprehensive data on this subset of the transcriptome can only be obtained by application of high-throughput sequencing, which yields data that are inherently complex and multidimensional, as sequence composition, length, and abundance will all inform to the small RNA function. Subsequent data analysis, hypothesis testing, and presentation/visualization of the results are correspondingly challenging. We have constructed small RNA libraries derived from different cellular compartments, including the nucleolus, and asked whether small RNAs exist in the nucleolus and whether they are distinct from cytoplasmic and nuclear small RNAs, the miRNAs. Here, we present a workflow for analysis of small RNA sequencing data generated by the Ion Torrent PGM sequencer from samples derived from different cellular compartments. PMID:27576724

  12. Small RNA Sequencing Based Identification of MiRNAs in Daphnia magna.

    PubMed

    Ünlü, Ercan Selçuk; Gordon, Donna M; Telli, Murat

    2015-01-01

    Small RNA molecules are short, non-coding RNAs identified for their crucial role in post-transcriptional regulation. A well-studied example includes miRNAs (microRNAs) which have been identified in several model organisms including the freshwater flea and planktonic crustacean Daphnia. A model for epigenetic-based studies with an available genome database, the identification of miRNAs and their potential role in regulating Daphnia gene expression has only recently garnered interest. Computational-based work using Daphnia pulex, has indicated the existence of 45 miRNAs, 14 of which have been experimentally verified. To extend this study, we took a sequencing approach towards identifying miRNAs present in a small RNA library isolated from Daphnia magna. Using Perl codes designed for comparative genomic analysis, 815,699 reads were obtained from 4 million raw reads and run against a database file of known miRNA sequences. Using this approach, we have identified 205 putative mature miRNA sequences belonging to 188 distinct miRNA families. Data from this study provides critical information necessary to begin an investigation into a role for these transcripts in the epigenetic regulation of Daphnia magna.

  13. Small RNA Sequencing Based Identification of MiRNAs in Daphnia magna

    PubMed Central

    2015-01-01

    Small RNA molecules are short, non-coding RNAs identified for their crucial role in post-transcriptional regulation. A well-studied example includes miRNAs (microRNAs) which have been identified in several model organisms including the freshwater flea and planktonic crustacean Daphnia. A model for epigenetic-based studies with an available genome database, the identification of miRNAs and their potential role in regulating Daphnia gene expression has only recently garnered interest. Computational-based work using Daphnia pulex, has indicated the existence of 45 miRNAs, 14 of which have been experimentally verified. To extend this study, we took a sequencing approach towards identifying miRNAs present in a small RNA library isolated from Daphnia magna. Using Perl codes designed for comparative genomic analysis, 815,699 reads were obtained from 4 million raw reads and run against a database file of known miRNA sequences. Using this approach, we have identified 205 putative mature miRNA sequences belonging to 188 distinct miRNA families. Data from this study provides critical information necessary to begin an investigation into a role for these transcripts in the epigenetic regulation of Daphnia magna. PMID:26367422

  14. Longitudinal RNA-Seq Analysis of Vertebrate Aging Identifies Mitochondrial Complex I as a Small-Molecule-Sensitive Modifier of Lifespan.

    PubMed

    Baumgart, Mario; Priebe, Steffen; Groth, Marco; Hartmann, Nils; Menzel, Uwe; Pandolfini, Luca; Koch, Philipp; Felder, Marius; Ristow, Michael; Englert, Christoph; Guthke, Reinhard; Platzer, Matthias; Cellerino, Alessandro

    2016-02-24

    Mutations and genetic variability affect gene expression and lifespan, but the impact of variations in gene expression within individuals on their aging-related mortality is poorly understood. We performed a longitudinal study in the short-lived killifish, Nothobranchius furzeri, and correlated quantitative variations in gene expression during early adult life with lifespan. Shorter- and longer-lived individuals differ in their gene expression before the onset of aging-related mortality; differences in gene expression are more pronounced early in life. We identified mitochondrial respiratory chain complex I as a hub in a module of genes whose expression is negatively correlated with lifespan. Accordingly, partial pharmacological inhibition of complex I by the small molecule rotenone reversed aging-related regulation of gene expression and extended lifespan in N. furzeri by 15%. These results support the use of N. furzeri as a vertebrate model for identifying the protein targets, pharmacological modulators, and individual-to-individual variability associated with aging.

  15. Longitudinal RNA-Seq Analysis of Vertebrate Aging Identifies Mitochondrial Complex I as a Small-Molecule-Sensitive Modifier of Lifespan.

    PubMed

    Baumgart, Mario; Priebe, Steffen; Groth, Marco; Hartmann, Nils; Menzel, Uwe; Pandolfini, Luca; Koch, Philipp; Felder, Marius; Ristow, Michael; Englert, Christoph; Guthke, Reinhard; Platzer, Matthias; Cellerino, Alessandro

    2016-02-24

    Mutations and genetic variability affect gene expression and lifespan, but the impact of variations in gene expression within individuals on their aging-related mortality is poorly understood. We performed a longitudinal study in the short-lived killifish, Nothobranchius furzeri, and correlated quantitative variations in gene expression during early adult life with lifespan. Shorter- and longer-lived individuals differ in their gene expression before the onset of aging-related mortality; differences in gene expression are more pronounced early in life. We identified mitochondrial respiratory chain complex I as a hub in a module of genes whose expression is negatively correlated with lifespan. Accordingly, partial pharmacological inhibition of complex I by the small molecule rotenone reversed aging-related regulation of gene expression and extended lifespan in N. furzeri by 15%. These results support the use of N. furzeri as a vertebrate model for identifying the protein targets, pharmacological modulators, and individual-to-individual variability associated with aging. PMID:27135165

  16. Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes

    SciTech Connect

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer We use MEL-A-containing cationic liposomes for siRNA delivery. Black-Right-Pointing-Pointer MEL-A-containing cationic liposomes can efficiently and rapidly deliver siRNA into the cytoplasm. Black-Right-Pointing-Pointer Rapid delivery of siRNA is due to the membrane fusion between liposomes and plasma membrane. -- Abstract: The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine Trade-Mark-Sign RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic

  17. RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes

    PubMed Central

    Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle

    2016-01-01

    ABSTRACT Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation. PMID:26580233

  18. Discovery and visualization of miRNA-mRNA functional modules within integrated data using bicluster analysis.

    PubMed

    Bryan, Kenneth; Terrile, Marta; Bray, Isabella M; Domingo-Fernandéz, Raquel; Watters, Karen M; Koster, Jan; Versteeg, Rogier; Stallings, Raymond L

    2014-02-01

    MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at a post-transcriptional level. An miRNA may target many messenger RNA (mRNA) transcripts, and each transcript may be targeted by multiple miRNAs. Our understanding of miRNA regulation is evolving to consider modules of miRNAs that regulate groups of functionally related mRNAs. Here we expand the model of miRNA functional modules and use it to guide the integration of miRNA and mRNA expression and target prediction data. We present evidence of cooperativity between miRNA classes within this integrated miRNA-mRNA association matrix. We then apply bicluster analysis to uncover miRNA functional modules within this integrated data set and develop a novel application to visualize and query these results. We show that this wholly unsupervised approach can discover a network of miRNA-mRNA modules that are enriched for both biological processes and miRNA classes. We apply this method to investigate the interplay of miRNAs and mRNAs in integrated data sets derived from neuroblastoma and human immune cells. This study is the first to apply the technique of biclustering to model functional modules within an integrated miRNA-mRNA association matrix. Results provide evidence of an extensive modular miRNA functional network and enable characterization of miRNA function and dysregulation in disease.

  19. Unraveling the conformational determinants of LARP7 and 7SK small nuclear RNA by theoretical approaches.

    PubMed

    Xu, Lei; Kong, Ren; Zhu, Jingyu; Sun, Huiyong; Chang, Shan

    2016-07-19

    LARP7, a member of the La-related proteins (LARPs), shares a conserved La module comprising the La-motif (LAM) and the RNA-recognition motif (RRM1), binding exclusively to the non-coding RNA 7SK. LARP7 is a component of the small nuclear ribonucleoprotein (7SKsnRNP) required for the stability and function of the RNA, and implicated in the transcription termination and regulation of translation. In the current work, molecular dynamics simulations were employed to investigate the recently determined crystal structures of the La module of LARP7 in complexs with a stretch of uridines at the 3'-end of 7SK in the presence and absence of RNA and two different mutants. The structural stabilities of the four systems provided by the simulations are consistent with the experimental data. Principal component analysis (PCA) and free energy landscape (FEL) were used to explore the dominant motions and the functional dynamics between the two ends of the superhelical structures in both RNA-bound and RNA-free systems. The final values of the intramolecular angle formed by the Cα atoms of Arg30, Lys53 and Pro189 are ∼96° and 125° for the RNA-bound and RNA-free systems, highlighting the importance of the binding of the 3'-end of RNA 7SK for system stability. The dynamic cross-correlation maps (DCCM) were utilized to evaluate the conformational changes in different mutants, and small values were found around the residues 29-50 and 100-120 in the F168A system, whereas large values were found around the residues 120-160 and 170-189 in the E130A system. The time evolutions of the hydrogen-bond distances of the terminal uridine U-1 and Asp54 and that of the penultimate residue U-2 and Gln41 were monitored to compare their conformational changes, and the results suggest that the E130A mutant may have an important effect on the RNA binding, which is consistent with site-directed mutagenesis. This study provides some new insights into the understanding of the recognition mechanism

  20. New perspectives on the diversification of the RNA interference system: insights from comparative genomics and small RNA sequencing.

    PubMed

    Burroughs, Alexander Maxwell; Ando, Yoshinari; Aravind, L

    2014-01-01

    Our understanding of the pervasive involvement of small RNAs in regulating diverse biological processes has been greatly augmented by recent application of deep-sequencing technologies to small RNA across diverse eukaryotes. We review the currently known small RNA classes and place them in context of the reconstructed evolutionary history of the RNA interference (RNAi) protein machinery. This synthesis indicates that the earliest versions of eukaryotic RNAi systems likely utilized small RNA processed from three types of precursors: (1) sense-antisense transcriptional products, (2) genome-encoded, imperfectly complementary hairpin sequences, and (3) larger noncoding RNA precursor sequences. Structural dissection of PIWI proteins along with recent discovery of novel families (including Med13 of the Mediator complex) suggest that emergence of a distinct architecture with the N-terminal domains (also occurring separately fused to endoDNases in prokaryotes) formed via duplication of an ancestral unit was key to their recruitment as primary RNAi effectors and use of small RNAs of certain preferred lengths. Prokaryotic PIWI proteins are typically components of several RNA-directed DNA restriction or CRISPR/Cas systems. However, eukaryotic versions appear to have emerged from a subset that evolved RNA-directed RNAi. They were recruited alongside RNaseIII domains and RNA-dependent RNA polymerase (RdRP) domains, also from prokaryotic systems, to form the core eukaryotic RNAi system. Like certain regulatory systems, RNAi diversified into two distinct but linked arms concomitant with eukaryotic nucleocytoplasmic compartmentalization. Subsequent elaboration of RNAi proceeded via diversification of the core protein machinery through lineage-specific expansions and recruitment of new components from prokaryotes (nucleases and small RNA-modifying enzymes), allowing for diversification of associating small RNAs. PMID:24311560

  1. Inhibition of pre-mRNA splicing by a synthetic Blom7α-interacting small RNA.

    PubMed

    Löscher, Marlies; Schosserer, Markus; Dausse, Eric; Lee, Kiseok; Ajuh, Paul; Grillari-Voglauer, Regina; Lamond, Angus I; Toulmé, Jean-Jacques; Grillari, Johannes

    2012-01-01

    Originally the novel protein Blom7α was identified as novel pre-mRNA splicing factor that interacts with SNEV(Prp19/Pso4), an essential protein involved in extension of human endothelial cell life span, DNA damage repair, the ubiquitin-proteasome system, and pre-mRNA splicing. Blom7α belongs to the heteronuclear ribonucleoprotein K homology (KH) protein family, displaying 2 KH domains, a well conserved and widespread RNA-binding motif. In order to identify specific sequence binding motifs, we here used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) with a synthetic RNA library. Besides sequence motifs like (U/A)(1-4) C(2-6) (U/A)(1-5), we identified an AC-rich RNA-aptamer that we termed AK48 (Aptamer KH-binding 48), binding to Blom7α with high affinity. Addition of AK48 to pre-mRNA splicing reactions in vitro inhibited the formation of mature spliced mRNA and led to a slight accumulation of the H complex of the spliceosome. These results suggest that the RNA binding activity of Blom7α might be required for pre-mRNA splicing catalysis. The inhibition of in-vitro splicing by the small RNA AK48 indicates the potential use of small RNA molecules in targeting the spliceosome complex as a novel target for drug development. PMID:23144703

  2. Upregulation of a small vault RNA (svtRNA2-1a) is an early event in Parkinson disease and induces neuronal dysfunction

    PubMed Central

    Miñones-Moyano, Elena; Friedländer, Marc R.; Pallares, Joan; Kagerbauer, Birgit; Porta, Sílvia; Escaramís, Georgia; Ferrer, Isidre; Estivill, Xavier; Martí, Eulàlia

    2013-01-01

    MicroRNAs (miRNAs) and other small non-coding RNAs (sncRNAs) are post-transcriptional regulators of gene expression, playing key roles in neuronal development, plasticity, and disease. Transcriptome deregulation caused by miRNA dysfunction has been associated to neurodegenerative diseases. Parkinson disease (PD) is the second most common neurodegenerative disease showing deregulation of the coding and small non-coding transcriptome. On profiling sncRNA in PD brain areas differently affected, we found that upregulation of a small vault RNA (svtRNA2-1a) is widespread in PD brains, occurring early in the course of the disease (at pre-motor stages). SvtRNA2-1a biogenesis was dependent on Dicer activity on its precursor (vtRNA2-1) but independent of Drosha endonuclease, unlike the canonical miRNAs. Although endogenous svtRNA2-1a was enriched in Ago-2 immunoprecipitates in differentiated SH-SY5Y neuronal cells, overexpression of svtRNA2-1a induced subtle transcriptomic changes, suggesting that gene expression regulation may involve other mechanisms than mRNA decay only. Function enrichment analysis of the genes deregulated by svtRNA2-1a overexpression or svtRNA2-1a predicted targets identified pathways related to nervous system development and cell type specification. The expression pattern of svtRNA2-1a during development and aging of the human brain and the detrimental consequences of a svtRNA2-1a mimic overexpression in neuronal cells further indicate that low svtRNA2-1a levels may be important for the maintenance of neurons. Our results suggest that early svtRNA2-1a upregulation in PD may contribute to perturbations of gene expression networks, underlying metabolic impairment and cell dysfunction. A better understanding of the pathways regulated by svtRNA2-a, and also the mechanisms regulating its expression should facilitate the identification of new targets for therapeutic approaches in PD. PMID:23673382

  3. PlantMirnaT: miRNA and mRNA integrated analysis fully utilizing characteristics of plant sequencing data.

    PubMed

    Rhee, S; Chae, H; Kim, S

    2015-07-15

    miRNA is known to regulate up to several hundreds coding genes, thus the integrated analysis of miRNA and mRNA expression data is an important problem. Unfortunately, the integrated analysis is challenging since it needs to consider expression data of two different types, miRNA and mRNA, and target relationship between miRNA and mRNA is not clear, especially when microarray data is used. Fortunately, due to the low sequencing cost, small RNA and RNA sequencing are routinely processed and we may be able to infer regulation relationships between miRNAs and mRNAs more accurately by using sequencing data. However, no method is developed specifically for sequencing data. Thus we developed PlantMirnaT, a new miRNA-mRNA integrated analysis system. To fully leverage the power of sequencing data, three major features are developed and implemented in PlantMirnaT. First, we implemented a plant-specific short read mapping tool based on recent discoveries on miRNA target relationship in plant. Second, we designed and implemented an algorithm considering miRNA targets in the full intragenic region, not just 3' UTR. Lastly but most importantly, our algorithm is designed to consider quantity of miRNA expression and its distribution on target mRNAs. The new algorithm was used to characterize rice under drought condition using our proprietary data. Our algorithm successfully discovered that two miRNAs, miRNA1425-5p, miRNA 398b, that are involved in suppression of glucose pathway in a naturally drought resistant rice, Vandana. The system can be downloaded at https://sites.google.com/site/biohealthinformaticslab/resources. PMID:25863133

  4. Substitution rate calibration of small subunit ribosomal RNA identifies chlorarachniophyte endosymbionts as remnants of green algae.

    PubMed Central

    Van de Peer, Y; Rensing, S A; Maier, U G; De Wachter, R

    1996-01-01

    Chlorarachniophytes are amoeboid algae with chlorophyll a and b containing plastids that are surrounded by four membranes instead of two as in plants and green algae. These extra membranes form important support for the hypothesis that chlorarachniophytes have acquired their plastids by the ingestion of another eukaryotic plastid-containing alga. Chlorarachniophytes also contain a small nucleus-like structure called the nucleomorph situated between the two inner and the two outer membranes surrounding the plastid. This nucleomorph is a remnant of the endosymbiont's nucleus and encodes, among other molecules, small subunit ribosomal RNA. Previous phylogenetic analyses on the basis of this molecule provided unexpected and contradictory evidence for the origin of the chlorarachniophyte endosymbiont. We developed a new method for measuring the substitution rates of the individual nucleotides of small subunit ribosomal RNA. From the resulting substitution rate distribution, we derived an equation that gives a more realistic relationship between sequence dissimilarity and evolutionary distance than equations previously available. Phylogenetic trees constructed on the basis of evolutionary distances computed by this new method clearly situate the chlorarachniophyte nucleomorphs among the green algae. Moreover, this relationship is confirmed by transversion analysis of the Chlorarachnion plastid small subunit ribosomal RNA. PMID:8755544

  5. Identification and annotation of small RNA genes using ShortStack

    PubMed Central

    Shahid, Saima; Axtell, Michael J.

    2013-01-01

    Highly parallel sequencing of cDNA derived from endogenous small RNAs (small RNA-seq) is a key method that has accelerated understanding of regulatory small RNAs in eukaryotes. Eukaryotic regulatory small RNAs, which include microRNAs (miRNAs), short interfering RNAs (siRNAs), and Piwi-associated RNAs (piRNAs), typically derive from the processing of longer precursor RNAs. Alignment of small RNA-seq data to a reference genome allows the inference of the longer precursor and thus the annotation of small RNA producing genes. ShortStack is a program that was developed to comprehensively analyze reference-aligned small RNA-seq data, and output detailed and useful annotations of the causal small RNA-producing genes. Here, we provide a step- by-step tutorial of ShortStack usage with the goal of introducing new users to the software and pointing out some common pitfalls. PMID:24139974

  6. Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.

    PubMed

    Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide

    2010-07-30

    Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway.

  7. RNA Secondary Structure Analysis Using RNAstructure.

    PubMed

    Mathews, David H

    2014-06-17

    RNAstructure is a user-friendly program for the prediction and analysis of RNA secondary structure. It is available as a Web server, as a program with a graphical user interface, or as a set of command-line tools. The programs are available for Microsoft Windows, Macintosh OS X, or Linux. This unit provides protocols for RNA secondary structure prediction (using the Web server or the graphical user interface) and prediction of high-affinity oligonucleotide biding sites to a structured RNA target (using the graphical user interface).

  8. Myelin basic protein synthesis is regulated by small non-coding RNA 715.

    PubMed

    Bauer, Nina M; Moos, Christina; van Horssen, Jack; Witte, Maarten; van der Valk, Paul; Altenhein, Benjamin; Luhmann, Heiko J; White, Robin

    2012-09-01

    Oligodendroglial Myelin Basic Protein (MBP) synthesis is essential for myelin formation in the central nervous system. During oligodendrocyte differentiation, MBP mRNA is kept in a translationally silenced state while intracellularly transported, until neuron-derived signals initiate localized MBP translation. Here we identify the small non-coding RNA 715 (sncRNA715) as an inhibitor of MBP translation. SncRNA715 localizes to cytoplasmic granular structures and associates with MBP mRNA transport granule components. We also detect increased levels of sncRNA715 in demyelinated chronic human multiple sclerosis lesions, which contain MBP mRNA but lack MBP protein.

  9. Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts

    PubMed Central

    Turowski, Tomasz W.; Leśniewska, Ewa; Delan-Forino, Clementine; Sayou, Camille; Boguta, Magdalena; Tollervey, David

    2016-01-01

    RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5′ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential “housekeeping” roles. Many tRNA genes were found to generate long, 3′-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3′-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5′-exonuclease Rat1. PMID:27206856

  10. Development and utilization of non-coding RNA-small molecule interactions.

    PubMed

    Georgianna, Wesleigh E; Young, Douglas D

    2011-12-01

    RNA plays a crucial role in cellular biology as a carrier of genetic information. However, beyond this passive role, RNA has been shown to regulate various cellular processes in a form that is not translated into protein. Non-coding RNA (ncRNA) has been shown to be important in gene regulation, and its aberrant activity has been associated with several disease states. As such, ncRNAs represent a novel target for small molecule regulation and recently, significant advances have been made towards elucidating small molecule regulators of ncRNAs. Herein, we provide an overview of miRNA, siRNA, RNA aptamers, riboswitches, and ribozymes, within the context of recent findings regarding the exogenous regulation of these ncRNAs by small molecules. The development of these small molecule tools has far-reaching applications in the advancement of molecular therapeutics.

  11. Molecular identification of nanoplanktonic protists based on small subunit ribosomal RNA gene sequences for ecological studies.

    PubMed

    Lim, E L

    1996-01-01

    Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.

  12. New perspectives on the diversification of the RNA interference system: insights from comparative genomics and small RNA sequencing

    PubMed Central

    Burroughs, Alexander Maxwell; Ando, Yoshinari; Aravind, L

    2014-01-01

    Our understanding of the pervasive involvement of small RNAs in regulating diverse biological processes has been greatly augmented by recent application of deep-sequencing technologies to small RNA across diverse eukaryotes. We review the currently-known small RNA classes and place them in context of the reconstructed evolutionary history of the RNAi protein machinery. This synthesis indicates the earliest versions of eukaryotic RNAi systems likely utilized small RNA processed from three types of precursors: 1) sense-antisense transcriptional products, 2) genome-encoded, imperfectly-complementary hairpin sequences, and 3) larger non-coding RNA precursor sequences. Structural dissection of PIWI proteins along with recent discovery of novel families (including Med13 of the Mediator complex) suggest that emergence of a distinct architecture with the N-terminal domains (also occurring separately fused to endoDNases in prokaryotes) formed via duplication of an ancestral unit was key to their recruitment as primary RNAi effectors and use of small RNAs of certain preferred lengths. Prokaryotic PIWI proteins are typically components of several RNA-directed DNA restriction or CRISPR/Cas systems. However, eukaryotic versions appear to have emerged from a subset that evolved RNA-directed RNA interference. They were recruited alongside RNaseIII domains and RdRP domains, also from prokaryotic systems, to form the core eukaryotic RNAi system. Like certain regulatory systems, RNAi diversified into two distinct but linked arms concomitant with eukaryotic nucleo-cytoplasmic compartmentalization. Subsequent elaboration of RNAi proceeded via diversification of the core protein machinery through lineage-specific expansions and recruitment of new components from prokaryotes (nucleases and small RNA-modifying enzymes), allowing for diversification of associating small RNAs. PMID:24311560

  13. An assessment of bacterial small RNA target prediction programs.

    PubMed

    Pain, Adrien; Ott, Alban; Amine, Hamza; Rochat, Tatiana; Bouloc, Philippe; Gautheret, Daniel

    2015-01-01

    Most bacterial regulatory RNAs exert their function through base-pairing with target RNAs. Computational prediction of targets is a busy research field that offers biologists a variety of web sites and software. However, it is difficult for a non-expert to evaluate how reliable those programs are. Here, we provide a simple benchmark for bacterial sRNA target prediction based on trusted E. coli sRNA/target pairs. We use this benchmark to assess the most recent RNA target predictors as well as earlier programs for RNA-RNA hybrid prediction. Moreover, we consider how the definition of mRNA boundaries can impact overall predictions. Recent algorithms that exploit both conservation of targets and accessibility information offer improved accuracy over previous software. However, even with the best predictors, the number of true biological targets with low scores and non-targets with high scores remains puzzling. PMID:25760244

  14. Targeting Th17 Cells with Small Molecules and Small Interference RNA

    PubMed Central

    Lin, Hui; Song, Pingfang; Zhao, Yi; Xue, Li-Jia; Liu, Yi; Chu, Cong-Qiu

    2015-01-01

    T helper 17 (Th17) cells play a central role in inflammatory and autoimmune diseases via the production of proinflammatory cytokines interleukin- (IL-) 17, IL-17F, and IL-22. Anti-IL-17 monoclonal antibodies show potent efficacy in psoriasis but poor effect in rheumatoid arthritis (RA) and Crohn's disease. Alternative agents targeting Th17 cells may be a better way to inhibit the development and function of Th17 cells than antibodies of blocking a single effector cytokine. Retinoic acid-related orphan receptor gamma t (RORγt) which acts as the master transcription factor of Th17 differentiation has been an attractive pharmacologic target for the treatment of Th17-mediated autoimmune disease. Recent progress in technology of chemical screen and engineering nucleic acid enable two new classes of therapeutics targeting RORγt. Chemical screen technology identified several small molecule specific inhibitors of RORγt from a small molecule library. Systematic evolution of ligands by exponential enrichment (SELEX) technology enabled target specific aptamers to be isolated from a random sequence oligonucleotide library. In this review, we highlight the development and therapeutic potential of small molecules inhibiting Th17 cells by targeting RORγt and aptamer mediated CD4+ T cell specific delivery of small interference RNA against RORγt gene expression to inhibit pathogenic effector functions of Th17 lineage. PMID:26792955

  15. Targeting Th17 Cells with Small Molecules and Small Interference RNA.

    PubMed

    Lin, Hui; Song, Pingfang; Zhao, Yi; Xue, Li-Jia; Liu, Yi; Chu, Cong-Qiu

    2015-01-01

    T helper 17 (Th17) cells play a central role in inflammatory and autoimmune diseases via the production of proinflammatory cytokines interleukin- (IL-) 17, IL-17F, and IL-22. Anti-IL-17 monoclonal antibodies show potent efficacy in psoriasis but poor effect in rheumatoid arthritis (RA) and Crohn's disease. Alternative agents targeting Th17 cells may be a better way to inhibit the development and function of Th17 cells than antibodies of blocking a single effector cytokine. Retinoic acid-related orphan receptor gamma t (RORγt) which acts as the master transcription factor of Th17 differentiation has been an attractive pharmacologic target for the treatment of Th17-mediated autoimmune disease. Recent progress in technology of chemical screen and engineering nucleic acid enable two new classes of therapeutics targeting RORγt. Chemical screen technology identified several small molecule specific inhibitors of RORγt from a small molecule library. Systematic evolution of ligands by exponential enrichment (SELEX) technology enabled target specific aptamers to be isolated from a random sequence oligonucleotide library. In this review, we highlight the development and therapeutic potential of small molecules inhibiting Th17 cells by targeting RORγt and aptamer mediated CD4(+) T cell specific delivery of small interference RNA against RORγt gene expression to inhibit pathogenic effector functions of Th17 lineage. PMID:26792955

  16. Combinatorial analysis of interacting RNA molecules.

    PubMed

    Li, Thomas J X; Reidys, Christian M

    2011-09-01

    Recently several minimum free energy (MFE) folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Their folding targets are interaction structures, that can be represented as diagrams with two backbones drawn horizontally on top of each other such that (1) intramolecular and intermolecular bonds are noncrossing and (2) there is no "zigzag" configuration. This paper studies joint structures with arc-length at least four in which both, interior and exterior stack-lengths are at least two (no isolated arcs). The key idea in this paper is to consider a new type of shape, based on which joint structures can be derived via symbolic enumeration. Our results imply simple asymptotic formulas for the number of joint structures with surprisingly small exponential growth rates. They are of interest in the context of designing prediction algorithms for RNA-RNA interactions. PMID:21689666

  17. Distinct Small RNA Signatures in Extracellular Vesicles Derived from Breast Cancer Cell Lines

    PubMed Central

    Knutsen, Erik; Nikolaisen, Marlen Aas; Jørgensen, Tor Erik; Johansen, Steinar Daae; Perander, Maria; Seternes, Ole Morten

    2016-01-01

    Breast cancer is a heterogeneous disease, and different subtypes of breast cancer show distinct cellular morphology, gene expression, metabolism, motility, proliferation, and metastatic potential. Understanding the molecular features responsible for this heterogeneity is important for correct diagnosis and better treatment strategies. Extracellular vesicles (EVs) and their associated molecules have gained much attention as players in intercellular communication, ability to precondition specific organs for metastatic invasion, and for their potential role as circulating cancer biomarkers. EVs are released from the cells and contain proteins, DNA, and long and small RNA species. Here we show by high-throughput small RNA-sequencing that EVs from nine different breast cancer cell lines share common characteristics in terms of small RNA content that are distinct from their originating cells. Most strikingly, a highly abundant small RNA molecule derived from the nuclear 28S rRNA is vastly enriched in EVs. The miRNA profiles in EVs correlate with the cellular miRNA expression pattern, but with a few exceptions that includes miR-21. This cancer-associated miRNA is retained in breast cancer cell lines. Finally, we report that EVs from breast cancer cell lines cluster together based on their small RNA signature when compared to EVs derived from other cancer cell lines. Altogether, our data demonstrate that breast cancer cell lines manifest a specific small RNA signature in their released EVs. This opens up for further evaluation of EVs as breast cancer biomarkers. PMID:27579604

  18. Distinct Small RNA Signatures in Extracellular Vesicles Derived from Breast Cancer Cell Lines.

    PubMed

    Fiskaa, Tonje; Knutsen, Erik; Nikolaisen, Marlen Aas; Jørgensen, Tor Erik; Johansen, Steinar Daae; Perander, Maria; Seternes, Ole Morten

    2016-01-01

    Breast cancer is a heterogeneous disease, and different subtypes of breast cancer show distinct cellular morphology, gene expression, metabolism, motility, proliferation, and metastatic potential. Understanding the molecular features responsible for this heterogeneity is important for correct diagnosis and better treatment strategies. Extracellular vesicles (EVs) and their associated molecules have gained much attention as players in intercellular communication, ability to precondition specific organs for metastatic invasion, and for their potential role as circulating cancer biomarkers. EVs are released from the cells and contain proteins, DNA, and long and small RNA species. Here we show by high-throughput small RNA-sequencing that EVs from nine different breast cancer cell lines share common characteristics in terms of small RNA content that are distinct from their originating cells. Most strikingly, a highly abundant small RNA molecule derived from the nuclear 28S rRNA is vastly enriched in EVs. The miRNA profiles in EVs correlate with the cellular miRNA expression pattern, but with a few exceptions that includes miR-21. This cancer-associated miRNA is retained in breast cancer cell lines. Finally, we report that EVs from breast cancer cell lines cluster together based on their small RNA signature when compared to EVs derived from other cancer cell lines. Altogether, our data demonstrate that breast cancer cell lines manifest a specific small RNA signature in their released EVs. This opens up for further evaluation of EVs as breast cancer biomarkers. PMID:27579604

  19. Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine.

    PubMed

    Crossland, Rachel E; Norden, Jean; Bibby, Louis A; Davis, Joanna; Dickinson, Anne M

    2016-02-01

    MicroRNAs are small regulatory molecules that demonstrate useful biomarker potential. They have been recognised in biofluids, where they are protected from degradation by encapsulation into extracellular vesicles (EVs). A number of commercial products are available for the isolation of EVs and their RNA content; however, extensive protocol comparisons are lacking. Furthermore, robust qRT-PCR assessment of microRNA expression within EVs is problematic, as endogenous controls (ECs) previously used in cellular samples may not be present. This study compares EV isolation and RNA extraction methods (EV precipitation reagents, RNA isolation kits and ultracentrifugation) from serum or urine samples and evaluates suitable ECs for incorporation into qRT-PCR analysis. Results were assessed by electron microscopy, nanoparticle tracking analysis and bioanalyzer concentrations. The stability of 8 ECs was compared for both serum and urine EV RNA and retrospectively validated in independent cohorts (serum n=55, urine n=50). The Life Technologies precipitation reagent gave superior serum EV recovery compared to SBI reagent, as assessed by NTA size distribution, increased RNA concentration, and lower small RNA Ct values. Similarly, the Norgen Biotek Urine Exosome RNA Isolation Kit gave improved results for urine EV isolation compared to ultracentrifugation, when determined by the same parameters. The Qiagen miRNeasy™ RNA isolation kit gave suitable serum EV RNA concentrations compared to other kits, as assessed by Bioanalyzer and small RNA qRT-PCR. Small RNAs HY3 (S.D=1.77, CoV=6.2%) and U6 (S.D=2.14, CoV=8.6%) were selected as optimal ECs for serum EV microRNA expression analysis, while HY3 (S.D=1.67, CoV=6.5%) and RNU48 (S.D=1.85, CoV=5.3%) were identified as suitable for urine studies. In conclusion, this study identifies optimal methods for isolation of serum and urine EV RNA, and suitable ECs for normalisation of qRT-PCR studies. Such reports should aid in the

  20. Systemic delivery of siRNA in pumpkin by a plant PHLOEM SMALL RNA-BINDING PROTEIN 1-ribonucleoprotein complex.

    PubMed

    Ham, Byung-Kook; Li, Gang; Jia, Weitao; Leary, Julie A; Lucas, William J

    2014-11-01

    In plants, the vascular system, specifically the phloem, functions in delivery of small RNA (sRNA) to exert epigenetic control over developmental and defense-related processes. Although the importance of systemic sRNA delivery has been established, information is currently lacking concerning the nature of the protein machinery involved in this process. Here, we show that a PHLOEM SMALL-RNA BINDING PROTEIN 1 (PSRP1) serves as the basis for formation of an sRNA ribonucleoprotein complex (sRNPC) that delivers sRNA (primarily 24 nt) to sink organs. Assembly of this complex is facilitated through PSRP1 phosphorylation by a phloem-localized protein kinase, PSRPK1. During long-distance transport, PSRP1-sRNPC is stable against phloem phosphatase activity. Within target tissues, phosphatase activity results in disassembly of PSRP1-sRNPC, a process that is probably required for unloading cargo sRNA into surrounding cells. These findings provide an insight into the mechanism involved in delivery of sRNA associated with systemic gene silencing in plants.

  1. Diverse evolutionary trajectories for small RNA biogenesis genes in the oomycete genus Phytophthora

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, ...

  2. Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inconsistent detection of plant-based dietary small RNAs in circulation has thwarted the use of dietary RNA therapeutics. Here we demonstrate mice consuming diets rich in vegetables displayed enhanced serum levels of the plant specific small RNA MIR2911. Differential centrifugation, size-exclusion c...

  3. Small-Signal ac Analysis

    NASA Technical Reports Server (NTRS)

    Jagielski, James M.; Chen, Jess

    1987-01-01

    Program simulates power circuits and systems. Small Signal A.C. Analysis program (SSAC) valuable tool for design and analysis of electrical-power-system circuits. By combining "black box" power-system components operating in specified manner, user characterizes system modeled. Menu-driven program proved simple and cost effective in development and modification of arbitrary power-system configurations. Package includes sample data from Dynamic Explorer satellite family. Results compared favorable to calculations from such general circuit-analysis programs as SPICE. Written in FORTRAN 77.

  4. Highly efficient ligation of small RNA molecules for microRNA quantitation by high-throughput sequencing.

    PubMed

    Lee, Jerome E; Yi, Rui

    2014-01-01

    MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3' and 5' oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3' and 5' ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples. PMID:25490151

  5. Genetic Characterization of Clinical Acanthamoeba Isolates from Japan using Nuclear and Mitochondrial Small Subunit Ribosomal RNA

    PubMed Central

    Rahman, Md Moshiur; Yagita, Kenji; Kobayashi, Akira; Oikawa, Yosaburo; Hussein, Amjad I.A.; Matsumura, Takahiro

    2013-01-01

    Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype (AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba. PMID:24039282

  6. Identification and expression profiling of Vigna mungo microRNAs from leaf small RNA transcriptome by deep sequencing.

    PubMed

    Paul, Sujay; Kundu, Anirban; Pal, Amita

    2014-01-01

    MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that play a crucial role in post-transcriptional gene regulation. Several conserved and species-specific miRNAs have been characterized to date, predominantly from the plant species whose genome is well characterized. However, information on the variability of these regulatory RNAs in economically important but genetically less characterized crop species are limited. Vigna mungo is an important grain legume, which is grown primarily for its protein-rich edible seeds. miRNAs from this species have not been identified to date due to lack of genome sequence information. To identify miRNAs from V. mungo, a small RNA library was constructed from young leaves. High-throughput Illumina sequencing technology and bioinformatic analysis of the small RNA reads led to the identification of 66 miRNA loci represented by 45 conserved miRNAs belonging to 19 families and eight non-conserved miRNAs belonging to seven families. Besides, 13 novel miRNA candidates in V. mungo were also identified. Expression patterns of selected conserved, non-conserved, and novel miRNA candidates have been demonstrated in leaf, stem, and root tissues by quantitative polymerase chain reaction, and potential target genes were predicted for most of the conserved miRNAs. This information offers genomic resources for better understanding of miRNA mediated post-transcriptional gene regulation.

  7. Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

    PubMed

    Kopechek, Jonathan A; Carson, Andrew R; McTiernan, Charles F; Chen, Xucai; Klein, Edwin C; Villanueva, Flordeliza S

    2016-01-01

    RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease. PMID:27471848

  8. Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound

    PubMed Central

    McTiernan, Charles F.; Chen, Xucai; Klein, Edwin C.; Villanueva, Flordeliza S.

    2016-01-01

    RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease. PMID:27471848

  9. miR-isomiRExp: a web-server for the analysis of expression of miRNA at the miRNA/isomiR levels

    PubMed Central

    Guo, Li; Yu, Jiafeng; Liang, Tingming; Zou, Quan

    2016-01-01

    MicroRNA (miRNA) locus has been found that can generate a series of varied isomiR sequences. Most studies always focus on determining miRNA level, however, the canonical miRNA sequence is only a specific member in the multiple isomiRs. Some studies have shown that isomiR sequences play versatile roles in biological progress, and the analysis and research should be simultaneously performed at the miRNA/isomiR levels. Based on the biological characteristics of miRNA and isomiR, we developed miR-isomiRExp to analyze expression pattern of miRNA at the miRNA/isomiR levels, provide insights into tracking miRNA/isomiR maturation and processing mechanisms, and reveal functional characteristics of miRNA/isomiR. Simultaneously, we also performed expression analysis of specific human diseases using public small RNA sequencing datasets based on the analysis platform, which may help in surveying the potential deregulated miRNA/isomiR expression profiles, especially sequence and function-related isomiRs for further interaction analysis and study. The miR-isomiRExp platform provides miRNA/isomiR expression patterns and more information to study deregulated miRNA loci and detailed isomiR sequences. This comprehensive analysis will enrich experimental miRNA studies. miR-isomiRExp is available at http://mirisomirexp.aliapp.com. PMID:27009551

  10. Effects of low temperature on mRNA and small RNA transcriptomes in Solanum lycopersicoides leaf revealed by RNA-Seq.

    PubMed

    Chen, Hongyu; Chen, Xiuling; Chai, Xinfeng; Qiu, Youwen; Gong, Chao; Zhang, Zhenzhu; Wang, Tingting; Zhang, Yue; Li, Jingfu; Wang, Aoxue

    2015-08-28

    The plant low temperature tolerance mechanisms have been studied in the Arabidopsis, tomato, Solanum commersonii, Solanum tuberosum, Chorispora bungeana, and Chinese cabbage at the transcriptional level. Some genome-wide works to identify cold-regulated genes, but no comprehensive research of the Solanum lycopersicoides transcriptome under low temperature stress have been performed. S. lycopersicoides is more freeze-tolerant than the cultivated tomato. We analyzed the low temperature transcriptomes and small RNA fractions of S. lycopersicoides leaf tissue using an Illumina platform for high-throughput RNA sequencing (RNA-seq). There were 59,286 unigenes obtained using de novo assembly, and 2052 down-regulated and 2409 up-regulated unigenes were identified in response to chilling. The expression of six cold-regulated genes was confirmed by qPCR. Some biological processes were showed, by gene ontology term enrichment analysis of the cold-regulated genes, including 'response to stimulus', 'signaling', and 'cell killing' in the response of S. lycopersicoides to chilling. In addition, we identified a total of 952 novel miRNA candidates that may regulate relevant target genes. Our data indicated that certain miRNAs (e.g., sly-miR156a, sly-miR397, and unconservative_SL2.50ch00_21686) play roles in response to low temperature stress. Sequencing of mRNAs and miRNAs revealed new genes and allowed us to have new assumptions for a low temperature tolerance mechanism. PMID:26187671

  11. Mouse nucleolin binds to 4.5S RNAH, a small noncoding RNA

    SciTech Connect

    Hirose, Yutaka Harada, Fumio

    2008-01-04

    4.5S RNAH is a rodent-specific small noncoding RNA that exhibits extensive homology to the B1 short interspersed element. Although 4.5S RNAH is known to associate with cellular poly(A)-terminated RNAs and retroviral genomic RNAs, its function remains unclear. In this study, we analyzed 4.5S RNAH-binding proteins in mouse nuclear extracts using gel mobility shift and RNA-protein UV cross-linking assays. We found that at least nine distinct polypeptides (p170, p110, p93, p70, p48, p40, p34, p20, and p16.5) specifically interacted with 4.5S RNAHin vitro. Using anti-La antibody, p48 was identified as mouse La protein. To identify the other 4.5S RNAH-binding proteins, we performed expression cloning from a mouse cDNA library and obtained cDNA clones derived from nucleolin mRNA. We identified p110 as nucleolin using nucleolin-specific antibodies. UV cross-linking analysis using various deletion mutants of nucleolin indicated that the third of four tandem RNA recognition motifs is a major determinant for 4.5S RNAH recognition. Immunoprecipitation of nucleolin from the subcellular fractions of mouse cell extracts revealed that a portion of the endogenous 4.5S RNAH was associated with nucleolin and that this complex was located in both the nucleoplasm and nucleolus.

  12. Recent In Vivo Evidences of Particle-Based Delivery of Small-Interfering RNA (siRNA) into Solid Tumors

    PubMed Central

    2014-01-01

    Small-interfering RNA (siRNA) is both a powerful tool in research and a promising therapeutic platform to modulate expression of disease-related genes. Malignant tumors are attractive disease targets for nucleic acid-based therapies. siRNA directed against oncogenes, and genes driving metastases or angiogenesis have been evaluated in animal models and in some cases, in humans. The outcomes of these studies indicate that drug delivery is a significant limiting factor. This review provides perspectives on in vivo validated nanoparticle-based siRNA delivery systems. Results of recent advances in liposomes and polymeric and inorganic formulations illustrate the need for mutually optimized attributes for performance in systemic circulation, tumor interstitial space, plasma membrane, and endosomes. Physiochemical properties conducive to efficient siRNA delivery are summarized and directions for future research are discussed. PMID:25221632

  13. Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing.

    PubMed

    Hafner, Markus; Renwick, Neil; Farazi, Thalia A; Mihailović, Aleksandra; Pena, John T G; Tuschl, Thomas

    2012-10-01

    The characterization of post-transcriptional gene regulation by small regulatory (20-30 nt) RNAs, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for characterizing small RNAs is their identification and quantification across different developmental stages, and in normal and disease tissues, as well as model cell lines. Here we present a step-by-step protocol for generating barcoded small RNA cDNA libraries compatible with Illumina HiSeq sequencing, thereby facilitating miRNA and other small RNA profiling of large sample collections.

  14. Phylogenetic position of Cryothecomonas inferred from nuclear-encoded small subunit ribosomal RNA.

    PubMed

    Kühn, S; Lange, M; Medlin, L K

    2000-12-01

    The systematic position of the genus Cryothecomonas has been determined from an analysis of the nuclear-encoded small subunit ribosomal RNA gene of Cryothecomonas longipes and two strains of Cryothecomonas aestivalis. Our phylogenetic trees inferred from maximum likelihood, distance and maximum parsimony methods robustly show that the genus Cryothecomonas clusters within the phylum Cercozoa, and is related to the sarcomonad flagellate Heteromita globosa. Morphological data supporting the taxonomic placement of Cryothecomonas near the sarcomonad flagellates has been compiled from the literature. The high number of nucleotide substitutions found between two morphologically indistinguishable strains of Cryothecomonas aestivalis suggests the possibility of cryptic species within Cryothecomonas aestivalis. PMID:11212894

  15. Studying a Drug-like, RNA-Focused Small Molecule Library Identifies Compounds That Inhibit RNA Toxicity in Myotonic Dystrophy.

    PubMed

    Rzuczek, Suzanne G; Southern, Mark R; Disney, Matthew D

    2015-12-18

    There are many RNA targets in the transcriptome to which small molecule chemical probes and lead therapeutics are desired. However, identifying compounds that bind and modulate RNA function in cellulo is difficult. Although rational design approaches have been developed, they are still in their infancies and leave many RNAs "undruggable". In an effort to develop a small molecule library that is biased for binding RNA, we computationally identified "drug-like" compounds from screening collections that have favorable properties for binding RNA and for suitability as lead drugs. As proof-of-concept, this collection was screened for binding to and modulating the cellular dysfunction of the expanded repeating RNA (r(CUG)(exp)) that causes myotonic dystrophy type 1. Hit compounds bind the target in cellulo, as determined by the target identification approach Competitive Chemical Cross-Linking and Isolation by Pull-down (C-ChemCLIP), and selectively improve several disease-associated defects. The best compounds identified from our 320-member library are more potent in cellulo than compounds identified by high-throughput screening (HTS) campaigns against this RNA. Furthermore, the compound collection has a higher hit rate (9% compared to 0.01-3%), and the bioactive compounds identified are not charged; thus, RNA can be "drugged" with compounds that have favorable pharmacological properties. Finally, this RNA-focused small molecule library may serve as a useful starting point to identify lead "drug-like" chemical probes that affect the biological (dys)function of other RNA targets by direct target engagement.

  16. LPEseq: Local-Pooled-Error Test for RNA Sequencing Experiments with a Small Number of Replicates

    PubMed Central

    Gim, Jungsoo; Won, Sungho; Park, Taesung

    2016-01-01

    RNA-Sequencing (RNA-Seq) provides valuable information for characterizing the molecular nature of the cells, in particular, identification of differentially expressed transcripts on a genome-wide scale. Unfortunately, cost and limited specimen availability often lead to studies with small sample sizes, and hypothesis testing on differential expression between classes with a small number of samples is generally limited. The problem is especially challenging when only one sample per each class exists. In this case, only a few methods among many that have been developed are applicable for identifying differentially expressed transcripts. Thus, the aim of this study was to develop a method able to accurately test differential expression with a limited number of samples, in particular non-replicated samples. We propose a local-pooled-error method for RNA-Seq data (LPEseq) to account for non-replicated samples in the analysis of differential expression. Our LPEseq method extends the existing LPE method, which was proposed for microarray data, to allow examination of non-replicated RNA-Seq experiments. We demonstrated the validity of the LPEseq method using both real and simulated datasets. By comparing the results obtained using the LPEseq method with those obtained from other methods, we found that the LPEseq method outperformed the others for non-replicated datasets, and showed a similar performance with replicated samples; LPEseq consistently showed high true discovery rate while not increasing the rate of false positives regardless of the number of samples. Our proposed LPEseq method can be effectively used to conduct differential expression analysis as a preliminary design step or for investigation of a rare specimen, for which a limited number of samples is available. PMID:27532300

  17. Genome-scale mRNA and small RNA transcriptomic insights into initiation of citrus apomixis

    PubMed Central

    Long, Jian-Mei; Liu, Zheng; Wu, Xiao-Meng; Fang, Yan-Ni; Jia, Hui-Hui; Xie, Zong-Zhou; Deng, Xiu-Xin; Guo, Wen-Wu

    2016-01-01

    Nucellar embryony (NE) is an adventitious form of apomixis common in citrus, wherein asexual embryos initiate directly from nucellar cells surrounding the embryo sac. NE enables the fixation of desirable agronomic traits and the production of clonal offspring of virus-free rootstock, but impedes progress in hybrid breeding. In spite of the great importance of NE in citrus breeding and commercial production, little is understood about the underlying molecular mechanisms. In this study, the stages of nucellar embryo initiation (NEI) were determined for two polyembryonic citrus cultivars via histological observation. To explore the genes and regulatory pathways involved in NEI, we performed mRNA-seq and sRNA-seq analyses of ovules immediately prior to and at stages during NEI in the two pairs of cultivars. A total of 305 differentially expressed genes (DEGs) were identified between the poly- and monoembryonic ovules. Gene ontology (GO) analysis revealed that several processes are significantly enriched based on DEGs. In particular, response to stress, and especially response to oxidative stress, was over-represented in polyembryonic ovules. Nearly 150 miRNAs, comprising ~90 conserved and ~60 novel miRNAs, were identified in the ovules of either cultivar pair. Only two differentially expressed miRNAs (DEMs) were identified, of which the novel miRN23-5p was repressed whereas the targets accumulated in the polyembryonic ovules. This integrated study on the transcriptional and post-transcriptional regulatory profiles between poly- and monoembryonic citrus ovules provides new insights into the mechanism of NE, which should contribute to revealing the regulatory mechanisms of plant apomixis. PMID:27619233

  18. MicroRNA Expression Profile in Penile Cancer Revealed by Next-Generation Small RNA Sequencing

    PubMed Central

    Zhang, Yuanwei; Xu, Bo; Zhou, Jun; Fan, Song; Hao, Zongyao; Shi, Haoqiang; Zhang, Xiansheng; Kong, Rui; Xu, Lingfan; Gao, Jingjing; Zou, Duohong; Liang, Chaozhao

    2015-01-01

    Penile cancer (PeCa) is a relatively rare tumor entity but possesses higher morbidity and mortality rates especially in developing countries. To date, the concrete pathogenic signaling pathways and core machineries involved in tumorigenesis and progression of PeCa remain to be elucidated. Several studies suggested miRNAs, which modulate gene expression at posttranscriptional level, were frequently mis-regulated and aberrantly expressed in human cancers. However, the miRNA profile in human PeCa has not been reported before. In this present study, the miRNA profile was obtained from 10 fresh penile cancerous tissues and matched adjacent non-cancerous tissues via next-generation sequencing. As a result, a total of 751 and 806 annotated miRNAs were identified in normal and cancerous penile tissues, respectively. Among which, 56 miRNAs with significantly different expression levels between paired tissues were identified. Subsequently, several annotated miRNAs were selected randomly and validated using quantitative real-time PCR. Compared with the previous publications regarding to the altered miRNAs expression in various cancers and especially genitourinary (prostate, bladder, kidney, testis) cancers, the most majority of deregulated miRNAs showed the similar expression pattern in penile cancer. Moreover, the bioinformatics analyses suggested that the putative target genes of differentially expressed miRNAs between cancerous and matched normal penile tissues were tightly associated with cell junction, proliferation, growth as well as genomic instability and so on, by modulating Wnt, MAPK, p53, PI3K-Akt, Notch and TGF-β signaling pathways, which were all well-established to participate in cancer initiation and progression. Our work presents a global view of the differentially expressed miRNAs and potentially regulatory networks of their target genes for clarifying the pathogenic transformation of normal penis to PeCa, which research resource also provides new insights

  19. Small things considered: the small accessory subunits of RNA polymerase in Gram-positive bacteria

    PubMed Central

    Weiss, Andy; Shaw, Lindsey N.

    2015-01-01

    The DNA-dependent RNA polymerase core enzyme in Gram-positive bacteria consists of seven subunits. Whilst four of them (α2ββ′) are essential, three smaller subunits, δ, ε and ω (∼9–21.5 kDa), are considered accessory. Both δ and ω have been viewed as integral components of RNAP for several decades; however, ε has only recently been described. Functionally these three small subunits carry out a variety of tasks, imparting important, supportive effects on the transcriptional process of Gram-positive bacteria. While ω is thought to have a wide range of roles, reaching from maintaining structural integrity of RNAP to σ factor recruitment, the only suggested function for ε thus far is in protecting cells from phage infection. The third subunit, δ, has been shown to have distinct influences in maintaining transcriptional specificity, and thus has a key role in cellular fitness. Collectively, all three accessory subunits, although dispensable under laboratory conditions, are often thought to be crucial for proper RNAP function. Herein we provide an overview of the available literature on each subunit, summarizing landmark findings that have deepened our understanding of these proteins and their function, and outline future challenges in understanding the role of these small subunits in the transcriptional process. PMID:25878038

  20. Repurposed Transcriptomic Data Reveal Small Viral RNA Produced by Influenza Virus during Infection in Mice

    PubMed Central

    Koire, Amanda; Gilbert, Brian E.; Sucgang, Richard

    2016-01-01

    Influenza virus, a highly infectious ssRNA virus, replicates in the nucleus of host cells. This unusual feature brings the possibility that the virus may hijack host small noncoding RNA metabolism. Influenza small viral RNA production has been examined in vitro but has not yet been studied in an in vivo setting. We assessed small RNA species from influenza virus during mouse infection by mining publicly available mouse small RNA transcriptome data. We uncovered 26 nt reads corresponding to svRNA, a small viral RNA previously detected in vitro that regulates the transition from transcription to replication during infection, and found a strong positive correlation between svRNA production and host susceptibility to influenza virus infection. We also detected significant overrepresentation of a non-coding 23 nt sequence that we speculate may behave like a miRNA and work with influenza protein NS1 to prevent the transcription and maturation of interferon-stimulated mRNAs. PMID:27788253

  1. A survey of best practices for RNA-seq data analysis.

    PubMed

    Conesa, Ana; Madrigal, Pedro; Tarazona, Sonia; Gomez-Cabrero, David; Cervera, Alejandra; McPherson, Andrew; Szcześniak, Michał Wojciech; Gaffney, Daniel J; Elo, Laura L; Zhang, Xuegong; Mortazavi, Ali

    2016-01-01

    RNA-sequencing (RNA-seq) has a wide variety of applications, but no single analysis pipeline can be used in all cases. We review all of the major steps in RNA-seq data analysis, including experimental design, quality control, read alignment, quantification of gene and transcript levels, visualization, differential gene expression, alternative splicing, functional analysis, gene fusion detection and eQTL mapping. We highlight the challenges associated with each step. We discuss the analysis of small RNAs and the integration of RNA-seq with other functional genomics techniques. Finally, we discuss the outlook for novel technologies that are changing the state of the art in transcriptomics. PMID:26813401

  2. A survey of best practices for RNA-seq data analysis.

    PubMed

    Conesa, Ana; Madrigal, Pedro; Tarazona, Sonia; Gomez-Cabrero, David; Cervera, Alejandra; McPherson, Andrew; Szcześniak, Michał Wojciech; Gaffney, Daniel J; Elo, Laura L; Zhang, Xuegong; Mortazavi, Ali

    2016-01-26

    RNA-sequencing (RNA-seq) has a wide variety of applications, but no single analysis pipeline can be used in all cases. We review all of the major steps in RNA-seq data analysis, including experimental design, quality control, read alignment, quantification of gene and transcript levels, visualization, differential gene expression, alternative splicing, functional analysis, gene fusion detection and eQTL mapping. We highlight the challenges associated with each step. We discuss the analysis of small RNAs and the integration of RNA-seq with other functional genomics techniques. Finally, we discuss the outlook for novel technologies that are changing the state of the art in transcriptomics.

  3. Insect small nuclear RNA gene promoters evolve rapidly yet retain conserved features involved in determining promoter activity and RNA polymerase specificity.

    PubMed

    Hernandez, Genaro; Valafar, Faramarz; Stumph, William E

    2007-01-01

    In animals, most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II (Pol II), but U6 snRNA is synthesized by RNA polymerase III (Pol III). In Drosophila melanogaster, the promoters for the Pol II-transcribed snRNA genes consist of approximately 21 bp PSEA and approximately 8 bp PSEB. U6 genes utilize a PSEA but have a TATA box instead of the PSEB. The PSEAs of the two classes of genes bind the same protein complex, DmSNAPc. However, the PSEAs that recruit Pol II and Pol III differ in sequence at a few nucleotide positions that play an important role in determining RNA polymerase specificity. We have now performed a bioinformatic analysis to examine the conservation and divergence of the snRNA gene promoter elements in other species of insects. The 5' half of the PSEA is well-conserved, but the 3' half is divergent. Moreover, within each species positions exist where the PSEAs of the Pol III-transcribed genes differ from those of the Pol II-transcribed genes. Interestingly, the specific positions vary among species. Nevertheless, we speculate that these nucleotide differences within the 3' half of the PSEA act similarly to induce conformational alterations in DNA-bound SNAPc that result in RNA polymerase specificity.

  4. A piRNA-like small RNA interacts with and modulates p-ERM proteins in human somatic cells

    PubMed Central

    Mei, Yuping; Wang, Yuyan; Kumari, Priti; Shetty, Amol Carl; Clark, David; Gable, Tyler; MacKerell, Alexander D.; Ma, Mark Z.; Weber, David J.; Yang, Austin J.; Edelman, Martin J.; Mao, Li

    2015-01-01

    PIWI-interacting RNAs (piRNAs) are thought to silence transposon and gene expression during development. However, the roles of piRNAs in somatic tissues are largely unknown. Here we report the identification of 555 piRNAs in human lung bronchial epithelial (HBE) and non-small cell lung cancer (NSCLC) cell lines, including 295 that do not exist in databases termed as piRNA-like sncRNAs or piRNA-Ls. Distinctive piRNA/piRNA-L expression patterns are observed between HBE and NSCLC cells. piRNA-like-163 (piR-L-163), the top downregulated piRNA-L in NSCLC cells, binds directly to phosphorylated ERM proteins (p-ERM), which is dependent on the central part of UUNNUUUNNUU motif in piR-L-163 and the RRRKPDT element in ERM. The piR-L-163/p-ERM interaction is critical for p-ERM's binding capability to filamentous actin (F-actin) and ERM-binding phosphoprotein 50 (EBP50). Thus, piRNA/piRNA-L may play a regulatory role through direct interaction with proteins in physiological and pathophysiological conditions. PMID:26095918

  5. Subcellular distribution of small interfering RNA: directed delivery through RNA polymerase III expression cassettes and localization by in situ hybridization.

    PubMed

    Paul, Cynthia P

    2005-01-01

    Reduction in the expression of specific genes through small interfering RNAs (siRNAs) is dependent on the colocalization of siRNAs with other components of the RNA interference (RNAi) pathways within the cell. The expression of siRNAs within cells from cassettes that are derived from genes transcribed by RNA polymerase III (pol III) and provide for selective subcellular distribution of their products can be used to direct siRNAs to the cellular pathways. Expression from the human U6 promoter, resulting in siRNA accumulation in the nucleus, is effective in reducing gene expression, whereas cytoplasmic and nucleolar localization of the siRNA when expressed from the 5S or 7 SL promoters is not effective. The distribution of siRNA within the cell is determined by fluorescence in situ hybridization. Although the long uninterrupted duplex of siRNA makes it difficult to detect with DNA oligonucleotide probes, labeled oligonucleotide probes with 2'-O-methyl RNA backbones provide the stability needed for a strong signal. These methods contribute to studies of the interconnected cellular RNAi pathways and are useful in adapting RNAi as a tool to determine gene function and develop RNA-based therapeutics. PMID:15644179

  6. The VP3 factor from viruses of Birnaviridae family suppresses RNA silencing by binding both long and small RNA duplexes.

    PubMed

    Valli, Adrian; Busnadiego, Idoia; Maliogka, Varvara; Ferrero, Diego; Castón, José R; Rodríguez, José Francisco; García, Juan Antonio

    2012-01-01

    RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV) displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

  7. The VP3 Factor from Viruses of Birnaviridae Family Suppresses RNA Silencing by Binding Both Long and Small RNA Duplexes

    PubMed Central

    Maliogka, Varvara; Ferrero, Diego; Castón, José R.; Rodríguez, José Francisco; García, Juan Antonio

    2012-01-01

    RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV) displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery. PMID:23049903

  8. Small interfering RNA delivery by polyethylenimine-functionalised porous silicon nanoparticles.

    PubMed

    Hasanzadeh Kafshgari, M; Alnakhli, M; Delalat, B; Apostolou, S; Harding, F J; Mäkilä, E; Salonen, J J; Kuss, B J; Voelcker, N H

    2015-12-01

    In this study, thermally hydrocarbonised porous silicon nanoparticles (THCpSiNPs) capped with polyethylenimine (PEI) were fabricated, and their potential for small interfering RNA (siRNA) delivery was investigated in an in vitro glioblastoma model. PEI coating following siRNA loading enhanced the sustained release of siRNA, and suppressed burst release effects. The positively-charged surface improved the internalisation of the nanoparticles across the cell membrane. THCpSiNP-mediated siRNA delivery reduced mRNA expression of the MRP1 gene, linked to the resistence of glioblastoma to chemotherapy, by 63% and reduced MRP1-protein levels by 70%. MRP1 siRNA loaded nanoparticles did not induce cytotoxicity in glioblastoma cells, but markedly reduced cell proliferation. In summary, the results demonstrated that non-cytotoxic cationic THCpSiNPs are promising vehicles for therapeutic siRNA delivery.

  9. A Small Molecule That Represses Translation of G-Quadruplex-Containing mRNA.

    PubMed

    Katsuda, Yousuke; Sato, Shin-Ichi; Asano, Lisa; Morimura, Yoshitaka; Furuta, Tomoyuki; Sugiyama, Hiroshi; Hagihara, Masaki; Uesugi, Motonari

    2016-07-27

    The G-quadruplexes form highly stable nucleic acid structures, which are implicated in various biological processes in both DNA and RNA. Although DNA G-quadruplexes have been studied in great detail, biological roles of RNA G-quadruplexes have received less attention. Here, a screening of a chemical library permitted identification of a small-molecule tool that binds selectively to RNA G-quadruplex structures. The polyaromatic molecule, RGB-1, stabilizes RNA G-quadruplex, but not DNA versions or other RNA structures. RGB-1 intensified the G-quadruplex-mediated inhibition of RNA translation in mammalian cells, decreased expression of the NRAS proto-oncogene in breast cancer cells, and permitted identification of a novel sequence that forms G-quadruplex in NRAS mRNA. RGB-1 may serve as a unique tool for understanding cellular roles of RNA G-quadruplex structures. PMID:27410677

  10. Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus

    PubMed Central

    Guo, Yusong R.; Toh, Yukimatsu; Poranen, Minna M.; Tao, Yizhi J.

    2016-01-01

    During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5’-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly. PMID:27078841

  11. Small noncoding RNA modulates japanese encephalitis virus replication and translation in trans

    PubMed Central

    2011-01-01

    Background Sequence and structural elements in the 3'-untranslated region (UTR) of Japanese encephalitis virus (JEV) are known to regulate translation and replication. We previously reported an abundant accumulation of small subgenomic flaviviral RNA (sfRNA) which is collinear with the highly conserved regions of the 3'-UTR in JEV-infected cells. However, function of the sfRNA in JEV life cycle remains unknown. Results Northern blot and real-time RT-PCR analyses indicated that the sfRNA becomes apparent at the time point at which minus-strand RNA (antigenome) reaches a plateau suggesting a role for sfRNA in the regulation of antigenome synthesis. Transfection of minus-sense sfRNA into JEV-infected cells, in order to counter the effects of plus-sense sfRNA, resulted in higher levels of antigenome suggesting that the presence of the sfRNA inhibits antigenome synthesis. Trans-acting effect of sfRNA on JEV translation was studied using a reporter mRNA containing the luciferase gene fused to partial coding regions of JEV and flanked by the respective JEV UTRs. In vivo and in vitro translation revealed that sfRNA inhibited JEV translation. Conclusions Our results indicate that sfRNA modulates viral translation and replication in trans. PMID:22040380

  12. Comparative analysis of dinoflagellate chloroplast genomes reveals rRNA and tRNA genes

    PubMed Central

    Barbrook, Adrian C; Santucci, Nicole; Plenderleith, Lindsey J; Hiller, Roger G; Howe, Christopher J

    2006-01-01

    Background Peridinin-containing dinoflagellates have a highly reduced chloroplast genome, which is unlike that found in other chloroplast containing organisms. Genome reduction appears to be the result of extensive transfer of genes to the nuclear genome. Unusually the genes believed to be remaining in the chloroplast genome are found on small DNA 'minicircles'. In this study we present a comparison of sets of minicircle sequences from three dinoflagellate species. Results PCR was used to amplify several minicircles from Amphidinium carterae so that a homologous set of gene-containing minicircles was available for Amphidinium carterae and Amphidinium operculatum, two apparently closely related peridinin-containing dinoflagellates. We compared the sequences of these minicircles to determine the content and characteristics of their chloroplast genomes. We also made comparisons with minicircles which had been obtained from Heterocapsa triquetra, another peridinin-containing dinoflagellate. These in silico comparisons have revealed several genetic features which were not apparent in single species analyses. The features include further protein coding genes, unusual rRNA genes, which we show are transcribed, and the first examples of tRNA genes from peridinin-containing dinoflagellate chloroplast genomes. Conclusion Comparative analysis of minicircle sequences has allowed us to identify previously unrecognised features of dinoflagellate chloroplast genomes, including additional protein and RNA genes. The chloroplast rRNA gene sequences are radically different from those in other organisms, and in many ways resemble the rRNA genes found in some highly reduced mitochondrial genomes. The retention of certain tRNA genes in the dinoflagellate chloroplast genome has important implications for models of chloroplast-mitochondrion interaction. PMID:17123435

  13. Cytoplasmic RNA viruses as potential vehicles for the delivery of therapeutic small RNAs

    PubMed Central

    2013-01-01

    Viral vectors have become the best option for the delivery of therapeutic genes in conventional and RNA interference-based gene therapies. The current viral vectors for the delivery of small regulatory RNAs are based on DNA viruses and retroviruses/lentiviruses. Cytoplasmic RNA viruses have been excluded as viral vectors for RNAi therapy because of the nuclear localization of the microprocessor complex and the potential degradation of the viral RNA genome during the excision of any virus-encoded pre-microRNAs. However, in the last few years, the presence of several species of small RNAs (e.g., virus-derived small interfering RNAs, virus-derived short RNAs, and unusually small RNAs) in animals and cell cultures that are infected with cytoplasmic RNA viruses has suggested the existence of a non-canonical mechanism of microRNA biogenesis. Several studies have been conducted on the tick-borne encephalitis virus and on the Sindbis virus in which microRNA precursors were artificially incorporated and demonstrated the production of mature microRNAs. The ability of these viruses to recruit Drosha to the cytoplasm during infection resulted in the efficient processing of virus-encoded microRNA without the viral genome entering the nucleus. In this review, we discuss the relevance of these findings with an emphasis on the potential use of cytoplasmic RNA viruses as vehicles for the efficient delivery of therapeutic small RNAs. PMID:23759022

  14. An Integrated Analysis of MicroRNA and mRNA Expression Profiles to Identify RNA Expression Signatures in Lambskin Hair Follicles in Hu Sheep.

    PubMed

    Lv, Xiaoyang; Sun, Wei; Yin, Jinfeng; Ni, Rong; Su, Rui; Wang, Qingzeng; Gao, Wen; Bao, Jianjun; Yu, Jiarui; Wang, Lihong; Chen, Ling

    2016-01-01

    Wave patterns in lambskin hair follicles are an important factor determining the quality of sheep's wool. Hair follicles in lambskin from Hu sheep, a breed unique to China, have 3 types of waves, designated as large, medium, and small. The quality of wool from small wave follicles is excellent, while the quality of large waves is considered poor. Because no molecular and biological studies on hair follicles of these sheep have been conducted to date, the molecular mechanisms underlying the formation of different wave patterns is currently unknown. The aim of this article was to screen the candidate microRNAs (miRNA) and genes for the development of hair follicles in Hu sheep. Two-day-old Hu lambs were selected from full-sib individuals that showed large, medium, and small waves. Integrated analysis of microRNA and mRNA expression profiles employed high-throughout sequencing technology. Approximately 13, 24, and 18 differentially expressed miRNAs were found between small and large waves, small and medium waves, and medium and large waves, respectively. A total of 54, 190, and 81 differentially expressed genes were found between small and large waves, small and medium waves, and medium and large waves, respectively, by RNA sequencing (RNA-seq) analysis. Differentially expressed genes were classified using gene ontology and pathway analyses. They were found to be mainly involved in cell differentiation, proliferation, apoptosis, growth, immune response, and ion transport, and were associated with MAPK and the Notch signaling pathway. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of differentially-expressed miRNA and genes were consistent with sequencing results. Integrated analysis of miRNA and mRNA expression indicated that, compared to small waves, large waves included 4 downregulated miRNAs that had regulatory effects on 8 upregulated genes and 3 upregulated miRNAs, which in turn influenced 13 downregulated genes. Compared to small waves, medium

  15. An Integrated Analysis of MicroRNA and mRNA Expression Profiles to Identify RNA Expression Signatures in Lambskin Hair Follicles in Hu Sheep

    PubMed Central

    Lv, Xiaoyang; Sun, Wei; Yin, Jinfeng; Ni, Rong; Su, Rui; Wang, Qingzeng; Gao, Wen; Bao, Jianjun; Yu, Jiarui; Wang, Lihong; Chen, Ling

    2016-01-01

    Wave patterns in lambskin hair follicles are an important factor determining the quality of sheep’s wool. Hair follicles in lambskin from Hu sheep, a breed unique to China, have 3 types of waves, designated as large, medium, and small. The quality of wool from small wave follicles is excellent, while the quality of large waves is considered poor. Because no molecular and biological studies on hair follicles of these sheep have been conducted to date, the molecular mechanisms underlying the formation of different wave patterns is currently unknown. The aim of this article was to screen the candidate microRNAs (miRNA) and genes for the development of hair follicles in Hu sheep. Two-day-old Hu lambs were selected from full-sib individuals that showed large, medium, and small waves. Integrated analysis of microRNA and mRNA expression profiles employed high-throughout sequencing technology. Approximately 13, 24, and 18 differentially expressed miRNAs were found between small and large waves, small and medium waves, and medium and large waves, respectively. A total of 54, 190, and 81 differentially expressed genes were found between small and large waves, small and medium waves, and medium and large waves, respectively, by RNA sequencing (RNA-seq) analysis. Differentially expressed genes were classified using gene ontology and pathway analyses. They were found to be mainly involved in cell differentiation, proliferation, apoptosis, growth, immune response, and ion transport, and were associated with MAPK and the Notch signaling pathway. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of differentially-expressed miRNA and genes were consistent with sequencing results. Integrated analysis of miRNA and mRNA expression indicated that, compared to small waves, large waves included 4 downregulated miRNAs that had regulatory effects on 8 upregulated genes and 3 upregulated miRNAs, which in turn influenced 13 downregulated genes. Compared to small waves

  16. Translational control of small heat shock genes in mesophilic and thermophilic cyanobacteria by RNA thermometers.

    PubMed

    Cimdins, Annika; Klinkert, Birgit; Aschke-Sonnenborn, Ursula; Kaiser, Friederike M; Kortmann, Jens; Narberhaus, Franz

    2014-01-01

    Cyanobacteria constitute a heterogeneous phylum of oxygen-producing, photosynthetic prokaryotes. They are susceptible to various stress conditions like heat, salt, or light stress, all inducing the cyanobacterial heat shock response (HSR). Cyanobacterial small heat shock proteins (sHsps) are known to preserve thylakoid membrane integrity under stress conditions, thereby protecting the photosynthesis machinery. In Synechocystis sp PCC 6803, synthesis of the sHsp Hsp17 is regulated by an RNA thermometer (RNAT) in the 5'-untranslated region (5'-UTR) of the hsp17 mRNA. RNATs are direct temperature sensors that control expression of many bacterial heat shock and virulence genes. They hinder translation at low temperatures by base pairing, thus blocking ribosome access to the mRNA.   To explore the temperature range in which RNATs act, we studied various RNAT candidates upstream of sHsp genes from mesophilic and thermophilic cyanobacteria. The mesophilic cyanobacteria Anabaena variabilis and Nostoc sp chromosomally encode two sHsps each. Reporter gene studies suggested RNAT-mediated post-transcriptional regulation of shsp expression in both organisms. Detailed structural analysis of the two A. variabilis candidates revealed two novel RNAT types. The first, avashort, regulates translation primarily by masking of the AUG translational start codon. The second, featuring an extended initial hairpin, thus named avalong, presumably makes use of complex tertiary interaction. The 5'-UTR of the small heat shock gene hspA in the thermophile Thermosynechococcus elongatus is predicted to adopt an extended secondary structure. Structure probing revealed that the ribosome binding site was blocked at temperatures below 55 °C. The results of this study demonstrate that cyanobacteria commonly use RNATs to control expression of their small heat shock genes.

  17. A distinct small RNA pathway silences selfish genetic elements in the germline.

    PubMed

    Vagin, Vasily V; Sigova, Alla; Li, Chengjian; Seitz, Hervé; Gvozdev, Vladimir; Zamore, Phillip D

    2006-07-21

    In the Drosophila germline, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Whereas small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appears not to require Dicer-1, which makes microRNAs (miRNAs), or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2',3' hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. Our data suggest that rasiRNAs protect the fly germline through a silencing mechanism distinct from both the miRNA and RNA interference pathways.

  18. Control of ruminant morbillivirus replication by small interfering RNA.

    PubMed

    Servan de Almeida, Renata; Keita, Djénéba; Libeau, Geneviève; Albina, Emmanuel

    2007-08-01

    Peste-des-petits-ruminants virus (PPRV) and rinderpest virus (RPV) are two morbilliviruses of economic relevance in African and Asian countries. Although efficient vaccines are available for both diseases, they cannot protect the animals before 14 days post-vaccination. In emergencies, it would be desirable to have efficient therapeutics for virus control. Here, two regions are described in the nucleocapsid genes of PPRV and RPV that can be targeted efficiently by synthetic short interfering RNAs (siRNAs), resulting in a >80 % reduction in virus replication. The effects of siRNAs on the production of viral RNA by real-time quantitative PCR, of viral proteins by flow cytometry and of virus particles by appreciation of the cytopathic effect and virus titration were monitored. The findings of this work highlight the potential for siRNA molecules to be developed as therapeutic agents for the treatment of PPRV and RPV infections.

  19. Kinetic Analysis of tRNA Methylfransferases

    PubMed Central

    Hou, Ya-Ming; Masuda, Isao

    2016-01-01

    Transfer RNA (tRNA) molecules contain many chemical modifications that are introduced after transcription. A major form of these modifications is methyl transfer to bases and backbone groups, using S-adenosyl methionine (AdoMet) as the methyl donor. Each methylation confers a specific advantage to tRNA in structure or in function. A remarkable methylation is to the G37 base on the 3' side of the anticodon to generate m1G37-tRNA, which suppresses frameshift errors during protein synthesis and is therefore essential for cell growth in all three domains of life. This methylation is catalyzed by TrmD in bacteria and by Trm5 in eukaryotes and archaea. Although TrmD and Trm5 catalyze the same methylation reaction, kinetic analysis reveal that these two enzymes are unrelated to each other and are distinct in their reaction mechanism. This chapter summarizes the kinetic assays that are used to reveal the distinction between TrmD and Trm5. Three types of assays are described, the steady-state, the pre-steady-state, and the single turnover assays, which collectively provide the basis for mechanistic investigation of AdoMet-dependent methyl transfer reactions. PMID:26253967

  20. The small RNA SgrS controls sugar–phosphate accumulation by regulating multiple PTS genes

    PubMed Central

    Rice, Jennifer B.; Vanderpool, Carin K.

    2011-01-01

    A number of bacterial small RNAs (sRNAs) act as global regulators of stress responses by controlling expression of multiple genes. The sRNA SgrS is expressed in response to glucose–phosphate stress, a condition associated with disruption of glycolytic flux and accumulation of sugar–phosphates. SgrS has been shown to stimulate degradation of the ptsG mRNA, encoding the major glucose transporter. This study demonstrates that SgrS regulates the genes encoding the mannose and secondary glucose transporter, manXYZ. Analysis of manXYZ mRNA stability and translation in the presence and absence of SgrS indicate that manXYZ is regulated by SgrS under stress conditions and when SgrS is ectopically expressed. In vitro footprinting and in vivo mutational analyses showed that SgrS base pairs with manXYZ within the manX coding sequence to prevent manX translation. Regulation of manX did not require the RNase E degradosome complex, suggesting that the primary mechanism of regulation is translational. An Escherichia coli ptsG mutant strain that is manXYZ+ experiences stress when exposed to the glucose analogs α-methyl glucoside or 2-deoxyglucose. A ptsG manXYZ double mutant is resistant to the stress, indicating that PTS transporters encoded by both SgrS targets are involved in taking up substrates that cause stress. PMID:21245045

  1. Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In eukaryotes, RNA silencing pathways utilize 20–30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focuse...

  2. Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination.

    PubMed

    Noto, Tomoko; Kataoka, Kensuke; Suhren, Jan H; Hayashi, Azusa; Woolcock, Katrina J; Gorovsky, Martin A; Mochizuki, Kazufumi

    2015-07-16

    Small RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymena are robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production.

  3. Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination

    PubMed Central

    Noto, Tomoko; Kataoka, Kensuke; Suhren, Jan H.; Hayashi, Azusa; Woolcock, Katrina J.; Gorovsky, Martin A.; Mochizuki, Kazufumi

    2015-01-01

    Summary Small RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymena are robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production. PMID:26095658

  4. Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

    DOEpatents

    Lucas, William J.; Yoo, Byung-Chun; Lough, Tony J.; Varkonyi-Gasic, Erika

    2007-03-13

    The present invention provides a polynucleotide sequence encoding a component of the protein machinery involved in small RNA trafficking, Cucurbita maxima phloem small RNA-binding protein (CmPSRB 1), and the corresponding polypeptide sequence. The invention also provides genetic constructs and transgenic plants comprising the polynucleotide sequence encoding a phloem small RNA-binding protein to alter (e.g., prevent, reduce or elevate) non-cell autonomous signaling events in the plants involving small RNA metabolism. These signaling events are involved in a broad spectrum of plant physiological and biochemical processes, including, for example, systemic resistance to pathogens, responses to environmental stresses, e.g., heat, drought, salinity, and systemic gene silencing (e.g., viral infections).

  5. Improving small-angle X-ray scattering data for structural analyses of the RNA world

    PubMed Central

    Rambo, Robert P.; Tainer, John A.

    2010-01-01

    Defining the shape, conformation, or assembly state of an RNA in solution often requires multiple investigative tools ranging from nucleotide analog interference mapping to X-ray crystallography. A key addition to this toolbox is small-angle X-ray scattering (SAXS). SAXS provides direct structural information regarding the size, shape, and flexibility of the particle in solution and has proven powerful for analyses of RNA structures with minimal requirements for sample concentration and volumes. In principle, SAXS can provide reliable data on small and large RNA molecules. In practice, SAXS investigations of RNA samples can show inconsistencies that suggest limitations in the SAXS experimental analyses or problems with the samples. Here, we show through investigations on the SAM-I riboswitch, the Group I intron P4-P6 domain, 30S ribosomal subunit from Sulfolobus solfataricus (30S), brome mosaic virus tRNA-like structure (BMV TLS), Thermotoga maritima asd lysine riboswitch, the recombinant tRNAval, and yeast tRNAphe that many problems with SAXS experiments on RNA samples derive from heterogeneity of the folded RNA. Furthermore, we propose and test a general approach to reducing these sample limitations for accurate SAXS analyses of RNA. Together our method and results show that SAXS with synchrotron radiation has great potential to provide accurate RNA shapes, conformations, and assembly states in solution that inform RNA biological functions in fundamental ways. PMID:20106957

  6. Inhibitory impact of 3'-terminal 2'-O-methylated small silencing RNA on target-primed polymerization and unbiased amplified quantification of the RNA in Arabidopsis thaliana.

    PubMed

    Chen, Feng; Fan, Chunhai; Zhao, Yongxi

    2015-09-01

    3'-terminal 2'-O-methylation has been found in several kinds of small silencing RNA, regarded as a protective mechanism against enzymatic 3' → 5' degradation and 3'-end uridylation. The influence of this modification on enzymatic polymerization, however, remains unknown. Herein, a systematic investigation is performed to explore this issue. We found these methylated small RNAs exhibited a suppression behavior in target-primed polymerization, revealing biased result for the manipulation of these small RNAs by conventional polymerization-based methodology. The related potential mechanism is investigated and discussed, which is probably ascribed to the big size of modified group and its close location to 3'-OH. Furthermore, two novel solutions each utilizing base-stacking hybridization and three-way junction structure have been proposed to realize unbiased recognition of small RNAs. On the basis of phosphorothioate against nicking, a creative amplified strategy, phosphorothioate-protected polymerization/binicking amplification, has also been developed for the unbiased quantification of methylated small RNA in Arabidopsis thaliana, demonstrating its promising potential for real sample analysis. Collectively, our studies uncover the polymerization inhibition by 3'-terminal 2'-O-methylated small RNAs with mechanistic discussion, and propose novel unbiased solutions for amplified quantification of small RNAs in real sample. PMID:26244621

  7. Inhibitory impact of 3'-terminal 2'-O-methylated small silencing RNA on target-primed polymerization and unbiased amplified quantification of the RNA in Arabidopsis thaliana.

    PubMed

    Chen, Feng; Fan, Chunhai; Zhao, Yongxi

    2015-09-01

    3'-terminal 2'-O-methylation has been found in several kinds of small silencing RNA, regarded as a protective mechanism against enzymatic 3' → 5' degradation and 3'-end uridylation. The influence of this modification on enzymatic polymerization, however, remains unknown. Herein, a systematic investigation is performed to explore this issue. We found these methylated small RNAs exhibited a suppression behavior in target-primed polymerization, revealing biased result for the manipulation of these small RNAs by conventional polymerization-based methodology. The related potential mechanism is investigated and discussed, which is probably ascribed to the big size of modified group and its close location to 3'-OH. Furthermore, two novel solutions each utilizing base-stacking hybridization and three-way junction structure have been proposed to realize unbiased recognition of small RNAs. On the basis of phosphorothioate against nicking, a creative amplified strategy, phosphorothioate-protected polymerization/binicking amplification, has also been developed for the unbiased quantification of methylated small RNA in Arabidopsis thaliana, demonstrating its promising potential for real sample analysis. Collectively, our studies uncover the polymerization inhibition by 3'-terminal 2'-O-methylated small RNAs with mechanistic discussion, and propose novel unbiased solutions for amplified quantification of small RNAs in real sample.

  8. Structural insights into mechanisms of the small RNA methyltransferase HEN1

    SciTech Connect

    Huang, Ying; Ji, Lijuan; Huang, Qichen; Vassylyev, Dmitry G.; Chen, Xuemei; Ma, Jin-Biao

    2010-02-22

    RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of {approx}20-30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-L-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 {angstrom} crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-L-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg{sup 2+}-dependent 2'-O-methylation mechanism.

  9. A highly expressed miR-101 isomiR is a functional silencing small RNA

    PubMed Central

    2013-01-01

    Background MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression usually producing translational repression and gene silencing. High-throughput sequencing technologies have revealed heterogeneity at length and sequence level for the majority of mature miRNAs (IsomiRs). Most isomiRs can be explained by variability in either Dicer1 or Drosha cleavage during miRNA biogenesis at 5’ or 3’ of the miRNA (trimming variants). Although isomiRs have been described in different tissues and organisms, their functional validation as modulators of gene expression remains elusive. Here we have characterized the expression and function of a highly abundant miR-101 5’-trimming variant (5’-isomiR-101). Results The analysis of small RNA sequencing data in several human tissues and cell lines indicates that 5’-isomiR-101 is ubiquitously detected and a highly abundant, especially in the brain. 5’-isomiR-101 was found in Ago-2 immunocomplexes and complementary approaches showed that 5’-isomiR-101 interacted with different members of the silencing (RISC) complex. In addition, 5’-isomiR-101 decreased the expression of five validated miR-101 targets, suggesting that it is a functional variant. Both the binding to RISC members and the degree of silencing were less efficient for 5’-isomiR-101 compared with miR-101. For some targets, both miR-101 and 5’-isomiR-101 significantly decreased protein expression with no changes in the respective mRNA levels. Although a high number of overlapping predicted targets suggest similar targeted biological pathways, a correlation analysis of the expression profiles of miR-101 variants and predicted mRNA targets in human brains at different ages, suggest specific functions for miR-101- and 5’-isomiR-101. Conclusions These results suggest that isomiRs are functional variants and further indicate that for a given miRNA, the different isomiRs may contribute to the overall effect as quantitative and

  10. Small RNA Profiling of Two Important Cultivars of Banana and Overexpression of miRNA156 in Transgenic Banana Plants.

    PubMed

    Ghag, Siddhesh B; Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2015-01-01

    Micro RNAs (miRNAs) are a class of non-coding, short RNAs having important roles in regulation of gene expression. Although plant miRNAs have been studied in detail in some model plants, less is known about these miRNAs in important fruit plants like banana. miRNAs have pivotal roles in plant growth and development, and in responses to diverse biotic and abiotic stress stimuli. Here, we have analyzed the small RNA expression profiles of two different economically significant banana cultivars by using high-throughput sequencing technology. We identified a total of 170 and 244 miRNAs in the two libraries respectively derived from cv. Grand Naine and cv. Rasthali leaves. In addition, several cultivar specific microRNAs along with their putative target transcripts were also detected in our studies. To validate our findings regarding the small RNA profiles, we also undertook overexpression of a common microRNA, MusamiRNA156 in transgenic banana plants. The transgenic plants overexpressing the stem-loop sequence derived from MusamiRNA156 gene were stunted in their growth together with peculiar changes in leaf anatomy. These results provide a foundation for further investigations into important physiological and metabolic pathways operational in banana in general and cultivar specific traits in particular. PMID:25962076

  11. Small RNA Profiling of Two Important Cultivars of Banana and Overexpression of miRNA156 in Transgenic Banana Plants

    PubMed Central

    Ganapathi, Thumballi R.

    2015-01-01

    Micro RNAs (miRNAs) are a class of non-coding, short RNAs having important roles in regulation of gene expression. Although plant miRNAs have been studied in detail in some model plants, less is known about these miRNAs in important fruit plants like banana. miRNAs have pivotal roles in plant growth and development, and in responses to diverse biotic and abiotic stress stimuli. Here, we have analyzed the small RNA expression profiles of two different economically significant banana cultivars by using high-throughput sequencing technology. We identified a total of 170 and 244 miRNAs in the two libraries respectively derived from cv. Grand Naine and cv. Rasthali leaves. In addition, several cultivar specific microRNAs along with their putative target transcripts were also detected in our studies. To validate our findings regarding the small RNA profiles, we also undertook overexpression of a common microRNA, MusamiRNA156 in transgenic banana plants. The transgenic plants overexpressing the stem-loop sequence derived from MusamiRNA156 gene were stunted in their growth together with peculiar changes in leaf anatomy. These results provide a foundation for further investigations into important physiological and metabolic pathways operational in banana in general and cultivar specific traits in particular. PMID:25962076

  12. RNA sequencing identifies specific PIWI-interacting small non-coding RNA expression patterns in breast cancer

    PubMed Central

    Marchese, Giovanna; Ravo, Maria; Tarallo, Roberta; Nassa, Giovanni; Giurato, Giorgio; Santamaria, Gianluca; Cordella, Angela; Cantarella, Concita; Weisz, Alessandro

    2014-01-01

    PIWI-interacting small non-coding RNAs (piRNAs) are genetic and epigenetic regulatory factors in germline cells, where they maintain genome stability, are involved in RNA silencing and regulate gene expression. We found that the piRNA biogenesis and effector pathway are present in human breast cancer (BC) cells and, analyzing smallRNA-Seq data generated from BC cell lines and tumor biopsies, we identified >100 BC piRNAs, including some very abundant and/or differentially expressed in mammary epithelial compared to BC cells, where this was influenced by estrogen or estrogen receptor β, and in cancer respect to normal breast tissues. A search for mRNAs targeted by the BC piRNome revealed that eight piRNAs showing a specific expression pattern in breast tumors target key cancer cell pathways. Evidence of an active piRNA pathway in BC suggests that these small non-coding RNAs do exert transcriptional and post-transcriptional gene regulatory actions also in cancer cells. PMID:25313140

  13. RNA sequencing identifies specific PIWI-interacting small non-coding RNA expression patterns in breast cancer.

    PubMed

    Hashim, Adnan; Rizzo, Francesca; Marchese, Giovanna; Ravo, Maria; Tarallo, Roberta; Nassa, Giovanni; Giurato, Giorgio; Santamaria, Gianluca; Cordella, Angela; Cantarella, Concita; Weisz, Alessandro

    2014-10-30

    PIWI-interacting small non-coding RNAs (piRNAs) are genetic and epigenetic regulatory factors in germline cells, where they maintain genome stability, are involved in RNA silencing and regulate gene expression. We found that the piRNA biogenesis and effector pathway are present in human breast cancer (BC) cells and, analyzing smallRNA-Seq data generated from BC cell lines and tumor biopsies, we identified >100 BC piRNAs, including some very abundant and/or differentially expressed in mammary epithelial compared to BC cells, where this was influenced by estrogen or estrogen receptor β, and in cancer respect to normal breast tissues. A search for mRNAs targeted by the BC piRNome revealed that eight piRNAs showing a specific expression pattern in breast tumors target key cancer cell pathways. Evidence of an active piRNA pathway in BC suggests that these small non-coding RNAs do exert transcriptional and post-transcriptional gene regulatory actions also in cancer cells. PMID:25313140

  14. Phytophthora Have Distinct Endogenous Small RNA Populations That Include Short Interfering and microRNAs

    PubMed Central

    Fahlgren, Noah; Bollmann, Stephanie R.; Kasschau, Kristin D.; Cuperus, Josh T.; Press, Caroline M.; Sullivan, Christopher M.; Chapman, Elisabeth J.; Hoyer, J. Steen; Gilbert, Kerrigan B.; Grünwald, Niklaus J.; Carrington, James C.

    2013-01-01

    In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work. PMID:24204767

  15. Comparative Analysis of miRNAs and Their Target Transcripts between a Spontaneous Late-Ripening Sweet Orange Mutant and Its Wild-Type Using Small RNA and Degradome Sequencing

    PubMed Central

    Wu, Juxun; Zheng, Saisai; Feng, Guizhi; Yi, Hualin

    2016-01-01

    Fruit ripening in citrus is not well-understood at the molecular level. Knowledge of the regulatory mechanism of citrus fruit ripening at the post-transcriptional level in particular is lacking. Here, we comparatively analyzed the miRNAs and their target genes in a spontaneous late-ripening mutant, “Fengwan” sweet orange (MT) (Citrus sinensis L. Osbeck), and its wild-type counterpart (“Fengjie 72-1,” WT). Using high-throughput sequencing of small RNAs and RNA degradome tags, we identified 107 known and 21 novel miRNAs, as well as 225 target genes. A total of 24 miRNAs (16 known miRNAs and 8 novel miRNAs) were shown to be differentially expressed between MT and WT. The expression pattern of several key miRNAs and their target genes during citrus fruit development and ripening stages was examined. Csi-miR156k, csi-miR159, and csi-miR166d suppressed specific transcription factors (GAMYBs, SPLs, and ATHBs) that are supposed to be important regulators involved in citrus fruit development and ripening. In the present study, miRNA-mediated silencing of target genes was found under complicated and sensitive regulation in citrus fruit. The identification of miRNAs and their target genes provide new clues for future investigation of mechanisms that regulate citrus fruit ripening. PMID:27708662

  16. Stand-alone rolling circle amplification combined with capillary electrophoresis for specific detection of small RNA.

    PubMed

    Li, Ni; Jablonowski, Carolyn; Jin, Hailing; Zhong, Wenwan

    2009-06-15

    Noncoding small RNAs play diverse, important biological roles through gene expression regulation. However, their low expression levels make it difficult to identify new small RNA species and study their functions, calling for the development of detection schemes with higher simplicity, sensitivity, and specificity. Herein, we reported a straightforward assay that combined the stand-alone rolling circle amplification (RCA) with capillary electrophoresis (CE) for specific and sensitive detection of small RNAs in biological samples. In order to enhance the overall reaction efficiency and simplify the procedure, RCA was not preceded with ligation, and a preformed circular probe was employed as the template for the target small RNA-primed isothermal amplification. The long RCA product was digested and analyzed by CE. Two DNA polymerases, the Phi29 and Bst, were compared for their detection performance. Bst is superior in the aspects of specificity, procedure simplicity, and reproducibility, while Phi29 leads to a 5-fold lower detection limit and is able to detect as low as 35 amol of the target small RNA. Coamplification of an internal standard with the target and employment of the RNase A digestion step allow accurate and reproducible quantification of low amounts of small RNA targets spiked into hundreds of nanograms of the plant total RNA extract with a recovery below 110% using either enzyme. Our assay can be adapted to a capillary array system for high-throughput screening of small RNA expression in biological samples. Also, the one-step isothermal process has the potential to conveniently amplify a very limited amount of the RNA samples, e.g., RNA extracted from only a few cells, inside the capillary column or on a microchip.

  17. Characterization by Small RNA Sequencing of Taro Bacilliform CH Virus (TaBCHV), a Novel Badnavirus.

    PubMed

    Kazmi, Syeda Amber; Yang, Zuokun; Hong, Ni; Wang, Guoping; Wang, Yanfen

    2015-01-01

    RNA silencing is an antiviral immunity that regulates gene expression through the production of small RNAs (sRNAs). In this study, deep sequencing of small RNAs was used to identify viruses infecting two taro plants. Blast searching identified five and nine contigs assembled from small RNAs of samples T1 and T2 matched onto the genome sequences of badnaviruses in the family Caulimoviridae. Complete genome sequences of two isolates of the badnavirus determined by sequence specific amplification comprised of 7,641 nucleotides and shared overall nucleotide similarities of 44.1%‒55.8% with other badnaviruses. Six open reading frames (ORFs) were identified on the plus strand, showed amino acid similarities ranging from 59.8% (ORF3) to 10.2% (ORF6) to the corresponding proteins encoded by other badnaviruses. Phylogenetic analysis also supports that the virus is a new member in the genus Badnavirus. The virus is tentatively named as Taro bacilliform CH virus (TaBCHV), and it is the second badnavirus infecting taro plants, following Taro bacilliform virus (TaBV). In addition, analyzes of viral derived small RNAs (vsRNAs) from TaBCHV showed that almost equivalent number of vsRNAs were generated from both strands and the most abundant vsRNAs were 21 nt, with uracil bias at 5' terminal. Furthermore, TaBCHV vsRNAs were asymmetrically distributed on its entire circular genome at both orientations with the hotspots mainly generated in the ORF5 region.

  18. Characterization by Small RNA Sequencing of Taro Bacilliform CH Virus (TaBCHV), a Novel Badnavirus

    PubMed Central

    Kazmi, Syeda Amber; Yang, Zuokun; Hong, Ni; Wang, Guoping; Wang, Yanfen

    2015-01-01

    RNA silencing is an antiviral immunity that regulates gene expression through the production of small RNAs (sRNAs). In this study, deep sequencing of small RNAs was used to identify viruses infecting two taro plants. Blast searching identified five and nine contigs assembled from small RNAs of samples T1 and T2 matched onto the genome sequences of badnaviruses in the family Caulimoviridae. Complete genome sequences of two isolates of the badnavirus determined by sequence specific amplification comprised of 7,641 nucleotides and shared overall nucleotide similarities of 44.1%‒55.8% with other badnaviruses. Six open reading frames (ORFs) were identified on the plus strand, showed amino acid similarities ranging from 59.8% (ORF3) to 10.2% (ORF6) to the corresponding proteins encoded by other badnaviruses. Phylogenetic analysis also supports that the virus is a new member in the genus Badnavirus. The virus is tentatively named as Taro bacilliform CH virus (TaBCHV), and it is the second badnavirus infecting taro plants, following Taro bacilliform virus (TaBV). In addition, analyzes of viral derived small RNAs (vsRNAs) from TaBCHV showed that almost equivalent number of vsRNAs were generated from both strands and the most abundant vsRNAs were 21 nt, with uracil bias at 5' terminal. Furthermore, TaBCHV vsRNAs were asymmetrically distributed on its entire circular genome at both orientations with the hotspots mainly generated in the ORF5 region. PMID:26207896

  19. Characterization by Small RNA Sequencing of Taro Bacilliform CH Virus (TaBCHV), a Novel Badnavirus.

    PubMed

    Kazmi, Syeda Amber; Yang, Zuokun; Hong, Ni; Wang, Guoping; Wang, Yanfen

    2015-01-01

    RNA silencing is an antiviral immunity that regulates gene expression through the production of small RNAs (sRNAs). In this study, deep sequencing of small RNAs was used to identify viruses infecting two taro plants. Blast searching identified five and nine contigs assembled from small RNAs of samples T1 and T2 matched onto the genome sequences of badnaviruses in the family Caulimoviridae. Complete genome sequences of two isolates of the badnavirus determined by sequence specific amplification comprised of 7,641 nucleotides and shared overall nucleotide similarities of 44.1%‒55.8% with other badnaviruses. Six open reading frames (ORFs) were identified on the plus strand, showed amino acid similarities ranging from 59.8% (ORF3) to 10.2% (ORF6) to the corresponding proteins encoded by other badnaviruses. Phylogenetic analysis also supports that the virus is a new member in the genus Badnavirus. The virus is tentatively named as Taro bacilliform CH virus (TaBCHV), and it is the second badnavirus infecting taro plants, following Taro bacilliform virus (TaBV). In addition, analyzes of viral derived small RNAs (vsRNAs) from TaBCHV showed that almost equivalent number of vsRNAs were generated from both strands and the most abundant vsRNAs were 21 nt, with uracil bias at 5' terminal. Furthermore, TaBCHV vsRNAs were asymmetrically distributed on its entire circular genome at both orientations with the hotspots mainly generated in the ORF5 region. PMID:26207896

  20. Transcriptional evidence for small RNA regulation of pupal diapause in the flesh fly, Sarcophaga bullata.

    PubMed

    Reynolds, Julie A; Clark, Jennifer; Diakoff, Stephen J; Denlinger, David L

    2013-10-01

    Understanding the molecular basis of diapause, a phenotypically plastic, alternative developmental pathway, is key to predicting the seasonal distribution of economically and medically important insect species. Small regulatory RNAs, including piwi-related RNAs, small-interfering RNAs, and miRNAs, represent one type of epigenetic process that can alter the phenotype of organisms independent of changes in genome sequence. We hypothesize that small RNAs regulate pupal diapause and a maternal block of diapause in the flesh fly Sarcophaga bullata. We assessed the relative abundance of eight genes related to small RNA biogenesis and function using qRT-PCR in pre-diapause and diapause stages compared to their non-diapause counterparts. Elevated mRNA expression of piwi and spindle-E, as well as argonaute2 and r2d2, in photosensitive 1st instar larvae reared in diapause-inducing conditions indicate involvement of the piwi-associated RNA and small-interfering RNA pathways, respectively, in programming the switch from direct development to a developmental pathway that includes diapause. Two genes, related to the microRNA pathway, argonaute1 and loquacious, are upregulated during pupal diapause, suggesting a role for this pathway in maintaining diapause. Substantial reduction in transcript abundance of small RNA-related genes in photosensitive 1st instar larvae from mothers with a diapause history compared to those from mothers with no diapause history also suggest a role for small RNA pathways in regulating a diapause maternal effect in S. bullata. Together, the results point to a role for small RNAs in regulating the developmental trajectory in this species.

  1. The mammalian response to virus infection is independent of small RNA silencing

    PubMed Central

    Backes, Simone; Langlois, Ryan A.; Schmid, Sonja; Varble, Andrew; Shim, Jaehee V.; Sachs, David

    2014-01-01

    Summary A successful cellular response to virus infection is essential for evolutionary survival. In plants, arthropods, and nematodes, cellular antiviral defenses rely on RNA interference (RNAi). Interestingly, the mammalian response to virus is predominantly orchestrated through interferon (IFN)-mediated induction of antiviral proteins. Despite the potency of the IFN system, it remains unclear whether mammals also have the capacity to employ antiviral RNAi. Here we investigate this by disabling either IFN, small RNA function or both activities in the context of virus infection. We find that loss of small RNAs in the context of an in vivo RNA virus infection lowers titers due to reduced transcriptional repression of the host antiviral response. In contrast, enabling a virus with the capacity to inhibit the IFN system results in increased titers. Taken together, we conclude that small RNA silencing is not a physiological contributor to the IFN-mediated cellular response to virus infection. PMID:24953656

  2. The Influence of Genotype and Environment on Small RNA Profiles in Grapevine Berry

    PubMed Central

    Paim Pinto, Daniela Lopes; Brancadoro, Lucio; Dal Santo, Silvia; De Lorenzis, Gabriella; Pezzotti, Mario; Meyers, Blake C.; Pè, Mario E.; Mica, Erica

    2016-01-01

    Understanding the molecular mechanisms involved in the interaction between the genetic composition and the environment is crucial for modern viticulture. We approached this issue by focusing on the small RNA transcriptome in grapevine berries of the two varieties Cabernet Sauvignon and Sangiovese, growing in adjacent vineyards in three different environments. Four different developmental stages were studied and a total of 48 libraries of small RNAs were produced and sequenced. Using a proximity-based pipeline, we determined the general landscape of small RNAs accumulation in grapevine berries. We also investigated the presence of known and novel miRNAs and analyzed their accumulation profile. The results showed that the distribution of small RNA-producing loci is variable between the two cultivars, and that the level of variation depends on the vineyard. Differently, the profile of miRNA accumulation mainly depends on the developmental stage. The vineyard in Riccione maximizes the differences between the varieties, promoting the production of more than 1000 specific small RNA loci and modulating their expression depending on the cultivar and the maturation stage. In total, 89 known vvi-miRNAs and 33 novel vvi-miRNA candidates were identified in our samples, many of them showing the accumulation profile modulated by at least one of the factors studied. The in silico prediction of miRNA targets suggests their involvement in berry development and in secondary metabolites accumulation such as anthocyanins and polyphenols. PMID:27761135

  3. Structural and functional characterization of mouse U7 small nuclear RNA active in 3' processing of histone pre-mRNA

    SciTech Connect

    Soldati, D.; Schumperli, D.

    1988-04-01

    Oligonucleotides derived from the spacer element of the histone RNA 3' processing signal were used to characterize mouse U7 small nuclear RNA (snRNA), i.e., the snRNA component active in 3' processing of histone pre-mRNA. Under RNase H conditions, such oligonucleotides inhibited the processing reaction, indicating the formation of a DNA-RNA hybrid with a functional ribonucleoprotein component. Moreover, these oligonucleotides hybridized to a single nuclear RNA species of approximately 65 nucleotides. The sequence of this RNA was determined by primer extension experiments and was found to bear several structural similarities with sea urchin U7 snRNA. The comparison of mouse and sea urchin U7 snRNA structure yields some further insight into the mechanism of histone RNA 3' processing.

  4. Phylogeny of trichomonads inferred from small-subunit rRNA sequences.

    PubMed

    Gunderson, J; Hinkle, G; Leipe, D; Morrison, H G; Stickel, S K; Odelson, D A; Breznak, J A; Nerad, T A; Müller, M; Sogin, M L

    1995-01-01

    Small subunit (16S-like) ribosomal RNA sequences were obtained from representatives of all four families constituting the order Trichomonadida. Comparative sequence analysis revealed that the Trichomonadida are a monophyletic lineage and a deep branch of the eukaryotic tree. Relative to the early divergent eukaryotic assemblages the branching pattern within the Trichomonadida is very shallow. This pattern suggests the Trichomonadida radiated recently, perhaps in conjunction with their animal hosts. From a morphological perspective the Devescovinidae and Calonymphidae are considered more derived than the Monocercomonadidae and Trichomonadidae. Molecular trees inferred by distance, parsimony and likelihood techniques consistently show the Devescovinidae and Calonymphidae are the earliest diverging lineages within the Trichomonadida, however bootstrap values do not strongly support a particular branching order. In an analysis of all known 16S-like ribosomal RNA sequences, the Trichomonadida share most recent common ancestry with unidentified protists from the hindgut of the termite Reticulitermes flavipes. The position of two putative free-living trichomonads in the tree is indicative of derivation from symbionts rather than direct descent from some free-living ancestral trichomonad.

  5. Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome

    PubMed Central

    Anda, Mizue; Ohtsubo, Yoshiyuki; Okubo, Takashi; Sugawara, Masayuki; Nagata, Yuji; Tsuda, Masataka; Minamisawa, Kiwamu; Mitsui, Hisayuki

    2015-01-01

    rRNA is essential for life because of its functional importance in protein synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is located on the “main” chromosome in all bacteria documented to date and is frequently used as a marker of chromosomes. Here, our genome analysis of a plant-associated alphaproteobacterium, Aureimonas sp. AU20, indicates that this strain has its sole rrn operon on a small (9.4 kb), high-copy-number replicon. We designated this unusual replicon carrying the rrn operon on the background of an rrn-lacking chromosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also had this RLC/rrn-plasmid organization. Phylogenetic analysis showed that those strains having the RLC/rrn-plasmid organization represented one clade within the genus Aureimonas. Our finding introduces a previously unaddressed viewpoint into studies of genetics, genomics, and evolution in microbiology and biology in general. PMID:26534993

  6. Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog

    PubMed Central

    Willkomm, Dagmar K.; Minnerup, Jens; Hüttenhofer, Alexander; Hartmann, Roland K.

    2005-01-01

    By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its γ-proteobacterial homologs (∼185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the γ-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the γ-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites. PMID:15814812

  7. Novel small RNA (sRNA) landscape of the starvation-stress response transcriptome of Salmonella enterica serovar typhimurium.

    PubMed

    Amin, Shivam V; Roberts, Justin T; Patterson, Dillon G; Coley, Alexander B; Allred, Jonathan A; Denner, Jason M; Johnson, Justin P; Mullen, Genevieve E; O'Neal, Trenton K; Smith, Jason T; Cardin, Sara E; Carr, Hank T; Carr, Stacie L; Cowart, Holly E; DaCosta, David H; Herring, Brendon R; King, Valeria M; Polska, Caroline J; Ward, Erin E; Wise, Alice A; McAllister, Kathleen N; Chevalier, David; Spector, Michael P; Borchert, Glen M

    2016-01-01

    Small RNAs (sRNAs) are short (∼50-200 nucleotides) noncoding RNAs that regulate cellular activities across bacteria. Salmonella enterica starved of a carbon-energy (C) source experience a host of genetic and physiological changes broadly referred to as the starvation-stress response (SSR). In an attempt to identify novel sRNAs contributing to SSR control, we grew log-phase, 5-h C-starved and 24-h C-starved cultures of the virulent Salmonella enterica subspecies enterica serovar Typhimurium strain SL1344 and comprehensively sequenced their small RNA transcriptomes. Strikingly, after employing a novel strategy for sRNA discovery based on identifying dynamic transcripts arising from "gene-empty" regions, we identify 58 wholly undescribed Salmonella sRNA genes potentially regulating SSR averaging an ∼1,000-fold change in expression between log-phase and C-starved cells. Importantly, the expressions of individual sRNA loci were confirmed by both comprehensive transcriptome analyses and northern blotting of select candidates. Of note, we find 43 candidate sRNAs share significant sequence identity to characterized sRNAs in other bacteria, and ∼70% of our sRNAs likely assume characteristic sRNA structural conformations. In addition, we find 53 of our 58 candidate sRNAs either overlap neighboring mRNA loci or share significant sequence complementarity to mRNAs transcribed elsewhere in the SL1344 genome strongly suggesting they regulate the expression of transcripts via antisense base-pairing. Finally, in addition to this work resulting in the identification of 58 entirely novel Salmonella enterica genes likely participating in the SSR, we also find evidence suggesting that sRNAs are significantly more prevalent than currently appreciated and that Salmonella sRNAs may actually number in the thousands.

  8. Genes and Small RNA Transcripts Exhibit Dosage-Dependent Expression Pattern in Maize Copy-Number Alterations.

    PubMed

    Zuo, Tao; Zhang, Jianbo; Lithio, Andrew; Dash, Sudhansu; Weber, David F; Wise, Roger; Nettleton, Dan; Peterson, Thomas

    2016-07-01

    Copy-number alterations are widespread in animal and plant genomes, but their immediate impact on gene expression is still unclear. In animals, copy-number alterations usually exhibit dosage effects, except for sex chromosomes which tend to be dosage compensated. In plants, genes within small duplications (<100 kb) often exhibit dosage-dependent expression, whereas large duplications (>50 Mb) are more often dosage compensated. However, little or nothing is known about expression in moderately-sized (1-50 Mb) segmental duplications, and about the response of small RNAs to dosage change. Here, we compared maize (Zea mays) plants with two, three, and four doses of a 14.6-Mb segment of chromosome 1 that contains ∼300 genes. Plants containing the duplicated segment exhibit dosage-dependent effects on ear length and flowering time. Transcriptome analyses using GeneChip and RNA-sequencing methods indicate that most expressed genes and unique small RNAs within the duplicated segments exhibit dosage-dependent transcript levels. We conclude that dosage effect is the predominant regulatory response for both genes and unique small RNA transcripts in the segmental dosage series we tested. To our knowledge this is the first analysis of small RNA expression in plant gene dosage variants. Because segmental duplications comprise a significant proportion of eukaryotic genomes, these findings provide important new insight into the regulation of genes and small RNAs in response to dosage changes. PMID:27129738

  9. Identification of MiRNA from Eggplant (Solanum melongena L.) by Small RNA Deep Sequencing and Their Response to Verticillium dahliae Infection

    PubMed Central

    Yang, Liu; Jue, Dengwei; Li, Wang; Zhang, Ruijie; Chen, Min; Yang, Qing

    2013-01-01

    MiRNAs are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data, including eggplant (Solanum melongena L.). To identify miRNAs in eggplant and their response to Verticillium dahliae infection, a fungal pathogen for which clear understanding of infection mechanisms and effective cure methods are currently lacking, we deep-sequenced two small RNA (sRNA) libraries prepared from mock-infected and infected seedlings of eggplants. Specifically, 30,830,792 reads produced 7,716,328 unique miRNAs representing 99 known miRNA families that have been identified in other plant species. Two novel putative miRNAs were predicted with eggplant ESTs. The potential targets of the identified known and novel miRNAs were also predicted based on sequence homology search. It was observed that the length distribution of obtained sRNAs and the expression of 6 miRNA families were obviously different between the two libraries. These results provide a framework for further analysis of miRNAs and their role in regulating plant response to fungal infection and Verticillium wilt in particular. PMID:24015279

  10. Identification of MiRNA from eggplant (Solanum melongena L.) by small RNA deep sequencing and their response to Verticillium dahliae infection.

    PubMed

    Yang, Liu; Jue, Dengwei; Li, Wang; Zhang, Ruijie; Chen, Min; Yang, Qing

    2013-01-01

    MiRNAs are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data, including eggplant (Solanum melongena L.). To identify miRNAs in eggplant and their response to Verticillium dahliae infection, a fungal pathogen for which clear understanding of infection mechanisms and effective cure methods are currently lacking, we deep-sequenced two small RNA (sRNA) libraries prepared from mock-infected and infected seedlings of eggplants. Specifically, 30,830,792 reads produced 7,716,328 unique miRNAs representing 99 known miRNA families that have been identified in other plant species. Two novel putative miRNAs were predicted with eggplant ESTs. The potential targets of the identified known and novel miRNAs were also predicted based on sequence homology search. It was observed that the length distribution of obtained sRNAs and the expression of 6 miRNA families were obviously different between the two libraries. These results provide a framework for further analysis of miRNAs and their role in regulating plant response to fungal infection and Verticillium wilt in particular.

  11. Detection of an abundant plant-based small RNA in consumers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mechanisms of delivery of plant small RNAs to consumers must be addressed in order to harness this technology to positively impact agbiotechnology. Two groups have used honeysuckle (Lonicera japonica) feeding regimes to detect a plant-based small RNA, termed MIR2911, in sera. Meanwhile, numerous gro...

  12. Silencing of natural transformation by an RNA chaperone and a multitarget small RNA.

    PubMed

    Attaiech, Laetitia; Boughammoura, Aïda; Brochier-Armanet, Céline; Allatif, Omran; Peillard-Fiorente, Flora; Edwards, Ross A; Omar, Ayat R; MacMillan, Andrew M; Glover, Mark; Charpentier, Xavier

    2016-08-01

    A highly conserved DNA uptake system allows many bacteria to actively import and integrate exogenous DNA. This process, called natural transformation, represents a major mechanism of horizontal gene transfer (HGT) involved in the acquisition of virulence and antibiotic resistance determinants. Despite evidence of HGT and the high level of conservation of the genes coding the DNA uptake system, most bacterial species appear non-transformable under laboratory conditions. In naturally transformable species, the DNA uptake system is only expressed when bacteria enter a physiological state called competence, which develops under specific conditions. Here, we investigated the mechanism that controls expression of the DNA uptake system in the human pathogen Legionella pneumophila We found that a repressor of this system displays a conserved ProQ/FinO domain and interacts with a newly characterized trans-acting sRNA, RocR. Together, they target mRNAs of the genes coding the DNA uptake system to control natural transformation. This RNA-based silencing represents a previously unknown regulatory means to control this major mechanism of HGT. Importantly, these findings also show that chromosome-encoded ProQ/FinO domain-containing proteins can assist trans-acting sRNAs and that this class of RNA chaperones could play key roles in post-transcriptional gene regulation throughout bacterial species. PMID:27432973

  13. In silico reconstruction of viral genomes from small RNAs improves virus-derived small interfering RNA profiling.

    PubMed

    Vodovar, Nicolas; Goic, Bertsy; Blanc, Hervé; Saleh, Maria-Carla

    2011-11-01

    RNA interference (RNAi) is the essential component of antiviral immunity in invertebrates and plants. One of the landmarks of the antiviral RNAi response is the production of virus-derived small interfering RNA (vsiRNA) from viral double-stranded RNA (dsRNA). vsiRNAs constitute a fragmented image of the viral genome sequence that results from Dicer cleavage. vsiRNA sequence profiling is used extensively as a surrogate to study the antiviral RNAi response by determining the nature of the viral dsRNA molecules exposed to and processed by the RNAi machinery. The accuracy of these profiles depends on the actual viral genome sequence used as a reference to align vsiRNA reads, and the interpretation of inaccurate profiles can be misleading. Using Flock house virus and Drosophila melanogaster as a model RNAi-competent organism, we show accurate reconstruction of full-length virus reference sequence from vsiRNAs and prediction of the structure of defective interfering particles (DIs). We developed a Perl script, named Paparazzi, that reconstitutes viral genomes through an iterative alignment/consensus call procedure using a related reference sequence as scaffold. As prevalent DI-derived reads introduce artifacts during reconstruction, Paparazzi eliminates DI-specific reads to improve the quality of the reconstructed genome. Paparazzi constitutes a promising alternative to Sanger sequencing in this context and an effective tool to study antiviral RNAi mechanisms by accurately quantifying vsiRNA along the replicating viral genome. We further discuss Paparazzi as a companion tool for virus discovery as it provides full-length genome sequences and corrects for potential artifacts of assembly. PMID:21880776

  14. In Silico Reconstruction of Viral Genomes from Small RNAs Improves Virus-Derived Small Interfering RNA Profiling ▿ † ‡

    PubMed Central

    Vodovar, Nicolas; Goic, Bertsy; Blanc, Hervé; Saleh, Maria-Carla

    2011-01-01

    RNA interference (RNAi) is the essential component of antiviral immunity in invertebrates and plants. One of the landmarks of the antiviral RNAi response is the production of virus-derived small interfering RNA (vsiRNA) from viral double-stranded RNA (dsRNA). vsiRNAs constitute a fragmented image of the viral genome sequence that results from Dicer cleavage. vsiRNA sequence profiling is used extensively as a surrogate to study the antiviral RNAi response by determining the nature of the viral dsRNA molecules exposed to and processed by the RNAi machinery. The accuracy of these profiles depends on the actual viral genome sequence used as a reference to align vsiRNA reads, and the interpretation of inaccurate profiles can be misleading. Using Flock house virus and Drosophila melanogaster as a model RNAi-competent organism, we show accurate reconstruction of full-length virus reference sequence from vsiRNAs and prediction of the structure of defective interfering particles (DIs). We developed a Perl script, named Paparazzi, that reconstitutes viral genomes through an iterative alignment/consensus call procedure using a related reference sequence as scaffold. As prevalent DI-derived reads introduce artifacts during reconstruction, Paparazzi eliminates DI-specific reads to improve the quality of the reconstructed genome. Paparazzi constitutes a promising alternative to Sanger sequencing in this context and an effective tool to study antiviral RNAi mechanisms by accurately quantifying vsiRNA along the replicating viral genome. We further discuss Paparazzi as a companion tool for virus discovery as it provides full-length genome sequences and corrects for potential artifacts of assembly. PMID:21880776

  15. Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors.

    PubMed

    Russo, Joseph; Harrington, Andrew W; Steiniger, Mindy

    2016-01-01

    Movement of transposons causes insertions, deletions, and chromosomal rearrangements potentially leading to premature lethality in Drosophila melanogaster. To repress these elements and combat genomic instability, eukaryotes have evolved several small RNA-mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. We show that most transposon families are transcribed in both the sense (S) and antisense (AS) direction in Dmel-2 cells. LTR retrotransposons Dm297, mdg1, and blood, and non-LTR retrotransposons juan and jockey transcripts, are generated from intraelement transcription start sites with canonical RNA polymerase II promoters. We also determined that retrotransposon antisense transcripts are less polyadenylated than sense. RNA-seq and small RNA-seq revealed that Dicer-2 RNA interference (RNAi) depletion causes a decrease in the number of esiRNAs mapping to retrotransposons and an increase in expression of both S and AS retrotransposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element-specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active.

  16. Antisense Transcription of Retrotransposons in Drosophila: An Origin of Endogenous Small Interfering RNA Precursors.

    PubMed

    Russo, Joseph; Harrington, Andrew W; Steiniger, Mindy

    2016-01-01

    Movement of transposons causes insertions, deletions, and chromosomal rearrangements potentially leading to premature lethality in Drosophila melanogaster. To repress these elements and combat genomic instability, eukaryotes have evolved several small RNA-mediated defense mechanisms. Specifically, in Drosophila somatic cells, endogenous small interfering (esi)RNAs suppress retrotransposon mobility. EsiRNAs are produced by Dicer-2 processing of double-stranded RNA precursors, yet the origins of these precursors are unknown. We show that most transposon families are transcribed in both the sense (S) and antisense (AS) direction in Dmel-2 cells. LTR retrotransposons Dm297, mdg1, and blood, and non-LTR retrotransposons juan and jockey transcripts, are generated from intraelement transcription start sites with canonical RNA polymerase II promoters. We also determined that retrotransposon antisense transcripts are less polyadenylated than sense. RNA-seq and small RNA-seq revealed that Dicer-2 RNA interference (RNAi) depletion causes a decrease in the number of esiRNAs mapping to retrotransposons and an increase in expression of both S and AS retrotransposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and processed by Dicer-2 into esiRNAs that silence both sense and antisense retrotransposon transcripts. Reduction of sense retrotransposon transcripts potentially lowers element-specific protein levels to prevent transposition. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active. PMID:26534950

  17. StarScan: a web server for scanning small RNA targets from degradome sequencing data

    PubMed Central

    Liu, Shun; Li, Jun-Hao; Wu, Jie; Zhou, Ke-Ren; Zhou, Hui; Yang, Jian-Hua; Qu, Liang-Hu

    2015-01-01

    Endogenous small non-coding RNAs (sRNAs), including microRNAs, PIWI-interacting RNAs and small interfering RNAs, play important gene regulatory roles in animals and plants by pairing to the protein-coding and non-coding transcripts. However, computationally assigning these various sRNAs to their regulatory target genes remains technically challenging. Recently, a high-throughput degradome sequencing method was applied to identify biologically relevant sRNA cleavage sites. In this study, an integrated web-based tool, StarScan (sRNA target Scan), was developed for scanning sRNA targets using degradome sequencing data from 20 species. Given a sRNA sequence from plants or animals, our web server performs an ultrafast and exhaustive search for potential sRNA–target interactions in annotated and unannotated genomic regions. The interactions between small RNAs and target transcripts were further evaluated using a novel tool, alignScore. A novel tool, degradomeBinomTest, was developed to quantify the abundance of degradome fragments located at the 9–11th nucleotide from the sRNA 5′ end. This is the first web server for discovering potential sRNA-mediated RNA cleavage events in plants and animals, which affords mechanistic insights into the regulatory roles of sRNAs. The StarScan web server is available at http://mirlab.sysu.edu.cn/starscan/. PMID:25990732

  18. A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions.

    PubMed

    Gerson-Gurwitz, Adina; Wang, Shaohe; Sathe, Shashank; Green, Rebecca; Yeo, Gene W; Oegema, Karen; Desai, Arshad

    2016-04-01

    Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here, we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by the density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to the emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition. PMID:27020753

  19. Comparative evaluation of different extraction and quantification methods for forensic RNA analysis.

    PubMed

    Grabmüller, Melanie; Madea, Burkhard; Courts, Cornelius

    2015-05-01

    Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material. In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol® Reagent, Invitrogen; NucleoSpin® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling. We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of 'RNA integrity number' (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates. By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin® miRNA Kit excelling at DNA co-extraction and RNA results, respectively. Overall, there was no 'best' method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand.

  20. Soybean ribulose bisphosphate carboxylase small subunit: Mechanisms and determinants of RNA turnover in higher plants

    SciTech Connect

    Meagher, R.B.

    1990-02-01

    The goals of examining the mechanisms and determinants of RNA turnover in higher plants remain the same. We will continue with two of the major approaches (1) in vivo chemical modification of RNA structure and (2) analysis of Rubisco SSU RNA structure and turnover in transgenic plants. We plan to reduce the amount of molecular physiology (studies of transcription and steady state levels) to a minimum and expand these efforts into the analysis of plant rebonucleases. We have also broadened our examination of light induced turnover of rubisco SSU RNA to include general RNA turnover. We plan to identify specific 3{prime}->5{prime} precessive ribonuclease by complementation of E. coli mutants. The activity of these novel RNases and their potential role in plant RNA turnover and processing will be characterized.

  1. Gene silencing in tick cell lines using small interfering or long double-stranded RNA.

    PubMed

    Barry, Gerald; Alberdi, Pilar; Schnettler, Esther; Weisheit, Sabine; Kohl, Alain; Fazakerley, John K; Bell-Sakyi, Lesley

    2013-03-01

    Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system.

  2. Optimized in vivo transfer of small interfering RNA targeting dermal tissue using in vivo surface electroporation.

    PubMed

    Broderick, Kate E; Chan, Amy; Lin, Feng; Shen, Xuefei; Kichaev, Gleb; Khan, Amir S; Aubin, Justin; Zimmermann, Tracy S; Sardesai, Niranjan Y

    2012-01-01

    Electroporation (EP) of mammalian tissue is a technique that has been used successfully in the clinic for the delivery of genetic-based vaccines in the form of DNA plasmids. There is great interest in platforms which efficiently deliver RNA molecules such as messenger RNA and small interfering RNA (siRNA) to mammalian tissue. However, the in vivo delivery of RNA enhanced by EP has not been extensively characterized. This paper details the optimization of electrical parameters for a novel low-voltage EP method to deliver oligonucleotides (both DNA and RNA) to dermal tissue in vivo. Initially, the electrical parameters were optimized for dermal delivery of plasmid DNA encoding green fluorescent protein (GFP) using this novel surface dermal EP device. While all investigated parameters resulted in visible transfection, voltage parameters in the 10 V range elicited the most robust signal. The parameters optimized for DNA, were then assessed for translation of successful electrotransfer of siRNA into dermal tissue. Robust tagged-siRNA transfection in skin was detected. We then assessed whether these parameters translated to successful transfer of siRNA resulting in gene knockdown in vivo. Using a reporter gene construct encoding GFP and tagged siRNA targeting the GFP message, we show simultaneous transfection of the siRNA to the skin via EP and the concomitant knockdown of the reporter gene signal. The siRNA delivery was accomplished with no evidence of injection site inflammation or local tissue damage. The minimally invasive low-voltage EP method is thus capable of efficiently delivering both DNA and RNA molecules to dermal tissue in a tolerable manner. PMID:23344722

  3. RNA-Seq of the nucleolus reveals abundant SNORD44-derived small RNAs.

    PubMed

    Bai, Baoyan; Yegnasubramanian, Srinivasan; Wheelan, Sarah J; Laiho, Marikki

    2014-01-01

    Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep) sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA) associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs had a unique size distribution consisting of 19-20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA). Sequences from 47 sdRNAs were identified, which mapped to both 5' and 3' ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha-mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs.

  4. A putative precursor for the small ribosomal RNA from mitochondria of Saccharomyces cerevisiae.

    PubMed Central

    Osinga, K A; Evers, R F; Van der Laan, J C; Tabak, H F

    1981-01-01

    We have characterized a putative precursor RNA (15.5S) for the 15S ribosomal RNA in mitochondria of Saccharomyces cerevisiae. Hybrids were formed with mitochondrial RNA and mtDNA fragments terminally labelled at restriction sites located within the gene coding for 15S ribosomal RNA and treated with S1 nuclease (Berk, A.J. and Sharp, J.A. (1977) 12, 721-732). Sites of resistant hybrids were measured by agarose gel electrophoresis and end points of RNAs determined. The 15.5S RNA is approximately 80 nucleotides longer than the 15S ribosomal RNA, with the extra sequences being located at the 5'-end. Both 15S ribosomal RNA and 15.5S RNA are fully localised within a 2000 base pair HapII fragment. This putative precursor and the mature 15S ribosomal RNA are also found in petite mutants which retain the 15S ribosomal RNA gene. The petite mutant with the smallest genetic complexity has its end point of deletion (junction) just outside the HapII site located in the 5' flank of the 15S ribosomal RNA genes as determined by S1 nuclease analysis. This leaves a DNA stretch approximately 300 base pairs long where an initiation signal for mitochondrial transcription may be present. Images PMID:6262728

  5. Small RNA-Based Antiviral Defense in the Phytopathogenic Fungus Colletotrichum higginsianum

    PubMed Central

    Carrington, James C.

    2016-01-01

    Even though the fungal kingdom contains more than 3 million species, little is known about the biological roles of RNA silencing in fungi. The Colletotrichum genus comprises fungal species that are pathogenic for a wide range of crop species worldwide. To investigate the role of RNA silencing in the ascomycete fungus Colletotrichum higginsianum, knock-out mutants affecting genes for three RNA-dependent RNA polymerase (RDR), two Dicer-like (DCL), and two Argonaute (AGO) proteins were generated by targeted gene replacement. No effects were observed on vegetative growth for any mutant strain when grown on complex or minimal media. However, Δdcl1, Δdcl1Δdcl2 double mutant, and Δago1 strains showed severe defects in conidiation and conidia morphology. Total RNA transcripts and small RNA populations were analyzed in parental and mutant strains. The greatest effects on both RNA populations was observed in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains, in which a previously uncharacterized dsRNA mycovirus [termed Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1)] was derepressed. Phylogenetic analyses clearly showed a close relationship between ChNRV1 and members of the segmented Partitiviridae family, despite the non-segmented nature of the genome. Immunoprecipitation of small RNAs associated with AGO1 showed abundant loading of 5’U-containing viral siRNA. C. higginsianum parental and Δdcl1 mutant strains cured of ChNRV1 revealed that the conidiation and spore morphology defects were primarily caused by ChNRV1. Based on these results, RNA silencing involving ChDCL1 and ChAGO1 in C. higginsianum is proposed to function as an antiviral mechanism. PMID:27253323

  6. Small RNA-Based Antiviral Defense in the Phytopathogenic Fungus Colletotrichum higginsianum.

    PubMed

    Campo, Sonia; Gilbert, Kerrigan B; Carrington, James C

    2016-06-01

    Even though the fungal kingdom contains more than 3 million species, little is known about the biological roles of RNA silencing in fungi. The Colletotrichum genus comprises fungal species that are pathogenic for a wide range of crop species worldwide. To investigate the role of RNA silencing in the ascomycete fungus Colletotrichum higginsianum, knock-out mutants affecting genes for three RNA-dependent RNA polymerase (RDR), two Dicer-like (DCL), and two Argonaute (AGO) proteins were generated by targeted gene replacement. No effects were observed on vegetative growth for any mutant strain when grown on complex or minimal media. However, Δdcl1, Δdcl1Δdcl2 double mutant, and Δago1 strains showed severe defects in conidiation and conidia morphology. Total RNA transcripts and small RNA populations were analyzed in parental and mutant strains. The greatest effects on both RNA populations was observed in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains, in which a previously uncharacterized dsRNA mycovirus [termed Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1)] was derepressed. Phylogenetic analyses clearly showed a close relationship between ChNRV1 and members of the segmented Partitiviridae family, despite the non-segmented nature of the genome. Immunoprecipitation of small RNAs associated with AGO1 showed abundant loading of 5'U-containing viral siRNA. C. higginsianum parental and Δdcl1 mutant strains cured of ChNRV1 revealed that the conidiation and spore morphology defects were primarily caused by ChNRV1. Based on these results, RNA silencing involving ChDCL1 and ChAGO1 in C. higginsianum is proposed to function as an antiviral mechanism. PMID:27253323

  7. Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases

    PubMed Central

    Krumm, Stefanie A.; Ndungu, J. Maina; Yoon, Jeong-Joong; Dochow, Melanie; Sun, Aiming; Natchus, Michael; Snyder, James P.; Plemper, Richard K.

    2011-01-01

    Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp) activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid adaptation to a pathogen

  8. Small RNA and transcriptome deep sequencing proffers insight into floral gene regulation in Rosa cultivars

    PubMed Central

    2012-01-01

    Background Roses (Rosa sp.), which belong to the family Rosaceae, are the most economically important ornamental plants—making up 30% of the floriculture market. However, given high demand for roses, rose breeding programs are limited in molecular resources which can greatly enhance and speed breeding efforts. A better understanding of important genes that contribute to important floral development and desired phenotypes will lead to improved rose cultivars. For this study, we analyzed rose miRNAs and the rose flower transcriptome in order to generate a database to expound upon current knowledge regarding regulation of important floral characteristics. A rose genetic database will enable comprehensive analysis of gene expression and regulation via miRNA among different Rosa cultivars. Results We produced more than 0.5 million reads from expressed sequences, totalling more than 110 million bp. From these, we generated 35,657, 31,434, 34,725, and 39,722 flower unigenes from Rosa hybrid: ‘Vital’, ‘Maroussia’, and ‘Sympathy’ and Rosa rugosa Thunb. , respectively. The unigenes were assigned functional annotations, domains, metabolic pathways, Gene Ontology (GO) terms, Plant Ontology (PO) terms, and MIPS Functional Catalogue (FunCat) terms. Rose flower transcripts were compared with genes from whole genome sequences of Rosaceae members (apple, strawberry, and peach) and grape. We also produced approximately 40 million small RNA reads from flower tissue for Rosa, representing 267 unique miRNA tags. Among identified miRNAs, 25 of them were novel and 242 of them were conserved miRNAs. Statistical analyses of miRNA profiles revealed both shared and species-specific miRNAs, which presumably effect flower development and phenotypes. Conclusions In this study, we constructed a Rose miRNA and transcriptome database, and we analyzed the miRNAs and transcriptome generated from the flower tissues of four Rosa cultivars. The database provides a comprehensive genetic

  9. The small 6C RNA of Corynebacterium glutamicum is involved in the SOS response.

    PubMed

    Pahlke, Jennifer; Dostálová, Hana; Holátko, Jiří; Degner, Ursula; Bott, Michael; Pátek, Miroslav; Polen, Tino

    2016-09-01

    The 6C RNA family is a class of small RNAs highly conserved in Actinobacteria, including the genera Mycobacterium, Streptomyces and Corynebacterium whose physiological function has not yet been elucidated. We found that strong transcription of the cgb_03605 gene, which encodes 6C RNA in C. glutamicum, was driven by the SigA- and SigB-dependent promoter Pcgb_03605. 6C RNA was detected at high level during exponential growth phase (180 to 240 molcules per cell) which even increased at the entry of the stationary phase. 6C RNA level did not decrease within 240 min after transcription had been stopped with rifampicin, which suggests high 6C RNA stability. The expression of cgb_03605 further increased approximately twofold in the presence of DNA-damaging mitomycin C (MMC) and nearly threefold in the absence of LexA. Deletion of the 6C RNA gene cgb_03605 resulted in a higher sensitivity of C. glutamicum toward MMC and UV radiation. These results indicate that 6C RNA is involved in the DNA damage response. Both 6C RNA level-dependent pausing of cell growth and branched cell morphology in response to MMC suggest that 6C RNA may also be involved in a control of cell division. PMID:27362471

  10. Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA.

    PubMed

    Hecke, Anneke; Brooks, Hilary; Meryet-Figuière, Matthieu; Minne, Stephanie; Konstantinides, Stavros; Hasenfuss, Gerd; Lebleu, Bernard; Schäfer, Katrin

    2006-05-01

    Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.

  11. Biogenesis of RNA polymerases II and III requires the conserved GPN small GTPases in Saccharomyces cerevisiae.

    PubMed

    Minaker, Sean W; Filiatrault, Megan C; Ben-Aroya, Shay; Hieter, Philip; Stirling, Peter C

    2013-03-01

    The GPN proteins are a poorly characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2, and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here we show that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1 and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition, and defects in RNA polymerase II nuclear localization. Importantly, we identify previously unrecognized RNA polymerase III localization defects in GPN2, GPN3, and IWR1 mutant backgrounds but find no localization defects of unrelated nuclear proteins or of RNA polymerase I. Previously, it was unclear whether the GPN proteins and Iwr1 had overlapping function in RNA polymerase II assembly or import. In this study, we show that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1, suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common, and likely essential, function in RNA polymerase assembly and transport.

  12. Biogenesis of RNA Polymerases II and III Requires the Conserved GPN Small GTPases in Saccharomyces cerevisiae

    PubMed Central

    Minaker, Sean W.; Filiatrault, Megan C.; Ben-Aroya, Shay; Hieter, Philip; Stirling, Peter C.

    2013-01-01

    The GPN proteins are a poorly characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2, and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here we show that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1 and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition, and defects in RNA polymerase II nuclear localization. Importantly, we identify previously unrecognized RNA polymerase III localization defects in GPN2, GPN3, and IWR1 mutant backgrounds but find no localization defects of unrelated nuclear proteins or of RNA polymerase I. Previously, it was unclear whether the GPN proteins and Iwr1 had overlapping function in RNA polymerase II assembly or import. In this study, we show that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1, suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common, and likely essential, function in RNA polymerase assembly and transport. PMID:23267056

  13. The small 6C RNA of Corynebacterium glutamicum is involved in the SOS response.

    PubMed

    Pahlke, Jennifer; Dostálová, Hana; Holátko, Jiří; Degner, Ursula; Bott, Michael; Pátek, Miroslav; Polen, Tino

    2016-09-01

    The 6C RNA family is a class of small RNAs highly conserved in Actinobacteria, including the genera Mycobacterium, Streptomyces and Corynebacterium whose physiological function has not yet been elucidated. We found that strong transcription of the cgb_03605 gene, which encodes 6C RNA in C. glutamicum, was driven by the SigA- and SigB-dependent promoter Pcgb_03605. 6C RNA was detected at high level during exponential growth phase (180 to 240 molcules per cell) which even increased at the entry of the stationary phase. 6C RNA level did not decrease within 240 min after transcription had been stopped with rifampicin, which suggests high 6C RNA stability. The expression of cgb_03605 further increased approximately twofold in the presence of DNA-damaging mitomycin C (MMC) and nearly threefold in the absence of LexA. Deletion of the 6C RNA gene cgb_03605 resulted in a higher sensitivity of C. glutamicum toward MMC and UV radiation. These results indicate that 6C RNA is involved in the DNA damage response. Both 6C RNA level-dependent pausing of cell growth and branched cell morphology in response to MMC suggest that 6C RNA may also be involved in a control of cell division.

  14. RNA structural analysis by evolving SHAPE chemistry.

    PubMed

    Spitale, Robert C; Flynn, Ryan A; Torre, Eduardo A; Kool, Eric T; Chang, Howard Y

    2014-01-01

    RNA is central to the flow of biological information. From transcription to splicing, RNA localization, translation, and decay, RNA is intimately involved in regulating every step of the gene expression program, and is thus essential for health and understanding disease. RNA has the unique ability to base-pair with itself and other nucleic acids to form complex structures. Hence the information content in RNA is not simply its linear sequence of bases, but is also encoded in complex folding of RNA molecules. A general chemical functionality that all RNAs have is a 2'-hydroxyl group in the ribose ring, and the reactivity of the 2'-hydroxyl in RNA is gated by local nucleotide flexibility. In other words, the 2'-hydroxyl is reactive at single-stranded and conformationally flexible positions but is unreactive at nucleotides constrained by base-pairing. Recent efforts have been focused on developing reagents that modify RNA as a function of RNA 2' hydroxyl group reactivity. Such RNA structure probing techniques can be read out by primer extension in experiments termed RNA SHAPE (selective 2'- hydroxyl acylation and primer extension). Herein, we describe the efforts devoted to the design and utilization of SHAPE probes for characterizing RNA structure. We also describe current technological advances that are being applied to utilize SHAPE chemistry with deep sequencing to probe many RNAs in parallel. The merging of chemistry with genomics is sure to open the door to genome-wide exploration of RNA structure and function.

  15. Small indels induced by CRISPR/Cas9 in the 5' region of microRNA lead to its depletion and Drosha processing retardance.

    PubMed

    Jiang, Qian; Meng, Xing; Meng, Lingwei; Chang, Nannan; Xiong, Jingwei; Cao, Huiqing; Liang, Zicai

    2014-01-01

    MicroRNA knockout by genome editing technologies is promising. In order to extend the application of the technology and to investigate the function of a specific miRNA, we used CRISPR/Cas9 to deplete human miR-93 from a cluster by targeting its 5' region in HeLa cells. Various small indels were induced in the targeted region containing the Drosha processing site and seed sequences. Interestingly, we found that even a single nucleotide deletion led to complete knockout of the target miRNA with high specificity. Functional knockout was confirmed by phenotype analysis. Furthermore, de novo microRNAs were not found by RNA-seq. Nevertheless, expression of the pri-microRNAs was increased. When combined with structural analysis, the data indicated that biogenesis was impaired. Altogether, we showed that small indels in the 5' region of a microRNA result in sequence depletion as well as Drosha processing retard.

  16. High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.)

    PubMed Central

    Donaire, Livia; Pedrola, Laia; de la Rosa, Raúl; Llave, César

    2011-01-01

    Small RNAs (sRNAs) of 20 to 25 nucleotides (nt) in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the sRNA component of more than 40 plant species. Here, we used deep sequencing and molecular methods to report the first inventory of sRNAs in olive (Olea europaea L.). sRNA libraries prepared from juvenile and adult shoots revealed that the 24-nt class dominates the sRNA transcriptome and atypically accumulates to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. A total of 18 known miRNA families were identified in the libraries. Also, 5 other sRNAs derived from potential hairpin-like precursors remain as plausible miRNA candidates. RNA blots confirmed miRNA expression and suggested tissue- and/or developmental-specific expression patterns. Target mRNAs of conserved miRNAs were computationally predicted among the olive cDNA collection and experimentally validated through endonucleolytic cleavage assays. Finally, we use expression data to uncover genetic components of the miR156, miR172 and miR390/TAS3-derived trans-acting small interfering RNA (tasiRNA) regulatory nodes, suggesting that these interactive networks controlling developmental transitions are fully operational in olive. PMID:22140484

  17. Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911

    PubMed Central

    Yang, Jian; Hotz, Tremearne; Broadnax, LaCassidy; Yarmarkovich, Mark; Elbaz-Younes, Ismail; Hirschi, Kendal D.

    2016-01-01

    Inconsistent detection of plant-based dietary small RNAs in circulation has thwarted the use of dietary RNA therapeutics. Here we demonstrate mice consuming diets rich in vegetables displayed enhanced serum levels of the plant specific small RNA MIR2911. Differential centrifugation, size-exclusion chromatography, and proteinase K treatment of plant extracts suggest this RNA resides within a proteinase K-sensitive complex. Plant derived MIR2911 was more bioavailable than the synthetic RNA. Furthermore, MIR2911 exhibited unusual digestive stability compared with other synthetic plant microRNAs. The characteristics of circulating MIR2911 were also unusual as it was not associated with exosomes and fractionated as a soluble complex that was insensitive to proteinase K treatment, consistent with MIR2911 being stabilized by modifications conferred by the host. These results indicate that intrinsic stability and plant-based modifications orchestrate consumer uptake of this anomalous plant based small RNA and invite revisiting plant-based microRNA therapeutic approaches. PMID:27251858

  18. Creation of transgenic rice plants producing small interfering RNA of Rice tungro spherical virus.

    PubMed

    Le, Dung Tien; Chu, Ha Duc; Sasaya, Takahide

    2015-01-01

    Rice tungro spherical virus (RTSV), also known as Rice waika virus, does not cause visible symptoms in infected rice plants. However, the virus plays a critical role in spreading Rice tungro bacilliform virus (RTBV), which is the major cause of severe symptoms of rice tungro disease. Recent studies showed that RNA interference (RNAi) can be used to develop virus-resistance transgenic rice plants. In this report, we presented simple procedures and protocols needed for the creation of transgenic rice plants capable of producing small interfering RNA specific against RTSV sequences. Notably, our study showed that 60 out of 64 individual hygromycin-resistant lines (putative transgenic lines) obtained through transformation carried transgenes designed for producing hairpin double-stranded RNA. Northern blot analyses revealed the presence of small interfering RNA of 21- to 24-mer in 46 out of 56 confirmed transgenic lines. Taken together, our study indicated that transgenic rice plants carrying an inverted repeat of 500-bp fragments encoding various proteins of RTSV can produce small interfering RNA from the hairpin RNA transcribed from that transgene. In light of recent studies with other viruses, it is possible that some of these transgenic rice lines might be resistant to RTSV.

  19. A molecular-beacon-based screen for small molecule inhibitors of miRNA maturation.

    PubMed

    Bose, Debojit; Jayaraj, Gopal Gunanathan; Kumar, Santosh; Maiti, Souvik

    2013-05-17

    miRNAs are small non-coding RNAs that regulate about 60% of mammalian genes by modulating their transcript levels. Network scale studies of miRNA-mediated regulatory circuits demonstrate the central importance of this class of small RNA in the maintenance of biological robustness. More recently, several reports have described the deregulation of numerous miRNA to be causally associated with many diseases, including cancer. These studies have highlighted the potential for development of therapeutic modalities against miRNA. Previous screening protocols, for small molecules targeting miRNA function, are either costly or technically too complex to be applied in a high-throughput manner in standard chemical laboratories. We describe a simple in vitro screening method using a DNA-based molecular beacon that overcomes the limitations associated with earlier screens. We used this method to identify inhibitors of miR-27a function from a library of 14 aminoglycosides as a pilot study. Inhibitory molecules identified were further scrutinized to identify the validity of screen. With this proof of concept we illustrate the utility of a scalable molecular-beacon-based screening strategy for miRNA inhibitors.

  20. Creation of transgenic rice plants producing small interfering RNA of Rice tungro spherical virus

    PubMed Central

    Le, Dung Tien; Chu, Ha Duc; Sasaya, Takahide

    2015-01-01

    ABSTRACT Rice tungro spherical virus (RTSV), also known as Rice waika virus, does not cause visible symptoms in infected rice plants. However, the virus plays a critical role in spreading Rice tungro bacilliform virus (RTBV), which is the major cause of severe symptoms of rice tungro disease. Recent studies showed that RNA interference (RNAi) can be used to develop virus-resistance transgenic rice plants. In this report, we presented simple procedures and protocols needed for the creation of transgenic rice plants capable of producing small interfering RNA specific against RTSV sequences. Notably, our study showed that 60 out of 64 individual hygromycin-resistant lines (putative transgenic lines) obtained through transformation carried transgenes designed for producing hairpin double-stranded RNA. Northern blot analyses revealed the presence of small interfering RNA of 21- to 24-mer in 46 out of 56 confirmed transgenic lines. Taken together, our study indicated that transgenic rice plants carrying an inverted repeat of 500-bp fragments encoding various proteins of RTSV can produce small interfering RNA from the hairpin RNA transcribed from that transgene. In light of recent studies with other viruses, it is possible that some of these transgenic rice lines might be resistant to RTSV. PMID:25984767

  1. Nanopore-Based Conformational Analysis of a Viral RNA Drug Target

    PubMed Central

    Stoloff, Daniel H.; Rynearson, Kevin D.; Hermann, Thomas; Wanunu, Meni

    2016-01-01

    Nanopores are single-molecule sensors that show exceptional promise as a biomolecular analysis tool by enabling label-free detection of small amounts of sample. In this paper, we demonstrate that nanopores are capable of detecting the conformation of an antiviral RNA drug target. The hepatitis C virus uses an internal ribosome entry site (IRES) motif in order to initiate translation by docking to ribosomes in its host cell. The IRES is therefore a viable and important drug target. Drug-induced changes to the conformation of the HCV IRES motif, from a bent to a straight conformation, have been shown to inhibit HCV replication. However, there is presently no straightforward method to analyze the effect of candidate small-molecule drugs on the RNA conformation. In this paper, we show that RNA translocation dynamics through a 3 nm diameter nanopore is conformation-sensitive by demonstrating a difference in transport times between bent and straight conformations of a short viral RNA motif. Detection is possible because bent RNA is stalled in the 3 nm pore, resulting in longer molecular dwell times than straight RNA. Control experiments show that binding of a weaker drug does not produce a conformational change, as consistent with independent fluorescence measurements. Nanopore measurements of RNA conformation can thus be useful for probing the structure of various RNA motifs, as well as structural changes to the RNA upon small-molecule binding. PMID:24861167

  2. Exploratory factor analysis for small samples.

    PubMed

    Jung, Sunho; Lee, Soonmook

    2011-09-01

    Traditionally, two distinct approaches have been employed for exploratory factor analysis: maximum likelihood factor analysis and principal component analysis. A third alternative, called regularized exploratory factor analysis, was introduced recently in the psychometric literature. Small sample size is an important issue that has received considerable discussion in the factor analysis literature. However, little is known about the differential performance of these three approaches to exploratory factor analysis in a small sample size scenario. A simulation study and an empirical example demonstrate that regularized exploratory factor analysis may be recommended over the two traditional approaches, particularly when sample sizes are small (below 50) and the sample covariance matrix is near singular.

  3. Characterization and structure of the mitochondrial small rRNA gene of the entomopathogenic fungus Beauveria bassiana.

    PubMed

    Hegedus, D D; Pfeifer, T A; Mulyk, D S; Khachatourians, G G

    1998-06-01

    The entire mitochondrial (mt) small ribosomal RNA (srRNA) gene from the entomopathogenic fungus Beauveria bassiana was sequenced. Alignment of the sequence to those of other filamentous fungi revealed gross length differences in their respective products. Construction of a secondary structural model showed that these differences were restricted to known variable srRNA subdomains. Several features were identified that were common only to the hyphomycetous fungi examined. Phylogenetic analysis indicated that the anamorph B. bassiana was more closely related to the pyrenomycete than to the plectomycete ascomycetous fungi. Based on our previous comparison of mt gene arrangement in filamentous fungi, this was unexpected. The possibility that the smaller mt genomes reflect the ancestral arrangement of genes is discussed.

  4. Comparative analysis of RNA silencing suppression activities between viral suppressors and an endogenous plant RNA-dependent RNA polymerase.

    PubMed

    Yoon, Ju-Yeon; Han, Kyoung-Sik; Park, Han-Yong; Choi, Seung-Kook

    2012-06-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in eukaryotes, including higher plants. To counteract this, several plant viruses express silencing suppressors that inhibit RNA silencing in host plants. Here, we show that both 2b protein from peanut stunt virus (PSV) and a hairpin construct (designated hp-RDR6) that silences endogenous RNA-dependent RNA polymerase 6 (RDR6) strongly suppress RNA silencing. The Agrobacterium infiltration system was used to demonstrate that both PSV 2b and hp-RDR6 suppressed local RNA silencing as strongly as helper component (HC-Pro) from potato virus Y (PVY) and P19 from tomato bush stunt virus (TBSV). The 2b protein from PSV eliminated the small-interfering RNAs (siRNAs) associated with RNA silencing and prevented systemic silencing, similar to 2b protein from cucumber mosaic virus (CMV). On the other hand, hp-RDR6 suppressed RNA silencing by inhibiting the generation of secondary siRNAs. The small coat protein (SCP) of squash mosaic virus (SqMV) also displayed weak suppression activity of RNA silencing. Agrobacterium-mediated gene transfer was used to investigate whether viral silencing suppressors or hp-RDR6 enhanced accumulations of green fluorescence protein (GFP) and β-glucuronidase (GUS) as markers of expression in leaf tissues of Nicotina benthamiana. Expression of both GFP and GUS was significantly enhanced in the presence of PSV 2b or CMV 2b, compared to no suppression or the weak SqMV SCP suppressor. Co-expression with hp-RDR6 also significantly increased the expression of GFP and GUS to levels similar to those induced by PVY HC-Pro and TBSV P19.

  5. 7SK small nuclear RNA inhibits cancer cell proliferation through apoptosis induction.

    PubMed

    Keramati, Farid; Seyedjafari, Ehsan; Fallah, Parviz; Soleimani, Masoud; Ghanbarian, Hossein

    2015-04-01

    7SK small nuclear RNA (snRNA) is a 331-333-bp non-coding RNA, which recruits HEXIM 1/2 protein to inhibit positive elongation factor b (P-TEFb) activity. P-TEFb is an essential factor in alleviating promoter-proximal paused RNA polymerase II (Pol II) and initiating the productive elongation phase of gene transcription. Without this protein, Pol II will remain in its hypophosphorylated state, and no transcription occurs. In this study, we inhibited P-TEFb activity by over-expressing 7SK snRNA in human embryonic kidney (HEK) 293T cancer cell line. This inhibition led to a significant decrease in cell viability, which can be due to the transcription inhibition. Moreover, 7SK snRNA over-expression promoted apoptosis in cancerous cells. Our results suggest 7SK snRNA as a potential endogenous anti-cancer agent, and to the best of our knowledge, this is the first study that uses a long non-coding RNA's over-expression against cancer cell growth and proliferation.

  6. Natural-abundance stable carbon isotopes of small-subunit ribosomal RNA (SSU rRNA) from Guaymas Basin (Mexico)

    NASA Astrophysics Data System (ADS)

    MacGregor, B. J.; Mendlovitz, H.; Albert, D.; Teske, A. P.

    2012-12-01

    Small-subunit ribosomal RNA (SSU rRNA) is a phylogenetically informative molecule found in all species. Because it is poorly preserved in most environments, it is a useful marker for active microbial populations. We are using the natural-abundance stable carbon isotopic composition of specific microbial groups to help identify the carbon substrates contributing to microbial biomass in a variety of marine environments. At Guaymas Basin, hydrothermal fluids interact with abundant sedimentary organic carbon to produce natural gas and petroleum. Where this reaches the sediment surface, it can support dense patches of seafloor life, including Beggiatoa mats. We report here on the stable carbon isotopic composition of SSU rRNA from a Beggiatoa mat transect, a cold background site, a warm site with high oil concentration, and a second Beggiatoa mat. The central part of the transect mat overlay the steepest temperature gradient, and was visually dominated by orange Beggiatoa. This was fringed by white Beggiatoa mat and bare, but still warm, sediment. Methane concentrations were saturating beneath the orange and white mats and at the oily site, lower beneath bare sediment, and below detection at the background site. Our initial hypotheses were that rRNA isotopic composition would be strongly influenced by methane supply, and that archaeal rRNA might be lighter than bacterial due to contributions from methanogens and anaerobic methane oxidizers. We used biotin-labeled oligonucleotides to capture Bacterial and Archaeal SSU rRNA for isotopic determination. Background-site rRNA was isotopically heaviest, and bacterial RNA from below 2 cm at the oily site was lightest, consistent with control by methane. Within the transect mat, however, the pattern was more complicated; at some sediment depths, rRNA from the mat periphery was isotopically lightest. Part of this may be due to the spatially and temporally variable paths followed by hydrothermal fluid, which can include horizontal

  7. Human metapneumovirus infection induces significant changes in small noncoding RNA expression in airway epithelial cells.

    PubMed

    Deng, Junfang; Ptashkin, Ryan N; Wang, Qingrong; Liu, Guangliang; Zhang, Guanping; Lee, Inhan; Lee, Yong Sun; Bao, Xiaoyong

    2014-05-20

    Small noncoding RNAs (sncRNAs), such as microRNAs (miRNA), virus-derived sncRNAs, and more recently identified tRNA-derived RNA fragments, are critical to posttranscriptional control of genes. Upon viral infection, host cells alter their sncRNA expression as a defense mechanism, while viruses can circumvent host defenses and promote their own propagation by affecting host cellular sncRNA expression or by expressing viral sncRNAs. Therefore, characterizing sncRNA profiles in response to viral infection is an important tool for understanding host-virus interaction, and for antiviral strategy development. Human metapneumovirus (hMPV), a recently identified pathogen, is a major cause of lower respiratory tract infections in infants and children. To investigate whether sncRNAs play a role in hMPV infection, we analyzed the changes in sncRNA profiles of airway epithelial cells in response to hMPV infection using ultrahigh-throughput sequencing. Of the cloned sncRNAs, miRNA was dominant in A549 cells, with the percentage of miRNA increasing in a time-dependent manner after the infection. In addition, several hMPV-derived sncRNAs and corresponding ribonucleases for their biogenesis were identified. hMPV M2-2 protein was revealed to be a key viral protein regulating miRNA expression. In summary, this study revealed several novel aspects of hMPV-mediated sncRNA expression, providing a new perspective on hMPV-host interactions.

  8. Intact MicroRNA Analysis Using High Resolution Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Kullolli, Majlinda; Knouf, Emily; Arampatzidou, Maria; Tewari, Muneesh; Pitteri, Sharon J.

    2014-01-01

    MicroRNAs (miRNAs) are small single-stranded non-coding RNAs that post-transcriptionally regulate gene expression, and play key roles in the regulation of a variety of cellular processes and in disease. New tools to analyze miRNAs will add understanding of the physiological origins and biological functions of this class of molecules. In this study, we investigate the utility of high resolution mass spectrometry for the analysis of miRNAs through proof-of-concept experiments. We demonstrate the ability of mass spectrometry to resolve and separate miRNAs and corresponding 3' variants in mixtures. The mass accuracy of the monoisotopic deprotonated peaks from various miRNAs is in the low ppm range. We compare fragmentation of miRNA by collision-induced dissociation (CID) and by higher-energy collisional dissociation (HCD) which yields similar sequence coverage from both methods but additional fragmentation by HCD versus CID. We measure the linear dynamic range, limit of detection, and limit of quantitation of miRNA loaded onto a C18 column. Lastly, we explore the use of data-dependent acquisition of MS/MS spectra of miRNA during online LC-MS and demonstrate that multiple charge states can be fragmented, yielding nearly full sequence coverage of miRNA on a chromatographic time scale. We conclude that high resolution mass spectrometry allows the separation and measurement of miRNAs in mixtures and a standard LC-MS setup can be adapted for online analysis of these molecules.

  9. Detection of an Abundant Plant-Based Small RNA in Healthy Consumers

    PubMed Central

    Yang, Jian; Farmer, Lisa M.; Agyekum, Abia A. A.; Elbaz-Younes, Ismail; Hirschi, Kendal D.

    2015-01-01

    The mechanisms of delivery of plant small RNAs to consumers must be investigated in order to harness this technology to positively impact biotechnology. Two groups have used honeysuckle (Lonicera japonica) feeding regimes to detect a plant-based small RNA, termed MIR2911, in sera. Meanwhile, numerous groups have failed to detect dietary plant-based small RNAs in consumers. Here we catalog levels of MIR2911 in different herbs, and suggest that in particular herb MIR2911 levels are elevated. Feeding these different herb-based diets to mice, we found MIR2911 levels in the sera and urine were associated with dietary intake levels. Abundance was not the sole determinate of apparent RNA bioavailability, as gavage-feeding large-doses of synthetic MIR2911 permitted only small transient increases in serum levels. Dietary MIR2911 were not modified in circulation by association with the host’s RNA-induced silencing complex, as the RNA did not co-immunoprecipitate with AGO2. The stability of dietary MIR2911 in circulation differed from synthesized small RNAs, as tail vein administration of various synthetic plant-based small RNAs resulted in rapid clearance. However, synthetic MIR2911 appeared to be more stable than the other plant miRNAs tested. Notably, this uptake of dietary MIR2911 was not related to perturbations in the host’s microbiome or gut permeability. We suggest dietary uptake of MIR2911 is commonplace in healthy consumers, and reproducible detection of plant-based small RNAs in consumers depends on dietary abundance, RNA stability and digestion from within the food-matrix. PMID:26335106

  10. Detection of an Abundant Plant-Based Small RNA in Healthy Consumers.

    PubMed

    Yang, Jian; Farmer, Lisa M; Agyekum, Abia A A; Elbaz-Younes, Ismail; Hirschi, Kendal D

    2015-01-01

    The mechanisms of delivery of plant small RNAs to consumers must be investigated in order to harness this technology to positively impact biotechnology. Two groups have used honeysuckle (Lonicera japonica) feeding regimes to detect a plant-based small RNA, termed MIR2911, in sera. Meanwhile, numerous groups have failed to detect dietary plant-based small RNAs in consumers. Here we catalog levels of MIR2911 in different herbs, and suggest that in particular herb MIR2911 levels are elevated. Feeding these different herb-based diets to mice, we found MIR2911 levels in the sera and urine were associated with dietary intake levels. Abundance was not the sole determinate of apparent RNA bioavailability, as gavage-feeding large-doses of synthetic MIR2911 permitted only small transient increases in serum levels. Dietary MIR2911 were not modified in circulation by association with the host's RNA-induced silencing complex, as the RNA did not co-immunoprecipitate with AGO2. The stability of dietary MIR2911 in circulation differed from synthesized small RNAs, as tail vein administration of various synthetic plant-based small RNAs resulted in rapid clearance. However, synthetic MIR2911 appeared to be more stable than the other plant miRNAs tested. Notably, this uptake of dietary MIR2911 was not related to perturbations in the host's microbiome or gut permeability. We suggest dietary uptake of MIR2911 is commonplace in healthy consumers, and reproducible detection of plant-based small RNAs in consumers depends on dietary abundance, RNA stability and digestion from within the food-matrix. PMID:26335106

  11. Strand-asymmetric endogenous Tetrahymena small RNA production requires a previously uncharacterized uridylyltransferase protein partner

    PubMed Central

    Talsky, Kristin Benjamin; Collins, Kathleen

    2012-01-01

    Many eukaryotes initiate pathways of Argonaute-bound small RNA (sRNA) production with a step that specifically targets sets of aberrant and/or otherwise deleterious transcripts for recognition by an RNA-dependent RNA polymerase complex (RDRC). The biogenesis of 23- to 24-nt sRNAs in growing Tetrahymena occurs by physical and functional coupling of the growth-expressed Dicer, Dcr2, with one of three RDRCs each containing the single genome-encoded RNA-dependent RNA polymerase, Rdr1. Tetrahymena RDRCs contain an active uridylyltransferase, either Rdn1 or Rdn2, and Rdn1 RDRCs also contain the Rdf1 and Rdf2 proteins. Although Rdn2 is nonessential and RDRC-specific, Rdn1 is genetically essential and interacts with a non-RDRC protein of 124 kDa. Here we characterize this 124-kDa protein, designated RNA silencing protein 1 (Rsp1), using endogenous locus tagging, affinity purification, and functional assays, as well as gene-knockout studies. We find that Rsp1 associates with Rdn1-Rdf1 or Rdn1-Rdf2 subcomplexes as an alternative to Rdr1, creating Rsp1 complexes (RSPCs) that are physically separate from RDRCs. The uridylyltransferase activity of Rdn1 is greatly reduced in RSPCs compared with RDRCs, suggesting enzyme regulation by the alternative partners. Surprisingly, despite the loss of all known RDRC-generated classes of endogenous sRNAs, RSP1 gene knockout was tolerated in growing cells. A minority class of Dcr2-dependent sRNAs persists in cells lacking Rsp1 with increased size heterogeneity. These findings bring new insights about the essential and nonessential functions of RNA silencing in Tetrahymena, about mechanisms of endogenous small interfering RNA production, and about the roles of cellular uridylyltransferases. PMID:22706992

  12. Modified Method of rRNA Structure Analysis Reveals Novel Characteristics of Box C/D RNA Analogues.

    PubMed

    Filippova, J A; Stepanov, G A; Semenov, D V; Koval, O A; Kuligina, E V; Rabinov, I V; Richter, V A

    2015-01-01

    Ribosomal RNA (rRNA) maturation is a complex process that involves chemical modifications of the bases or sugar residues of specific nucleotides. One of the most abundant types of rRNA modifications, ribose 2'-O-methylation, is guided by ribonucleoprotein complexes containing small nucleolar box C/D RNAs. Since the majority of 2'-O-methylated nucleotides are located in the most conserved regions of rRNA that comprise functionally important centers of the ribosome, an alteration in a 2'-O-methylation profile can affect ribosome assembly and function. One of the key approaches for localization of 2'-O-methylated nucleotides in long RNAs is a method based on the termination of reverse transcription. The current study presents an adaptation of this method for the use of fluorescently labeled primers and analysis of termination products by capillary gel electrophoresis on an automated genetic analyzer. The developed approach allowed us to analyze the influence of the synthetic analogues of box C/D RNAs on post-transcriptional modifications of human 28S rRNA in MCF-7 cells. It has been established that the transfection of MCF-7 cells with a box C/D RNA analogue leads to an enhanced modification level of certain native sites of 2'-O-methylation in the target rRNA. The observed effect of synthetic RNAs on the 2'-O-methylation of rRNA in human cells demonstrates a path towards targeted regulation of rRNA post-transcriptional maturation. The described approach can be applied in the development of novel diagnostic methods for detecting diseases in humans. PMID:26085946

  13. Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism.

    PubMed

    van der Meulen, Sjoerd B; de Jong, Anne; Kok, Jan

    2016-01-01

    RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species. PMID:26950529

  14. Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism

    PubMed Central

    van der Meulen, Sjoerd B.; de Jong, Anne; Kok, Jan

    2016-01-01

    ABSTRACT RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species. PMID:26950529

  15. Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation

    PubMed Central

    Zhang, Guoqiang; Estève, Pierre-Olivier; Chin, Hang Gyeong; Terragni, Jolyon; Dai, Nan; Corrêa, Ivan R.; Pradhan, Sriharsa

    2015-01-01

    Mammalian cells contain copious amounts of RNA including both coding and noncoding RNA (ncRNA). Generally the ncRNAs function to regulate gene expression at the transcriptional and post-transcriptional level. Among ncRNA, the long ncRNA and small ncRNA can affect histone modification, DNA methylation targeting and gene silencing. Here we show that endogenous DNA methyltransferase 1 (DNMT1) co-purifies with inhibitory ncRNAs. MicroRNAs (miRNAs) bind directly to DNMT1 with high affinity. The binding of miRNAs, such as miR-155-5p, leads to inhibition of DNMT1 enzyme activity. Exogenous miR-155-5p in cells induces aberrant DNA methylation of the genome, resulting in hypomethylation of low to moderately methylated regions. And small shift of hypermethylation of previously hypomethylated region was also observed. Furthermore, hypomethylation led to activation of genes. Based on these observations, overexpression of miR-155-5p resulted in aberrant DNA methylation by inhibiting DNMT1 activity, resulting in altered gene expression. PMID:25990724

  16. The Crystal Structure and Small-Angle X-Ray Analysis of CsdL/TcdA Reveal a New tRNA Binding Motif in the MoeB/E1 Superfamily

    PubMed Central

    López-Estepa, Miguel; Ardá, Ana; Savko, Martin; Round, Adam; Shepard, William E.; Bruix, Marta; Coll, Miquel; Fernández, Francisco J.; Jiménez-Barbero, Jesús; Vega, M. Cristina

    2015-01-01

    Cyclic N6-threonylcarbamoyladenosine (‘cyclic t6A’, ct6A) is a non-thiolated hypermodification found in transfer RNAs (tRNAs) in bacteria, protists, fungi and plants. In bacteria and yeast cells ct6A has been shown to enhance translation fidelity and efficiency of ANN codons by improving the faithful discrimination of aminoacylated tRNAs by the ribosome. To further the understanding of ct6A biology we have determined the high-resolution crystal structures of CsdL/TcdA in complex with AMP and ATP, an E1-like activating enzyme from Escherichia coli, which catalyzes the ATP-dependent dehydration of t6A to form ct6A. CsdL/TcdA is a dimer whose structural integrity and dimer interface depend critically on strongly bound K+ and Na+ cations. By using biochemical assays and small-angle X-ray scattering we show that CsdL/TcdA can associate with tRNA with a 1:1 stoichiometry and with the proper position and orientation for the cyclization of t6A. Furthermore, we show by nuclear magnetic resonance that CsdL/TcdA engages in transient interactions with CsdA and CsdE, which, in the latter case, involve catalytically important residues. These short-lived interactions may underpin the precise channeling of sulfur atoms from cysteine to CsdL/TcdA as previously characterized. In summary, the combination of structural, biophysical and biochemical methods applied to CsdL/TcdA has afforded a more thorough understanding of how the structure of this E1-like enzyme has been fine tuned to accomplish ct6A synthesis on tRNAs while providing support for the notion that CsdA and CsdE are able to functionally interact with CsdL/TcdA. PMID:25897750

  17. The crystal structure and small-angle X-ray analysis of CsdL/TcdA reveal a new tRNA binding motif in the MoeB/E1 superfamily.

    PubMed

    López-Estepa, Miguel; Ardá, Ana; Savko, Martin; Round, Adam; Shepard, William E; Bruix, Marta; Coll, Miquel; Fernández, Francisco J; Jiménez-Barbero, Jesús; Vega, M Cristina

    2015-01-01

    Cyclic N6-threonylcarbamoyladenosine ('cyclic t6A', ct(6)A) is a non-thiolated hypermodification found in transfer RNAs (tRNAs) in bacteria, protists, fungi and plants. In bacteria and yeast cells ct(6)A has been shown to enhance translation fidelity and efficiency of ANN codons by improving the faithful discrimination of aminoacylated tRNAs by the ribosome. To further the understanding of ct(6)A biology we have determined the high-resolution crystal structures of CsdL/TcdA in complex with AMP and ATP, an E1-like activating enzyme from Escherichia coli, which catalyzes the ATP-dependent dehydration of t6A to form ct(6)A. CsdL/TcdA is a dimer whose structural integrity and dimer interface depend critically on strongly bound K+ and Na+ cations. By using biochemical assays and small-angle X-ray scattering we show that CsdL/TcdA can associate with tRNA with a 1:1 stoichiometry and with the proper position and orientation for the cyclization of t6A. Furthermore, we show by nuclear magnetic resonance that CsdL/TcdA engages in transient interactions with CsdA and CsdE, which, in the latter case, involve catalytically important residues. These short-lived interactions may underpin the precise channeling of sulfur atoms from cysteine to CsdL/TcdA as previously characterized. In summary, the combination of structural, biophysical and biochemical methods applied to CsdL/TcdA has afforded a more thorough understanding of how the structure of this E1-like enzyme has been fine tuned to accomplish ct(6)A synthesis on tRNAs while providing support for the notion that CsdA and CsdE are able to functionally interact with CsdL/TcdA.

  18. Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection.

    PubMed

    Qiao, Yongli; Shi, Jinxia; Zhai, Yi; Hou, Yingnan; Ma, Wenbo

    2015-05-01

    A broad range of parasites rely on the functions of effector proteins to subvert host immune response and facilitate disease development. The notorious Phytophthora pathogens evolved effectors with RNA silencing suppression activity to promote infection in plant hosts. Here we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) can bind to an evolutionarily conserved nuclear protein containing the aspartate-glutamate-alanine-histidine-box RNA helicase domain in plants. This protein, designated PSR1-Interacting Protein 1 (PINP1), regulates the accumulation of both microRNAs and endogenous small interfering RNAs in Arabidopsis. A null mutation of PINP1 causes embryonic lethality, and silencing of PINP1 leads to developmental defects and hypersusceptibility to Phytophthora infection. These phenotypes are reminiscent of transgenic plants expressing PSR1, supporting PINP1 as a direct virulence target of PSR1. We further demonstrate that the localization of the Dicer-like 1 protein complex is impaired in the nucleus of PINP1-silenced or PSR1-expressing cells, indicating that PINP1 may facilitate small RNA processing by affecting the assembly of dicing complexes. A similar function of PINP1 homologous genes in development and immunity was also observed in Nicotiana benthamiana. These findings highlight PINP1 as a previously unidentified component of RNA silencing that regulates distinct classes of small RNAs in plants. Importantly, Phytophthora has evolved effectors to target PINP1 in order to promote infection.

  19. Endogenous Small RNA Mediates Meiotic Silencing of a Novel DNA Transposon

    PubMed Central

    Wang, Yizhou; Smith, Kristina M.; Taylor, John W.; Freitag, Michael; Stajich, Jason E.

    2015-01-01

    Genome defense likely evolved to curtail the spread of transposable elements and invading viruses. A combination of effective defense mechanisms has been shown to limit colonization of the Neurospora crassa genome by transposable elements. A novel DNA transposon named Sly1-1 was discovered in the genome of the most widely used laboratory “wild-type” strain FGSC 2489 (OR74A). Meiotic silencing by unpaired DNA, also simply called meiotic silencing, prevents the expression of regions of the genome that are unpaired during karyogamy. This mechanism is posttranscriptional and is proposed to involve the production of small RNA, so-called masiRNAs, by proteins homologous to those involved in RNA interference−silencing pathways in animals, fungi, and plants. Here, we demonstrate production of small RNAs when Sly1-1 was unpaired in a cross between two wild-type strains. These small RNAs are dependent on SAD-1, an RNA-dependent RNA polymerase necessary for meiotic silencing. We present the first case of endogenously produced masiRNA from a novel N. crassa DNA transposable element. PMID:26109355

  20. Analysis of the miRNA-mRNA-lncRNA networks in ER+ and ER- breast cancer cell lines.

    PubMed

    Wu, Qian; Guo, Li; Jiang, Fei; Li, Lei; Li, Zhong; Chen, Feng

    2015-12-01

    Recently, rapid advances in bioinformatics analysis have expanded our understanding of the transcriptome to a genome-wide level. miRNA-mRNA-lncRNA interactions have been shown to play critical regulatory role in cancer biology. In this study, we discussed the use of an integrated systematic approach to explore new facets of the oestrogen receptor (ER)-regulated transcriptome. The identification of RNAs that are related to the expression status of the ER may be useful in clinical therapy and prognosis. We used a network modelling strategy. First, microarray expression profiling of mRNA, lncRNA and miRNA was performed in MCF-7 (ER-positive) and MDA-MB-231 cells (ER- negative). A co-expression network was then built using co-expression relationships of the differentially expressed mRNAs and lncRNAs. Finally, the selected miRNA-mRNA network was added to the network. The key miRNA-mRNA-lncRNA interaction can be inferred from the network. The mRNA and non-coding RNA expression profiles of the cells with different ER phenotypes were distinct. Among the aberrantly expressed miRNAs, the expression levels of miR-19a-3p, miR-19b-3p and miR-130a-3p were much lower in the MCF-7 cells, whereas that of miR-148b-3p was much higher. In a cluster of miR-17-92, the expression levels of six of seven miRNAs were lower in the MCF-7 cells, in addition to miR-20b in the miR-106a-363 cluster. However, the levels of all the miRNAs in the miR-106a-25 cluster were higher in the MCF-7 cells. In the co-expression networking, CD74 and FMNL2 gene which is involved in the immune response and metastasis, respectively, had a stronger correlation with ER. Among the aberrantly expressed lncRNAs, lncRNA-DLEU1 was highly expressed in the MCF-7 cells. A statistical analysis revealed that there was a co-expression relationship between ESR1 and lncRNA-DLEU1. In addition, miR-19a and lncRNA-DLEU1 are both located on the human chromosome 13q. We speculate that miR-19a might be co-expressed with lncRNA-DLEU1

  1. Genome-Wide Analysis of miRNA targets in Brachypodium and Biomass Energy Crops

    SciTech Connect

    Green, Pamela J.

    2015-08-11

    MicroRNAs (miRNAs) contribute to the control of numerous biological processes through the regulation of specific target mRNAs. Although the identities of these targets are essential to elucidate miRNA function, the targets are much more difficult to identify than the small RNAs themselves. Before this work, we pioneered the genome-wide identification of the targets of Arabidopsis miRNAs using an approach called PARE (German et al., Nature Biotech. 2008; Nature Protocols, 2009). Under this project, we applied PARE to Brachypodium distachyon (Brachypodium), a model plant in the Poaceae family, which includes the major food grain and bioenergy crops. Through in-depth global analysis and examination of specific examples, this research greatly expanded our knowledge of miRNAs and target RNAs of Brachypodium. New regulation in response to environmental stress or tissue type was found, and many new miRNAs were discovered. More than 260 targets of new and known miRNAs with PARE sequences at the precise sites of miRNA-guided cleavage were identified and characterized. Combining PARE data with the small RNA data also identified the miRNAs responsible for initiating approximately 500 phased loci, including one of the novel miRNAs. PARE analysis also revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. The project included generation of small RNA and PARE resources for bioenergy crops, to facilitate ongoing discovery of conserved miRNA-target RNA regulation. By associating specific miRNA-target RNA pairs with known physiological functions, the research provides insights about gene regulation in different tissues and in response to environmental stress. This, and release of new PARE and small RNA data sets should contribute basic knowledge to enhance breeding and may suggest new strategies for improvement of biomass energy crops.

  2. MicroRNA-Like Small RNAs Prediction in the Development of Antrodia cinnamomea

    PubMed Central

    Lin, Yan-Liang; Ma, Li-Ting; Lee, Yi-Ru; Lin, Shih-Shun; Wang, Sheng-Yang; Chang, Tun-Tschu; Shaw, Jei-Fu; Li, Wen-Hsiung; Chu, Fang-Hua

    2015-01-01

    Antrodia cinnamomea, a precious, host-specific brown-rot fungus that has been used as a folk medicine in Taiwan for centuries is known to have diverse bioactive compounds with potent pharmaceutical activity. In this study, different fermentation states of A. cinnamomea (wild-type fruiting bodies and liquid cultured mycelium) were sequenced using the next-generation sequencing (NGS) technique. A 45.58 Mb genome encoding 6,522 predicted genes was obtained. High quality reads were assembled into a total of 13,109 unigenes. Using a previously constructed pipeline to search for microRNAs (miRNAs), we then identified 4 predicted conserved miRNA and 63 novel predicted miRNA-like small RNA (milRNA) candidates. Target prediction revealed several interesting proteins involved in tri-terpenoid synthesis, mating type recognition, chemical or physical sensory protein and transporters predicted to be regulated by the miRNAs and milRNAs. PMID:25860872

  3. Systematic coarse-grained modeling of complexation between small interfering RNA and polycations

    SciTech Connect

    Wei, Zonghui; Luijten, Erik

    2015-12-28

    All-atom molecular dynamics simulations can provide insight into the properties of polymeric gene-delivery carriers by elucidating their interactions and detailed binding patterns with nucleic acids. However, to explore nanoparticle formation through complexation of these polymers and nucleic acids and study their behavior at experimentally relevant time and length scales, a reliable coarse-grained model is needed. Here, we systematically develop such a model for the complexation of small interfering RNA (siRNA) and grafted polyethyleneimine copolymers, a promising candidate for siRNA delivery. We compare the predictions of this model with all-atom simulations and demonstrate that it is capable of reproducing detailed binding patterns, charge characteristics, and water release kinetics. Since the coarse-grained model accelerates the simulations by one to two orders of magnitude, it will make it possible to quantitatively investigate nanoparticle formation involving multiple siRNA molecules and cationic copolymers.

  4. Zebrafish U6 small nuclear RNA gene promoters contain a SPH element in an unusual location.

    PubMed

    Halbig, Kari M; Lekven, Arne C; Kunkel, Gary R

    2008-09-15

    Promoters for vertebrate small nuclear RNA (snRNA) genes contain a relatively simple array of transcriptional control elements, divided into proximal and distal regions. Most of these genes are transcribed by RNA polymerase II (e.g., U1, U2), whereas the U6 gene is transcribed by RNA polymerase III. Previously identified vertebrate U6 snRNA gene promoters consist of a proximal sequence element (PSE) and TATA element in the proximal region, plus a distal region with octamer (OCT) and SphI postoctamer homology (SPH) elements. We have found that zebrafish U6 snRNA promoters contain the SPH element in a novel proximal position immediately upstream of the TATA element. The zebrafish SPH element is recognized by SPH-binding factor/selenocysteine tRNA gene transcription activating factor/zinc finger protein 143 (SBF/Staf/ZNF143) in vitro. Furthermore, a zebrafish U6 promoter with a defective SPH element is inefficiently transcribed when injected into embryos.

  5. A Small RNA-Based Immune System Defends Germ Cells against Mobile Genetic Elements

    PubMed Central

    2016-01-01

    Transposons are mobile genetic elements that threaten the survival of species by destabilizing the germline genomes. Limiting the spread of these selfish elements is imperative. Germ cells employ specialized small regulatory RNA pathways to restrain transposon activity. PIWI proteins and Piwi-interacting RNAs (piRNAs) silence transposons at the transcriptional and posttranscriptional level with loss-of-function mutant animals universally exhibiting sterility often associated with germ cell defects. This short review aims to illustrate basic strategies of piRNA-guided defense against transposons. Mechanisms of piRNA silencing are most readily studied in Drosophila melanogaster, which serves as a model to delineate molecular concepts and as a reference for mammalian piRNA systems. PiRNA pathways utilize two major strategies to handle the challenges of transposon control: (1) the hard-wired molecular memory of prior transpositions enables recognition of mobile genetic elements and discriminates transposons from host genes; (2) a feed-forward adaptation mechanism shapes piRNA populations to selectively combat the immediate threat of transposon transcripts. In flies, maternally contributed PIWI-piRNA complexes bolster both of these lines of defense and ensure transgenerational immunity. While recent studies have provided a conceptual framework of what could be viewed as an ancient immune system, we are just beginning to appreciate its many molecular innovations. PMID:26681955

  6. Targeted Delivery of Small Interfering RNA Using Reconstituted High-Density Lipoprotein Nanoparticles12

    PubMed Central

    Shahzad, Mian MK; Mangala, Lingegowda S; Han, Hee Dong; Lu, Chunhua; Bottsford-Miller, Justin; Nishimura, Masato; Mora, Edna M; Lee, Jeong-Won; Stone, Rebecca L; Pecot, Chad V; Thanapprapasr, Duangmani; Roh, Ju-Won; Gaur, Puja; Nair, Maya P; Park, Yun-Yong; Sabnis, Nirupama; Deavers, Michael T; Lee, Ju-Seog; Ellis, Lee M; Lopez-Berestein, Gabriel; McConathy, Walter J; Prokai, Laszlo; Lacko, Andras G; Sood, Anil K

    2011-01-01

    RNA interference holds tremendous potential as a therapeutic approach, especially in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA). To maintain a high level of growth, tumor cells scavenge high-density lipoprotein (HDL) particles by overexpressing its receptor: scavenger receptor type B1 (SR-B1). In this study, we exploited this cellular characteristic to achieve efficient siRNA delivery and established a novel formulation of siRNA by incorporating it into reconstituted HDL (rHDL) nanoparticles. Here, we demonstrate that rHDL nanoparticles facilitate highly efficient systemic delivery of siRNA in vivo, mediated by the SR-B1. Moreover, in therapeutic proof-of-concept studies, these nanoparticles were effective in silencing the expression of two proteins that are key to cancer growth and metastasis (signal transducer and activator of transcription 3 and focal adhesion kinase) in orthotopic mouse models of ovarian and colorectal cancer. These data indicate that an rHDL nanoparticle is a novel and highly efficient siRNA carrier, and therefore, this novel technology could serve as the foundation for new cancer therapeutic approaches. PMID:21472135

  7. An engineered small RNA-mediated genetic switch based on a ribozyme expression platform

    PubMed Central

    Klauser, Benedikt; Hartig, Jörg S.

    2013-01-01

    An important requirement for achieving many goals of synthetic biology is the availability of a large repertoire of reprogrammable genetic switches and appropriate transmitter molecules. In addition to engineering genetic switches, the interconnection of individual switches becomes increasingly important for the construction of more complex genetic networks. In particular, RNA-based switches of gene expression have become a powerful tool to post-transcriptionally program genetic circuits. RNAs used for regulatory purposes have the advantage to transmit, sense, process and execute information. We have recently used the hammerhead ribozyme to control translation initiation in a small molecule-dependent fashion. In addition, riboregulators have been constructed in which a small RNA acts as transmitter molecule to control translation of a target mRNA. In this study, we combine both concepts and redesign the hammerhead ribozyme to sense small trans-acting RNAs (taRNAs) as input molecules resulting in repression of translation initiation in Escherichia coli. Importantly, our ribozyme-based expression platform is compatible with previously reported artificial taRNAs, which were reported to act as inducers of gene expression. In addition, we provide several insights into key requirements of riboregulatory systems, including the influences of varying transcriptional induction of the taRNA and mRNA transcripts, 5′-processing of taRNAs, as well as altering the secondary structure of the taRNA. In conclusion, we introduce an RNA-responsive ribozyme-based expression system to the field of artificial riboregulators that can serve as reprogrammable platform for engineering higher-order genetic circuits. PMID:23585277

  8. DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function.

    PubMed

    Soltanieh, Sahar; Osheim, Yvonne N; Spasov, Krasimir; Trahan, Christian; Beyer, Ann L; Dragon, François

    2015-03-01

    DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.

  9. Divergent patterns of endogenous small RNA populations from seed and vegetative tissues of Glycine max

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Small non-coding RNAs (smRNAs) are known to have major roles in gene regulation in eukaryotes. In plants, knowledge of the biogenesis and mechanisms of action of smRNA classes including microRNAs (miRNAs), short interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs) has been gaine...

  10. Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7

    PubMed Central

    Uchikawa, Emiko; Natchiar, Kundhavai S.; Han, Xiao; Proux, Florence; Roblin, Pierre; Zhang, Elodie; Durand, Alexandre; Klaholz, Bruno P.; Dock-Bregeon, Anne-Catherine

    2015-01-01

    The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3′-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3′ hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5′-end with the foot of the 3′ hairpin and has thus functional implications. PMID:25753663

  11. Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ.

    PubMed

    Mank, Nils N; Berghoff, Bork A; Hermanns, Yannick N; Klug, Gabriele

    2012-10-01

    The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.

  12. Unique gene program of rat small resistance mesenteric arteries as revealed by deep RNA sequencing

    PubMed Central

    Reho, John J; Shetty, Amol; Dippold, Rachael P; Mahurkar, Anup; Fisher, Steven A

    2015-01-01

    Deep sequencing of RNA samples from rat small mesenteric arteries (MA) and aorta (AO) identified common and unique features of their gene programs. ∼5% of mRNAs were quantitatively differentially expressed in MA versus AO. Unique transcriptional control in MA smooth muscle is suggested by the selective or enriched expression of transcription factors Nkx2-3, HAND2, and Tcf21 (Capsulin). Enrichment in AO of PPAR transcription factors and their target genes of mitochondrial function, lipid metabolism, and oxidative phosphorylation is consistent with slow (oxidative) tonic smooth muscle. In contrast MA was enriched in contractile and calcium channel mRNAs suggestive of components of fast (glycolytic) phasic smooth muscle. Myosin phosphatase regulatory subunit paralogs Mypt1 and p85 were expressed at similar levels, while smooth muscle MLCK was the only such kinase expressed, suggesting functional redundancy of the former but not the latter in accordance with mouse knockout studies. With regard to vaso-regulatory signals, purinergic receptors P2rx1 and P2rx5 were reciprocally expressed in MA versus AO, while the olfactory receptor Olr59 was enriched in MA. Alox15, which generates the EDHF HPETE, was enriched in MA while eNOS was equally expressed, consistent with the greater role of EDHF in the smaller arteries. mRNAs that were not expressed at a level consistent with impugned function include skeletal myogenic factors, IKK2, nonmuscle myosin, and Gnb3. This screening analysis of gene expression in the small mesenteric resistance arteries suggests testable hypotheses regarding unique aspects of small artery function in the regional control of blood flow. PMID:26156969

  13. Role of a neuronal small non-messenger RNA: behavioural alterations in BC1 RNA-deleted mice.

    PubMed

    Lewejohann, L; Skryabin, B V; Sachser, N; Prehn, C; Heiduschka, P; Thanos, S; Jordan, U; Dell'Omo, G; Vyssotski, A L; Pleskacheva, M G; Lipp, H-P; Tiedge, H; Brosius, J; Prior, H

    2004-09-23

    BC1 RNA is a small non-messenger RNA common in dendritic microdomains of neurons in rodents. In order to investigate its possible role in learning and behaviour, we compared controls and knockout mice from three independent founder lines established from separate embryonic stem cells. Mutant mice were healthy with normal brain morphology and appeared to have no neurological deficits. A series of tests for exploration and spatial memory was carried out in three different laboratories. The tests were chosen as to ensure that different aspects of spatial memory and exploration could be separated and that possible effects of confounding variables could be minimised. Exploration was studied in a barrier test, in an open-field test, and in an elevated plus-maze test. Spatial memory was investigated in a Barnes maze and in a Morris water maze (memory for a single location), in a multiple T-maze and in a complex alley maze (route learning), and in a radial maze (working memory). In addition to these laboratory tasks, exploratory behaviour and spatial memory were assessed under semi-naturalistic conditions in a large outdoor pen. The combined results indicate that BC1 RNA-deficient animals show behavioural changes best interpreted in terms of reduced exploration and increased anxiety. In contrast, spatial memory was not affected. In the outdoor pen, the survival rates of BC1-depleted mice were lower than in controls. Thus, we conclude that the neuron-specific non-messenger BC1 RNA contributes to the aptive modulation of behaviour.

  14. Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization.

    PubMed

    Locati, Mauro D; Terpstra, Inez; de Leeuw, Wim C; Kuzak, Mateusz; Rauwerda, Han; Ensink, Wim A; van Leeuwen, Selina; Nehrdich, Ulrike; Spaink, Herman P; Jonker, Martijs J; Breit, Timo M; Dekker, Rob J

    2015-08-18

    There is an increasing interest in complementing RNA-seq experiments with small-RNA (sRNA) expression data to obtain a comprehensive view of a transcriptome. Currently, two main experimental challenges concerning sRNA-seq exist: how to check the size distribution of isolated sRNAs, given the sensitive size-selection steps in the protocol; and how to normalize data between samples, given the low complexity of sRNA types. We here present two separate sets of synthetic RNA spike-ins for monitoring size-selection and for performing data normalization in sRNA-seq. The size-range quality control (SRQC) spike-in set, consisting of 11 oligoribonucleotides (10-70 nucleotides), was tested by intentionally altering the size-selection protocol and verified via several comparative experiments. We demonstrate that the SRQC set is useful to reproducibly track down biases in the size-selection in sRNA-seq. The external reference for data-normalization (ERDN) spike-in set, consisting of 19 oligoribonucleotides, was developed for sample-to-sample normalization in differential-expression analysis of sRNA-seq data. Testing and applying the ERDN set showed that it can reproducibly detect differential expression over a dynamic range of 2(18). Hence, biological variation in sRNA composition and content between samples is preserved while technical variation is effectively minimized. Together, both spike-in sets can significantly improve the technical reproducibility of sRNA-seq.

  15. Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation.

    PubMed

    Stutika, Catrin; Mietzsch, Mario; Gogol-Döring, Andreas; Weger, Stefan; Sohn, Madlen; Chen, Wei; Heilbronn, Regine

    2016-01-01

    Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic. PMID:27611072

  16. Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation

    PubMed Central

    Stutika, Catrin; Mietzsch, Mario; Gogol-Döring, Andreas; Weger, Stefan; Sohn, Madlen; Chen, Wei; Heilbronn, Regine

    2016-01-01

    Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic. PMID:27611072

  17. Identification of conserved and novel microRNAs in Aquilaria sinensis based on small RNA sequencing and transcriptome sequence data.

    PubMed

    Gao, Zhi-Hui; Wei, Jian-He; Yang, Yun; Zhang, Zheng; Xiong, Huan-Ying; Zhao, Wen-Ting

    2012-08-15

    Agarwood is in great demand for its high value in medicine, incense, and perfume across Asia, Middle East, and Europe. As agarwood is formed only when the Aquilaria trees are wounded or infected by some microbes, overharvesting and habitat loss are threatening some populations of agarwood-producing species. Aquilaria sinensis is such a significant economic tree species. To promote the production efficiency and protect the resource of A. sinensis, it would be critical to reveal the regulation mechanisms of stress-induced agarwood formation. MicroRNAs (miRNAs), a key gene expression regulator involved in various plant stress response and metabolic processes, might function in agarwood formation, but no report concerning miRNAs in Aquilaria is available. In this study, the small RNA high-throughput sequencing and 454 transcriptome data were adopted to identify both conserved and novel miRNAs in A. sinensis. Deep sequencing showed that the small RNA (sRNA) population of A. sinensis was complex and the length of sRNAs varied. By in silico analysis of the small RNA deep sequencing data and transcriptome data, we discovered 27 novel miRNAs in A. sinensis. Based on the mature miRNA sequence conservation, we identified 74 putative conserved miRNAs from A. sinensis and 10 of them were confirmed with hairpin forming precursor. Interestingly, a novel miRNA sequence was determined to be the miRNA of asi-miR408, but with accumulation much higher than asi-miR408. The expression levels of ten stress-responsive miRNAs were examined during the time-course after wound treatment. Eight were shown to be wound-responsive. This not only shows the existence of miRNAs in this Asian economically significant tree species but also indicated its critical role in stress-induced agarwood formation. The highly accumulated miRNA of asi-miR408 implied miRNAs would be functional as well as miRNAs in plants.

  18. Joint analysis of miRNA and mRNA expression data.

    PubMed

    Muniategui, Ander; Pey, Jon; Planes, Francisco J; Rubio, Angel

    2013-05-01

    miRNAs are small RNA molecules ('22 nt) that interact with their target mRNAs inhibiting translation or/and cleavaging the target mRNA. This interaction is guided by sequence complentarity and results in the reduction of mRNA and/or protein levels. miRNAs are involved in key biological processes and different diseases. Therefore, deciphering miRNA targets is crucial for diagnostics and therapeutics. However, miRNA regulatory mechanisms are complex and there is still no high-throughput and low-cost miRNA target screening technique. In recent years, several computational methods based on sequence complementarity of the miRNA and the mRNAs have been developed. However, the predicted interactions using these computational methods are inconsistent and the expected false positive rates are still large. Recently, it has been proposed to use the expression values of miRNAs and mRNAs (and/or proteins) to refine the results of sequence-based putative targets for a particular experiment. These methods have shown to be effective identifying the most prominent interactions from the databases of putative targets. Here, we review these methods that combine both expression and sequence-based putative targets to predict miRNA targets. PMID:22692086

  19. Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells – Evidence of unique microRNA cargos

    PubMed Central

    Lunavat, Taral R; Cheng, Lesley; Kim, Dae-Kyum; Bhadury, Joydeep; Jang, Su Chul; Lässer, Cecilia; Sharples, Robyn A; López, Marcela Dávila; Nilsson, Jonas; Gho, Yong Song; Hill, Andrew F; Lötvall, Jan

    2015-01-01

    Melanoma cells release different types of extracellular vesicles (EVs) into the extracellular milieu that are involved with communication and signaling in the tumor microenvironment. Subsets of EVs include exosomes, microvesicles, and apoptotic bodies that carry protein and genetic (RNA) cargos. To define the contribution of the RNA cargo of melanoma cell derived EVs we performed small RNA sequencing to identify different small RNAs in the EV subsets. Using validated centrifugation protocols, we separated these EV subsets released by the melanoma cell line MML-1, and performed RNA sequencing with the Ion Torrent platform. Various, but different, non-coding RNAs were detected in the EV subsets, including microRNA, mitochondrial associated tRNA, small nucleolar RNA, small nuclear RNA, Ro associated Y-RNA, vault RNA and Y-RNA. We identified in total 1041 miRNAs in cells and EV subsets. Hierarchical clustering showed enrichment of specific miRNAs in exosomes, including hsa-miR-214-3p, hsa-miR-199a-3p and hsa-miR-155-5p, all being associated with melanoma progression. Comparison of exosomal miRNAs with miRNAs in clinical melanoma samples indicate that multiple miRNAs in exosomes also are expressed specifically in melanoma tissues, but not in benign naevi. This study shows for the first time the presence of distinct small RNAs in subsets of EVs released by melanoma cells, with significant similarities to clinical melanoma tissue, and provides unique insights into the contribution of EV associated extracellular RNA in cancer. PMID:26176991

  20. A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD.

    PubMed

    Fröhlich, Kathrin S; Papenfort, Kai; Berger, Allison A; Vogel, Jörg

    2012-04-01

    A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, σ(S) (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by σ(S). We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3'-RACE, an experimental approach that promises to be of use in predicting other sRNA-target interactions in bacteria.

  1. Suppression of Breast Cancer Cell Migration by Small Interfering RNA Delivered by Polyethylenimine-Functionalized Graphene Oxide

    NASA Astrophysics Data System (ADS)

    Huang, Yuan-Pin; Hung, Chao-Ming; Hsu, Yi-Chiang; Zhong, Cai-Yan; Wang, Wan-Rou; Chang, Chi-Chang; Lee, Mon-Juan

    2016-05-01

    The carbon-based nanomaterial graphene can be chemically modified to associate with various molecules such as chemicals and biomolecules and developed as novel carriers for drug and gene delivery. In this study, a nonviral gene transfection reagent was produced by functionalizing graphene oxide (GO) with a polycationic polymer, polyethylenimine (PEI), to increase the biocompatibility of GO and to transfect small interfering RNA (siRNA) against C-X-C chemokine receptor type 4 (CXCR4), a biomarker associated with cancer metastasis, into invasive breast cancer cells. PEI-functionalized GO (PEI-GO) was a homogeneous aqueous solution that remained in suspension during storage at 4 °C for at least 6 months. The particle size of PEI-GO was 172 ± 4.58 and 188 ± 5.00 nm at 4 and 25 °C, respectively, and increased slightly to 262 ± 17.6 nm at 37 °C, but remained unaltered with time. Binding affinity of PEI-GO toward siRNA was assessed by electrophoretic mobility shift assay (EMSA), in which PEI-GO and siRNA were completely associated at a PEI-GO:siRNA weight ratio of 2:1 and above. The invasive breast cancer cell line, MDA-MB-231, was transfected with PEI-GO in complex with siRNAs against CXCR4 (siCXCR4). Suppression of the mRNA and protein expression of CXCR4 by the PEI-GO/siCXCR4 complex was confirmed by real-time PCR and western blot analysis. In addition, the metastatic potential of MDA-MB-231 cells was attenuated by the PEI-GO/siCXCR4 complex as demonstrated in wound healing assay. Our results suggest that PEI-GO is effective in the delivery of siRNA and may contribute to targeted gene therapy to suppress cancer metastasis.

  2. Discovery and small RNA profile of Pecan mosaic-associated virus, a novel potyvirus of pecan trees

    PubMed Central

    Su, Xiu; Fu, Shuai; Qian, Yajuan; Zhang, Liqin; Xu, Yi; Zhou, Xueping

    2016-01-01

    A novel potyvirus was discovered in pecan (Carya illinoensis) showing leaf mosaic symptom through the use of deep sequencing of small RNAs. The complete genome of this virus was determined to comprise of 9,310 nucleotides (nt), and shared 24.0% to 58.9% nucleotide similarities with that of other Potyviridae viruses. The genome was deduced to encode a single open reading frame (polyprotein) on the plus strand. Phylogenetic analysis based on the whole genome sequence and coat protein amino acid sequence showed that this virus is most closely related to Lettuce mosaic virus. Using electron microscopy, the typical Potyvirus filamentous particles were identified in infected pecan leaves with mosaic symptoms. Our results clearly show that this virus is a new member of the genus Potyvirus in the family Potyviridae. The virus is tentatively named Pecan mosaic-associated virus (PMaV). Additionally, profiling of the PMaV-derived small RNA (PMaV-sRNA) showed that the most abundant PMaV-sRNAs were 21-nt in length. There are several hotspots for small RNA production along the PMaV genome; two 21-nt PMaV-sRNAs starting at 811 nt and 610 nt of the minus-strand genome were highly repeated. PMID:27226228

  3. Discovery and small RNA profile of Pecan mosaic-associated virus, a novel potyvirus of pecan trees.

    PubMed

    Su, Xiu; Fu, Shuai; Qian, Yajuan; Zhang, Liqin; Xu, Yi; Zhou, Xueping

    2016-01-01

    A novel potyvirus was discovered in pecan (Carya illinoensis) showing leaf mosaic symptom through the use of deep sequencing of small RNAs. The complete genome of this virus was determined to comprise of 9,310 nucleotides (nt), and shared 24.0% to 58.9% nucleotide similarities with that of other Potyviridae viruses. The genome was deduced to encode a single open reading frame (polyprotein) on the plus strand. Phylogenetic analysis based on the whole genome sequence and coat protein amino acid sequence showed that this virus is most closely related to Lettuce mosaic virus. Using electron microscopy, the typical Potyvirus filamentous particles were identified in infected pecan leaves with mosaic symptoms. Our results clearly show that this virus is a new member of the genus Potyvirus in the family Potyviridae. The virus is tentatively named Pecan mosaic-associated virus (PMaV). Additionally, profiling of the PMaV-derived small RNA (PMaV-sRNA) showed that the most abundant PMaV-sRNAs were 21-nt in length. There are several hotspots for small RNA production along the PMaV genome; two 21-nt PMaV-sRNAs starting at 811 nt and 610 nt of the minus-strand genome were highly repeated. PMID:27226228

  4. Small molecules targeting microRNA for cancer therapy: Promises and obstacles.

    PubMed

    Wen, Di; Danquah, Michael; Chaudhary, Amit Kumar; Mahato, Ram I

    2015-12-10

    Aberrant expression of miRNAs is critically implicated in cancer initiation and progression. Therapeutic approaches focused on regulating miRNAs are therefore a promising approach for treating cancer. Antisense oligonucleotides, miRNA sponges, and CRISPR/Cas9 genome editing systems are being investigated as tools for regulating miRNAs. Despite the accruing insights in the use of these tools, delivery concerns have mitigated clinical application of such systems. In contrast, little attention has been given to the potential of small molecules to modulate miRNA expression for cancer therapy. In these years, many researches proved that small molecules targeting cancer-related miRNAs might have greater potential for cancer treatment. Small molecules targeting cancer related miRNAs showed significantly promising results in different cancer models. However, there are still several obstacles hindering the progress and clinical application in this area. This review discusses the development, mechanisms and application of small molecules for modulating oncogenic miRNAs (oncomiRs). Attention has also been given to screening technologies and perspectives aimed to facilitate clinical translation for small molecule-based miRNA therapeutics.

  5. Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes

    PubMed Central

    Nicolás, Francisco E.; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M.

    2003-01-01

    Transformation of Mucor circinelloides with self-replicative plasmids containing a wild-type copy of the carotenogenic gene carB causes silencing of the carB function in 3% of transformants. Genomic analyses revealed a relationship between silenced phenotype and number of copies of plasmids. This phenotype results from a reduction of the steady-state levels of carB mRNA, a reduction that is not due to differences in the level of transcription, indicating that silencing is post-transcriptional. Small sense and antisense RNAs have been found to be associated with gene silencing in M.circinelloides. Two size classes of small antisense RNAs, differentially accumulated during the vegetative growth of silenced transformants, have been detected: a long 25-nucleotide RNA and a short 21-nucleotide RNA. Secondary sense and antisense RNAs corresponding to sequences of the endogenous gene downstream of the initial triggering molecule have also been detected, revealing the existence of spreading of RNA targeting in fungi. These findings, together with the self-replicative nature of the triggering molecules, make M.circinelloides a suitable organism for investigating some unresolved questions in RNA silencing. PMID:12881432

  6. Identification of microRNA-like small RNAs from fungal parasite Nosema ceranae.

    PubMed

    Huang, Qiang; Evans, Jay D

    2016-01-01

    We previously found transcripts encoding Dicer and Argonaute which are involved in the production of microRNAs, in the honey bee parasite Nosema ceranae. In order to identify microRNAs in N. ceranae, we sequenced small RNAs from midgut tissues of infected honey bees at 24 h intervals for 6 days post infection, covering the complete reproduction cycle for this intracellular parasite. We predicted six microRNA-like small RNAs, all of which were confirmed via RT-qPCR assays. This is the first evidence for microRNA-like small RNAs generated by a microsporidian species, providing new insights into host-parasite interactions involving this widespread taxonomic group. PMID:26678507

  7. Nanoparticle-based delivery of small interfering RNA: challenges for cancer therapy

    PubMed Central

    Miele, Evelina; Spinelli, Gian Paolo; Miele, Ermanno; Di Fabrizio, Enzo; Ferretti, Elisabetta; Tomao, Silverio; Gulino, Alberto

    2012-01-01

    During recent decades there have been remarkable advances and profound changes in cancer therapy. Many therapeutic strategies learned at the bench, including monoclonal antibodies and small molecule inhibitors, have been used at the bedside, leading to important successes. One of the most important advances in biology has been the discovery that small interfering RNA (siRNA) is able to regulate the expression of genes, by a phenomenon known as RNA interference (RNAi). RNAi is one of the most rapidly growing fields of research in biology and therapeutics. Much research effort has gone into the application of this new discovery in the treatment of various diseases, including cancer. However, even though these molecules may have potential and strong utility, some limitations make their clinical application difficult, including delivery problems, side effects due to off-target actions, disturbance of physiological functions of the cellular machinery involved in gene silencing, and induction of the innate immune response. Many researchers have attempted to overcome these limitations and to improve the safety of potential RNAi-based therapeutics. Nanoparticles, which are nanostructured entities with tunable size, shape, and surface, as well as biological behavior, provide an ideal opportunity to modify current treatment regimens in a substantial way. These nanoparticles could be designed to surmount one or more of the barriers encountered by siRNA. Nanoparticle drug formulations afford the chance to improve drug bioavailability, exploiting superior tissue permeability, payload protection, and the “stealth” features of these entities. The main aims of this review are: to explain the siRNA mechanism with regard to potential applications in siRNA-based cancer therapy; to discuss the possible usefulness of nanoparticle-based delivery of certain molecules for overcoming present therapeutic limitations; to review the ongoing relevant clinical research with its pitfalls and

  8. U1 small nuclear RNA and spliceosomal introns in Euglena gracilis

    PubMed Central

    Breckenridge, David G.; Watanabe, Yoh-ichi; Greenwood, Spencer J.; Gray, Michael W.; Schnare, Murray N.

    1999-01-01

    In the flagellated protozoon Euglena gracilis, characterized nuclear genes harbor atypical introns that usually are flanked by short repeats, adopt complex secondary structures in pre-mRNA, and do not obey the GT-AG rule of conventional cis-spliced introns. In the nuclear fibrillarin gene of E. gracilis, we have identified three spliceosomal-type introns that have GT-AG consensus borders. Furthermore, we have isolated a small RNA from E. gracilis and propose, on the basis of primary and secondary structure comparisons, that it is a homolog of U1 small nuclear RNA, an essential component of the cis-spliceosome in higher eukaryotes. Conserved sequences at the 5′ splice sites of the fibrillarin introns can potentially base pair with Euglena U1 small nuclear RNA. Our observations demonstrate that spliceosomal GT-AG cis-splicing occurs in Euglena, in addition to the nonconventional cis-splicing and spliced leader trans-splicing previously recognized in this early diverging unicellular eukaryote. PMID:9927657

  9. DeAnnIso: a tool for online detection and annotation of isomiRs from small RNA sequencing data

    PubMed Central

    Zhang, Yuanwei; Zang, Qiguang; Zhang, Huan; Ban, Rongjun; Yang, Yifan; Iqbal, Furhan; Li, Ao; Shi, Qinghua

    2016-01-01

    Small RNA (sRNA) Sequencing technology has revealed that microRNAs (miRNAs) are capable of exhibiting frequent variations from their canonical sequences, generating multiple variants: the isoforms of miRNAs (isomiRs). However, integrated tool to precisely detect and systematically annotate isomiRs from sRNA sequencing data is still in great demand. Here, we present an online tool, DeAnnIso (Detection and Annotation of IsomiRs from sRNA sequencing data). DeAnnIso can detect all the isomiRs in an uploaded sample, and can extract the differentially expressing isomiRs from paired or multiple samples. Once the isomiRs detection is accomplished, detailed annotation information, including isomiRs expression, isomiRs classification, SNPs in miRNAs and tissue specific isomiR expression are provided to users. Furthermore, DeAnnIso provides a comprehensive module of target analysis and enrichment analysis for the selected isomiRs. Taken together, DeAnnIso is convenient for users to screen for isomiRs of their interest and useful for further functional studies. The server is implemented in PHP + Perl + R and available to all users for free at: http://mcg.ustc.edu.cn/bsc/deanniso/ and http://mcg2.ustc.edu.cn/bsc/deanniso/. PMID:27179030

  10. DeAnnIso: a tool for online detection and annotation of isomiRs from small RNA sequencing data.

    PubMed

    Zhang, Yuanwei; Zang, Qiguang; Zhang, Huan; Ban, Rongjun; Yang, Yifan; Iqbal, Furhan; Li, Ao; Shi, Qinghua

    2016-07-01

    Small RNA (sRNA) Sequencing technology has revealed that microRNAs (miRNAs) are capable of exhibiting frequent variations from their canonical sequences, generating multiple variants: the isoforms of miRNAs (isomiRs). However, integrated tool to precisely detect and systematically annotate isomiRs from sRNA sequencing data is still in great demand. Here, we present an online tool, DeAnnIso (Detection and Annotation of IsomiRs from sRNA sequencing data). DeAnnIso can detect all the isomiRs in an uploaded sample, and can extract the differentially expressing isomiRs from paired or multiple samples. Once the isomiRs detection is accomplished, detailed annotation information, including isomiRs expression, isomiRs classification, SNPs in miRNAs and tissue specific isomiR expression are provided to users. Furthermore, DeAnnIso provides a comprehensive module of target analysis and enrichment analysis for the selected isomiRs. Taken together, DeAnnIso is convenient for users to screen for isomiRs of their interest and useful for further functional studies. The server is implemented in PHP + Perl + R and available to all users for free at: http://mcg.ustc.edu.cn/bsc/deanniso/ and http://mcg2.ustc.edu.cn/bsc/deanniso/. PMID:27179030

  11. Small interfering RNA pathway modulates persistent infection of a plant virus in its insect vector

    PubMed Central

    Lan, Hanhong; Wang, Haitao; Chen, Qian; Chen, Hongyan; Jia, Dongsheng; Mao, Qianzhuo; Wei, Taiyun

    2016-01-01

    Plant reoviruses, rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent-propagative manner. How such persistent infection of plant viruses in insect vectors is established and maintained remains poorly understood. In this study, we used rice gall dwarf virus (RGDV), a plant reovirus, and its main vector leafhopper Recilia dorsalis as a virus–insect system to determine how the small interference (siRNA) pathway modulates persistent infection of a plant virus in its insect vector. We showed that a conserved siRNA antiviral response was triggered by the persistent replication of RGDV in cultured leafhopper cells and in intact insects, by appearance of virus-specific siRNAs, primarily 21-nt long, and the increased expression of siRNA pathway core components Dicer-2 and Argonaute-2. Silencing of Dicer-2 using RNA interference strongly suppressed production of virus-specific siRNAs, promoted viral accumulation, and caused cytopathological changes in vitro and in vivo. When the viral accumulation level rose above a certain threshold of viral genome copy (1.32 × 1014 copies/μg insect RNA), the infection of the leafhopper by RGDV was lethal rather than persistent. Taken together, our results revealed a new finding that the siRNA pathway in insect vector can modulate persistent infection of plant viruses. PMID:26864546

  12. Development of a software tool and criteria evaluation for efficient design of small interfering RNA

    SciTech Connect

    Chaudhary, Aparna; Srivastava, Sonam; Garg, Sanjeev

    2011-01-07

    Research highlights: {yields} The developed tool predicted siRNA constructs with better thermodynamic stability and total score based on positional and other criteria. {yields} Off-target silencing below score 30 were observed for the best siRNA constructs for different genes. {yields} Immunostimulation and cytotoxicity motifs considered and penalized in the developed tool. {yields} Both positional and compositional criteria were observed to be important. -- Abstract: RNA interference can be used as a tool for gene silencing mediated by small interfering RNAs (siRNA). The critical step in effective and specific RNAi processing is the selection of suitable constructs. Major design criteria, i.e., Reynolds's design rules, thermodynamic stability, internal repeats, immunostimulatory motifs were emphasized and implemented in the siRNA design tool. The tool provides thermodynamic stability score, GC content and a total score based on other design criteria in the output. The viability of the tool was established with different datasets. In general, the siRNA constructs produced by the tool had better thermodynamic score and positional properties. Comparable thermodynamic scores and better total scores were observed with the existing tools. Moreover, the results generated had comparable off-target silencing effect. Criteria evaluations with additional criteria were achieved in WEKA.

  13. Soybean ribulose bisphosphate carboxylase small subunit: Mechanisms and determinants of RNA turnover. Annual progress report

    SciTech Connect

    Meagher, R.B.

    1993-12-31

    An in vitro degradation system has been developed from petunia and soybean polysomes in order to investigate the mechanisms and determinants controlling RNA turnover in higher plants. This system faithfully degrades soybean ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) mRNA into the same products observed in total RNA preparations. In previous years it was shown that the most stable products represent a nested constellation of fragments, which are shortened from their 3{prime} ends, and have intact 5{prime} ends. Exogenous rbcS RNA tagged with novel 5{prime} sequence 15 or 56 bp long were synthesized in vitro as Sp6 and T7 runoff transcripts, respectively. When added to the system they were degraded faithfully into constellation of products which were 15 or 56 bp longer than the endogenous products, respectively. Detailed kinetics on the appearance of these exogenous products confirmed degradation proceeds in an overall 3{prime} to 5{prime} direction but suggested that there are multiple pathways through which the RNA may be degraded. To further demonstrate a precursor product relationships, in vitro synthesized transcripts truncated at their 3{prime} ends were shown to degrade into the expected smaller fragments previously mapped in the 5{prime} portion of the rbcS RNA.

  14. Small interfering RNA pathway modulates persistent infection of a plant virus in its insect vector.

    PubMed

    Lan, Hanhong; Wang, Haitao; Chen, Qian; Chen, Hongyan; Jia, Dongsheng; Mao, Qianzhuo; Wei, Taiyun

    2016-01-01

    Plant reoviruses, rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent-propagative manner. How such persistent infection of plant viruses in insect vectors is established and maintained remains poorly understood. In this study, we used rice gall dwarf virus (RGDV), a plant reovirus, and its main vector leafhopper Recilia dorsalis as a virus-insect system to determine how the small interference (siRNA) pathway modulates persistent infection of a plant virus in its insect vector. We showed that a conserved siRNA antiviral response was triggered by the persistent replication of RGDV in cultured leafhopper cells and in intact insects, by appearance of virus-specific siRNAs, primarily 21-nt long, and the increased expression of siRNA pathway core components Dicer-2 and Argonaute-2. Silencing of Dicer-2 using RNA interference strongly suppressed production of virus-specific siRNAs, promoted viral accumulation, and caused cytopathological changes in vitro and in vivo. When the viral accumulation level rose above a certain threshold of viral genome copy (1.32 × 10(14) copies/μg insect RNA), the infection of the leafhopper by RGDV was lethal rather than persistent. Taken together, our results revealed a new finding that the siRNA pathway in insect vector can modulate persistent infection of plant viruses. PMID:26864546

  15. [Small interfering RNA delivery mediated by mPEG-PCL-g-PEI polymer nanoparticles].

    PubMed

    Huang, Wei; Lü, Ming; Gao, Zhong-Gao; Jin, Ming-Ji; Yang, Chang-Qing

    2011-03-01

    The aim of this paper is to report the synthesis of the mPEG-PCL-g-PEI copolymers as small interfering RNA (siRNA) delivery vector, and exploration of the siRNA delivery potential of mPEG-PCL-g-PEI in vitro. The diblock copolymers mPEG-PCL-OH was prepared through the ring-opening polymerization. Then, the hydroxyl terminal (-OH) of mPEG-PCL-OH was chemically converted into the carboxy (-COOH) and N-hydroxysuccinimide (NHS) in turn to prepare mPEG-PCL-NHS. The branched PEI was reacted with mPEG-PCL-NHS to synthesize the ternary copolymers mPEG-PCL-g-PEI. The structure of mPEG-PCL-g-PEI copolymers was characterized with Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The mPEG-PCL-g-PEI/siRNA nanoparticles were prepared by complex coacervation, and the nanoparticles size and zeta potential were determined, separately. The cytotoxicities of mPEG-PCL-g-PEI/siRNA nanoparticles and PEI/siRNA nanoparticles were compared through cells MTT assays in vitro. The inhibition efficiencies of firefly luciferase gene expression by mPEG-PCL-g-PEI/ siRNA nanoparticle at various N/P ratios were investigated through cell transfection in vitro. The experimental results suggested that the ternary (mPEG5k-PCL(1.2k))1.4-g-PEI(10k) copolymers were successfully synthesized. (mPEG(5k)-PCL(1.2k))1.4-g-PEI(10k) could condense siRNA into nanoparticles (50-200 nm) with positive zeta potential. MTT assay results showed that the cytotoxicity of (mPEG(5k)-PCL(1.2k))1.4-g-PEI(10k)/siRNA nanoparticles was significantly lower than that of PEI(10k)/siRNA nanoparticles (P < 0.05). The expression of firefly luciferase gene could be significantly down-regulated at a range of N/P ratio from 50 to 150 (P < 0.01), and maximally inhibited at the N/P ratio of 125. The mPEG-PCL-g-PEI polymers could delivery siRNA into cells to inhibit the expression of target gene with very low cytotoxicity, which suggested that mPEG-PCL-g-PEI could serve

  16. Small self-RNA generated by RNase L amplifies antiviral innate immunity.

    PubMed

    Malathi, Krishnamurthy; Dong, Beihua; Gale, Michael; Silverman, Robert H

    2007-08-16

    Antiviral innate immunity is initiated in response to RNA molecules that are produced in virus-infected cells. These RNAs activate signalling cascades that activate the genes that encode alpha- and beta-interferon (IFN). Signalling occurs through the interaction of the RNAs with either of two pathogen recognition receptors, retinoic acid-inducible gene-I (RIG-I, also known as DDX58) and melanoma differentiation associated gene-5 (MDA5, also known as IFIH1), which contain amino-terminal caspase activation and recruitment domains (CARD) and carboxy-terminal DExD/H Box RNA helicase motifs. RIG-I and MDA5 interact with another CARD protein, interferon-beta promotor stimulator protein-1 (IPS-1, also known as MAVS, VISA and Cardif), in the mitochondrial membrane, which relays the signal through the transcription factors interferon regulatory factor 3 (IRF-3) and nuclear factor (NF)-kappaB to the IFN-beta gene. Although the signalling pathway is well understood, the origin of the RNA molecules that initiate these processes is not. Here we show that activation of the antiviral endoribonuclease, RNase L, by 2',5'-linked oligoadenylate (2-5A) produces small RNA cleavage products from self-RNA that initiate IFN production. Accordingly, mouse embryonic fibroblasts lacking RNase L were resistant to the induction of IFN-beta expression in response to 2-5A, dsRNA or viral infection. Single-stranded regions of RNA are cleaved 3' of UpUp and UpAp sequences by RNase L during viral infections, resulting in small, often duplex, RNAs. We show that small self-RNAs produced by the action of RNase L on cellular RNA induce IFN-beta expression and that the signalling involves RIG-I, MDA5 and IPS-1. Mice lacking RNase L produce significantly less IFN-beta during viral infections than infected wild-type mice. Furthermore, activation of RNase L with 2-5A in vivo induced the expression of IFN-beta in wild-type but not RNase L-deficient mice. Our results indicate that RNase L has an essential

  17. Ancient Origin of the U2 Small Nuclear RNA Gene-Targeting Non-LTR Retrotransposons Utopia

    PubMed Central

    Kojima, Kenji K.

    2015-01-01

    Most non-long terminal repeat (non-LTR) retrotransposons encoding a restriction-like endonuclease show target-specific integration into repetitive sequences such as ribosomal RNA genes and microsatellites. However, only a few target-specific lineages of non-LTR retrotransposons are distributed widely and no lineage is found across the eukaryotic kingdoms. Here we report the most widely distributed lineage of target sequence-specific non-LTR retrotransposons, designated Utopia. Utopia is found in three supergroups of eukaryotes: Amoebozoa, SAR, and Opisthokonta. Utopia is inserted into a specific site of U2 small nuclear RNA genes with different strength of specificity for each family. Utopia families from oomycetes and wasps show strong target specificity while only a small number of Utopia copies from reptiles are flanked with U2 snRNA genes. Oomycete Utopia families contain an “archaeal” RNase H domain upstream of reverse transcriptase (RT), which likely originated from a plant RNase H gene. Analysis of Utopia from oomycetes indicates that multiple lineages of Utopia have been maintained inside of U2 genes with few copy numbers. Phylogenetic analysis of RT suggests the monophyly of Utopia, and it likely dates back to the early evolution of eukaryotes. PMID:26556480

  18. Ancient Origin of the U2 Small Nuclear RNA Gene-Targeting Non-LTR Retrotransposons Utopia.

    PubMed

    Kojima, Kenji K; Jurka, Jerzy

    2015-01-01

    Most non-long terminal repeat (non-LTR) retrotransposons encoding a restriction-like endonuclease show target-specific integration into repetitive sequences such as ribosomal RNA genes and microsatellites. However, only a few target-specific lineages of non-LTR retrotransposons are distributed widely and no lineage is found across the eukaryotic kingdoms. Here we report the most widely distributed lineage of target sequence-specific non-LTR retrotransposons, designated Utopia. Utopia is found in three supergroups of eukaryotes: Amoebozoa, SAR, and Opisthokonta. Utopia is inserted into a specific site of U2 small nuclear RNA genes with different strength of specificity for each family. Utopia families from oomycetes and wasps show strong target specificity while only a small number of Utopia copies from reptiles are flanked with U2 snRNA genes. Oomycete Utopia families contain an "archaeal" RNase H domain upstream of reverse transcriptase (RT), which likely originated from a plant RNase H gene. Analysis of Utopia from oomycetes indicates that multiple lineages of Utopia have been maintained inside of U2 genes with few copy numbers. Phylogenetic analysis of RT suggests the monophyly of Utopia, and it likely dates back to the early evolution of eukaryotes. PMID:26556480

  19. Ancient Origin of the U2 Small Nuclear RNA Gene-Targeting Non-LTR Retrotransposons Utopia.

    PubMed

    Kojima, Kenji K; Jurka, Jerzy

    2015-01-01

    Most non-long terminal repeat (non-LTR) retrotransposons encoding a restriction-like endonuclease show target-specific integration into repetitive sequences such as ribosomal RNA genes and microsatellites. However, only a few target-specific lineages of non-LTR retrotransposons are distributed widely and no lineage is found across the eukaryotic kingdoms. Here we report the most widely distributed lineage of target sequence-specific non-LTR retrotransposons, designated Utopia. Utopia is found in three supergroups of eukaryotes: Amoebozoa, SAR, and Opisthokonta. Utopia is inserted into a specific site of U2 small nuclear RNA genes with different strength of specificity for each family. Utopia families from oomycetes and wasps show strong target specificity while only a small number of Utopia copies from reptiles are flanked with U2 snRNA genes. Oomycete Utopia families contain an "archaeal" RNase H domain upstream of reverse transcriptase (RT), which likely originated from a plant RNase H gene. Analysis of Utopia from oomycetes indicates that multiple lineages of Utopia have been maintained inside of U2 genes with few copy numbers. Phylogenetic analysis of RT suggests the monophyly of Utopia, and it likely dates back to the early evolution of eukaryotes.

  20. Filamentous pathogen effectors interfering with small RNA silencing in plant hosts.

    PubMed

    Ye, Wenwu; Ma, Wenbo

    2016-08-01

    Filamentous eukaryotic pathogens including fungi and oomycetes are major threats of plant health. During the co-evolutionary arms race with the hosts, these pathogens have evolved a large repertoire of secreted virulence proteins, called effectors, to facilitate colonization and infection. Many effectors are believed to directly manipulate targeted processes inside the host cells; and a fundamental function of the effectors is to dampen immunity. Recent evidence suggests that the destructive oomycete pathogens in the genus Phytophthora encode RNA silencing suppressors. These effectors play an important virulence role during infection, likely through their inhibitory effect on host small RNA-mediated defense. PMID:27104934

  1. A novel albumin nanocomplex containing both small interfering RNA and gold nanorods for synergetic anticancer therapy

    NASA Astrophysics Data System (ADS)

    Choi, Jin-Ha; Hwang, Hai-Jin; Shin, Seung Won; Choi, Jeong-Woo; Um, Soong Ho; Oh, Byung-Keun

    2015-05-01

    Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au nanorods per BSA complex and were successively functionalized with polyethylene glycol (PEG) and anti-ErbB-2 antibodies to facilitate active targeting. The synergetic therapeutic activity originating from the two components effectively induced cell death (~80% reduction in viability compared with control cells) in target breast cancer cells after a single dose of laser irradiation. Intracellular SREB nanocomplex decomposition by proteolytic enzymes resulted in simultaneous RNA interference and thermal ablation, thus leading to apoptosis in the targeted cancer cells. Moreover, these therapeutic effects were sustained for approximately 72 hours. The intrinsic biocompatibility, multifunctionality, and potent in vitro anticancer properties of these SREB nanocomplexes indicate that they have great therapeutic potential for in vivo targeted cancer therapy, in addition to other areas of nanomedicine.Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au

  2. Identification of Novel Small RNAs and Characterization of the 6S RNA of Coxiella burnetii

    PubMed Central

    Warrier, Indu; Hicks, Linda D.; Battisti, James M.; Raghavan, Rahul; Minnick, Michael F.

    2014-01-01

    Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and a dormant small cell variant (SCV). As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs) have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs). Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated (≥2-fold) during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS) was mapped to the 5′ UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella’s 6S RNA interacts with RNA polymerase (RNAP) in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium’s intracellular stress response by interacting with RNAP during

  3. CCR5 small interfering RNA ameliorated joint inflammation in rats with adjuvant-induced arthritis.

    PubMed

    Duan, Hongmei; Yang, Pingting; Fang, Fang; Ding, Shuang; Xiao, Weiguo

    2014-12-01

    Rheumatoid arthritis (RA) is a systemic inflammatory disease. C-C chemokine receptor type 5 (CCR5) is found in inflamed synovium of RA patients and is necessary for formation of RA. We aimed to check whether delivery of CCR5-specific small interfering RNA (siRNA) via electroporation suppresses local inflammation in arthritis rats. Vectors encoding siRNA that target CCR5 or negative control siRNA were constructed for gene silencing and the silencing effects of suppressing CCR5 expression in synovium examined by western blot. The vector with strongest effect was delivered into the knee joint of adjuvant-induced arthritis (AIA) rats by the in vivo electroporation method 7, 10, 13, and 16 days after immunization with Complete Freund's adjuvant. During an observation of 28 days, behavior, paw swelling, arthritis and histopathologic scoring were estimated. The expression level of CCR5 in synovium was evaluated by western blot and real-time PCR. Anti-CCR5 D1 siRNA was effectively inhibited CCR5 expression in vitro. Moreover, delivery of the siRNA into inflammatory joint also suppressed the expression of CCR5 in vivo and markedly suppressed paw swelling and inflammation. Local electroporation of anti-CCR5 siRNA into the left inflamed joints could achieve the silencing of CCR5 gene and alleviate local inflammation just in the knee joint injected with siRNA other than the opposite joint. Inhibition of CCR5 expression may provide a potential for treatment of RA.

  4. Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

    PubMed Central

    Bartlett, J S; Sethna, M; Ramamurthy, L; Gowen, S A; Samulski, R J; Marzluff, W F

    1996-01-01

    Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8799116

  5. A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics

    NASA Technical Reports Server (NTRS)

    Winker, S.; Woese, C. R.

    1991-01-01

    The number of small subunit rRNA sequences is now great enough that the three domains Archaea, Bacteria and Eucarya (Woese et al., 1990) can be reliably defined in terms of their sequence "signatures". Approximately 50 homologous positions (or nucleotide pairs) in the small subunit rRNA characterize and distinguish among the three. In addition, the three can be recognized by a variety of nonhomologous rRNA characters, either individual positions and/or higher-order structural features. The Crenarchaeota and the Euryarchaeota, the two archaeal kingdoms, can also be defined and distinguished by their characteristic compositions at approximately fifteen positions in the small subunit rRNA molecule.

  6. Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing.

    PubMed

    Falaleeva, Marina; Pages, Amadis; Matuszek, Zaneta; Hidmi, Sana; Agranat-Tamir, Lily; Korotkov, Konstantin; Nevo, Yuval; Eyras, Eduardo; Sperling, Ruth; Stamm, Stefan

    2016-03-22

    C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2'-O-methylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex,SNORD27 regulates the alternative splicing of the transcription factor E2F7p re-mRNA through direct RNA-RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously "silent" exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes. PMID:26957605

  7. Statistical Analysis Techniques for Small Sample Sizes

    NASA Technical Reports Server (NTRS)

    Navard, S. E.

    1984-01-01

    The small sample sizes problem which is encountered when dealing with analysis of space-flight data is examined. Because of such a amount of data available, careful analyses are essential to extract the maximum amount of information with acceptable accuracy. Statistical analysis of small samples is described. The background material necessary for understanding statistical hypothesis testing is outlined and the various tests which can be done on small samples are explained. Emphasis is on the underlying assumptions of each test and on considerations needed to choose the most appropriate test for a given type of analysis.

  8. Insights into the therapeutic potential of hypoxia-inducible factor-1α small interfering RNA in malignant melanoma delivered via folate-decorated cationic liposomes

    PubMed Central

    Chen, Zhongjian; Zhang, Tianpeng; Wu, Baojian; Zhang, Xingwang

    2016-01-01

    Malignant melanoma (MM) represents the most dangerous form of skin cancer, and its incidence is expected to rise in the coming time. However, therapy for MM is limited by low topical drug concentration and multidrug resistance. This article aimed to develop folate-decorated cationic liposomes (fc-LPs) for hypoxia-inducible factor-1α (HIF-1α) small interfering (siRNA) delivery, and to evaluate the potential of such siRNA/liposome complexes in MM therapy. HIF-1α siRNA-loaded fc-LPs (siRNA-fc-LPs) were prepared by a film hydration method followed by siRNA incubation. Folate decoration of liposomes was achieved by incorporation of folate/oleic acid-diacylated oligochitosans. The resulting siRNA-fc-LPs were 95.3 nm in size with a ζ potential of 2.41 mV. The liposomal vectors exhibited excellent loading capacity and protective effect toward siRNA. The in vitro cell transfection efficiency was almost parallel to the commercially available Lipofectamine™ 2000. Moreover, the anti-melanoma activity of HIF-1α siRNA was significantly enhanced through fc-LPs. Western blot analysis and apoptosis test demonstrated that siRNA-fc-LPs substantially reduced the production of HIF-1α-associated protein and induced the apoptosis of hypoxia-tolerant melanoma cells. Our designed liposomal vectors might be applicable as siRNA delivery vehicle to systemically or topically treat MM. PMID:27042054

  9. mRNA and microRNA expression profiles of radioresistant NCI-H520 non-small cell lung cancer cells

    PubMed Central

    GUO, WEI; XIE, LI; ZHAO, LONG; ZHAO, YUEHUAN

    2015-01-01

    To elucidate the mechanism of radioresistance in non-small cell lung cancer (NSCLC) cells and to identify key molecules conferring radioresistance, the radioresistant subclone NCI-H520/R, derived from the NCI-H520 NSCLC cell line, was established with eight rounds of sublethal irradiation. The radioresistant features were subsequently assessed using a clonogenic assay, analysis of apoptosis and an MTT assay, the gene expression levels were examined using an Agilent Whole Human Genome 4×44 k Oligo microarray and Agilent Human miRCURY™ LNA array, and confirmed by reverse transcription-quantitative polymerase chain reaction. Pathway analysis and Gene Ontology (GO) analysis were performed to determine the biological functions of the subset of differentially expressed genes. miRNA-mRNA correlation analysis between the expression levels of each miRNA and all its predicted target genes was performed to further understand the radioresistance in the NCI-H520 cells. Following eight rounds of sublethal irradiation, a total of 2,862 mRNAs were significantly differentially expressed in the NCI-H520/R cells, including 893 upregulated genes and 1,969 downregulated genes. A total of 162 upregulated miRNAs and 274 downregulated miRNAs were significantly deregulated in the NCI-H520/R cells. Multiple core regulatory processes and signaling pathways were identified as being of likely relevance to radioresistance in NCI-H520/R cells, including the mitogen-activated protein kinase signaling pathway and neurotrophin signaling pathway. The expression of genes associated with radioresistance reflects the complex biological processes involved in clinical cancer cell eradication and requires further investigation for future enhancement of therapy. PMID:25873351

  10. Expression of vascular endothelial growth factor mRNA in non-small-cell lung carcinomas.

    PubMed

    Fontanini, G; Boldrini, L; Chinè, S; Pisaturo, F; Basolo, F; Calcinai, A; Lucchi, M; Mussi, A; Angeletti, C A; Bevilacqua, G

    1999-01-01

    The vascular endothelial growth factor (VEGF) has been shown to be strictly related to vascular permeability and endothelial cell growth under physiological and pathological conditions. In tumour development and progression, VEGF plays a pivotal role in the development of the tumoral vascular network, and useful information in the progression of human cancer can be obtained by analysing the vascular endothelial growth factor expression of the tumours. In this study, we investigated the vascular endothelial growth factor transcript expression in non-small-cell lung carcinomas to evaluate the significance of this factor in a group of cancers in which the vascular pattern has been shown to significantly affect progression. Surgical samples of 42 patients with NSCLC were studied using reverse transcription polymerase chain reaction (PCR) analysis and in situ hybridization. Thirty-three out of 42 cases (78.6%) showed VEGF transcript expression predominantly as transcripts for the secretory forms of VEGF (isoforms 121 and 165). In situ hybridization, performed on 24 out of 42 samples, showed that the VEGF transcript expression was in several cases present in the cytoplasm both of neoplastic and normal cells, even if the VEGF mRNA was less expressed in the corresponding non-tumoral part. The VEGF 121 expression was associated with hilar and/or mediastinal nodal involvement (P = 0.02), and, taken together, the VEGF isoforms were shown to significantly influence overall (P = 0.02) and disease-free survival (P = 0.03). As a regulator of tumour angiogenesis, VEGF may represent a useful indicator of progression and poor prognosis in non-small-cell lung carcinomas.

  11. Nodamura virus B2 amino terminal domain sensitivity to small interfering RNA.

    PubMed

    Ali, P Shaik Syed; John, Jasmine; Selvaraj, Manikandan; Kek, Teh Lay; Salleh, Mohd Zaki

    2015-05-01

    Nodamura virus (NoV) B2, a suppressor of RNA interference, binds double stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) corresponding to Dicer substrates and products. Here, we report that the amino terminal domain of NoV B2 (NoV B2 79) specifically binds siRNAs but not dsRNAs. NoV B2 79 oligomerizes on binding to 27 nucleotide siRNA. Mutation of the residues phenylalanine49 and alanine60 to cysteine and methionine, respectively enhances the RNA binding affinity of NoV B2 79. Circular dichroism spectra demonstrated that the wild type and mutant NoV B2 79 have similar secondary structure conformations.

  12. Elucidation of the Mechanism of Gene Silencing using Small Interferin RNA: DNA Hybrid Molecules

    SciTech Connect

    Dugan, L

    2006-02-08

    The recent discovery that short hybrid RNA:DNA molecules (siHybrids) induce long-term silencing of gene expression in mammalian cells conflicts with the currently hypothesized mechanisms explaining the action of small, interfering RNA (siRNA). As a first step to elucidating the mechanism for this effect, we set out to quantify the delivery of siHybrids and determine their cellular localization in mammalian cells. We then tracked the segregation of the siHybrids into daughter cells after cell division. Markers for siHybrid delivery were shown to enter cells with and without the use of a transfection agent. Furthermore, delivery without transfection agent only occurred after a delay of 2-4 hours, suggesting a degradation process occurring in the cell culture media. Therefore, we studied the effects of nucleases and backbone modifications on the stability of siHybrids under cell culture conditions.

  13. Phylogeny of lobose amoebae based on actin and small-subunit ribosomal RNA genes.

    PubMed

    Fahrni, José F; Bolivar, Ignacio; Berney, Cédric; Nassonova, Elena; Smirnov, Alexey; Pawlowski, Jan

    2003-11-01

    Lobose amoebae are abundant free-living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyze actin and small-subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well-defined clades of amoebae, the "Gymnamoebia sensu stricto" and the Archamoebae (pelobionts + entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa.

  14. Therapeutic potential of small interfering RNAs/micro interfering RNA in hepatocellular carcinoma

    PubMed Central

    Farra, Rossella; Grassi, Mario; Grassi, Gabriele; Dapas, Barbara

    2015-01-01

    Hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer and represents the third leading cause of cancer-related death worldwide. Current available therapeutic approaches are poorly effective, especially for the advanced forms of the disease. In the last year, short double stranded RNA molecules termed small interfering RNAs (siRNAs) and micro interfering RNAs (miRNA), emerged as interesting molecules with potential therapeutic value for HCC. The practical use of these molecules is however limited by the identification of optimal molecular targets and especially by the lack of effective and targeted HCC delivery systems. Here we focus our discussion on the most recent advances in the identification of siRNAs/miRNAs molecular targets and on the development of suitable siRNA/miRNAs delivery systems. PMID:26290628

  15. Deep sequencing reveals unique small RNA repertoire that is regulated during head regeneration in Hydra magnipapillata

    PubMed Central

    Krishna, Srikar; Nair, Aparna; Cheedipudi, Sirisha; Poduval, Deepak; Dhawan, Jyotsna; Palakodeti, Dasaradhi; Ghanekar, Yashoda

    2013-01-01

    Small non-coding RNAs such as miRNAs, piRNAs and endo-siRNAs fine-tune gene expression through post-transcriptional regulation, modulating important processes in development, differentiation, homeostasis and regeneration. Using deep sequencing, we have profiled small non-coding RNAs in Hydra magnipapillata and investigated changes in small RNA expression pattern during head regeneration. Our results reveal a unique repertoire of small RNAs in hydra. We have identified 126 miRNA loci; 123 of these miRNAs are unique to hydra. Less than 50% are conserved across two different strains of Hydra vulgaris tested in this study, indicating a highly diverse nature of hydra miRNAs in contrast to bilaterian miRNAs. We also identified siRNAs derived from precursors with perfect stem–loop structure and that arise from inverted repeats. piRNAs were the most abundant small RNAs in hydra, mapping to transposable elements, the annotated transcriptome and unique non-coding regions on the genome. piRNAs that map to transposable elements and the annotated transcriptome display a ping–pong signature. Further, we have identified several miRNAs and piRNAs whose expression is regulated during hydra head regeneration. Our study defines different classes of small RNAs in this cnidarian model system, which may play a role in orchestrating gene expression essential for hydra regeneration. PMID:23166307

  16. Developmental regulation of cryptdin, a corticostatin/defensin precursor mRNA in mouse small intestinal crypt epithelium

    PubMed Central

    1989-01-01

    Cryptdin mRNA codes for the apparent precursor to a corticostatin/defensin-related peptide that accumulates to high levels in mouse intestinal crypt epithelium during postnatal development. The primary structure, intestinal cell distribution, and developmental appearance of cryptdin mRNA have been determined. Cryptdin mRNA is 450- 480 nucleotides long. Translation of the partial cryptdin cDNA sequence reveals a 70-amino acid open reading frame that includes 32 carboxy- terminal residues that align with those in the consensus sequence, C.CR...C....ER..G.C....CCR, which is a common feature of leukocyte defensins and lung corticostatins (Selsted, M. E., D. M. Brown, R. J. DeLange, S. S. L. Harwig, and R. I. Lehrer. 1985. J. Biol. Chem. 260:4579-4584; Zhu, Q., J. Hu, S. Mulay, F. Esch, S. Shimasaki, and S. Solomon. 1988. Proc. Natl. Acad. Sci. USA. 85:592-596). In situ hybridization of cryptdin cDNA to paraformaldehyde-fixed, frozen sections of adult jejunum and ileum showed intense and specific labeling of epithelial cells in the base of all crypts. Analysis of sections from suckling mice showed that cryptdin mRNA is detectable in 10-20% of crypts in 10-d-old mice, in approximately 80% of crypts in 16- d-old mice, and in all crypts of mice 20 d and older. During the fourth week, the sequence accumulates in crypts to the maximal adult level. Cryptdin mRNA content in adult small intestine is independent both of T cell involvement and luminal bacteria. The role of cryptdin in small bowel physiology remains to be determined: cryptdin may inhibit bacterial translocation, modulate intestinal hormone synthesis, influence hormonal sensitivity of the intestinal epithelium, or exhibit a multiplicity of related activities. PMID:2715173

  17. Quality Control and Analysis of NGS RNA Sequencing Data.

    PubMed

    Quinn, Emma M; McManus, Ross

    2015-01-01

    Transcriptome sequencing, where RNA is isolated, converted to library of cDNA fragments, and sequenced using next-generation sequencing technology, has become the method of choice for the genome-wide characterization of mRNA levels. It offers a more accurate quantification of transcript levels than array-based methods, but also has the added benefit of allowing the discovery of novel gene/transcripts, alternative splice junctions, and novel RNAs. In addition, RNA sequencing may be used to investigate differential gene expression, allelic imbalance, eQTL mapping, RNA editing, RNA-protein interactions, and alternative splicing. A number of statistical methods and tools are available for differential expression analysis using RNA sequencing data and these are continually being developed and improved to handle more complex experimental designs. This chapter describes an example workflow for the quality control and analysis of raw RNA sequencing reads for the purposes of differential gene expression analysis, followed by pathway/enrichment analysis of significantly different genes. The methods and tools described are just one example of how this analysis can be conducted, but they can be applied to most standard RNA sequencing studies of differential gene expression. The methods covered are based on Illumina HiSeq single-end 50 bp reads. However, all programs used are capable of working with paired-end data, subsequent to minor adaptations.

  18. miARma-Seq: a comprehensive tool for miRNA, mRNA and circRNA analysis

    PubMed Central

    Andrés-León, Eduardo; Núñez-Torres, Rocío; Rojas, Ana M.

    2016-01-01

    Large-scale RNAseq has substantially changed the transcriptomics field, as it enables an unprecedented amount of high resolution data to be acquired. However, the analysis of these data still poses a challenge to the research community. Many tools have been developed to overcome this problem, and to facilitate the study of miRNA expression profiles and those of their target genes. While a few of these enable both kinds of analysis to be performed, they also present certain limitations in terms of their requirements and/or the restrictions on data uploading. To avoid these restraints, we have developed a suite that offers the identification of miRNA, mRNA and circRNAs that can be applied to any sequenced organism. Additionally, it enables differential expression, miRNA-mRNA target prediction and/or functional analysis. The miARma-Seq pipeline is presented as a stand-alone tool that is both easy to install and flexible in terms of its use, and that brings together well-established software in a single bundle. Our suite can analyze a large number of samples due to its multithread design. By testing miARma-Seq in validated datasets, we demonstrate here the benefits that can be gained from this tool by making it readily accessible to the research community. PMID:27167008

  19. Molecular Evolution of Multi-subunit RNA Polymerases: Sequence Analysis

    PubMed Central

    Lane, William J.; Darst, Seth A.

    2009-01-01

    Transcription in all cellular organisms is performed by multi-subunit, DNA-dependent RNA polymerases that synthesize RNA from DNA templates. Previous sequence and structural studies have elucidated the importance of shared regions common to all multi-subunit RNA polymerases. In addition RNA polymerases contain multiple lineage-specific domain insertions involved in protein-protein and protein-nucleic acid interactions. We have created comprehensive multiple sequence alignments using all available sequence data for the multi-subunit RNA polymerase large subunits, including the bacterial β and β′ subunits and their homologues from archaebacterial RNA polymerases, the eukaryotic RNA polymerases I, II, and III, the nuclear-cytoplasmic large double-stranded DNA Virus RNA polymerases, and plant plastid RNA polymerases. In order to overcome technical difficulties inherent to the large subunit sequences, including large sequence length, small and large lineage-specific insertions, split subunits, and fused proteins, we created an automated and customizable sequence retrieval and processing system. In addition, we used our alignments to create a more expansive set of shared sequence regions and bacterial lineage-specific domain insertions. We also analyzed the intergenic gap between the bacterial β and β′ genes. PMID:19895820

  20. Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

    PubMed Central

    Wroblewska, Zuzanna; Olejniczak, Mikolaj

    2016-01-01

    The bacterial protein Hfq participates in the regulation of translation by small noncoding RNAs (sRNAs). Several mechanisms have been proposed to explain the role of Hfq in the regulation by sRNAs binding to the 5′-untranslated mRNA regions. However, it remains unknown how Hfq affects those sRNAs that target the coding sequence. Here, the contribution of Hfq to the annealing of three sRNAs, RybB, SdsR, and MicC, to the coding sequence of Salmonella ompD mRNA was investigated. Hfq bound to ompD mRNA with tight, subnanomolar affinity. Moreover, Hfq strongly accelerated the rates of annealing of RybB and MicC sRNAs to this mRNA, and it also had a small effect on the annealing of SdsR. The experiments using truncated RNAs revealed that the contributions of Hfq to the annealing of each sRNA were individually adjusted depending on the structures of interacting RNAs. In agreement with that, the mRNA structure probing revealed different structural contexts of each sRNA binding site. Additionally, the annealing of RybB and MicC sRNAs induced specific conformational changes in ompD mRNA consistent with local unfolding of mRNA secondary structure. Finally, the mutation analysis showed that the long AU-rich sequence in the 5′-untranslated mRNA region served as an Hfq binding site essential for the annealing of sRNAs to the coding sequence. Overall, the data showed that the functional specificity of Hfq in the annealing of each sRNA to the ompD mRNA coding sequence was determined by the sequence and structure of the interacting RNAs. PMID:27154968

  1. Small angle scattering study of the structure and organization of RNA and protein in Brome Mosaic Virus (BMV)

    NASA Astrophysics Data System (ADS)

    Das, Narayan C.; Warren, Garfield T.; Cheng, Si; Kao, C. Cheng; Ni, Peng; Dragnea, Bogdan; Sokol, Paul E.

    2012-02-01

    Brome mosaic virus (BMV) is a small icosahedral of the alpha virus-like superfamily of RNA with a segmented positive-strand RNA genome and a mean diameter ˜ 268å that offers high levels of RNA synthesis and virus production in plants. BMV also tightly regulates the packaging of its four RNAs (RNA1 through RNA4) into three separate particles; RNA1 and RNA2 are encapsidated separately while one copy each of RNA3 and RNA4 are normally packaged together. Small angle neutron scattering (SANS) and small angle X-ray scattering (SAXS) were applied to study the size, shape and protein-RNA organization of BMV. D2O/H2O mixture was used to enhance contrast in SANS measurement. The radial distribution of BMV from the Fourier transform of scattering spectrum gives a clear indication of RNA packing, and distribution and their structure in the BMV. The result reveals that the virus is about 266 å in diameter and is composed of RNA inside the virion coated with a protein shell.

  2. Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase

    PubMed Central

    Xu, Hong-Tao; Colby-Germinario, Susan P.; Hassounah, Said; Quashie, Peter K.; Han, Yingshan; Oliveira, Maureen; Stranix, Brent R.

    2015-01-01

    The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 μM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor. PMID:26574011

  3. miEAA: microRNA enrichment analysis and annotation

    PubMed Central

    Backes, Christina; Khaleeq, Qurratulain T.; Meese, Eckart; Keller, Andreas

    2016-01-01

    Similar to the development of gene set enrichment and gene regulatory network analysis tools over a decade ago, microRNA enrichment tools are currently gaining importance. Building on our experience with the gene set analysis toolkit GeneTrail, we implemented the miRNA Enrichment Analysis and Annotation tool (miEAA). MiEAA is a web-based application that offers a variety of commonly applied statistical tests such as over-representation analysis and miRNA set enrichment analysis, which is similar to Gene Set Enrichment Analysis. Besides the different statistical tests, miEAA also provides rich functionality in terms of miRNA categories. Altogether, over 14 000 miRNA sets have been added, including pathways, diseases, organs and target genes. Importantly, our tool can be applied for miRNA precursors as well as mature miRNAs. To make the tool as useful as possible we additionally implemented supporting tools such as converters between different miRBase versions and converters from miRNA names to precursor names. We evaluated the performance of miEAA on two sets of miRNAs that are affected in lung adenocarcinomas and have been detected by array analysis. The web-based application is freely accessible at: http://www.ccb.uni-saarland.de/mieaa_tool/. PMID:27131362

  4. tRF2Cancer: A web server to detect tRNA-derived small RNA fragments (tRFs) and their expression in multiple cancers

    PubMed Central

    Zheng, Ling-Ling; Xu, Wei-Lin; Liu, Shun; Sun, Wen-Ju; Li, Jun-Hao; Wu, Jie; Yang, Jian-Hua; Qu, Liang-Hu

    2016-01-01

    tRNA-derived small RNA fragments (tRFs) are one class of small non-coding RNAs derived from transfer RNAs (tRNAs). tRFs play important roles in cellular processes and are involved in multiple cancers. High-throughput small RNA (sRNA) sequencing experiments can detect all the cellular expressed sRNAs, including tRFs. However, distinguishing genuine tRFs from RNA fragments generated by random degradation remains a major challenge. In this study, we developed an integrated web-based computing system, tRF2Cancer, to accurately identify tRFs from sRNA deep-sequencing data and evaluate their expression in multiple cancers. The binomial test was introduced to evaluate whether reads from a small RNA-seq data set represent tRFs or degraded fragments. A classification method was then used to annotate the types of tRFs based on their sites of origin in pre-tRNA or mature tRNA. We applied the pipeline to analyze 10 991 data sets from 32 types of cancers and identified thousands of expressed tRFs. A tool called ‘tRFinCancer’ was developed to facilitate the users to inspect the expression of tRFs across different types of cancers. Another tool called ‘tRFBrowser’ shows both the sites of origin and the distribution of chemical modification sites in tRFs on their source tRNA. The tRF2Cancer web server is available at http://rna.sysu.edu.cn/tRFfinder/. PMID:27179031

  5. tRF2Cancer: A web server to detect tRNA-derived small RNA fragments (tRFs) and their expression in multiple cancers.

    PubMed

    Zheng, Ling-Ling; Xu, Wei-Lin; Liu, Shun; Sun, Wen-Ju; Li, Jun-Hao; Wu, Jie; Yang, Jian-Hua; Qu, Liang-Hu

    2016-07-01

    tRNA-derived small RNA fragments (tRFs) are one class of small non-coding RNAs derived from transfer RNAs (tRNAs). tRFs play important roles in cellular processes and are involved in multiple cancers. High-throughput small RNA (sRNA) sequencing experiments can detect all the cellular expressed sRNAs, including tRFs. However, distinguishing genuine tRFs from RNA fragments generated by random degradation remains a major challenge. In this study, we developed an integrated web-based computing system, tRF2Cancer, to accurately identify tRFs from sRNA deep-sequencing data and evaluate their expression in multiple cancers. The binomial test was introduced to evaluate whether reads from a small RNA-seq data set represent tRFs or degraded fragments. A classification method was then used to annotate the types of tRFs based on their sites of origin in pre-tRNA or mature tRNA. We applied the pipeline to analyze 10 991 data sets from 32 types of cancers and identified thousands of expressed tRFs. A tool called 'tRFinCancer' was developed to facilitate the users to inspect the expression of tRFs across different types of cancers. Another tool called 'tRFBrowser' shows both the sites of origin and the distribution of chemical modification sites in tRFs on their source tRNA. The tRF2Cancer web server is available at http://rna.sysu.edu.cn/tRFfinder/.

  6. tRF2Cancer: A web server to detect tRNA-derived small RNA fragments (tRFs) and their expression in multiple cancers.

    PubMed

    Zheng, Ling-Ling; Xu, Wei-Lin; Liu, Shun; Sun, Wen-Ju; Li, Jun-Hao; Wu, Jie; Yang, Jian-Hua; Qu, Liang-Hu

    2016-07-01

    tRNA-derived small RNA fragments (tRFs) are one class of small non-coding RNAs derived from transfer RNAs (tRNAs). tRFs play important roles in cellular processes and are involved in multiple cancers. High-throughput small RNA (sRNA) sequencing experiments can detect all the cellular expressed sRNAs, including tRFs. However, distinguishing genuine tRFs from RNA fragments generated by random degradation remains a major challenge. In this study, we developed an integrated web-based computing system, tRF2Cancer, to accurately identify tRFs from sRNA deep-sequencing data and evaluate their expression in multiple cancers. The binomial test was introduced to evaluate whether reads from a small RNA-seq data set represent tRFs or degraded fragments. A classification method was then used to annotate the types of tRFs based on their sites of origin in pre-tRNA or mature tRNA. We applied the pipeline to analyze 10 991 data sets from 32 types of cancers and identified thousands of expressed tRFs. A tool called 'tRFinCancer' was developed to facilitate the users to inspect the expression of tRFs across different types of cancers. Another tool called 'tRFBrowser' shows both the sites of origin and the distribution of chemical modification sites in tRFs on their source tRNA. The tRF2Cancer web server is available at http://rna.sysu.edu.cn/tRFfinder/. PMID:27179031

  7. Inhibition of Non-ATG Translational Events in Cells via Covalent Small Molecules Targeting RNA.

    PubMed

    Yang, Wang-Yong; Wilson, Henry D; Velagapudi, Sai Pradeep; Disney, Matthew D

    2015-04-29

    One major class of disease-causing RNAs is expanded repeating transcripts. These RNAs cause diseases via multiple mechanisms, including: (i) gain-of-function, in which repeating RNAs bind and sequester proteins involved in RNA biogenesis and (ii) repeat associated non-ATG (RAN) translation, in which repeating transcripts are translated into toxic proteins without use of a canonical, AUG, start codon. Herein, we develop and study chemical probes that bind and react with an expanded r(CGG) repeat (r(CGG)(exp)) present in a 5' untranslated region that causes fragile X-associated tremor/ataxia syndrome (FXTAS). Reactive compounds bind to r(CGG)(exp) in cellulo as shown with Chem-CLIP-Map, an approach to map small molecule binding sites within RNAs in cells. Compounds also potently improve FXTAS-associated pre-mRNA splicing and RAN translational defects, while not affecting translation of the downstream open reading frame. In contrast, oligonucleotides affect both RAN and canonical translation when they bind to r(CGG)(exp), which is mechanistically traced to a decrease in polysome loading. Thus, designer small molecules that react with RNA targets can be used to profile the RNAs to which they bind in cells, including identification of binding sites, and can modulate several aspects of RNA-mediated disease pathology in a manner that may be more beneficial than oligonucleotides.

  8. Novel small Cajal-body-specific RNAs identified in Drosophila: probing guide RNA function

    PubMed Central

    Deryusheva, Svetlana; Gall, Joseph G.

    2013-01-01

    The spliceosomal small nuclear RNAs (snRNAs) are modified post-transcriptionally by introduction of pseudouridines and 2′-O-methyl modifications, which are mediated by box H/ACA and box C/D guide RNAs, respectively. Because of their concentration in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific (sca) RNAs. In the cell, scaRNAs are associated with the WD-repeat protein WDR79. We used coimmunoprecipitation with WDR79 to recover seven new scaRNAs from Drosophila cell lysates. We demonstrated concentration of these new scaRNAs in the CB by in situ hybridization, and we verified experimentally that they can modify their putative target RNAs. Surprisingly, one of the new scaRNAs targets U6 snRNA, whose modification is generally assumed to occur in the nucleolus, not in the CB. Two other scaRNAs have dual guide functions, one for an snRNA and one for 28S rRNA. Again, the modification of 28S rRNA is assumed to take place in the nucleolus. These findings suggest that canonical scaRNAs may have functions in addition to their established role in modifying U1, U2, U4, and U5 snRNAs. We discuss the likelihood that processing by scaRNAs is not limited to the CB. PMID:24149844

  9. Delivery of small interfering RNA for inhibition of endothelial cell apoptosis by hypoxia and serum deprivation

    SciTech Connect

    Cho, Seung-Woo; Hartle, Lauren; Son, Sun Mi; Yang, Fan; Goldberg, Michael; Xu, Qiaobing; Langer, Robert; Anderson, Daniel G.

    2008-11-07

    RNA interference (RNAi) for anti-angiogenic or pro-apoptotic factors in endothelial cells (ECs) has great potential for the treatment of ischemic diseases by promoting angiogenesis or inhibiting apoptosis. Here, we report the utility of small interfering RNA (siRNA) in inhibiting EC apoptosis induced by tumor necrosis factor-{alpha} (TNF-{alpha}). siRNA was designed and synthesized targeting tumor necrosis factor-{alpha} receptor-1 (TNFR-1) and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were cultured under in vitro hypoxic and serum-deprived conditions to simulate in vivo ischemic conditions. Two days after liposomal delivery of siRNA targeting TNFR-1 and SHP-1, significant silencing of each target (TNFR-1; 76.5% and SHP-1; 97.2%) was detected. Under serum-deprived hypoxic (1% oxygen) conditions, TNF-{alpha} expression in HUVECs increased relative to normoxic (20% oxygen) and serum-containing conditions. Despite enhanced TNF-{alpha} expression, suppression of TNFR-1 or SHP-1 by siRNA delivery not only enhanced expression of angiogenic factors (KDR/Flk-1 and eNOS) and anti-apoptotic factor (Bcl-xL) but also reduced expression of a pro-apoptotic factor (Bax). Transfection of TNFR-1 or SHP-1 siRNA significantly decreased the HUVEC apoptosis while significantly enhancing HUVEC proliferation and capillary formation. The present study demonstrates that TNFR-1 and SHP-1 may be useful targets for the treatment of myocardial or hindlimb ischemia.

  10. Infrared multiphoton dissociation of small-interfering RNA anions and cations.

    PubMed

    Gardner, Myles W; Li, Na; Ellington, Andrew D; Brodbelt, Jennifer S

    2010-04-01

    Infrared multiphoton dissociation (IRMPD) on a linear ion trap mass spectrometer is applied for the sequencing of small interfering RNA (siRNA). Both single-strand siRNAs and duplex siRNA were characterized by IRMPD, and the results were compared with that obtained by traditional ion trap-based collision induced dissociation (CID). The single-strand siRNA anions were observed to dissociate via cleavage of the 5' P-O bonds yielding c- and y-type product ions as well as undergo neutral base loss. Full sequence coverage of the siRNA anions was obtained by both IRMPD and CID. While the CID mass spectra were dominated by base loss ions, accounting for approximately 25% to 40% of the product ion current, these ions were eliminated through secondary dissociation by increasing the irradiation time in the IRMPD mass spectra to produce higher abundances of informative sequence ions. With longer irradiation times, however, internal ions corresponding to cleavage of two 5' P-O bonds began to populate the product ion mass spectra as well as higher abundances of [a - Base] and w-type ions. IRMPD of siRNA cations predominantly produced c- and y-type ions with minimal contributions of [a - Base] and w-type ions to the product ion current; the presence of only two complementary series of product ions in the IRMPD mass spectra simplified spectral interpretation. In addition, IRMPD produced high abundances of protonated nucleobases, [G + H](+), [A + H](+), and [C + H](+), which were not detected in the CID mass spectra due to the low-mass cut-off associated with conventional CID in ion traps. CID and IRMPD using short irradiation times of duplex siRNA resulted in strand separation, similar to the dissociation trends observed for duplex DNA. With longer irradiation times, however, the individual single-strands underwent secondary dissociation to yield informative sequence ions not obtained by CID.

  11. A Stress-Induced Small RNA Modulates Alpha-Rhizobial Cell Cycle Progression

    PubMed Central

    Robledo, Marta; Frage, Benjamin; Wright, Patrick R.; Becker, Anke

    2015-01-01

    Mechanisms adjusting replication initiation and cell cycle progression in response to environmental conditions are crucial for microbial survival. Functional characterization of the trans-encoded small non-coding RNA (trans-sRNA) EcpR1 in the plant-symbiotic alpha-proteobacterium Sinorhizobium meliloti revealed a role of this class of riboregulators in modulation of cell cycle regulation. EcpR1 is broadly conserved in at least five families of the Rhizobiales and is predicted to form a stable structure with two defined stem-loop domains. In S. meliloti, this trans-sRNA is encoded downstream of the divK-pleD operon. ecpR1 belongs to the stringent response regulon, and its expression was induced by various stress factors and in stationary phase. Induced EcpR1 overproduction led to cell elongation and increased DNA content, while deletion of ecpR1 resulted in reduced competitiveness. Computationally predicted EcpR1 targets were enriched with cell cycle-related mRNAs. Post-transcriptional repression of the cell cycle key regulatory genes gcrA and dnaA mediated by mRNA base-pairing with the strongly conserved loop 1 of EcpR1 was experimentally confirmed by two-plasmid differential gene expression assays and compensatory changes in sRNA and mRNA. Evidence is presented for EcpR1 promoting RNase E-dependent degradation of the dnaA mRNA. We propose that EcpR1 contributes to modulation of cell cycle regulation under detrimental conditions. PMID:25923724

  12. A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum.

    PubMed

    Carbone, I; Anderson, J B; Kohn, L M

    1995-01-01

    A 1,380-bp intervening sequence within the mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of the fungus Sclerotinia sclerotiorum has been sequenced and identified as a group-I intron. This is the first report of an intron in the mt SSU rRNA gene. The intron shows close similarity in secondary structure to the subgroup-IC2 introns from Podospora (ND3i1, ND5i2, and COIi5) and Neurospora (ND5i1). The intron has an open reading frame (ORF) that encodes a putative protein of 420 amino acids which contains two copies of the LAGLI-DADG motif. The ORF belongs to a family of ORFs identified in Podospora (ND3i1, ND4Li1, ND4Li2, ND5i2, and COIi5) and Neurospora (ND5i1). The putative 420-aa polypeptide is also similar to a site-specific endonuclease in the chloroplast large subunit ribosomal RNA (LSU rRNA) gene of the green alga Chlamydomonas eugametos. In each clone of S. sclerotiorum examined, including several clones which were sampled over a 3-year period from geographically separated sites, all isolates either had the intron or lacked the intron within the mt SSU rRNA gene. Screening by means of Southern hybridization and PCR amplification detected the intron in the mt SSU rRNA genes of S. minor, S. trifoliorum and Sclerotium cepivorum, but not in other members of the Sclerotiniaceae, such as Botrytis anamorphs of Botryotinia spp., or in other ascomycetous and basidiomycetous fungi. PMID:7788720

  13. Identification of Survival Genes in Human Glioblastoma Cells by Small Interfering RNA Screening

    PubMed Central

    Thaker, Nikhil G.; Zhang, Fang; McDonald, Peter R.; Shun, Tong Ying; Lewen, Michael D.; Pollack, Ian F.

    2009-01-01

    Target identification and validation remain difficult steps in the drug discovery process, and uncovering the core genes and pathways that are fundamental for cancer cell survival may facilitate this process. Glioblastoma represents a challenging form of cancer for chemotherapy. Therefore, we assayed 16,560 short interfering RNA (siRNA) aimed at identifying which of the 5520 unique therapeutically targetable gene products were important for the survival of human glioblastoma. We analyzed the viability of T98G glioma cells 96 h after siRNA transfection with two orthogonal statistical methods and identified 55 survival genes that encoded proteases, kinases, and transferases. It is noteworthy that 22% (12/55) of the survival genes were constituents of the 20S and 26S proteasome subunits. An expression survey of a panel of glioma cell lines demonstrated expression of the proteasome component PSMB4, and the validity of the proteasome complex as a target for survival inhibition was confirmed in a series of glioma and nonglioma cell lines by pharmacological inhibition and RNA interference. Biological networks were built with the other survival genes using a protein-protein interaction network, which identified clusters of cellular processes, including protein ubiquitination, purine and pyrimidine metabolism, nucleotide excision repair, and NF-κB signaling. The results of this study should broaden our understanding of the core genes and pathways that regulate cell survival; through either small molecule inhibition or RNA interference, we highlight the potential significance of proteasome inhibition. PMID:19783622

  14. Inhibition of pathologic immunoglobulin free light chain production by small interfering RNA molecules

    PubMed Central

    Phipps, Jonathan E.; Kestler, Daniel P.; Foster, James S.; Kennel, Stephen J.; Donnell, Robert; Weiss, Deborah T.; Solomon, Alan; Wall, Jonathan S.

    2010-01-01

    Objectives Morbidity and mortality occurring in patients with multiple myeloma, AL amyloidosis, and light chain deposition disease can result from the pathologic deposition of monoclonal Ig light chains (LCs) in kidneys and other organs. To reduce synthesis of such components, therapy for these disorders typically has involved anti-plasma cell agents; however, this approach is not always effective and can have adverse consequences. We have investigated another means to achieve this objective; namely, RNA interference (RNAi). Materials and Methods SP2/O mouse myeloma cells were stably transfected with a construct encoding a λ6 LC (Wil) under control of the CMV promoter, while λ2-producing myeloma cell line RPMI 8226 was purchased from the ATCC. Both were treated with small interfering RNA (siRNA) directed specifically to the V, J, or C portions of the molecules and then analyzed by ELISA, flow cytometry and real time PCR. Results Transfected cells were found to constitutively express detectable quantities of mRNA and protein Wil and, after exposure to siRNAs, an ~40% reduction in mRNA and LC production was evidenced at 48 hours. An even greater effect was seen with the 8226 cells. Conclusion Our results have shown that RNAi can markedly reduce LC synthesis and provide the basis for testing the therapeutic potential of this strategy using in vivo experimental models of multiple myeloma. PMID:20637260

  15. The RNA-Seq Analysis pipeline on Galaxy

    SciTech Connect

    Meng, Xiandong; Martin, Jeffrey; Wang, Zhong

    2011-05-31

    Q: How do I know my RNA-Seq experiments worked well A: RNA-Seq QC PipelineQ: How do I detect transcripts which are over expressed or under expressed in my samples A: Counting and Statistic AnalysisQ: What do I do if I don't have a reference genome A: Rnnotator de novo Assembly.

  16. A comparison of two cellular delivery mechanisms for small interfering RNA.

    PubMed

    Valiunas, Virginijus; Wang, Hong-Zhang; Li, Ling; Gordon, Chris; Valiuniene, Laima; Cohen, Ira S; Brink, Peter R

    2015-02-01

    Cellular delivery of small interfering RNAs to target cells of a tissue has the potential to travel by two intercellular pathways. For intimately apposed cells gap junctions allow transport exclusive of the extracellular space. For cells not in intimate contact, exocytotic release of vesicular contents and subsequent retrieval via endocytosis of exosomes and other vesicular contents represent an alternative intercellular delivery system that utilizes the extracellular space. Previous studies have shown siRNA/miRNA transfer from a delivery cell to a target cell via gap junction channels. We hypothesized that siRNA can be delivered via gap junctions and downregulate the expression of a reporter gene, the cyclic nucleotide-gated cation channel gene (mHCN2), in the recipient cells of cell pairs. Whole-cell patch clamp was used to measure the mHCN2-induced current and junctional conductance. The target cells were HEK293 cells that endogenously express Cx43 or HeLaCx43 cells, both transfected with mHCN2. The source cells were HEK293 or HeLaCx43 cells transfected with fluorescent-labeled siRNA targeting mHCN2. We found that siRNA targeting mHCN2 resulted in significant downregulation of mHCN2 currents both in single cells and the recipient cell of a cell pair. In addition we also documented downregulation in target cells that were not in contact with source cells suggesting an extracellular-mediated delivery. To test further for extracellular delivery HEK293/HCN2 or HeLaCx43/HCN2 cells were cultured in medium collected from HEK293 or HeLaCx43 cells transfected with fluorescent-labeled siRNA or fluorescent-labeled morpholino designed to target HCN2. After 24 h single HEK293/HCN2 or HeLaCx43cells showed accumulation of siRNA. The mHCN2 currents were also down regulated in cells with siRNA uptake. Application of 200 nmol/L Bafilomycin A1, which has been shown to affect endosome acidification and endocytotic activity, resulted in a smaller accumulation of fluorescent

  17. The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti

    PubMed Central

    Baumgardt, Kathrin; Šmídová, Klára; Rahn, Helen; Lochnit, Günter; Robledo, Marta; Evguenieva-Hackenberg, Elena

    2016-01-01

    ABSTRACT Quorum sensing is a cell density-dependent communication system of bacteria relying on autoinducer molecules. During the analysis of the post-transcriptional regulation of quorum sensing in the nitrogen fixing plant symbiont Sinorhizobium meliloti, we predicted and verified a direct interaction between the 5'-UTR of sinI mRNA encoding the autoinducer synthase and a small RNA (sRNA), which we named RcsR1. In vitro, RcsR1 prevented cleavage in the 5'-UTR of sinI by RNase E and impaired sinI translation. In line with low ribosomal occupancy and transcript destabilization upon binding of RcsR1 to sinI, overproduction of RcsR1 in S. meliloti resulted in lower level and shorter half-life of sinI mRNA, and in decreased autoinducer amount. Although RcsR1 can influence quorum sensing via sinI, its level did not vary at different cell densities, but decreased under salt stress and increased at low temperature. We found that RcsR1 and its stress-related expression pattern, but not the interaction with sinI homologs, are conserved in Sinorhizobium, Rhizobium and Agrobacterium. Consistently, overproduction of RcsR1 in S. meliloti and Agrobacterium tumefaciens inhibited growth at high salinity. We identified conserved targets of RcsR1 and showed that most conserved interactions and the effect on growth under salt stress are mediated by the first stem-loop of RcsR1, while its central part is responsible for the species-specific interaction with sinI. We conclude that RcsR1 is an ancient, stress-related riboregulator in rhizobia and propose that it links stress responses to quorum sensing in S. meliloti. PMID:26588798

  18. The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti.

    PubMed

    Baumgardt, Kathrin; Šmídová, Klára; Rahn, Helen; Lochnit, Günter; Robledo, Marta; Evguenieva-Hackenberg, Elena

    2016-05-01

    Quorum sensing is a cell density-dependent communication system of bacteria relying on autoinducer molecules. During the analysis of the post-transcriptional regulation of quorum sensing in the nitrogen fixing plant symbiont Sinorhizobium meliloti, we predicted and verified a direct interaction between the 5'-UTR of sinI mRNA encoding the autoinducer synthase and a small RNA (sRNA), which we named RcsR1. In vitro, RcsR1 prevented cleavage in the 5'-UTR of sinI by RNase E and impaired sinI translation. In line with low ribosomal occupancy and transcript destabilization upon binding of RcsR1 to sinI, overproduction of RcsR1 in S. meliloti resulted in lower level and shorter half-life of sinI mRNA, and in decreased autoinducer amount. Although RcsR1 can influence quorum sensing via sinI, its level did not vary at different cell densities, but decreased under salt stress and increased at low temperature. We found that RcsR1 and its stress-related expression pattern, but not the interaction with sinI homologs, are conserved in Sinorhizobium, Rhizobium and Agrobacterium. Consistently, overproduction of RcsR1 in S. meliloti and Agrobacterium tumefaciens inhibited growth at high salinity. We identified conserved targets of RcsR1 and showed that most conserved interactions and the effect on growth under salt stress are mediated by the first stem-loop of RcsR1, while its central part is responsible for the species-specific interaction with sinI. We conclude that RcsR1 is an ancient, stress-related riboregulator in rhizobia and propose that it links stress responses to quorum sensing in S. meliloti. PMID:26588798

  19. Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

    PubMed Central

    Falaleeva, Marina; Pages, Amadis; Matuszek, Zaneta; Hidmi, Sana; Agranat-Tamir, Lily; Korotkov, Konstantin; Nevo, Yuval; Sperling, Ruth; Stamm, Stefan

    2016-01-01

    C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2′-O-methylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex, SNORD27 regulates the alternative splicing of the transcription factor E2F7 pre-mRNA through direct RNA–RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously “silent” exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes. PMID:26957605

  20. SURVIV for survival analysis of mRNA isoform variation

    PubMed Central

    Shen, Shihao; Wang, Yuanyuan; Wang, Chengyang; Wu, Ying Nian; Xing, Yi

    2016-01-01

    The rapid accumulation of clinical RNA-seq data sets has provided the opportunity to associate mRNA isoform variations to clinical outcomes. Here we report a statistical method SURVIV (Survival analysis of mRNA Isoform Variation), designed for identifying mRNA isoform variation associated with patient survival time. A unique feature and major strength of SURVIV is that it models the measurement uncertainty of mRNA isoform ratio in RNA-seq data. Simulation studies suggest that SURVIV outperforms the conventional Cox regression survival analysis, especially for data sets with modest sequencing depth. We applied SURVIV to TCGA RNA-seq data of invasive ductal carcinoma as well as five additional cancer types. Alternative splicing-based survival predictors consistently outperform gene expression-based survival predictors, and the integration of clinical, gene expression and alternative splicing profiles leads to the best survival prediction. We anticipate that SURVIV will have broad utilities for analysing diverse types of mRNA isoform variation in large-scale clinical RNA-seq projects. PMID:27279334

  1. Small RNA Profile in Moso Bamboo Root and Leaf Obtained by High Definition Adapters

    PubMed Central

    Yang, Li; Zhao, Hansheng; Gao, Zhimin; Dalmay, Tamas

    2014-01-01

    Moso bamboo (Phyllostachy heterocycla cv. pubescens L.) is an economically important fast-growing tree. In order to gain better understanding of gene expression regulation in this important species we used next generation sequencing to profile small RNAs in leaf and roots of young seedlings. Since standard kits to produce cDNA of small RNAs are biased for certain small RNAs, we used High Definition adapters that reduce ligation bias. We identified and experimentally validated five new microRNAs and a few other small non-coding RNAs that were not microRNAs. The biological implication of microRNA expression levels and targets of microRNAs are discussed. PMID:25079776

  2. Analysis of microRNA expression by in situ hybridization with RNA oligonucleotide probes

    PubMed Central

    Thompson, Robert C.; Deo, Monika; Turner, David L.

    2007-01-01

    In situ hybridization is an important tool for analyzing gene expression and developing hypotheses about gene functions. The discovery of hundreds of microRNA (miRNA) genes in animals has provided new challenges for analyzing gene expression and functions. The small size of the mature miRNAs (∼20-24 nucleotides in length) presents difficulties for conventional in situ hybridization methods. However, we have developed a modified in situ hybridization method for detection of mammalian miRNAs in tissue sections, based upon the use of RNA oligonucleotide probes in combination with highly specific wash conditions. Here we present detailed procedures for detection of miRNAs in tissue sections or cultured cells. The methods described can utilize either nonradioactive hapten-conjugated probes that are detected by enzyme-coupled antibodies, or radioactively labeled probes that are detected by autoradiography. The ability to visualize miRNA expression patterns in tissue sections provides an additional tool for the analyses of miRNA expression and function. In addition, the use of radioactively labeled probes should facilitate quantitative analyses of changes in miRNA gene expression. PMID:17889803

  3. A Significant Fraction of 21-Nucleotide Small RNA Originates from Phased Degradation of Resistance Genes in Several Perennial Species1[C][W][OA

    PubMed Central

    Källman, Thomas; Chen, Jun; Gyllenstrand, Niclas; Lagercrantz, Ulf

    2013-01-01

    Small RNAs (sRNAs), including microRNA (miRNA) and short-interfering RNA (siRNA), are important in the regulation of diverse biological processes. Comparative studies of sRNAs from plants have mainly focused on miRNA, even though they constitute a mere fraction of the total sRNA diversity. In this study, we report results from an in-depth analysis of the sRNA population from the conifer spruce (Picea abies) and compared the results with those of a range of plant species. The vast majority of sRNA sequences in spruce can be assigned to 21-nucleotide-long siRNA sequences, of which a large fraction originate from the degradation of transcribed sequences related to nucleotide-binding site-leucine-rich repeat-type resistance genes. Over 90% of all genes predicted to contain either a Toll/interleukin-1 receptor or nucleotide-binding site domain showed evidence of siRNA degradation. The data further suggest that this phased degradation of resistance-related genes is initiated from miRNA-guided cleavage, often by an abundant 22-nucleotide miRNA. Comparative analysis over a range of plant species revealed a huge variation in the abundance of this phenomenon. The process seemed to be virtually absent in several species, including Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and nonvascular plants, while particularly high frequencies were observed in spruce, grape (Vitis vinifera), and poplar (Populus trichocarpa). This divergent pattern might reflect a mechanism to limit runaway transcription of these genes in species with rapidly expanding nucleotide-binding site-leucine-rich repeat gene families. Alternatively, it might reflect variation in a counter-counter defense mechanism between plant species. PMID:23580593

  4. miRTarVis: an interactive visual analysis tool for microRNA-mRNA expression profile data

    PubMed Central

    2015-01-01

    Background MicroRNAs (miRNA) are short nucleotides that down-regulate its target genes. Various miRNA target prediction algorithms have used sequence complementarity between miRNA and its targets. Recently, other algorithms tried to improve sequence-based miRNA target prediction by exploiting miRNA-mRNA expression profile data. Some web-based tools are also introduced to help researchers predict targets of miRNAs from miRNA-mRNA expression profile data. A demand for a miRNA-mRNA visual analysis tool that features novel miRNA prediction algorithms and more interactive visualization techniques exists. Results We designed and implemented miRTarVis, which is an interactive visual analysis tool that predicts targets of miRNAs from miRNA-mRNA expression profile data and visualizes the resulting miRNA-target interaction network. miRTarVis has intuitive interface design in accordance with the analysis procedure of load, filter, predict, and visualize. It predicts targets of miRNA by adopting Bayesian inference and MINE analyses, as well as conventional correlation and mutual information analyses. It visualizes a resulting miRNA-mRNA network in an interactive Treemap, as well as a conventional node-link diagram. miRTarVis is available at http://hcil.snu.ac.kr/~rati/miRTarVis/index.html. Conclusions We reported findings from miRNA-mRNA expression profile data of asthma patients using miRTarVis in a case study. miRTarVis helps to predict and understand targets of miRNA from miRNA-mRNA expression profile data. PMID:26361498

  5. U20, a novel small nucleolar RNA, is encoded in an intron of the nucleolin gene in mammals.

    PubMed Central

    Nicoloso, M; Caizergues-Ferrer, M; Michot, B; Azum, M C; Bachellerie, J P

    1994-01-01

    We have found that intron 11 of the nucleolin gene in humans and rodents encodes a previously unidentified small nucleolar RNA, termed U20. The single-copy U20 sequence is located on the same DNA strand as the nucleolin mRNA. U20 RNA, which does not possess a trimethyl cap, appears to result from intronic RNA processing and not from transcription of an independent gene. In mammals, U20 RNA is an 80-nucleotide-long, metabolically stable species, present at about 7 x 10(3) molecules per exponentially growing HeLa cell. It has a nucleolar localization, as indicated by fluorescence microscopy following in situ hybridization with digoxigenin-labeled oligonucleotides. U20 RNA contains the box C and box D sequence motifs, hallmarks of most small nucleolar RNAs reported to date, and is immunoprecipitated by antifibrillarin antibodies. It also exhibits a 5'-3' terminal stem bracketing the box C-box D motifs like U14, U15, U16, or Y RNA. A U20 homolog of similar size has been detected in all vertebrate classes by Northern (RNA) hybridization with mammalian oligonucleotide probes. U20 RNA contains an extended region (21 nucleotides) of perfect complementarity with a phylogenetically conserved sequence in 18S rRNA. This complementarity is strongly preserved among distant vertebrates, suggesting that U20 RNA may be involved in the formation of the small ribosomal subunit like nucleolin, the product of its host gene. Images PMID:8065311

  6. P-SAMS: a web site for plant artificial microRNA and synthetic trans-acting small interfering RNA design

    PubMed Central

    Fahlgren, Noah; Hill, Steven T.; Carrington, James C.; Carbonell, Alberto

    2016-01-01

    Summary: The Plant Small RNA Maker Site (P-SAMS) is a web tool for the simple and automated design of artificial miRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) for efficient and specific targeted gene silencing in plants. P-SAMS includes two applications, P-SAMS amiRNA Designer and P-SAMS syn-tasiRNA Designer. The navigation through both applications is wizard-assisted, and the job runtime is relatively short. Both applications output the sequence of designed small RNA(s), and the sequence of the two oligonucleotides required for cloning into ‘B/c’ compatible vectors. Availability and implementation: The P-SAMS website is available at http://p-sams.carringtonlab.org. Contact: acarbonell@ibmcp.upv.es or nfahlgren@danforthcenter.org PMID:26382195

  7. Effects of long DNA folding and small RNA stem–loop in thermophoresis

    PubMed Central

    Maeda, Yusuke T.; Tlusty, Tsvi; Libchaber, Albert

    2012-01-01

    In thermophoresis, with the fluid at rest, suspensions move along a gradient of temperature. In an aqueous solution, a PEG polymer suspension is depleted from the hot region and builds a concentration gradient. In this gradient, DNA polymers of different sizes can be separated. In this work the effect of the polymer structure for genomic DNA and small RNA is studied. For genome-size DNA, individual single T4 DNA is visualized and tracked in a PEG solution under a temperature gradient built by infrared laser focusing. We find that T4 DNA follows steps of depletion, ring-like localization, and accumulation patterns as the PEG volume fraction is increased. Furthermore, a coil–globule transition for DNA is observed for a large enough PEG volume fraction. This drastically affects the localization position of T4 DNA. In a similar experiment, with small RNA such as ribozymes we find that the stem–loop folding of such polymers has important consequences. The RNA polymers having a long and rigid stem accumulate, whereas a polymer with stem length less than 4 base pairs shows depletion. Such measurements emphasize the crucial contribution of the double-stranded parts of RNA for thermal separation and selection under a temperature gradient. Because huge temperature gradients are present around hydrothermal vents in the deep ocean seafloor, this process might be relevant, at the origin of life, in an RNA world hypothesis. Ribozymes could be selected from a pool of random sequences depending on the length of their stems. PMID:23071341

  8. Annotation of primate miRNAs by high throughput sequencing of small RNA libraries

    PubMed Central

    2012-01-01

    Background In addition to genome sequencing, accurate functional annotation of genomes is required in order to carry out comparative and evolutionary analyses between species. Among primates, the human genome is the most extensively annotated. Human miRNA gene annotation is based on multiple lines of evidence including evidence for expression as well as prediction of the characteristic hairpin structure. In contrast, most miRNA genes in non-human primates are annotated based on homology without any expression evidence. We have sequenced small-RNA libraries from chimpanzee, gorilla, orangutan and rhesus macaque from multiple individuals and tissues. Using patterns of miRNA expression in conjunction with a model of miRNA biogenesis we used these high-throughput sequencing data to identify novel miRNAs in non-human primates. Results We predicted 47 new miRNAs in chimpanzee, 240 in gorilla, 55 in orangutan and 47 in rhesus macaque. The algorithm we used was able to predict 64% of the previously known miRNAs in chimpanzee, 94% in gorilla, 61% in orangutan and 71% in rhesus macaque. We therefore added evidence for expression in between one and five tissues to miRNAs that were previously annotated based only on homology to human miRNAs. We increased from 60 to 175 the number miRNAs that are located in orthologous regions in humans and the four non-human primate species studied here. Conclusions In this study we provide expression evidence for homology-based annotated miRNAs and predict de novo miRNAs in four non-human primate species. We increased the number of annotated miRNA genes and provided evidence for their expression in four non-human primates. Similar approaches using different individuals and tissues would improve annotation in non-human primates and allow for further comparative studies in the future. PMID:22453055

  9. Effective Small RNA Destruction by the Expression of a Short Tandem Target Mimic in Arabidopsis[C][W

    PubMed Central

    Yan, Jun; Gu, Yiyou; Jia, Xiaoyun; Kang, Wenjun; Pan, Shangjin; Tang, Xiaoqing; Chen, Xuemei; Tang, Guiliang

    2012-01-01

    MicroRNAs (miRNAs) and other endogenous small RNAs act as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. The interrogation of small RNA functions requires an effective, widely applicable method to specifically block small RNA function. Here, we report the development of a highly effective technology that targets specific endogenous miRNAs or small interfering RNAs for destruction in Arabidopsis thaliana. We show that the expression of a short tandem target mimic (STTM), which is composed of two short sequences mimicking small RNA target sites, separated by a linker of an empirically determined optimal size, leads to the degradation of targeted small RNAs by small RNA degrading nucleases. The efficacy of the technology was demonstrated by the strong and specific developmental defects triggered by STTMs targeting three miRNAs and an endogenous siRNA. In summary, we developed an effective approach for the destruction of endogenous small RNAs, thereby providing a powerful tool for functional genomics of small RNA molecules in plants and potentially animals. PMID:22345490

  10. Statistical Methods for Analysis of High-Throughput RNA Interference Screens

    PubMed Central

    Birmingham, Amanda; Selfors, Laura M.; Forster, Thorsten; Wrobel, David; Kennedy, Caleb J.; Shanks, Emma; Santoyo-Lopez, Javier; Dunican, Dara J.; Long, Aideen; Kelleher, Dermot; Smith, Queta; Beijersbergen, Roderick L.; Ghazal, Peter; Shamu, Caroline E.

    2009-01-01

    RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery and, in both of these areas, large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from small-molecule screening; however small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow. PMID:19644458

  11. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  12. Preparation and Analysis of RNA Crystals

    NASA Technical Reports Server (NTRS)

    Todd, Paul

    2000-01-01

    The crystallization of RiboNucleic Acids (RNA) was studied from the standpoint of mechanisms of crystal growth in three tasks: (1) preparation of high-quality crystals of oligonuclotides for X-ray diffraction, (2) finding pathways to the growth of high-quality crystals for X-ray diffraction and (3) investigation of mechanisms of action of inertial acceleration on crystal growth. In these tasks: (1) RNA crystals were prepared and studied by X-ray diffraction; (2) a pathway to high-quality crystals was discovered and characterized; a combination of kinetic and equilibrium factors could be optimized as described below; and (3) an interplay between purity and gravity was found in a combination of space and ground experiments with nucleic acids and proteins. Most significantly, the rate of concentration of precipitant and RNA can be controlled by membrane-based methods of water removal or by diffusion of multivalent cations across an interface stabilized by a membrane. Oligonucleotide solutions are electrokinetically stabilized colloids, and crystals can form by the controlled addition of multivalent cations.

  13. Comprehensive Annotation of Physcomitrella patens Small RNA Loci Reveals That the Heterochromatic Short Interfering RNA Pathway Is Largely Conserved in Land Plants.

    PubMed

    Coruh, Ceyda; Cho, Sung Hyun; Shahid, Saima; Liu, Qikun; Wierzbicki, Andrzej; Axtell, Michael J

    2015-08-01

    Many plant small RNAs are sequence-specific negative regulators of target mRNAs and/or chromatin. In angiosperms, the two most abundant endogenous small RNA populations are usually 21-nucleotide microRNAs (miRNAs) and 24-nucleotide heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs are derived from repetitive regions and reinforce DNA methylation at targeted loci. The existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Using small RNA-sequencing (RNA-seq) of the moss Physcomitrella patens, we identified 1090 loci that produce mostly 23- to 24-nucleotide siRNAs. These loci are mostly in intergenic regions with dense DNA methylation. Accumulation of siRNAs from these loci depends upon P. patens homologs of DICER-LIKE3 (DCL3), RNA-DEPENDENT RNA POLYMERASE2, and the largest subunit of DNA-DEPENDENT RNA POLYMERASE IV, with the largest subunit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23-nucleotide siRNA accumulation. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and P. patens, with the notable exception of the P. patens-specific use of mDCL to produce 23-nucleotide siRNAs.

  14. Comprehensive Annotation of Physcomitrella patens Small RNA Loci Reveals That the Heterochromatic Short Interfering RNA Pathway Is Largely Conserved in Land Plants[OPEN

    PubMed Central

    Coruh, Ceyda; Cho, Sung Hyun; Shahid, Saima; Liu, Qikun; Wierzbicki, Andrzej; Axtell, Michael J.

    2015-01-01

    Many plant small RNAs are sequence-specific negative regulators of target mRNAs and/or chromatin. In angiosperms, the two most abundant endogenous small RNA populations are usually 21-nucleotide microRNAs (miRNAs) and 24-nucleotide heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs are derived from repetitive regions and reinforce DNA methylation at targeted loci. The existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Using small RNA-sequencing (RNA-seq) of the moss Physcomitrella patens, we identified 1090 loci that produce mostly 23- to 24-nucleotide siRNAs. These loci are mostly in intergenic regions with dense DNA methylation. Accumulation of siRNAs from these loci depends upon P. patens homologs of DICER-LIKE3 (DCL3), RNA-DEPENDENT RNA POLYMERASE2, and the largest subunit of DNA-DEPENDENT RNA POLYMERASE IV, with the largest subunit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23-nucleotide siRNA accumulation. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and P. patens, with the notable exception of the P. patens-specific use of mDCL to produce 23-nucleotide siRNAs. PMID:26209555

  15. The gene for human E2 small nucleolar RNA resides in an intron of a laminin-binding protein gene

    SciTech Connect

    Selvamurugan, N.; Eliceiri, G.L.

    1995-11-20

    Several of the known small nucleolar RNA (snoRNA) species have been shown to be required for processing of ribosomal RNA precursors (pre-rRNA). The genes of most of the known vertebrate snoRNA species are located in introns of genes for messenger RNA precursors. E2 RNA is a nucleolar species that is 154 nucleotides long in human; it belongs to a new family of snoRNAs because it does not have the sequences named box C, C{prime} or D that are present in most vertebrate snoRNA species, and it does not bind fibrillarin, the nucleolar protein associated with most snoRNAs. E2 snoRNA is found in all tissues tested and in all vertebrates analyzed. E2 snoRNA is expected to have a unique function in ribosome formation, because it psoralen-photocrosslinks in vivo to a unique internal segment of the 28S rRNA sequence of pre-rRNA. Two observations are compatible with the possibility that the human E2 RNA gene may be intronic. First, the human E2 RNA gene lacks the intragenic or flanking sequences that are functional in other genes. Second, the 5{prime} end of E2 RNA is monophosphorylated, suggesting that is formed by RNA processing. Intron-encoded snoRNAs have monophosphorylated 5{prime}termini. Until now, it was not known whether the E2 RNA gene resides in an intron. This information is important for studying the biosynthesis of E2 RNA. 13 refs., 1 fig.

  16. Trans-acting small RNA determines dominance relationships in Brassica self-incompatibility.

    PubMed

    Tarutani, Yoshiaki; Shiba, Hiroshi; Iwano, Megumi; Kakizaki, Tomohiro; Suzuki, Go; Watanabe, Masao; Isogai, Akira; Takayama, Seiji

    2010-08-19

    A diploid organism has two copies of each gene, one inherited from each parent. The expression of two inherited alleles is sometimes biased by the effects known as dominant/recessive relationships, which determine the final phenotype of the organism. To explore the mechanisms underlying these relationships, we have examined the monoallelic expression of S-locus protein 11 genes (SP11), which encode the male determinants of self-incompatibility in Brassica. We previously reported that SP11 expression was monoallelic in some S heterozygotes, and that the promoter regions of recessive SP11 alleles were specifically methylated in the anther tapetum. Here we show that this methylation is controlled by trans-acting small non-coding RNA (sRNA). We identified inverted genomic sequences that were similar to the recessive SP11 promoters in the flanking regions of dominant SP11 alleles. These sequences were specifically expressed in the anther tapetum and processed into 24-nucleotide sRNA, named SP11 methylation inducer (Smi). Introduction of the Smi genomic region into the recessive S homozygotes triggered the methylation of the promoter of recessive SP11 alleles and repressed their transcription. This is an example showing sRNA encoded in the flanking region of a dominant allele acts in trans to induce transcriptional silencing of the recessive allele. Our finding may provide new insights into the widespread monoallelic gene expression systems. PMID:20725042

  17. U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer’s disease

    PubMed Central

    Bai, Bing; Hales, Chadwick M.; Chen, Ping-Chung; Gozal, Yair; Dammer, Eric B.; Fritz, Jason J.; Wang, Xusheng; Xia, Qiangwei; Duong, Duc M.; Street, Craig; Cantero, Gloria; Cheng, Dongmei; Jones, Drew R.; Wu, Zhiping; Li, Yuxin; Diner, Ian; Heilman, Craig J.; Rees, Howard D.; Wu, Hao; Lin, Li; Szulwach, Keith E.; Gearing, Marla; Mufson, Elliott J.; Bennett, David A.; Montine, Thomas J.; Seyfried, Nicholas T.; Wingo, Thomas S.; Sun, Yi E.; Jin, Peng; Hanfelt, John; Willcock, Donna M.; Levey, Allan; Lah, James J.; Peng, Junmin

    2013-01-01

    Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer’s disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis. PMID:24023061

  18. The highly conserved U small nuclear RNA 3'-end formation signal is quite tolerant to mutation.

    PubMed Central

    Ach, R A; Weiner, A M

    1987-01-01

    Formation of the 3' end of U1 and U2 small nuclear RNA (snRNA) precursors is directed by a conserved sequence called the 3' box located 9 to 28 nucleotides downstream of all metazoan U1 to U4 snRNA genes sequenced so far. Deletion of part or all of the 3' box from human U1 and U2 genes drastically reduces 3'-end formation. To define the essential nucleotides within this box that direct 3'-end formation, we constructed a set of point mutations in the conserved residues of the human U1 3' box. The ability of the various mutations to direct 3'-end formation was tested by microinjection into Xenopus oocytes and transfection into HeLa cells. We found that the point mutations had diverse effects on 3'-end formation, ranging from no effect at all to severe inhibition; however, no single or double point mutation we tested completely eliminated 3'-end formation. We also showed that a rat U3 3' flank can effectively substitute for the human U1 3' flank, indicating that the 3' boxes of the different U snRNA genes are functionally equivalent. Images PMID:3037343

  19. Identification and validation of novel small molecule disruptors of HuR-mRNA interaction.

    PubMed

    Wu, Xiaoqing; Lan, Lan; Wilson, David Michael; Marquez, Rebecca T; Tsao, Wei-Chung; Gao, Philip; Roy, Anuradha; Turner, Benjamin Andrew; McDonald, Peter; Tunge, Jon A; Rogers, Steven A; Dixon, Dan A; Aubé, Jeffrey; Xu, Liang

    2015-06-19

    HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3'-untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR-ARE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of ∼6000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR-ARE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers.

  20. One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9

    PubMed Central

    Kannan, Krishna; Tsvetanova, Billyana; Chuang, Ray-Yuan; Noskov, Vladimir N.; Assad-Garcia, Nacyra; Ma, Li; Hutchison III, Clyde A.; Smith, Hamilton O.; Glass, John I.; Merryman, Chuck; Venter, J. Craig; Gibson, Daniel G.

    2016-01-01

    Bacteria are indispensable for the study of fundamental molecular biology processes due to their relatively simple gene and genome architecture. The ability to engineer bacterial chromosomes is quintessential for understanding gene functions. Here we demonstrate the engineering of the small-ribosomal subunit (16S) RNA of Mycoplasma mycoides, by combining the CRISPR/Cas9 system and the yeast recombination machinery. We cloned the entire genome of M. mycoides in yeast and used constitutively expressed Cas9 together with in vitro transcribed guide-RNAs to introduce engineered 16S rRNA genes. By testing the function of the engineered 16S rRNA genes through genome transplantation, we observed surprising resilience of this gene to addition of genetic elements or helix substitutions with phylogenetically-distant bacteria. While this system could be further used to study the function of the 16S rRNA, one could envision the “simple” M. mycoides genome being used in this setting to study other genetic structures and functions to answer fundamental questions of life. PMID:27489041

  1. One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9.

    PubMed

    Kannan, Krishna; Tsvetanova, Billyana; Chuang, Ray-Yuan; Noskov, Vladimir N; Assad-Garcia, Nacyra; Ma, Li; Hutchison Iii, Clyde A; Smith, Hamilton O; Glass, John I; Merryman, Chuck; Venter, J Craig; Gibson, Daniel G

    2016-01-01

    Bacteria are indispensable for the study of fundamental molecular biology processes due to their relatively simple gene and genome architecture. The ability to engineer bacterial chromosomes is quintessential for understanding gene functions. Here we demonstrate the engineering of the small-ribosomal subunit (16S) RNA of Mycoplasma mycoides, by combining the CRISPR/Cas9 system and the yeast recombination machinery. We cloned the entire genome of M. mycoides in yeast and used constitutively expressed Cas9 together with in vitro transcribed guide-RNAs to introduce engineered 16S rRNA genes. By testing the function of the engineered 16S rRNA genes through genome transplantation, we observed surprising resilience of this gene to addition of genetic elements or helix substitutions with phylogenetically-distant bacteria. While this system could be further used to study the function of the 16S rRNA, one could envision the "simple" M. mycoides genome being used in this setting to study other genetic structures and functions to answer fundamental questions of life. PMID:27489041

  2. Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells.

    PubMed

    Slanina, H; Schmutzler, M; Christodoulides, M; Kim, K S; Schubert-Unkmeir, A

    2012-01-01

    Expression of exogenous DNA or small interfering RNA (siRNA) in vitro is significantly affected by the particular delivery system utilized. In this study, we evaluated the transfection efficiency of plasmid DNA and siRNA into human brain microvascular endothelial cells (HBMEC) and meningioma cells, which constitute the blood-cerebrospinal fluid barrier, a target of meningitis-causing pathogens. Chemical transfection methods and various lipofection reagents including Lipofectamin™, FuGene™, or jetPRIME®, as well as physical transfection methods and electroporation techniques were applied. To monitor the transfection efficiencies, HBMEC and meningioma cells were transfected with the reporter plasmid pTagGFP2-actin vector, and efficiency of transfection was estimated by fluorescence microscopy and flow cytometry. We established protocols based on electroporation using Cell Line Nucleofector® Kit V with the Amaxa® Nucleofector® II system from Lonza and the Neon® Transfection system from Invitrogen resulting in up to 41 and 82% green fluorescent protein-positive HBMEC, respectively. Optimal transfection solutions, pulse programs and length were evaluated. We furthermore demonstrated that lipofection is an efficient method to transfect meningioma cells with a transfection efficiency of about 81%. Finally, we applied the successful electroporation protocols to deliver synthetic siRNA to HBMEC and analyzed the role of the actin-binding protein cortactin in Neisseria meningitidis pathogenesis. PMID:23036990

  3. Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction

    PubMed Central

    Wu, Xiaoqing; Lan, Lan; Wilson, David Michael; Marquez, Rebecca T.; Tsao, Wei-chung; Gao, Philip; Roy, Anuradha; Turner, Benjamin Andrew; McDonald, Peter; Tunge, Jon A; Rogers, Steven A; Dixon, Dan A.; Aubé, Jeffrey; Xu, Liang

    2015-01-01

    HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3′-untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR–ARE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of ~6000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR–ARE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers. PMID:25750985

  4. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

    PubMed

    Meyer, Sarai; Chappell, James; Sankar, Sitara; Chew, Rebecca; Lucks, Julius B

    2016-01-01

    Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA engineering strategies can improve fold activation from 5.4-fold to 13.4-fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1-fold to 14.6-fold for an unrelated STAR regulator, opening the door to creating a range of additional STARs to use in a broad array of biotechnologies. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of RNA transcriptional repressors, enabling these optimized STARs to be used in sophisticated circuits. PMID:26134708

  5. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

    PubMed

    Meyer, Sarai; Chappell, James; Sankar, Sitara; Chew, Rebecca; Lucks, Julius B

    2016-01-01

    Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA engineering strategies can improve fold activation from 5.4-fold to 13.4-fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1-fold to 14.6-fold for an unrelated STAR regulator, opening the door to creating a range of additional STARs to use in a broad array of biotechnologies. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of RNA transcriptional repressors, enabling these optimized STARs to be used in sophisticated circuits.

  6. Small-Angle Neutron Scattering for Structural Biology of Protein-RNA Complexes.

    PubMed

    Gabel, Frank

    2015-01-01

    This chapter deals with the applications of small-angle neutron scattering (SANS) for the structural study of protein-RNA complexes in solution. After a brief historical introduction, the basic theory and practical requirements (e.g., sample state) for SANS experiments will be treated. Next, model-free parameters, such as the molecular mass and the radius of gyration, which can be obtained without a priori structural information, will be introduced. A more detailed section on the specific properties of SANS (with respect to its sister technique, small-angle X-ray scattering), and their implications on possibilities and limits of model building and interpretation will be discussed with a focus on protein-RNA systems. A practical illustration of the information content of SANS data will be given by applying ab initio modeling to a tRNA-synthetase system of known high-resolution structure. Finally, two present state-of-the-art examples that combine SANS data with complementary structural biology techniques (NMR and crystallography) will be presented and possible future developments and applications will be discussed.

  7. Small RNA Control of Cell-to-Cell Communication in Vibrio Harveyi and Vibrio Cholerae

    NASA Astrophysics Data System (ADS)

    Svenningsen, Sine Lo

    Quorum sensing is a process of cell-to-cell communication, by which bacteria coordinate gene expression and behavior on a population-wide scale. Quorum sensing is accomplished through production, secretion, and subsequent detection of chemical signaling molecules termed autoinducers. The human pathogen Vibrio cholerae and the marine bioluminescent bacterium Vibrio harveyi incorporate information from multiple autoinducers, and also environmental signals and metabolic cues into their quorum-sensing pathways. At the core of these pathways lie several homologous small regulatory RNA molecules, the Quorum Regulatory RNAs. Small noncoding RNAs have emerged throughout the bacterial and eukaryotic kingdoms as key regulators of behavioral and developmental processes. Here, I review our present understanding of the role of the Qrr small RNAs in integrating quorum-sensing signals and in regulating the individual cells response to this information.

  8. The small regulatory RNA FasX controls pilus expression and adherence in the human bacterial pathogen group A Streptococcus

    PubMed Central

    Liu, Zhuyun; Treviño, Jeanette; Ramirez-Peña, Esmeralda; Sumby, Paul

    2012-01-01

    Summary Bacterial pathogens use cell-surface-associated adhesion molecules to promote host attachment and colonization, and the ability to modulate adhesion expression is critical to pathogen success. Here, we show that the human-specific pathogen the group A Streptococcus (GAS) uses a small regulatory RNA (sRNA) to regulate the expression of adhesive pili. The fibronectin / fibrinogen-binding / haemolytic-activity / streptokinase-regulator-X (FasX) sRNA, previously shown to positively regulate expression of the secreted virulence factor streptokinase (SKA), negatively regulates the production of pili on the GAS cell surface. FasX base-pairs to the extreme 5’ end of mRNA from the pilus biosynthesis operon, and this RNA:RNA interaction reduces the stability of the mRNA, while also inhibiting translation of at least the first gene in the pilus biosynthesis operon (cpa, which encodes a minor pilin protein). The negative regulation of pilus expression by FasX reduces the ability of GAS to adhere to human keratinocytes. Our findings cement FasX sRNA as an important regulator of virulence factor production in GAS and identify that FasX uses at least three distinct mechanisms, positive (ska mRNA) and negative (pilus operon mRNA) regulation of mRNA stability, and negative regulation of mRNA translation (cpa mRNA), to post-transcriptionally regulate target mRNAs during infection. PMID:22882718

  9. Small RNA Regulators of Plant-Hemipteran Interactions: Micromanagers with Versatile Roles.

    PubMed

    Sattar, Sampurna; Thompson, Gary A

    2016-01-01

    Non-coding small RNAs (sRNAs) in plants have important roles in regulating biological processes, including development, reproduction, and stress responses. Recent research indicates significant roles for sRNA-mediated gene silencing during plant-hemipteran interactions that involve all three of these biological processes. Plant responses to hemipteran feeding are determined by changes in the host transcriptome that appear to be fine-tuned by sRNAs. The role of sRNA in plant defense responses is complex. Different forms of sRNAs, with specific modes of action, regulate changes in the host transcriptome primarily through post-transcriptional gene silencing and occasionally through translational repression. Plant genetic resistance against hemipterans provides a model to explore the regulatory roles of sRNAs in plant defense. Aphid-induced sRNA expression in resistance genotypes delivers a new paradigm in understanding the regulation of R gene-mediated resistance in host plants. Unique sRNA profiles, including changes in sRNA biogenesis and expression can also provide insights into susceptibility to insect herbivores. Activation of phytohormone-mediated defense responses against insect herbivory is another hallmark of this interaction, and recent studies have shown that regulation of phytohormone signaling is under the control of sRNAs. Hemipterans feeding on resistant plants also show changes in insect sRNA profiles, possibly influencing insect development and reproduction. Changes in insect traits such as fecundity, host range, and resistance to insecticides are impacted by sRNAs and can directly contribute to the success of certain insect biotypes. In addition to causing direct damage to the host plant, hemipteran insects are often vectors of viral pathogens. Insect anti-viral RNAi machinery is activated to limit virus accumulation, suggesting a role in insect immunity. Virus-derived long sRNAs strongly resemble insect piRNAs, leading to the speculation that the piRNA

  10. Small RNA Regulators of Plant-Hemipteran Interactions: Micromanagers with Versatile Roles

    PubMed Central

    Sattar, Sampurna; Thompson, Gary A.

    2016-01-01

    Non-coding small RNAs (sRNAs) in plants have important roles in regulating biological processes, including development, reproduction, and stress responses. Recent research indicates significant roles for sRNA-mediated gene silencing during plant-hemipteran interactions that involve all three of these biological processes. Plant responses to hemipteran feeding are determined by changes in the host transcriptome that appear to be fine-tuned by sRNAs. The role of sRNA in plant defense responses is complex. Different forms of sRNAs, with specific modes of action, regulate changes in the host transcriptome primarily through post-transcriptional gene silencing and occasionally through translational repression. Plant genetic resistance against hemipterans provides a model to explore the regulatory roles of sRNAs in plant defense. Aphid-induced sRNA expression in resistance genotypes delivers a new paradigm in understanding the regulation of R gene-mediated resistance in host plants. Unique sRNA profiles, including changes in sRNA biogenesis and expression can also provide insights into susceptibility to insect herbivores. Activation of phytohormone-mediated defense responses against insect herbivory is another hallmark of this interaction, and recent studies have shown that regulation of phytohormone signaling is under the control of sRNAs. Hemipterans feeding on resistant plants also show changes in insect sRNA profiles, possibly influencing insect development and reproduction. Changes in insect traits such as fecundity, host range, and resistance to insecticides are impacted by sRNAs and can directly contribute to the success of certain insect biotypes. In addition to causing direct damage to the host plant, hemipteran insects are often vectors of viral pathogens. Insect anti-viral RNAi machinery is activated to limit virus accumulation, suggesting a role in insect immunity. Virus-derived long sRNAs strongly resemble insect piRNAs, leading to the speculation that the piRNA

  11. Small RNA Regulators of Plant-Hemipteran Interactions: Micromanagers with Versatile Roles

    PubMed Central

    Sattar, Sampurna; Thompson, Gary A.

    2016-01-01

    Non-coding small RNAs (sRNAs) in plants have important roles in regulating biological processes, including development, reproduction, and stress responses. Recent research indicates significant roles for sRNA-mediated gene silencing during plant-hemipteran interactions that involve all three of these biological processes. Plant responses to hemipteran feeding are determined by changes in the host transcriptome that appear to be fine-tuned by sRNAs. The role of sRNA in plant defense responses is complex. Different forms of sRNAs, with specific modes of action, regulate changes in the host transcriptome primarily through post-transcriptional gene silencing and occasionally through translational repression. Plant genetic resistance against hemipterans provides a model to explore the regulatory roles of sRNAs in plant defense. Aphid-induced sRNA expression in resistance genotypes delivers a new paradigm in understanding the regulation of R gene-mediated resistance in host plants. Unique sRNA profiles, including changes in sRNA biogenesis and expression can also provide insights into susceptibility to insect herbivores. Activation of phytohormone-mediated defense responses against insect herbivory is another hallmark of this interaction, and recent studies have shown that regulation of phytohormone signaling is under the control of sRNAs. Hemipterans feeding on resistant plants also show changes in insect sRNA profiles, possibly influencing insect development and reproduction. Changes in insect traits such as fecundity, host range, and resistance to insecticides are impacted by sRNAs and can directly contribute to the success of certain insect biotypes. In addition to causing direct damage to the host plant, hemipteran insects are often vectors of viral pathogens. Insect anti-viral RNAi machinery is activated to limit virus accumulation, suggesting a role in insect immunity. Virus-derived long sRNAs strongly resemble insect piRNAs, leading to the speculation that the piRNA

  12. Analysis of Small Ions with Capillary Electrophoresis.

    PubMed

    Aulakh, Jatinder Singh; Kaur, Ramandeep; Malik, Ashok Kumar

    2016-01-01

    Small inorganic ions are easily separated through capillary electrophoresis because they have a high charge-to-mass ratio and suffer little from some of the undesired phenomenon affecting higher molecular weight species like adsorption to the capillary wall, decomposition, and precipitation. This chapter is focused on the analysis of small ions other than metal ions using capillary electrophoresis. Methods are described for the determination of ions of nitrogen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine. PMID:27645739

  13. Quantitative Northern Blot Analysis of Mammalian rRNA Processing.

    PubMed

    Wang, Minshi; Pestov, Dimitri G

    2016-01-01

    Assembly of eukaryotic ribosomes is an elaborate biosynthetic process that begins in the nucleolus and requires hundreds of cellular factors. Analysis of rRNA processing has been instrumental for studying the mechanisms of ribosome biogenesis and effects of stress conditions on the molecular milieu of the nucleolus. Here, we describe the quantitative analysis of the steady-state levels of rRNA precursors, applicable to studies in mammalian cells and other organisms. We include protocols for gel electrophoresis and northern blotting of rRNA precursors using procedures optimized for the large size of these RNAs. We also describe the ratio analysis of multiple precursors, a technique that facilitates the accurate assessment of changes in the efficiency of individual pre-rRNA processing steps. PMID:27576717

  14. RNA-Seq Analysis Pipeline Based on Oshell Environment.

    PubMed

    Li, Jing; Hu, Jun; Newman, Matthew; Liu, Kejun; Ge, Huanying

    2014-01-01

    Advances in transcriptome sequencing (RNA-Seq) have revolutionized the way to characterize and quantify transcripts. The breakthroughs in RNA-Seq technologies give rise to the ever-increasing volumes of data, making data processing the bottleneck of transcriptome research. It becomes crucial to develop an efficient analysis pipeline to automate RNA-Seq data analysis. Based on Oshell environment, we present here an ultra-fast and powerful RNA-Seq analysis pipeline for quality control, sequence alignment, variation detection, expression quantification and junction discovery. The pipeline runs on both Linux and Windows operating systems, with either stand-alone or cluster computing environment. Parallel computing is also supported for improved processing speed. Oshell is free for non-commercial use at http://omicsoft.com/oshell. PMID:26356868

  15. Resolving Individual Compo