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Sample records for small rna analysis

  1. 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. PMID:20478825

  2. RNA SHAPE Analysis of Small RNAs and Riboswitches

    PubMed Central

    Rice, Greggory M.; Busan, Steven; Karabiber, Fethullah; Favorov, Oleg V.; Weeks, Kevin M.

    2016-01-01

    We describe structural analysis of RNAs by SHAPE chemical probing. RNAs are treated with 1-methyl-7-nitroisatoic anhydride (1M7), a reagent that detects local nucleotides flexibility, and N-methylisatoic anhydride (NMIA) and 1-methyl-6-nitroisatoic anhydride (1M6), reagents which together detect higher-order and non-canonical interactions. Chemical adducts are detected as stops during reverse transcriptase-mediated primer extension. Probing information can be used to infer conformational changes and ligand binding, and to develop highly accurate models of RNA secondary structures. PMID:25432749

  3. Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments

    PubMed Central

    2011-01-01

    Prior to the advent of new, deep sequencing methods, small RNA (sRNA) discovery was dependent on Sanger sequencing, which was time-consuming and limited knowledge to only the most abundant sRNA. The innovation of large-scale, next-generation sequencing has exponentially increased knowledge of the biology, diversity and abundance of sRNA populations. In this review, we discuss issues involved in the design of sRNA sequencing experiments, including choosing a sequencing platform, inherent biases that affect sRNA measurements and replication. We outline the steps involved in preprocessing sRNA sequencing data and review both the principles behind and the current options for normalization. Finally, we discuss differential expression analysis in the absence and presence of biological replicates. While our focus is on sRNA sequencing experiments, many of the principles discussed are applicable to the sequencing of other RNA populations. PMID:21356093

  4. Expression of Porphyromonas gingivalis small RNA in response to hemin availability identified using microarray and RNA-seq analysis.

    PubMed

    Phillips, Priscilla; Progulske-Fox, Ann; Grieshaber, Scott; Grieshaber, Nicole

    2014-02-01

    There is a significant body of work suggesting that sRNA-mediated post-transcriptional regulation is a conserved mechanism among pathogenic bacteria to modulate bacterial virulence and survival. Porphyromonas gingivalis is recognized as an etiological agent of periodontitis and implicated in contributing to the development of multiple inflammatory diseases including cardiovascular disease. Using NimbleGen microarray analysis and a strand-specific method to sequence cDNA libraries of small RNA-enriched P. gingivalis transcripts using Illumina's high-throughput sequencing technology, we identified putative sRNA and generated sRNA expression profiles in response to growth phase, hemin availability after hemin starvation, or both. We identified transcripts that mapped to intergenic sequences as well as antisense transcripts that mapped to open reading frames of the annotated genome. Overall, this approach provided a comprehensive way to survey transcriptional activity to discover functionally linked RNA transcripts, responding to specific environmental cues, that merit further investigation. PMID:24245974

  5. 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

  6. 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

  7. 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

  8. Deep Sequencing Analysis of Small Noncoding RNA and mRNA Targets of the Global Post-Transcriptional Regulator, Hfq

    PubMed Central

    Sittka, Alexandra; Lucchini, Sacha; Papenfort, Kai; Sharma, Cynthia M.; Rolle, Katarzyna; Binnewies, Tim T.; Hinton, Jay C. D.; Vogel, Jörg

    2008-01-01

    Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co-immunoprecipitation (coIP) with epitope-tagged Hfq or control coIP, identified 727 mRNAs that are Hfq-bound in vivo. The cDNA analysis discovered new, small noncoding RNAs (sRNAs) and more than doubled the number of sRNAs known to be expressed in Salmonella to 64; about half of these are associated with Hfq. Our analysis explained aspects of the pleiotropic effects of Hfq loss-of-function. Specifically, we found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes) and flhDC (flagellar master regulator) were bound by Hfq. We predicted that defective SPI-1 secretion and flagellar phenotypes of the hfq mutant would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella. Our approach overcomes the limited availability of high-density microarrays that have impeded expression-based sRNA discovery in microorganisms. We present a generic strategy that is ideal for the systems-level analysis of the post-transcriptional regulons of RNA-binding proteins and for sRNA discovery in a wide range of bacteria. PMID:18725932

  9. DRME: Count-based differential RNA methylation analysis at small sample size scenario.

    PubMed

    Liu, Lian; Zhang, Shao-Wu; Gao, Fan; Zhang, Yixin; Huang, Yufei; Chen, Runsheng; Meng, Jia

    2016-04-15

    Differential methylation, which concerns difference in the degree of epigenetic regulation via methylation between two conditions, has been formulated as a beta or beta-binomial distribution to address the within-group biological variability in sequencing data. However, a beta or beta-binomial model is usually difficult to infer at small sample size scenario with discrete reads count in sequencing data. On the other hand, as an emerging research field, RNA methylation has drawn more and more attention recently, and the differential analysis of RNA methylation is significantly different from that of DNA methylation due to the impact of transcriptional regulation. We developed DRME to better address the differential RNA methylation problem. The proposed model can effectively describe within-group biological variability at small sample size scenario and handles the impact of transcriptional regulation on RNA methylation. We tested the newly developed DRME algorithm on simulated and 4 MeRIP-Seq case-control studies and compared it with Fisher's exact test. It is in principle widely applicable to several other RNA-related data types as well, including RNA Bisulfite sequencing and PAR-CLIP. The code together with an MeRIP-Seq dataset is available online (https://github.com/lzcyzm/DRME) for evaluation and reproduction of the figures shown in this article. PMID:26851340

  10. 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

  11. Comprehensive transcript analysis in small quantities of mRNA by SAGE-lite.

    PubMed

    Peters, D G; Kassam, A B; Yonas, H; O'Hare, E H; Ferrell, R E; Brufsky, A M

    1999-12-15

    Serial analysis of gene expression (SAGE) is a powerful technique that can be used for global analysis of gene expression. Its chief advantage over other methods is that SAGE does not require prior knowledge of the genes of interest and provides quantitative and qualitative data of potentially every transcribed sequence in a particular tissue or cell type. Furthermore, SAGE can quantify low-abundance transcripts and reliably detect relatively small differences in transcript abundance between cell populations. However, SAGE demands high input levels of mRNA which are often unavailable, particularly when studying human disease. To overcome this limitation, we have developed a modification of SAGE that allows detailed global analysis of gene expression in extremely small quantities of tissue or cultured cells. We have called this approach 'SAGE-Lite'. This technique was used for the global analysis of transcription in samples of normal and pathological human cerebrovasculature to study the molecular pathology of intracranial aneurysms. These samples, which are obtained during operative surgical repair, are typically no bigger than 1 or 2 mm and yield <100 ng of total RNA. In addition, we show that SAGE-Lite allows simple and rapid isolation of long cDNAs from short (15 bp) SAGE sequence tags. PMID:10572191

  12. 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

  13. 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

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

    PubMed Central

    Mukherjee, Sunil K.

    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

  15. Structural and functional analysis of chicken U4 small nuclear RNA genes.

    PubMed Central

    Hoffman, M L; Korf, G M; McNamara, K J; Stumph, W E

    1986-01-01

    Two distinct chicken U4 RNA genes have been cloned and characterized. They are closely linked within 465 base pairs of each other and have the same transcriptional orientation. The downstream U4 homology is a true gene, based on the criteria that it is colinear with chicken U4B RNA and is expressed when injected into Xenopus laevis oocytes. The upstream U4 homology, however, contains seven base substitutions relative to U4B RNA. This sequence may be a nonexpressed pseudogene, but the pattern of base substitutions suggests that it more probably encodes a variant yet functional U4 RNA product not yet characterized at the RNA level. In support of this, the two U4 genes have regions of homology with each other in their 5'-flanking DNA at two positions known to be essential for the efficient expression of vertebrate U1 and U2 small nuclear RNA genes. In the case of U1 and U2 RNA genes, the more distal region (located near position-200 with respect to the RNA cap site) is known to function as a transcriptional enhancer. Although this region is highly conserved in overall structure and sequence among U1 and U2 RNA genes, it is much less conserved in the chicken U4 RNA genes reported here. Interestingly, short sequence elements present in the -200 region of the U4 RNA genes are inverted (i.e., on the complementary strand) relative to their usual orientation upstream of U1 and U2 RNA genes. Thus, the -200 region of the U4 RNA genes may represent a natural evolutionary occurrence of an enhancer sequence inversion. Images PMID:3025618

  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. 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

  18. Nematode endogenous small RNA pathways

    PubMed Central

    Hoogstrate, Suzanne W; Volkers, Rita JM; Sterken, Mark G; Kammenga, Jan E; Snoek, L Basten

    2014-01-01

    The discovery of small RNA silencing pathways has greatly extended our knowledge of gene regulation. Small RNAs have been presumed to play a role in every field of biology because they affect many biological processes via regulation of gene expression and chromatin remodeling. Most well-known examples of affected processes are development, fertility, and maintenance of genome stability. Here we review the role of the three main endogenous small RNA silencing pathways in Caenorhabditis elegans: microRNAs, endogenous small interfering RNAs, and PIWI-interacting RNAs. After providing an entry-level overview on how these pathways function, we discuss research on other nematode species providing insight into the evolution of these small RNA pathways. In understanding the differences between the endogenous small RNA pathways and their evolution, a more comprehensive picture is formed of the functions and effects of small RNAs. PMID:25340013

  19. 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

  20. 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

  1. RNA-seq analysis of small RNPs in Trypanosoma brucei reveals a rich repertoire of non-coding RNAs

    PubMed Central

    Michaeli, Shulamit; Doniger, Tirza; Gupta, Sachin Kumar; Wurtzel, Omri; Romano, Mali; Visnovezky, Damian; Sorek, Rotem; Unger, Ron; Ullu, Elisabetta

    2012-01-01

    The discovery of a plethora of small non-coding RNAs (ncRNAs) has fundamentally changed our understanding of how genes are regulated. In this study, we employed the power of deep sequencing of RNA (RNA-seq) to examine the repertoire of ncRNAs present in small ribonucleoprotein particles (RNPs) of Trypanosoma brucei, an important protozoan parasite. We identified new C/D and H/ACA small nucleolar RNAs (snoRNAs), as well as tens of putative novel non-coding RNAs; several of these are processed from trans-spliced and polyadenylated transcripts. The RNA-seq analysis provided information on the relative abundance of the RNAs, and their 5′- and 3′-termini. The study demonstrated that three highly abundant snoRNAs are involved in rRNA processing and highlight the unique trypanosome-specific repertoire of these RNAs. Novel RNAs were studied using in situ hybridization, association in RNP complexes, and ‘RNA walk’ to detect interaction with their target RNAs. Finally, we showed that the abundance of certain ncRNAs varies between the two stages of the parasite, suggesting that ncRNAs may contribute to gene regulation during the complex parasite’s life cycle. This is the first study to provide a whole-genome analysis of the large repertoire of small RNPs in trypanosomes. PMID:21976736

  2. Preliminary Analysis of High-Throughput Expression Data and Small RNA in Soybean Stem Tissue Infected with Sclerotinia sclerotiorum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We recently published a report on transcriptome changes in soybean stem tissue challenged with Sclerotinia sclerotiorum based on cDNA microarray analysis. We are now advancing this study by examining the differential expression of small RNA (miRNAs and siRNAs) and gene transcripts using the Illumin...

  3. Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

    PubMed Central

    Van Nieuwerburgh, Filip; Kolliopoulou, Anna; Apostolou-Karampelis, Konstantinos; Head, Steven R.; Deforce, Dieter; Smagghe, Guy; Swevers, Luc

    2015-01-01

    ABSTRACT The lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera. IMPORTANCE This work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented double-stranded RNA (dsRNA) virus (CPV; Reoviridae) and highlights the importance of viral small-RNA (vsRNA) analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20-nucleotide vsRNAs or based on the activity of an uncharacterized endo-RNase that cleaves the viral RNA substrate at a degenerate motif. The analysis also indicates the existence of a degradation pathway that targets the positive strand of segment 10. PMID

  4. 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

  5. Small RNA Analysis in Sindbis Virus Infected Human HEK293 Cells

    PubMed Central

    Dalmay, Tamas; Powell, Penny P.

    2013-01-01

    Introduction In contrast to the defence mechanism of RNA interference (RNAi) in plants and invertebrates, its role in the innate response to virus infection of mammals is a matter of debate. Since RNAi has a well-established role in controlling infection of the alphavirus Sindbis virus (SINV) in insects, we have used this virus to investigate the role of RNAi in SINV infection of human cells. Results SINV AR339 and TR339-GFP were adapted to grow in HEK293 cells. Deep sequencing of small RNAs (sRNAs) early in SINV infection (4 and 6 hpi) showed low abundance (0.8%) of viral sRNAs (vsRNAs), with no size, sequence or location specific patterns characteristic of Dicer products nor did they possess any discernible pattern to ascribe to a specific RNAi biogenesis pathway. This was supported by multiple variants for each sequence, and lack of hot spots along the viral genome sequence. The abundance of the best defined vsRNAs was below the limit of Northern blot detection. The adaptation of the virus to HEK293 cells showed little sequence changes compared to the reference; however, a SNP in E1 gene with a preference from G to C was found. Deep sequencing results showed little variation of expression of cellular microRNAs (miRNAs) at 4 and 6 hpi compared to uninfected cells. Twelve miRNAs exhibiting some minor differential expression by sequencing, showed no difference in expression by Northern blot analysis. Conclusions We show that, unlike SINV infection of invertebrates, generation of Dicer-dependent svRNAs and change in expression of cellular miRNAs were not detected as part of the Human response to SINV. PMID:24391886

  6. 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

  7. Comparative analysis of the small RNA transcriptomes of miiuy croaker revealed microRNA-mediated regulation of TLR signaling pathway response to Vibrio anguillarum infection.

    PubMed

    Xu, Guoliang; Han, Jingjing; Xu, Tianjun

    2016-05-01

    MicroRNAs (miRNAs) are an abundant class of endogenous noncoding small RNAs (sRNAs) that are partially complementary to their target messenger RNA (mRNA), which post-transcriptionally regulate various biological processes by repressing mRNA translation or inducing mRNA degradation. MiRNAs have been demonstrated to play crucial roles in the host response to infection by diverse pathogens. As an important bacterial pathogen, Vibrio anguillarum has been caused great economic losses in miiuy croaker aquaculture. To identify immune-related miRNAs of miiuy croaker in response to V. anguillarum infection, we constructed two sRNA libraries with or without bacterial infection. High-throughput deep sequencing and subsequent bioinformatic analysis identified 241 conserved and 137 novel miRNA precursors in miiuy croaker based on its whole genome sequences, encoding 293 and 124 mature miRNAs, respectively. Then we compared the expression patterns of miRNAs in the two libraries. There were significant differences in the expression of 12 miRNAs between the infection group (IG) and control group (CG). Further, the expressions of six miRNAs were validated by real-time quantitative PCR. The target gene prediction and function analysis were conducted for the 12 differential miRNAs. This analysis revealed that these miRNAs participated in the regulation multiple immune-related signaling pathways. Transcription factors in TLR signaling, such as AP-1, IRF5, NF-κB and IRF3, were activated by these miRNAs via post-transcriptionally regulating the expression of TLRs and TLR-associated signaling proteins, inducing effective host immune response to eradicate infectious pathogens. This is the first study of the identification and characterization of miiuy croaker miRNAs in response to V. anguillarum infection. The comprehensive analysis of the expression of miRNAs and the target gene and function prediction of differently expressed miRNAs may help to understand the regulatory mechanisms of

  8. Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis.

    PubMed

    Rao, S Nageswara; Nath, B Surendra; Saratchandra, B

    2005-06-01

    This study is the first report on the genetic characterization and relationships among different microsporidia infecting the silkworm, Bombyx mori, using inter simple sequence repeat PCR (ISSR-PCR) analysis. Six different microsporidians were distinguished through molecular DNA typing using ISSR-PCR. Thus, ISSR-PCR analysis can be a powerful tool to detect polymorphisms and identify microsporidians, which are difficult to study with microscopy because of their extremely small size. Of the 100 ISSR primers tested, only 28 primers had reproducibility and high polymorphism (93%). A total of 24 ISSR primers produced 55 unique genetic markers, which could be used to differentiate the microsporidians from each other. Among the 28 SSRs tested, the most abundant were (CA)n, (GA)n, and (GT)n repeats. The degree of band sharing was used to evaluate genetic similarity between different microsporidian isolates and to construct a phylogenetic tree using Jaccard's similarity coefficient. The results indicate that the DNA profiles based on ISSR markers can be used as diagnostic tools to identify different microsporidia with considerable accuracy. In addition, the small subunit ribosomal RNA (SSU-rRNA) sequence gene was amplified, cloned, and sequenced from each of the 6 microsporidian isolates. These sequences were compared with 20 other microsporidian SSU-rRNA sequences to develop a phylogenetic tree for the microsporidia isolated from the silkworms. This method was found to be useful in establishing the phylogenetic relationships among the different microsporidians isolated from silkworms. Of the 6 microsporidian isolates, NIK-1s revealed an SSU-rRNA gene sequence similar to Nosema bombycis, indicating that NIK-1s is similar to N. bombycis; the remaining 5 isolates, which differed from each other and from N. bombycis, were considered to be different variants belonging to the species N. bombycis. PMID:16121233

  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. Molecular characterization and phylogenetic relationships among microsporidian isolates infecting silkworm, Bombyx mori using small subunit rRNA (SSU-rRNA) gene sequence analysis.

    PubMed

    Nath, B Surendra; Gupta, S K; Bajpai, A K

    2012-12-01

    The life cycle, spore morphology, pathogenicity, tissue specificity, mode of transmission and small subunit rRNA (SSU-rRNA) gene sequence analysis of the five new microsporidian isolates viz., NIWB-11bp, NIWB-12n, NIWB-13md, NIWB-14b and NIWB-15mb identified from the silkworm, Bombyx mori have been studied along with type species, NIK-1s_mys. The life cycle of the microsporidians identified exhibited the sequential developmental cycles that are similar to the general developmental cycle of the genus, Nosema. The spores showed considerable variations in their shape, length and width. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIWB-15mb was found to be more virulent than other isolates. All of the microsporidians were found to infect most of the tissues examined and showed gonadal infection and transovarial transmission in the infected silkworms. SSU-rRNA sequence based phylogenetic tree placed NIWB-14b, NIWB-12n and NIWB-11bp in a separate branch along with other Nosema species and Nosema bombycis; while NIWB-15mb and NIWB-13md together formed another cluster along with other Nosema species. NIK-1s_mys revealed a signature sequence similar to standard type species, N. bombycis, indicating that NIK-1s_mys is similar to N. bombycis. Based on phylogenetic relationships, branch length information based on genetic distance and nucleotide differences, we conclude that the microsporidian isolates identified are distinctly different from the other known species and belonging to the genus, Nosema. This SSU-rRNA gene sequence analysis method is found to be more useful approach in detecting different and closely related microsporidians of this economically important domestic insect. PMID:23129193

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

    PubMed Central

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

    2015-01-01

    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. PMID:26697336

  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. PMID:26697336

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

    PubMed Central

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

    2013-01-01

    The conventional small RNA isolation and detection methods for yeast cells have been designed for a small number of samples. In order to conduct a genome-wide assessment of how each gene product impacts upon small non-coding RNAs, we developed a rapid method for analyzing small RNAs from Saccharomyces cerevisiae wild-type 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 nonradioactive Northern method for RNA detection. The RNA isolation procedure is highly reproducible and requires only 4 hours for processing 96 samples, and yields RNA of good quality and quantity. The nonradioactive Northern method employs digoxigenin (DIG)-labeled DNA probes and chemiluminescence. It detects femtomole-level small RNAs within 1-minute exposure time. We minimized the processing time for large-scale analysis and optimized the stripping and re-probing procedures for analysis 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. PMID:23417998

  16. Transfer RNA-derived small RNAs in the cancer transcriptome.

    PubMed

    Green, Darrell; Fraser, William D; Dalmay, Tamas

    2016-06-01

    The cellular lifetime includes stages such as differentiation, proliferation, division, senescence and apoptosis. These stages are driven by a strictly ordered process of transcription dynamics. Molecular disruption to RNA polymerase assembly, chromatin remodelling and transcription factor binding through to RNA editing, splicing, post-transcriptional regulation and ribosome scanning can result in significant costs arising from genome instability. Cancer development is one example of when such disruption takes place. RNA silencing is a term used to describe the effects of post-transcriptional gene silencing mediated by a diverse set of small RNA molecules. Small RNAs are crucial for regulating gene expression and microguarding genome integrity. RNA silencing studies predominantly focus on small RNAs such as microRNAs, short-interfering RNAs and piwi-interacting RNAs. We describe an emerging renewal of interest in a 'larger' small RNA, the transfer RNA (tRNA). Precisely generated tRNA-derived small RNAs, named tRNA halves (tiRNAs) and tRNA fragments (tRFs), have been reported to be abundant with dysregulation associated with cancer. Transfection of tiRNAs inhibits protein translation by displacing eukaryotic initiation factors from messenger RNA (mRNA) and inaugurating stress granule formation. Knockdown of an overexpressed tRF inhibits cancer cell proliferation. Recovery of lacking tRFs prevents cancer metastasis. The dual oncogenic and tumour-suppressive role is typical of functional small RNAs. We review recent reports on tiRNA and tRF discovery and biogenesis, identification and analysis from next-generation sequencing data and a mechanistic animal study to demonstrate their physiological role in cancer biology. We propose tRNA-derived small RNA-mediated RNA silencing is an innate defence mechanism to prevent oncogenic translation. We expect that cancer cells are percipient to their ablated control of transcription and attempt to prevent loss of genome control

  17. 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...

  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. PMID:20008100

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

    PubMed Central

    Pantano, Lorena; Estivill, Xavier; Martí, Eulàlia

    2010-01-01

    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. Small RNA Deep Sequencing Reveals Role for Arabidopsis thaliana RNA-Dependent RNA Polymerases in Viral siRNA Biogenesis

    PubMed Central

    Qi, Xiaopeng; Bao, Forrest Sheng; Xie, Zhixin

    2009-01-01

    RNA silencing functions as an important antiviral defense mechanism in a broad range of eukaryotes. In plants, biogenesis of several classes of endogenous small interfering RNAs (siRNAs) requires RNA-dependent RNA Polymerase (RDR) activities. Members of the RDR family proteins, including RDR1and RDR6, have also been implicated in antiviral defense, although a direct role for RDRs in viral siRNA biogenesis has yet to be demonstrated. Using a crucifer-infecting strain of Tobacco Mosaic Virus (TMV-Cg) and Arabidopsis thaliana as a model system, we analyzed the viral small RNA profile in wild-type plants as well as rdr mutants by applying small RNA deep sequencing technology. Over 100,000 TMV-Cg-specific small RNA reads, mostly of 21- (78.4%) and 22-nucleotide (12.9%) in size and originating predominately (79.9%) from the genomic sense RNA strand, were captured at an early infection stage, yielding the first high-resolution small RNA map for a plant virus. The TMV-Cg genome harbored multiple, highly reproducible small RNA-generating hot spots that corresponded to regions with no apparent local hairpin-forming capacity. Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis. In addition, an informatics analysis showed that a large set of host genes could be potentially targeted by TMV-Cg-derived siRNAs for posttranscriptional silencing. Two of such predicted host targets, which encode a cleavage and polyadenylation specificity factor (CPSF30) and an unknown protein similar to translocon-associated protein alpha (TRAP α), respectively, yielded a positive result in cleavage validation by 5′RACE assays. Our data raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity. PMID:19308254

  3. Metatranscriptomic Analysis of Microbes in an Oceanfront Deep-Subsurface Hot Spring Reveals Novel Small RNAs and Type-Specific tRNA Degradation

    PubMed Central

    Murakami, Shinnosuke; Fujishima, Kosuke; Tomita, Masaru

    2012-01-01

    Studies of small noncoding RNAs (sRNAs) have been conducted predominantly using culturable organisms, and the acquisition of further information about sRNAs from global environments containing uncultured organisms now is very important. In this study, hot spring water (57°C, pH 8.1) was collected directly from the underground environment at depths of 250 to 1,000 m in Yunohama, Japan, and small RNA sequences obtained from the environment were analyzed. A phylogenetic analysis of both archaeal and bacterial 16S rRNA gene sequences was conducted, and the results suggested the presence of unique species in the environment, corresponding to the Archaeal Richmond Mine Acidophilic Nanoorganisms (ARMAN) group and three new Betaproteobacteria. A metatranscriptomic analysis identified 64,194 (20,057 nonredundant) cDNA sequences. Of these cDNAs, 90% were either tRNAs, tRNA fragments, rRNAs, or rRNA fragments, whereas 2,181 reads (10%) were classified as previously uncharacterized putative candidate sRNAs. Among these, 15 were particularly abundant, 14 of which showed no sequence similarity to any known noncoding RNA, and at least six of which form very stable RNA secondary structures. The analysis of a large number of tRNA fragments suggested that unique relationships exist between the anticodons of the tRNAs and the sites of tRNA degradation. Previous bacterial tRNA degradation studies have been limited to specific organisms, such as Escherichia coli and Streptomyces coelicolor, and the current results suggest that specific tRNA decay occurs more frequently than previously expected. PMID:22156430

  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

    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. Genome-Wide Analysis of Small RNA and Novel MicroRNA Discovery in Human Acute Lymphoblastic Leukemia Based on Extensive Sequencing Approach

    PubMed Central

    Zhang, Peng; Chen, Xiao; Wu, Jun; Xu, Ling; Luo, Xue-Qun; Ke, Zhi-Yong; Zhou, Hui; Qu, Liang-Hu; Chen, Yue-Qin

    2009-01-01

    Background MicroRNAs (miRNAs) have been proved to play an important role in various cellular processes and function as tumor suppressors or oncogenes in cancers including leukemia. The identification of a large number of novel miRNAs and other small regulatory RNAs will provide valuable insights into the roles they play in tumorgenesis. Methodology/Principal Findings To gain further understanding of the role of miRNAs relevant to acute lymphoblastic leukemia (ALL), we employed the sequencing-by-synthesis (SBS) strategy to sequence small RNA libraries prepared from ALL patients and normal donors. In total we identified 159 novel miRNAs and 116 novel miRNA*s from both libraries. Among the 159 novel miRNAs, 42 were identified with high stringency in our data set. Furthermore, we demonstrated the different expression patterns of 20 newly identified and several known miRNAs between ALL patients and normal donors, suggesting these miRNAs may be associated with ALL and could constitute an ALL-specific miRNA signature. Interestingly, GO “biological process” classifications revealed that a set of significantly abnormally expressed miRNAs are associated with disease relapse, which implies that these dysregulated miRNAs might promote the progression of ALL by regulating genes involved in the pathway of the disease development. Conclusion/Significance The study presents a comprehensive picture of the expression of small RNAs in human acute lymphoblastic leukemia and highlights novel and known miRNAs differentially expressed between ALL patients and normal donors. To our knowledge, this is the first study to look at genome-wide known and novel miRNA expression patterns in in human acute lymphoblastic leukemia. Our data revealed that these deregulated miRNAs may be associated with ALL or the onset of relapse. PMID:19724645

  6. A Systemic Small RNA Signaling System in Plants

    PubMed Central

    Yoo, Byung-Chun; Kragler, Friedrich; Varkonyi-Gasic, Erika; Haywood, Valerie; Archer-Evans, Sarah; Lee, Young Moo; Lough, Tony J.; Lucas, William J.

    2004-01-01

    Systemic translocation of RNA exerts non-cell-autonomous control over plant development and defense. Long-distance delivery of mRNA has been proven, but transport of small interfering RNA and microRNA remains to be demonstrated. Analyses performed on phloem sap collected from a range of plants identified populations of small RNA species. The dynamic nature of this population was reflected in its response to growth conditions and viral infection. The authenticity of these phloem small RNA molecules was confirmed by bioinformatic analysis; potential targets for a set of phloem small RNA species were identified. Heterografting studies, using spontaneously silencing coat protein (CP) plant lines, also established that transgene-derived siRNA move in the long-distance phloem and initiate CP gene silencing in the scion. Biochemical analysis of pumpkin (Cucurbita maxima) phloem sap led to the characterization of C. maxima Phloem SMALL RNA BINDING PROTEIN1 (CmPSRP1), a unique component of the protein machinery probably involved in small RNA trafficking. Equivalently sized small RNA binding proteins were detected in phloem sap from cucumber (Cucumis sativus) and lupin (Lupinus albus). PSRP1 binds selectively to 25-nucleotide single-stranded RNA species. Microinjection studies provided direct evidence that PSRP1 could mediate the cell-to-cell trafficking of 25-nucleotide single-stranded, but not double-stranded, RNA molecules. The potential role played by PSRP1 in long-distance transmission of silencing signals is discussed with respect to the pathways and mechanisms used by plants to exert systemic control over developmental and physiological processes. PMID:15258266

  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. 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. PMID:27077039

  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. 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

  11. Genome-Wide Analysis of leafbladeless1-Regulated and Phased Small RNAs Underscores the Importance of the TAS3 ta-siRNA Pathway to Maize Development

    PubMed Central

    Dotto, Marcela C.; Petsch, Katherine A.; Aukerman, Milo J.; Beatty, Mary; Hammell, Molly; Timmermans, Marja C. P.

    2014-01-01

    Maize leafbladeless1 (lbl1) encodes a key component in the trans-acting short-interfering RNA (ta-siRNA) biogenesis pathway. Correlated with a great diversity in ta-siRNAs and the targets they regulate, the phenotypes conditioned by mutants perturbing this small RNA pathway vary extensively across species. Mutations in lbl1 result in severe developmental defects, giving rise to plants with radial, abaxialized leaves. To investigate the basis for this phenotype, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects the accumulation of small RNAs in all major classes, and reveal unexpected crosstalk between ta-siRNA biogenesis and other small RNA pathways regulating transposons. Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model. Our analysis identified nine TAS loci, all belonging to the conserved TAS3 family. Information from RNA deep sequencing and PARE analyses identified the tasiR-ARFs as the major functional ta-siRNAs in the maize vegetative apex where they regulate expression of AUXIN RESPONSE FACTOR3 (ARF3) homologs. Plants expressing a tasiR-ARF insensitive arf3a transgene recapitulate the phenotype of lbl1, providing direct evidence that deregulation of ARF3 transcription factors underlies the developmental defects of maize ta-siRNA biogenesis mutants. The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3. Our data indicate that diversity in TAS pathways and their targets cannot fully account for the phenotypic differences conditioned by ta-siRNA biogenesis mutants across plant species. Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct

  12. Genomewide analysis of small RNAs in nonembryogenic and embryogenic tissues of citrus: microRNA- and siRNA-mediated transcript cleavage involved in somatic embryogenesis.

    PubMed

    Wu, Xiao-Meng; Kou, Shu-Jun; Liu, Yuan-Long; Fang, Yan-Ni; Xu, Qiang; Guo, Wen-Wu

    2015-04-01

    Somatic embryogenesis (SE) is a process of somatic cells becoming dedifferentiated and generating embryos. SE has been widely used in biotechnology as a powerful way of regeneration and a model system for studying plant embryogenesis, but the controlling mechanisms of SE are far from clear. Here, we show the genomewide profiles of miRNAs/siRNAs and their target genes in nonembryogenic and embryogenic tissues of 'Valencia' sweet orange. By high-throughput sequencing (HTS) of small RNAs and RNA degradome tags, we identified 50 known and 45 novel miRNAs, 130 miniature inverted-repeat transposable elements (MITEs) derived, 94 other and 235 phased small interfering RNAs (siRNAs), as well as 203 target genes. The majority of the abundantly expressed miRNAs/siRNAs exhibit lower expression levels in embryogenic callus (EC) or during SE process than in nonembryogenic callus (NEC), which is supposed to derepress the target genes that are involved in development and stress response, thus to activate the biological processes required for cell differentiation. However, the conserved csi-miR156a/b, miR164b and 171c directed suppression of specific transcription factors (TFs) are supposed to inactivate the postembryonic growth thus to maintain normal SE. In this study, miRNA- and siRNA-mediated silencing of target genes was found under sophisticated regulation in citrus SE system; the enhancement effect of specific conserved miRNAs on SE was discussed, providing new clues for future investigation of mechanisms that control SE. PMID:25615015

  13. 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

  14. Structural and functional analysis of Escherichia coli ribosomes containing small deletions around position 1760 in the 23S ribosomal RNA.

    PubMed Central

    Zweib, C; Dahlberg, A E

    1984-01-01

    Three different small deletions were produced at a single Pvu 2 restriction site in E. coli 23S rDNA of plasmid pKK 3535 using exonuclease Bal 31. The deletions were located around position 1760 in 23S rRNA and were characterized by DNA sequencing as well as by direct fingerprinting and S1-mapping of the rRNA. Two of the mutant plasmids, Pvu 2-32 and Pvu 2-33, greatly reduced the growth rate of transformed cells while the third mutant, Pvu 2-14 grew as fast as cells containing the wild-type plasmid pKK 3535. All three mutant 23S rRNAs were incorporated into 50S-like particles and were even found in 70S ribosomes and polysomes in vivo. The conformation of mutant 23S rRNA in 50S subunits was probed with a double-strand specific RNase from cobra venom. These analyses revealed changes in the accessibility of cleavage sites near the deletions around position 1760 and in the area around position 800 in all three mutant rRNAs. We suggest, that an altered conformation of the rRNAs at the site of the deletion is responsible for the slow growth of cells containing mutant plasmids Pvu 2-32 and Pvu 2-33. Images PMID:6091057

  15. Establishment of a continuous culture system for Entamoeba muris and analysis of the small subunit rRNA gene.

    PubMed

    Kobayashi, S; Suzuki, J; Takeuchi, T

    2009-06-01

    We established a culture system for Entamoeba muris (MG-EM-01 strain isolated from a Mongolian gerbil) using a modified Balamuth's egg yolk infusion medium supplemented with 4% adult bovine serum and Bacteroides fragilis cocultured with Escherichia coli. Further, encystation was observed in the culture medium. The morphological characteristics of E. muris are similar to those of Entamoeba coli (E. coli); moreover, the malic isoenzyme electrophoretic band, which shows species-specific electrophoretic mobility, of E. muris had almost the same mobility as that observed with the malic isoenzyme electrophorectic band of E. coli (UZG-EC-01 strain isolated from a gorilla). We determined the small subunit rRNA (SSU-rRNA) gene sequence of the MG-EM-01 strain, and this sequence was observed to show 82.7% homology with that of the UZG-EC-01 strain. Further, the resultant phylogenetic tree for molecular taxonomy based on the SSU-rRNA genes of the 21 strains of the intestinal parasitic amoeba species indicated that the MG-EM-01 strain was most closely related to E. coli. PMID:19585892

  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. Covalent small-molecule-RNA complex formation enables cellular profiling of small-molecule-RNA interactions

    PubMed Central

    Guan, Lirui

    2013-01-01

    Won’t let you go! A strategy is described to design small molecules that react with their cellular RNA targets. This approach not only improves the activity of compounds targeting RNA in cell culture by ≈2500-fold but also enables cell-wide profiling of its RNA targets. PMID:23913698

  18. Covalent small-molecule-RNA complex formation enables cellular profiling of small-molecule-RNA interactions.

    PubMed

    Guan, Lirui; Disney, Matthew D

    2013-09-16

    Won't let you go! A strategy is described to design small molecules that react with their cellular RNA targets. This approach not only improves the activity of compounds targeting RNA in cell culture by a factor of about 2500 but also enables cell-wide profiling of its RNA targets. PMID:23913698

  19. 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

  20. 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.

  1. 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.

  2. Genome-wide Medicago truncatula small RNA analysis revealed novel microRNAs and isoforms differentially regulated in roots and nodules.

    PubMed

    Lelandais-Brière, Christine; Naya, Loreto; Sallet, Erika; Calenge, Fanny; Frugier, Florian; Hartmann, Caroline; Gouzy, Jérome; Crespi, Martin

    2009-09-01

    Posttranscriptional regulation of a variety of mRNAs by small 21- to 24-nucleotide RNAs, notably the microRNAs (miRNAs), is emerging as a novel developmental mechanism. In legumes like the model Medicago truncatula, roots are able to develop a de novo meristem through the symbiotic interaction with nitrogen-fixing rhizobia. We used deep sequencing of small RNAs from root apexes and nodules of M. truncatula to identify 100 novel candidate miRNAs encoded by 265 hairpin precursors. New atypical precursor classes producing only specific 21- and 24-nucleotide small RNAs were found. Statistical analysis on sequencing reads abundance revealed specific miRNA isoforms in a same family showing contrasting expression patterns between nodules and root apexes. The differentially expressed conserved and nonconserved miRNAs may target a large variety of mRNAs. In root nodules, which show diverse cell types ranging from a persistent meristem to a fully differentiated central region, we discovered miRNAs spatially enriched in nodule meristematic tissues, vascular bundles, and bacterial infection zones using in situ hybridization. Spatial regulation of miRNAs may determine specialization of regulatory RNA networks in plant differentiation processes, such as root nodule formation. PMID:19767456

  3. 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

  4. 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...

  5. 'RNA walk' a novel approach to study RNA-RNA interactions between a small RNA and its target.

    PubMed

    Lustig, Yaniv; Wachtel, Chaim; Safro, Mark; Liu, Li; Michaeli, Shulamit

    2010-01-01

    In this study we describe a novel method to investigate the RNA-RNA interactions between a small RNA and its target that we termed 'RNA walk'. The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT-PCR or real-time PCR. Domains carrying the cross-linked adducts fail to efficiently amplify by PCR compared with non-cross-linked domains. This method was calibrated and used to study the interaction between a special tRNA-like molecule (sRNA-85) that is part of the trypanosome signal recognition particle (SRP) complex and the ribosome. Four contact sites between sRNA-85 and rRNA were identified by 'RNA walk' and were further fine-mapped by primer extension. Two of the contact sites are expected; one contact site mimics the interaction of the mammalian Alu domain of SRP with the ribosome and the other contact sites include a canonical tRNA interaction. The two other cross-linked sites could not be predicted. We propose that 'RNA walk, is a generic method to map target RNA small RNAs interactions in vivo. PMID:19854950

  6. 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

  7. RNA sequence analysis using covariance models.

    PubMed Central

    Eddy, S R; Durbin, R

    1994-01-01

    We describe a general approach to several RNA sequence analysis problems using probabilistic models that flexibly describe the secondary structure and primary sequence consensus of an RNA sequence family. We call these models 'covariance models'. A covariance model of tRNA sequences is an extremely sensitive and discriminative tool for searching for additional tRNAs and tRNA-related sequences in sequence databases. A model can be built automatically from an existing sequence alignment. We also describe an algorithm for learning a model and hence a consensus secondary structure from initially unaligned example sequences and no prior structural information. Models trained on unaligned tRNA examples correctly predict tRNA secondary structure and produce high-quality multiple alignments. The approach may be applied to any family of small RNA sequences. Images PMID:8029015

  8. 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

  9. 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

  10. Pulmonary administration of small interfering RNA: The route to go?

    PubMed

    Ruigrok, M J R; Frijlink, H W; Hinrichs, W L J

    2016-08-10

    Ever since the discovery of RNA interference (RNAi), which is a post-transcriptional gene silencing mechanism, researchers have been studying the therapeutic potential of using small interfering RNA (siRNA) to treat diseases that are characterized by excessive gene expression. Excessive gene expression can be particularly harmful if it occurs in a vulnerable organ such as the lungs as they are essential for physiological respiration. Consequently, RNAi could offer an approach to treat such lung diseases. Parenteral administration of siRNA has been shown to be difficult due to degradation by nucleases in the systemic circulation and excretion by the kidneys. To avoid these issues and to achieve local delivery and local effects, pulmonary administration has been proposed as an alternative administration route. Regarding this application, various animal studies have been conducted over the past few years. Therefore, this review presents a critical analysis of publications where pulmonary administration of siRNA in animals has been reported. Such an analysis is necessary to determine the feasibility of this administration route and to define directions for future research. First, we provide background information on lungs, pulmonary administration, and delivery vectors. Thereafter, we present and discuss relevant animal studies. Though nearly all publications reported positive outcomes, several reoccurring challenges were identified. They relate to 1) the necessity, efficacy, and safety of delivery vectors, 2) the biodistribution of siRNA in tissues other than the lungs, 3) the poor correlation between in vitro and in vivo models, and 4) the long-term effects upon (repeated) administration of siRNA. Finally, we present recommendations for future research to define the route to go: towards safer and more effective pulmonary administration of siRNA. PMID:27235976

  11. Recent advances in developing small molecules targeting RNA.

    PubMed

    Guan, Lirui; Disney, Matthew D

    2012-01-20

    RNAs are underexploited targets for small molecule drugs or chemical probes of function. This may be due, in part, to a fundamental lack of understanding of the types of small molecules that bind RNA specifically and the types of RNA motifs that specifically bind small molecules. In this review, we describe recent advances in the development and design of small molecules that bind to RNA and modulate function that aim to fill this void. PMID:22185671

  12. Small RNA in the nucleus: the RNA-chromatin ping-pong

    PubMed Central

    Olovnikov, Ivan; Aravin, Alexei A.; Toth, Katalin Fejes

    2012-01-01

    Eukaryotes use several classes of small RNA molecules to guide diverse protein machineries to target messenger RNA. The role of small RNA in post-transcriptional regulation of mRNA stability and translation is now well established. Small RNAs can also guide sequence-specific modification of chromatin structure and thus contribute to establishment and maintenance of distinct chromatin domains. In this review we summarize the model for the inter-dependent interaction between small RNA and chromatin that has emerged from studies on fission yeast and plants. We focus on recent results that link a distinct class of small RNAs, the piRNAs, to chromatin regulation in animals. PMID:22349141

  13. 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. PMID:27067812

  14. 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.

  15. Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development.

    PubMed

    Song, Xianwei; Wang, Dekai; Ma, Lijia; Chen, Zhiyu; Li, Pingchuan; Cui, Xia; Liu, Chunyan; Cao, Shouyun; Chu, Chengcai; Tao, Yuezhi; Cao, Xiaofeng

    2012-08-01

    Higher plants have evolved multiple RNA-dependent RNA polymerases (RDRs), which work with Dicer-like (DCL) proteins to produce different classes of small RNAs with specialized molecular functions. Here we report that OsRDR6, the rice (Oryza sativa L.) homolog of Arabidopsis RDR6, acts in the biogenesis of various types and sizes of small RNAs. We isolated a rice osrdr6-1 mutant, which was temperature sensitive and showed spikelet defects. This mutant displays reduced accumulation of tasiR-ARFs, the conserved trans-acting siRNAs (tasiRNAs) derived from the TAS3 locus, and ectopic expression of tasiR-ARF target genes, the Auxin Response Factors (including ARF2 and ARF3/ETTIN). The loss of tasiR-mediated repression of ARFs in osrdr6-1 can explain its morphological defects, as expression of two non-targeted ARF3 gene constructs (ARF3muts) in a wild-type background mimics the osrdr6 and osdcl4-1 mutant phenotypes. Small RNA high-throughput sequencing also reveals that besides tasiRNAs, 21-nucleotide (nt) phased small RNAs are also largely dependent on OsRDR6. Unexpectedly, we found that osrdr6-1 has a strong impact on the accumulation of 24-nt phased small RNAs, but not on unphased ones. Our work uncovers the key roles of OsRDR6 in small RNA biogenesis and directly illustrates the crucial functions of tasiR-ARFs in rice development. PMID:22443269

  16. Small Molecule Chemical Probes of MicroRNA Function

    PubMed Central

    Velagapudi, Sai Pradeep; Vummidi, Balayeshwanth R.; Disney, Matthew D.

    2015-01-01

    MicroRNAs (miRNAs) are small, non-coding RNAs that control protein expression. Aberrant miRNA expression has been linked to various human diseases, and thus miRNAs have been explored as diagnostic markers and therapeutic targets. Although it is challenging to target RNA with small molecules in general, there have been successful campaigns that have identified small molecule modulators of miRNA function by targeting various pathways. For example, small molecules that modulate transcription and target nuclease processing sites in miRNA precursors have been identified. Herein, we describe challenges in developing chemical probes that target miRNAs and highlight aspects of miRNA cellular biology elucidated by using small molecule chemical probes. We expect that this area will expand dramatically in the near future as strides are made to understand small molecule recognition of RNA from a fundamental perspective. PMID:25500006

  17. Small molecule chemical probes of microRNA function.

    PubMed

    Velagapudi, Sai Pradeep; Vummidi, Balayeshwanth R; Disney, Matthew D

    2015-02-01

    MicroRNAs (miRNAs) are small, non-coding RNAs that control protein expression. Aberrant miRNA expression has been linked to various human diseases, and thus miRNAs have been explored as diagnostic markers and therapeutic targets. Although it is challenging to target RNA with small molecules in general, there have been successful campaigns that have identified small molecule modulators of miRNA function by targeting various pathways. For example, small molecules that modulate transcription and target nuclease processing sites in miRNA precursors have been identified. Herein, we describe challenges in developing chemical probes that target miRNAs and highlight aspects of miRNA cellular biology elucidated by using small molecule chemical probes. We expect that this area will expand dramatically in the near future as progress is made in understanding small molecule recognition of RNA. PMID:25500006

  18. Exploiting the small RNA deep sequencing technology for identification of viruses and viroids in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Small RNAs (including miRNA and siRNA) are produced abundantly in plants and animals in regulating gene expression or in defense against virus or viroid infection. Analysis of a siRNA profile upon virus infection in plant may allow for de novo assembly of a virus genome. In the present study, four...

  19. 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

  20. 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

  1. 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

  2. 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

  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. 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

  5. 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

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

    PubMed Central

    Park, So Hae; Butcher, Bronwyn G.; Anderson, Zoe; Pellegrini, Nola; Bao, Zhongmeng; D’Amico, Katherine

    2013-01-01

    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 Pseudomonas syringae pv. tomato DC3000, P16, was investigated. We determined that RpoS regulates the expression of P16. We found that deletion of P16 results in increased sensitivity to hydrogen peroxide compared to the wild-type strain, suggesting that P16 plays a role in the bacteria’s susceptibility to oxidative stress. Additionally the P16 mutant displayed enhanced resistance to heat stress. Our findings provide new information on the regulation and role of this ncRNA in P. syringae. PMID:23258266

  7. 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

  8. Role of RNA polymerase IV in plant small RNA metabolism.

    PubMed

    Zhang, Xiaoyu; Henderson, Ian R; Lu, Cheng; Green, Pamela J; Jacobsen, Steven E

    2007-03-13

    In addition to the three RNA polymerases (RNAP I-III) shared by all eukaryotic organisms, plant genomes encode a fourth RNAP (RNAP IV) that appears to be specialized in the production of siRNAs. Available data support a model in which dsRNAs are generated by RNAP IV and RNA-dependent RNAP 2 (RDR2) and processed by DICER (DCL) enzymes into 21- to 24-nt siRNAs, which are associated with different ARGONAUTE (AGO) proteins for transcriptional or posttranscriptional gene silencing. However, it is not yet clear what fraction of genomic siRNA production is RNAP IV-dependent, and to what extent these siRNAs are preferentially processed by certain DCL(s) or associated with specific AGOs for distinct downstream functions. To address these questions on a genome-wide scale, we sequenced approximately 335,000 siRNAs from wild-type and RNAP IV mutant Arabidopsis plants by using 454 technology. The results show that RNAP IV is required for the production of >90% of all siRNAs, which are faithfully produced from a discrete set of genomic loci. Comparisons of these siRNAs with those accumulated in rdr2 and dcl2 dcl3 dcl4 and those associated with AGO1 and AGO4 provide important information regarding the processing, channeling, and functions of plant siRNAs. We also describe a class of RNAP IV-independent siRNAs produced from endogenous single-stranded hairpin RNA precursors. PMID:17360559

  9. A chemical screen for biological small molecule–RNA conjugates reveals CoA-linked RNA

    PubMed Central

    Kowtoniuk, Walter E.; Shen, Yinghua; Heemstra, Jennifer M.; Agarwal, Isha; Liu, David R.

    2009-01-01

    Compared with the rapidly expanding set of known biological roles for RNA, the known chemical diversity of cellular RNA has remained limited primarily to canonical RNA, 3′-aminoacylated tRNAs, nucleobase-modified RNAs, and 5′-capped mRNAs in eukaryotes. We developed two methods to detect in a broad manner chemically labile cellular small molecule–RNA conjugates. The methods were validated by the detection of known tRNA and rRNA modifications. The first method analyzes small molecules cleaved from RNA by base or nucleophile treatment. Application to Escherichia coli and Streptomyces venezuelae RNA revealed an RNA-linked hydroxyfuranone or succinyl ester group, in addition to a number of other putative small molecule–RNA conjugates not previously reported. The second method analyzes nuclease-generated mononucleotides before and after treatment with base or nucleophile and also revealed a number of new putative small molecule–RNA conjugates, including 3′-dephospho-CoA and its succinyl-, acetyl-, and methylmalonyl-thioester derivatives. Subsequent experiments established that these CoA species are attached to E. coli and S. venezuelae RNA at the 5′ terminus. CoA-linked RNA cannot be generated through aberrant transcriptional initiation by E. coli RNA polymerase in vitro, and CoA-linked RNA in E. coli is only found among smaller (≲200 nucleotide) RNAs that have yet to be identified. These results provide examples of small molecule-RNA conjugates and suggest that the chemical diversity of cellular RNA may be greater than previously understood. PMID:19416889

  10. A chemical screen for biological small molecule-RNA conjugates reveals CoA-linked RNA.

    PubMed

    Kowtoniuk, Walter E; Shen, Yinghua; Heemstra, Jennifer M; Agarwal, Isha; Liu, David R

    2009-05-12

    Compared with the rapidly expanding set of known biological roles for RNA, the known chemical diversity of cellular RNA has remained limited primarily to canonical RNA, 3'-aminoacylated tRNAs, nucleobase-modified RNAs, and 5'-capped mRNAs in eukaryotes. We developed two methods to detect in a broad manner chemically labile cellular small molecule-RNA conjugates. The methods were validated by the detection of known tRNA and rRNA modifications. The first method analyzes small molecules cleaved from RNA by base or nucleophile treatment. Application to Escherichia coli and Streptomyces venezuelae RNA revealed an RNA-linked hydroxyfuranone or succinyl ester group, in addition to a number of other putative small molecule-RNA conjugates not previously reported. The second method analyzes nuclease-generated mononucleotides before and after treatment with base or nucleophile and also revealed a number of new putative small molecule-RNA conjugates, including 3'-dephospho-CoA and its succinyl-, acetyl-, and methylmalonyl-thioester derivatives. Subsequent experiments established that these CoA species are attached to E. coli and S. venezuelae RNA at the 5' terminus. CoA-linked RNA cannot be generated through aberrant transcriptional initiation by E. coli RNA polymerase in vitro, and CoA-linked RNA in E. coli is only found among smaller (approximately < 200 nucleotide) RNAs that have yet to be identified. These results provide examples of small molecule-RNA conjugates and suggest that the chemical diversity of cellular RNA may be greater than previously understood. PMID:19416889

  11. Identification of discrete classes of small nucleolar RNA featuring different ends and RNA binding protein dependency

    PubMed Central

    Deschamps-Francoeur, Gabrielle; Garneau, Daniel; Dupuis-Sandoval, Fabien; Roy, Audrey; Frappier, Marie; Catala, Mathieu; Couture, Sonia; Barbe-Marcoux, Mélissa; Abou-Elela, Sherif; Scott, Michelle S.

    2014-01-01

    Small nucleolar RNAs (snoRNAs) are among the first discovered and most extensively studied group of small non-coding RNA. However, most studies focused on a small subset of snoRNAs that guide the modification of ribosomal RNA. In this study, we annotated the expression pattern of all box C/D snoRNAs in normal and cancer cell lines independent of their functions. The results indicate that C/D snoRNAs are expressed as two distinct forms differing in their ends with respect to boxes C and D and in their terminal stem length. Both forms are overexpressed in cancer cell lines but display a conserved end distribution. Surprisingly, the long forms are more dependent than the short forms on the expression of the core snoRNP protein NOP58, thought to be essential for C/D snoRNA production. In contrast, a subset of short forms are dependent on the splicing factor RBFOX2. Analysis of the potential secondary structure of both forms indicates that the k-turn motif required for binding of NOP58 is less stable in short forms which are thus less likely to mature into a canonical snoRNP. Taken together the data suggest that C/D snoRNAs are divided into at least two groups with distinct maturation and functional preferences. PMID:25074380

  12. Detection of small interfering RNA (siRNA) by mass spectrometry procedures in doping controls.

    PubMed

    Thomas, Andreas; Walpurgis, Katja; Delahaut, Philippe; Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

    2013-01-01

    Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging field in sports drug testing. Due to the potential to principally knock down every target gene in the organism by means of the RNA interference pathway, this facet of gene doping has become a realistic scenario. In the present study, two distinct model siRNAs comprising 21 nucleotides were designed as double strands which were perfect counterparts to a sequence of the respective messenger RNA coding the muscle regulator myostatin of Rattus norvegicus. Several modified nucleotides were introduced in both the sense and the antisense strand comprising phosphothioates, 2'-O-methylation, 2'-fluoro-nucleotides, locked nucleic acids and a cholesterol tag at the 3'-end. The model siRNAs were applied to rats at 1 mg/kg (i.v.) and blood as well as urine samples were collected. After isolation of the RNA by means of a RNA purification kit, the target analytes were detected by liquid chromatography - high resolution/high accuracy mass spectrometry (LC-HRMS). Analytes were detected as modified nucleotides after alkaline hydrolysis, as intact oligonucleotide strands (top-down) and by means of denaturing SDS-PAGE analysis. The gel-separated siRNA was further subjected to in-gel hydrolysis with different RNases and subsequent identification of the fragments by untargeted LC-HRMS analysis (bottom-up, 'experimental RNomics'). Combining the results of all approaches, the identification of several 3'-truncated urinary metabolites was accomplished and target analytes were detected up to 24 h after a single administration. Simultaneously collected blood samples yielded no promising results. The methods were validated and found fit-for-purpose for doping controls. PMID:23913913

  13. Characterization of a novel trypanosomatid small nucleolar RNA.

    PubMed Central

    Levitan, A; Xu, Y x; Ben-Dov, C; Ben-Shlomo, H; Zhang, Y; Michaeli, S

    1998-01-01

    Trypanosomes possess unique RNA processing mechanisms including trans- splicing of pre-mRNA and RNA editing of mitochondrial transcripts. The previous finding of a trimethylguanosine (TMG) capped U3 homologue in trypanosomes suggests that rRNA processing may be related to the processing in other eukaryotes. In this study, we describe the first trypanosomatid snoRNA that belongs to the snoRNAs that were shown to guide ribose methylation of rRNA. The RNA, identified in the monogenetic trypanosomatid Leptomonas collosoma, was termed snoRNA-2 and is encoded by a multi-copy gene. SnoRNA-2 is 85 nt long, it lacks a 5' cap and possesses the C and D boxes characteristic to all snoRNAs that bind fibrillarin. Computer analysis indicates a potential for base-pairing between snoRNA-2 and 5.8S rRNA, and 18S rRNA. The putative interaction domains obey the rules suggested for the interaction of guide snoRNA with its rRNA target for directing ribose methylation on the rRNA. However, mapping the methylated sites on the 5.8S rRNA and 18S rRNA indicates that the expected site on the 5.8S is methylated, whereas the site on the 18S is not. The proposed interaction with 5.8S rRNA is further supported by the presence of psoralen cross-link sites on snoRNA-2. GenBank search suggests that snoRNA-2 is not related to any published snoRNAs. Because of the early divergence of the Trypanosomatidae from the eukaryotic lineage, the presence of a methylating snoRNA that is encoded by a multi-copy gene suggests that methylating snoRNAs may have evolved in evolution from self-transcribed genes. PMID:9512552

  14. 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

  15. Global Analysis of the Small RNA Transcriptome in Different Ploidies and Genomic Combinations of a Vertebrate Complex – The Squalius alburnoides

    PubMed Central

    Inácio, Angela; Pinho, Joana; Pereira, Patrícia Matos; Comai, Luca; Coelho, Maria Manuela

    2012-01-01

    The Squalius alburnoides complex (Steindachner) is one of the most intricate hybrid polyploid systems known in vertebrates. In this complex, the constant switch of the genome composition in consecutive generations, very frequently involving a change on the ploidy level, promotes repetitive situations of potential genomic shock. Previously in this complex, it was showed that in response to the increase in genome dosage, triploids hybrids could regulate gene expression to a diploid state. In this work we compared the small RNA profiles in the different genomic compositions interacting in the complex in order to explore the miRNA involvement in gene expression regulation of triploids. Using high-throughput arrays and sequencing technologies we were able to verify that diploid and triploid hybrids shared most of their sequences and their miRNA expression profiles were high correlated. However, an overall view indicates an up-regulation of several miRNAs in triploids and a global miRNA expression in triploids higher than the predicted from an additive model. Those results point to a participation of miRNAs in the cellular functional stability needed when the ploidy change. PMID:22815952

  16. Small Molecule-Mediated Cleavage of RNA in Living Cells

    PubMed Central

    Guan, Lirui

    2013-01-01

    Antisense oligonucleotides and small interfering RNAs (siRNAs) control gene expression by triggering the degradation of a mRNA via recruitment of RNase H or the RNA-induced silencing complex (RISC), respectively.[1] These approaches are hampered, however, by the poor cellular permeability of oligonucleotides. A small molecule approach to cleave RNA targets could obviate uptake issues. Several compounds can induce RNA cleavage in vitro,[2] however, to the best of our knowledge no small molecules have been previously described to cleave RNA in living cells. Herein, we describe the development of a potentially general approach to design small molecules that specifically cleave an RNA in a living cell, affecting biological function. Specifically, a designed, modularly assembled small molecule that binds the RNA that causes myotonic dystrophy type 1 (DM1)[3] was appended with a moiety that generates hydroxyl radicals upon irradiation. Cleavage of the transcript improves DM1-associated defects in cell culture, and compounds are non-toxic at an efficacious dose as determined by a MTT viability assay. This approach may allow for the site-specific cleavage and inactivation of other cellular RNAs.[4] Compounds that bind to and cleave RNA have the potential to serve as chemical genetics probes of function or lead therapeutics with spatial and temporal control. PMID:23280953

  17. The Small Ribosomal Subunit RNA Isoforms in Plasmodium Cynomolgi

    PubMed Central

    Corredor, V.; Enea, V.

    1994-01-01

    We report the isolation, characterization and analysis of the small subunit rRNA genes in Plasmodium cynomolgi (Ceylon). As in other Plasmodium species, these genes are present in low copy number, are unlinked and form two types that are distinct in sequence and are expressed stage specifically. The asexually expressed (type A) genes are present in four copies in the Ceylon(-) and in five copies in the Berok(-) strain. Surprisingly, the sexually expressed (type B) gene is present in a single copy. The vast majority of the differences between gene types is confined to the variable regions. The pattern of divergence is different from that observed in Plasmodium berghei or in Plasmodium falciparum. Analysis of the small subunit rRNA sequences of P. cynomolgi, P. berghei and P. falciparum, indicates that the two gene types do not evolve independently but rather interact (through gene conversion or some form of recombination) to such an extent as to erase whatever stage-specific sequence signatures they may have had in the last common ancestor. PMID:8005440

  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. Identification of Biologically Active, HIV TAR RNA-Binding Small Molecules Using Small Molecule Microarrays

    PubMed Central

    2015-01-01

    Identifying small molecules that selectively bind to structured RNA motifs remains an important challenge in developing potent and specific therapeutics. Most strategies to find RNA-binding molecules have identified highly charged compounds or aminoglycosides that commonly have modest selectivity. Here we demonstrate a strategy to screen a large unbiased library of druglike small molecules in a microarray format against an RNA target. This approach has enabled the identification of a novel chemotype that selectively targets the HIV transactivation response (TAR) RNA hairpin in a manner not dependent on cationic charge. Thienopyridine 4 binds to and stabilizes the TAR hairpin with a Kd of 2.4 μM. Structure–activity relationships demonstrate that this compound achieves activity through hydrophobic and aromatic substituents on a heterocyclic core, rather than cationic groups typically required. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis was performed on a 365-nucleotide sequence derived from the 5′ untranslated region (UTR) of the HIV-1 genome to determine global structural changes in the presence of the molecule. Importantly, the interaction of compound 4 can be mapped to the TAR hairpin without broadly disrupting any other structured elements of the 5′ UTR. Cell-based anti-HIV assays indicated that 4 inhibits HIV-induced cytopathicity in T lymphocytes with an EC50 of 28 μM, while cytotoxicity was not observed at concentrations approaching 1 mM. PMID:24820959

  1. Small Molecule Ligands for Bulged RNA Secondary Structures

    PubMed Central

    Meyer, S. Todd; Hergenrother, Paul J.

    2016-01-01

    A class of wedge-shaped small molecules has been designed, synthesized, and shown to bind bulged RNA secondary structures. These minimally cationic ligands exhibit good affinity and selectivity for certain RNA bulges as demonstrated in a fluorescent intercalator displacement assay. PMID:19678613

  2. 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

  3. 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. PMID:25435401

  4. 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

  5. Unzipping and binding of small interfering RNA with single walled carbon nanotube: A platform for small interfering RNA delivery

    NASA Astrophysics Data System (ADS)

    Santosh, Mogurampelly; Panigrahi, Swati; Bhattacharyya, Dhananjay; Sood, A. K.; Maiti, Prabal K.

    2012-02-01

    In an effort to design efficient platform for siRNA delivery, we combine all atom classical and quantum simulations to study the binding of small interfering RNA (siRNA) by pristine single wall carbon nanotube (SWCNT). Our results show that siRNA strongly binds to SWCNT surface via unzipping its base-pairs and the propensity of unzipping increases with the increase in the diameter of the SWCNTs. The unzipping and subsequent wrapping events are initiated and driven by van der Waals interactions between the aromatic rings of siRNA nucleobases and the SWCNT surface. However, molecular dynamics (MD) simulations of double strand DNA (dsDNA) of the same sequence show that the dsDNA undergoes much less unzipping and wrapping on the SWCNT in the simulation time scale of 70 ns. This interesting difference is due to smaller interaction energy of thymidine of dsDNA with the SWCNT compared to that of uridine of siRNA, as calculated by dispersion corrected density functional theory (DFT) methods. After the optimal binding of siRNA to SWCNT, the complex is very stable which serves as one of the major mechanisms of siRNA delivery for biomedical applications. Since siRNA has to undergo unwinding process with the effect of RNA-induced silencing complex, our proposed delivery mechanism by SWCNT possesses potential advantages in achieving RNA interference.

  6. 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

  7. Peptides Used in the Delivery of Small Noncoding RNA

    PubMed Central

    2015-01-01

    RNA interference (RNAi) is an endogenous process in which small noncoding RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), post-transcriptionally regulate gene expressions. In general, siRNA and miRNA/miRNA mimics are similar in nature and activity except their origin and specificity. Although both siRNAs and miRNAs have been extensively studied as novel therapeutics for a wide range of diseases, the large molecular weight, anionic surface charges, instability in blood circulation, and intracellular trafficking to the RISC after cellular uptake have hindered the translation of these RNAs from bench to clinic. As a result, a great variety of delivery systems have been investigated for safe and effective delivery of small noncoding RNAs. Among these systems, peptides, especially cationic peptides, have emerged as a promising type of carrier due to their inherent ability to condense negatively charged RNAs, ease of synthesis, controllable size, and tunable structure. In this review, we will focus on three major types of cationic peptides, including poly(l-lysine) (PLL), protamine, and cell penetrating peptides (CPP), as well as peptide targeting ligands that have been extensively used in RNA delivery. The delivery strategies, applications, and limitations of these cationic peptides in siRNA/miRNA delivery will be discussed. PMID:25157701

  8. Suppressing RNA silencing with small molecules and the viral suppressor of RNA silencing protein p19.

    PubMed

    Danielson, Dana C; Filip, Roxana; Powdrill, Megan H; O'Hara, Shifawn; Pezacki, John P

    2015-08-01

    RNA silencing is a gene regulatory and host defense mechanism whereby small RNA molecules are engaged by Argonaute (AGO) proteins, which facilitate gene knockdown of complementary mRNA targets. Small molecule inhibitors of AGO represent a convenient method for reversing this effect and have applications in human therapy and biotechnology. Viral suppressors of RNA silencing, such as p19, can also be used to suppress the pathway. Here we assess the compatibility of these two approaches, by examining whether synthetic inhibitors of AGO would inhibit p19-siRNA interactions. We observe that aurintricarboxylic acid (ATA) is a potent inhibitor of p19's ability to bind siRNA (IC50 = 0.43 μM), oxidopamine does not inhibit p19:siRNA interactions, and suramin is a mild inhibitor of p19:siRNA interactions (IC50 = 430 μM). We observe that p19 and suramin are compatible inhibitors of RNA silencing in human hepatoma cells. Our data suggests that at least some inhibitors of AGO may be used in combination with p19 to inhibit RNA silencing at different points in the pathway. PMID:26079891

  9. A small molecule enhances RNA interference and promotes microRNA processing

    PubMed Central

    Shan, Ge; Li, Yujing; Zhang, Junliang; Li, Wendi; Szulwach, Keith E; Duan, Ranhui; Faghihi, Mohammad A; Khalil, Ahmad M; Lu, Lianghua; Paroo, Zain; Chan, Anthony W S; Shi, Zhangjie; Liu, Qinghua; Wahlestedt, Claes; He, Chuan; Jin, Peng

    2010-01-01

    Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are sequence-specific post-transcriptional regulators of gene expression. Although major components of the RNA interference (RNAi) pathway have been identified, regulatory mechanisms for this pathway remain largely unknown. Here we demonstrate that the RNAi pathway can be modulated intracellularly by small molecules. We have developed a cell-based assay to monitor the activity of the RNAi pathway and find that the small-molecule enoxacin (Penetrex) enhances siRNA-mediated mRNA degradation and promotes the biogenesis of endogenous miRNAs. We show that this RNAi-enhancing activity depends on the trans-activation-responsive region RNA-binding protein. Our results provide a proof-of-principle demonstration that small molecules can be used to modulate the activity of the RNAi pathway. RNAi enhancers may be useful in the development of research tools and therapeutics. PMID:18641635

  10. 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. PMID:27170187

  11. Current preclinical small interfering RNA (siRNA)-based conjugate systems for RNA therapeutics.

    PubMed

    Lee, Soo Hyeon; Kang, Yoon Young; Jang, Hyo-Eun; Mok, Hyejung

    2016-09-01

    Recent promising clinical results of RNA therapeutics have drawn big attention of academia and industries to RNA therapeutics and their carrier systems. To improve their feasibility in clinics, systemic evaluations of currently available carrier systems under clinical trials and preclinical studies are needed. In this review, we focus on recent noticeable preclinical studies and clinical results regarding siRNA-based conjugates for clinical translations. Advantages and drawbacks of siRNA-based conjugates are discussed, compared to particle-based delivery systems. Then, representative siRNA-based conjugates with aptamers, peptides, carbohydrates, lipids, polymers, and nanostructured materials are introduced. To improve feasibility of siRNA conjugates in preclinical studies, several considerations for the rational design of siRNA conjugates in terms of cleavability, immune responses, multivalent conjugations, and mechanism of action are also presented. Lastly, we discuss lessons from previous preclinical and clinical studies related to siRNA conjugates and perspectives of their clinical applications. PMID:26514375

  12. Global analysis of the mammalian RNA degradome reveals widespread miRNA-dependent and miRNA-independent endonucleolytic cleavage

    PubMed Central

    Bracken, Cameron P.; Szubert, Jan M.; Mercer, Tim R.; Dinger, Marcel E.; Thomson, Daniel W.; Mattick, John S.; Michael, Michael Z.; Goodall, Gregory J.

    2011-01-01

    The Ago2 component of the RNA-induced silencing complex (RISC) is an endonuclease that cleaves mRNAs that base pair with high complementarity to RISC-bound microRNAs. Many examples of such direct cleavage have been identified in plants, but not in vertebrates, despite the conservation of catalytic capacity in vertebrate Ago2. We performed parallel analysis of RNA ends (PAREs), a deep sequencing approach that identifies 5′-phosphorylated, polyadenylated RNAs, to detect potential microRNA-directed mRNA cleavages in mouse embryo and adult tissues. We found that numerous mRNAs are potentially targeted for cleavage by endogenous microRNAs, but at very low levels relative to the mRNA abundance, apart from miR-151-5p-guided cleavage of the N4BP1 mRNA. We also find numerous examples of non-miRNA-directed cleavage, including cleavage of a group of mRNAs within a CA-repeat consensus sequence. The PARE analysis also identified many examples of adenylated small non-coding RNAs, including microRNAs, tRNA processing intermediates and various other small RNAs, consistent with adenylation being part of a widespread proof-reading and/or degradation pathway for small RNAs. PMID:21427086

  13. Small RNA Detection by in Situ Hybridization Methods

    PubMed Central

    Urbanek, Martyna O.; Nawrocka, Anna U.; Krzyzosiak, Wlodzimierz J.

    2015-01-01

    Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics. PMID:26068454

  14. Small RNA Detection by in Situ Hybridization Methods.

    PubMed

    Urbanek, Martyna O; Nawrocka, Anna U; Krzyzosiak, Wlodzimierz J

    2015-01-01

    Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics. PMID:26068454

  15. 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

  16. 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

  17. 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.

  18. Empirical insights into the stochasticity of small RNA sequencing

    PubMed Central

    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

  19. 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.

  20. Arabidopsis RNA-dependent RNA polymerases and dicer-like proteins in antiviral defense and small interfering RNA biogenesis during Turnip Mosaic Virus infection.

    PubMed

    Garcia-Ruiz, Hernan; Takeda, Atsushi; Chapman, Elisabeth J; Sullivan, Christopher M; Fahlgren, Noah; Brempelis, Katherine J; Carrington, James C

    2010-02-01

    Plants respond to virus infections by activation of RNA-based silencing, which limits infection at both the single-cell and system levels. Viruses encode RNA silencing suppressor proteins that interfere with this response. Wild-type Arabidopsis thaliana is immune to silencing suppressor (HC-Pro)-deficient Turnip mosaic virus, but immunity was lost in the absence of DICER-LIKE proteins DCL4 and DCL2. Systematic analysis of susceptibility and small RNA formation in Arabidopsis mutants lacking combinations of RNA-dependent RNA polymerase (RDR) and DCL proteins revealed that the vast majority of virus-derived small interfering RNAs (siRNAs) were dependent on DCL4 and RDR1, although full antiviral defense also required DCL2 and RDR6. Among the DCLs, DCL4 was sufficient for antiviral silencing in inoculated leaves, but DCL2 and DCL4 were both involved in silencing in systemic tissues (inflorescences). Basal levels of antiviral RNA silencing and siRNA biogenesis were detected in mutants lacking RDR1, RDR2, and RDR6, indicating an alternate route to form double-stranded RNA that does not depend on the three previously characterized RDR proteins. PMID:20190077

  1. 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

  2. Computational and analytical framework for small RNA profiling by high-throughput sequencing.

    PubMed

    Fahlgren, Noah; Sullivan, Christopher M; Kasschau, Kristin D; Chapman, Elisabeth J; Cumbie, Jason S; Montgomery, Taiowa A; Gilbert, Sunny D; Dasenko, Mark; Backman, Tyler W H; Givan, Scott A; Carrington, James C

    2009-05-01

    The advent of high-throughput sequencing (HTS) methods has enabled direct approaches to quantitatively profile small RNA populations. However, these methods have been limited by several factors, including representational artifacts and lack of established statistical methods of analysis. Furthermore, massive HTS data sets present new problems related to data processing and mapping to a reference genome. Here, we show that cluster-based sequencing-by-synthesis technology is highly reproducible as a quantitative profiling tool for several classes of small RNA from Arabidopsis thaliana. We introduce the use of synthetic RNA oligoribonucleotide standards to facilitate objective normalization between HTS data sets, and adapt microarray-type methods for statistical analysis of multiple samples. These methods were tested successfully using mutants with small RNA biogenesis (miRNA-defective dcl1 mutant and siRNA-defective dcl2 dcl3 dcl4 triple mutant) or effector protein (ago1 mutant) deficiencies. Computational methods were also developed to rapidly and accurately parse, quantify, and map small RNA data. PMID:19307293

  3. 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

  4. MicroRNA: a small molecule with a big biological impact.

    PubMed

    Zhou, Xiaofeng; Yang, Pan-Chyr

    2012-01-01

    target-directed editing of mature microRNA (trimming and tailing by 3'-to-5' exonuclase and terminal nucleotide transferase) [2] further highlighted the complexity of microRNA processing and regulation mechanisms. Moreover, the wide range of microRNA expression, from tens of thousands to just few molecules per cell, complicated the detection of microRNAs expressed at low copy numbers. Hence, many novel microRNAs may exist even in well-explored species. Nevertheless, recent advances in genomic technologies and data analysis / bioinformatics approaches have made a significant impact on microRNA research. For example, next generation deep sequencing platforms are ideal for detecting and quantifying both known and novel microRNA sequences with high sensitivity and for a relatively low cost [3]. The microRNA field has experienced a major explosion in recent years. The microRNA gene family is continuously growing with novel members discovered in association with rapid advances in genomic technologies, and reports on the functional characterizations of specific microRNA genes have been dominating the recent literature. We devote this new journal, MicroRNA, to the rapidly advancing field of microRNA research. We dedicate our new journal to the scientists who work tirelessly on this family of small molecules, and their immense contributions to the biological sciences. MicroRNA publishes letters, full-length research articles, review articles, drug and clinical trial studies and thematic issues on all aspects of microRNA research. The scope of the journal covers all experimental microRNA research and applied research in the fields of health and disease, including therapeutic, biomarker, and diagnostic applications of microRNA. PMID:25048083

  5. Analysis of small-sample clinical genomics studies using multi-parameter shrinkage: application to high-throughput RNA interference screening

    PubMed Central

    2013-01-01

    High-throughput (HT) RNA interference (RNAi) screens are increasingly used for reverse genetics and drug discovery. These experiments are laborious and costly, hence sample sizes are often very small. Powerful statistical techniques to detect siRNAs that potentially enhance treatment are currently lacking, because they do not optimally use the amount of data in the other dimension, the feature dimension. We introduce ShrinkHT, a Bayesian method for shrinking multiple parameters in a statistical model, where 'shrinkage' refers to borrowing information across features. ShrinkHT is very flexible in fitting the effect size distribution for the main parameter of interest, thereby accommodating skewness that naturally occurs when siRNAs are compared with controls. In addition, it naturally down-weights the impact of nuisance parameters (e.g. assay-specific effects) when these tend to have little effects across siRNAs. We show that these properties lead to better ROC-curves than with the popular limma software. Moreover, in a 3 + 3 treatment vs control experiment with 'assay' as an additional nuisance factor, ShrinkHT is able to detect three (out of 960) significant siRNAs with stronger enhancement effects than the positive control. These were not detected by limma. In the context of gene-targeted (conjugate) treatment, these are interesting candidates for further research. PMID:23819807

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  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. 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

  16. 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.

  17. Constructing and characterizing a bioactive small molecule and microRNA association network for Alzheimer's disease

    PubMed Central

    Meng, Fanlin; Dai, Enyu; Yu, Xuexin; Zhang, Yan; Chen, Xiaowen; Liu, Xinyi; Wang, Shuyuan; Wang, Lihua; Jiang, Wei

    2014-01-01

    Alzheimer's disease (AD) is an incurable neurodegenerative disorder. Much effort has been devoted to developing effective therapeutic agents. Recently, targeting microRNAs (miRNAs) with small molecules has become a novel therapy for human diseases. In this study, we present a systematic computational approach to construct a bioactive Small molecule and miRNA association Network in AD (SmiRN-AD), which is based on the gene expression signatures of bioactive small molecule perturbation and AD-related miRNA regulation. We also performed topological and functional analysis of the SmiRN-AD from multiple perspectives. At the significance level of p ≤ 0.01, 496 small molecule–miRNA associations, including 25 AD-related miRNAs and 275 small molecules, were recognized and used to construct the SmiRN-AD. The drugs that were connected with the same miRNA tended to share common drug targets (p = 1.72 × 10−4) and belong to the same therapeutic category (p = 4.22 × 10−8). The miRNAs that were linked to the same small molecule regulated more common miRNA targets (p = 6.07 × 10−3). Further analysis of the positive connections (quinostatin and miR-148b, amantadine and miR-15a) and the negative connections (melatonin and miR-30e-5p) indicated that our large-scale predictions afforded specific biological insights into AD pathogenesis and therapy. This study proposes a holistic strategy for deciphering the associations between small molecules and miRNAs in AD, which may be helpful for developing a novel effective miRNA-associated therapeutic strategy for AD. A comprehensive database for the SmiRN-AD and the differential expression patterns of the miRNA targets in AD is freely available at http://bioinfo.hrbmu.edu.cn/SmiRN-AD/. PMID:24352679

  18. CoRAL: predicting non-coding RNAs from small RNA-sequencing data.

    PubMed

    Leung, Yuk Yee; Ryvkin, Paul; Ungar, Lyle H; Gregory, Brian D; Wang, Li-San

    2013-08-01

    The surprising observation that virtually the entire human genome is transcribed means we know little about the function of many emerging classes of RNAs, except their astounding diversities. Traditional RNA function prediction methods rely on sequence or alignment information, which are limited in their abilities to classify the various collections of non-coding RNAs (ncRNAs). To address this, we developed Classification of RNAs by Analysis of Length (CoRAL), a machine learning-based approach for classification of RNA molecules. CoRAL uses biologically interpretable features including fragment length and cleavage specificity to distinguish between different ncRNA populations. We evaluated CoRAL using genome-wide small RNA sequencing data sets from four human tissue types and were able to classify six different types of RNAs with ∼80% cross-validation accuracy. Analysis by CoRAL revealed that microRNAs, small nucleolar and transposon-derived RNAs are highly discernible and consistent across all human tissue types assessed, whereas long intergenic ncRNAs, small cytoplasmic RNAs and small nuclear RNAs show less consistent patterns. The ability to reliably annotate loci across tissue types demonstrates the potential of CoRAL to characterize ncRNAs using small RNA sequencing data in less well-characterized organisms. PMID:23700308

  19. The role of antisense long noncoding RNA in small RNA-triggered gene activation

    PubMed Central

    Zhang, Xizhe; Li, Haitang; Rossi, John J.

    2014-01-01

    Long noncoding RNAs (lncRNAs) are known to regulate neighboring protein-coding genes by directing chromatin remodeling complexes, imprinting, and X-chromosome inactivation. In this study, we explore the function of lncRNAs in small RNA-triggered transcriptional gene activation (TGA), a process in which microRNAs (miRNAs) or small interfering RNAs (siRNAs) associated with Argonaute (Ago) proteins induce chromatin remodeling and gene activation at promoters with sequence complementarity. We designed a model system with different lncRNA and chromatin environments to elucidate the molecular mechanisms required for mammalian TGA. Using RNA-fluorescence in situ hybridization (FISH) and rapid amplification of cDNA ends (RACE)-PCR, we demonstrated that small RNA-triggered TGA occurs at sites where antisense lncRNAs are transcribed through the reporter gene and promoter. Small RNA-induced TGA coincided with the enrichment of Ago2 at the promoter region, but Ago2-mediated cleavage of antisense lncRNAs was not observed. Moreover, we examined the allele-specific effects of lncRNAs through a Cre-induced inversion of a poly(A) sequence that was designed to block the transcription of antisense lncRNAs through the reporter gene region in an inducible and reversible manner. Termination of nascent antisense lncRNAs abrogated gene activation triggered by small RNAs, and only allele-specific cis-acting antisense lncRNAs, but not trans-acting lncRNAs, were capable of rescuing TGA. Hence, this model revealed that antisense lncRNAs can mediate TGA in cis and not in trans, serving as a molecular scaffold for a small RNA–Ago2 complex and chromatin remodeling. PMID:25344398

  20. 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

  1. 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

  2. Using small RNA (sRNA) deep sequencing to understand global virus distribution in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Small RNAs (sRNAs), a class of regulatory RNAs, have been used to serve as the specificity determinants of suppressing gene expression in plants and animals. Next generation sequencing (NGS) uncovered the sRNA landscape in most organisms including their associated microbes. In the current study, w...

  3. Small non-coding RNA biomarkers in sputum for lung cancer diagnosis.

    PubMed

    Su, Yun; Guarnera, Maria A; Fang, HongBin; Jiang, Feng

    2016-01-01

    The early detection of lung cancer can reduce the mortality. However, there is no effective means in clinical settings for noninvasively detecting lung cancer. We previously developed 3 sputum miRNA biomarkers and 2 sputum small nucleolar RNA (snoRNA) biomarkers that can potentially be used for noninvasively diagnosing lung cancer. Here we evaluate the individual and combined applications of the two types of biomarkers in different sets of lung cancer patients and controls. Combined analysis of the miRNAs and snoRNAs has a synergistic effect with 89 % sensitivity and 89 % specificity, and may provide a useful tool for lung cancer early detection. PMID:27176474

  4. 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

  5. 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

  6. 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. PMID:23378513

  7. Analysis of interactions between ribosomal proteins and RNA structural motifs

    PubMed Central

    2010-01-01

    Background One important goal of structural bioinformatics is to recognize and predict the interactions between protein binding sites and RNA. Recently, a comprehensive analysis of ribosomal proteins and their interactions with rRNA has been done. Interesting results emerged from the comparison of r-proteins within the small subunit in T. thermophilus and E. coli, supporting the idea of a core made by both RNA and proteins, conserved by evolution. Recent work showed also that ribosomal RNA is modularly composed. Motifs are generally single-stranded sequences of consecutive nucleotides (ssRNA) with characteristic folding. The role of these motifs in protein-RNA interactions has been so far only sparsely investigated. Results This work explores the role of RNA structural motifs in the interaction of proteins with ribosomal RNA (rRNA). We analyze composition, local geometries and conformation of interface regions involving motifs such as tetraloops, kink turns and single extruded nucleotides. We construct an interaction map of protein binding sites that allows us to identify the common types of shared 3-D physicochemical binding patterns for tetraloops. Furthermore, we investigate the protein binding pockets that accommodate single extruded nucleotides either involved in kink-turns or in arbitrary RNA strands. This analysis reveals a new structural motif, called tripod. It corresponds to small pockets consisting of three aminoacids arranged at the vertices of an almost equilateral triangle. We developed a search procedure for the recognition of tripods, based on an empirical tripod fingerprint. Conclusion A comparative analysis with the overall RNA surface and interfaces shows that contact surfaces involving RNA motifs have distinctive features that may be useful for the recognition and prediction of interactions. PMID:20122215

  8. Characterization of the small RNA component of leaves and fruits from four different cucurbit species

    PubMed Central

    2012-01-01

    Background MicroRNAs (miRNAs) are a class of non-coding small RNAs involved in post-transcriptional regulation of gene expression critical for plant growth and development, stress responses and other diverse biological processes in plants. The Cucurbitaceae or cucurbit family represents some of economically important species, particularly those with edible and medicinal fruits. Genomic tools for the molecular analysis of members of this family are just emerging. Partial draft genome sequence became available recently for cucumber and watermelon facilitating investigation of the small RNA component of the transcriptomes in cucurbits. Results We generated four small RNA libraries from bottle gourd (Lagenaria siceraria), Cucurbita moschata, Cucurbita pepo, and, watermelon (Citrullus lanatus var. lanatus) in order to identify conserved and novel lineage specific miRNAs in these cucurbits. Deep sequencing of small RNA libraries from these species resulted in 1,597,263, 532,948, 601,388, and 493,384 unique sRNA reads from bottle gourd, moschata, pepo and watermelon, respectively. Sequence analysis of these four libraries resulted in identification of 21 miRNA families that are highly conserved and 8 miRNA families that are moderately conserved in diverse dicots. We also identified 4 putative novel miRNAs in these plant species. Furthermore, the tasiRNAs were identified and their biogenesis was determined in these cucurbits. Small RNA blot analysis or q-PCR analyses of leaf and fruit tissues of these cucurbits showed differential expression of several conserved miRNAs. Interestingly, the abundance of several miRNAs in leaves and fruits of closely related C. moschata and C. pepo was also distinctly different. Target genes for the most conserved miRNAs are also predicted. Conclusion High-throughput sequencing of small RNA libraries from four cucurbit species has provided a glimpse of small RNA component in their transcriptomes. The analysis also showed considerable

  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. HrrF Is the Fur-Regulated Small RNA in Nontypeable Haemophilus influenzae

    PubMed Central

    Santana, Estevan A.; Harrison, Alistair; Zhang, Xinjun; Baker, Beth D.; Kelly, Benjamin J.; White, Peter; Liu, Yunlong; Munson, Robert S.

    2014-01-01

    Nontypeable Haemophilus influenzae (NTHi) are Gram-negative commensal bacteria that reside in the nasopharynx. NTHi can also cause multiple upper and lower respiratory tract diseases that include sinusitis, conjunctivitis, bronchitis, and otitis media. In numerous bacterial species the ferric uptake regulator (Fur) acts as a global regulator of iron homeostasis by negatively regulating the expression of iron uptake systems. However in NTHi strain 86-028NP and numerous other bacterial species there are multiple instances where Fur positively affects gene expression. It is known that many instances of positive regulation by Fur occur indirectly through a small RNA intermediate. However, no examples of small RNAs have been described in NTHi. Therefore we used RNA-Seq analysis to analyze the transcriptome of NTHi strain 86-028NPrpsL and an isogenic 86-028NPrpsLΔfur strain to identify Fur-regulated intergenic transcripts. From this analysis we identified HrrF, the first small RNA described in any Haemophilus species. Orthologues of this small RNA exist only among other Pasteurellaceae. Our analysis showed that HrrF is maximally expressed when iron levels are low. Additionally, Fur was shown to bind upstream of the hrrF promoter. RNA-Seq analysis was used to identify targets of HrrF which include genes whose products are involved in molybdate uptake, deoxyribonucleotide synthesis, and amino acid biosynthesis. The stability of HrrF is not dependent on the RNA chaperone Hfq. This study is the first step in an effort to investigate the role small RNAs play in altering gene expression in response to iron limitation in NTHi. PMID:25157846

  11. 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

  12. 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...

  13. Analysis of dsRNA from microbial communities identifies dsRNA virus-like elements

    PubMed Central

    Decker, Carolyn J.; Parker, Roy

    2014-01-01

    SUMMARY dsRNA can function as genetic information and may have served as genomic material before the existence of DNA-based life. By developing a method to purify dsRNA, we have investigated the diversity of dsRNA in microbial populations. We detect large dsRNAs in multiple microbial populations. Analysis of an aquatic microbial population reveals some dsRNA sequences match metagenomic DNA suggesting that microbes contain pools of sense-antisense transcripts. In addition, ~30% of the dsRNA sequences are not present in the corresponding DNA pool, and are strongly biased toward encoding novel proteins. Of these “dsRNA unique” sequences, only a small percentage share similarity to known viruses, a large fraction assemble into RNA-virus-like contigs, and the remaining fraction has an unexplained origin. These results have uncovered dsRNA virus-like elements and underscore that dsRNA potentially represents an additional reservoir of genetic information in microbial populations. PMID:24767992

  14. Structural features of microRNA (miRNA) precursors and their relevance to miRNA biogenesis and small interfering RNA/short hairpin RNA design.

    PubMed

    Krol, Jacek; Sobczak, Krzysztof; Wilczynska, Urszula; Drath, Maria; Jasinska, Anna; Kaczynska, Danuta; Krzyzosiak, Wlodzimierz J

    2004-10-01

    We have established the structures of 10 human microRNA (miRNA) precursors using biochemical methods. Eight of these structures turned out to be different from those that were computer-predicted. The differences localized in the terminal loop region and at the opposite side of the precursor hairpin stem. We have analyzed the features of these structures from the perspectives of miRNA biogenesis and active strand selection. We demonstrated the different thermodynamic stability profiles for pre-miRNA hairpins harboring miRNAs at their 5'- and 3'-sides and discussed their functional implications. Our results showed that miRNA prediction based on predicted precursor structures may give ambiguous results, and the success rate is significantly higher for the experimentally determined structures. On the other hand, the differences between the predicted and experimentally determined structures did not affect the stability of termini produced through "conceptual dicing." This result confirms the value of thermodynamic analysis based on mfold as a predictor of strand section by RNAi-induced silencing complex (RISC). PMID:15292246

  15. Global effects of the small RNA biogenesis machinery on the Arabidopsis thaliana transcriptome.

    PubMed

    Laubinger, Sascha; Zeller, Georg; Henz, Stefan R; Buechel, Sabine; Sachsenberg, Timo; Wang, Jia-Wei; Rätsch, Gunnar; Weigel, Detlef

    2010-10-12

    In Arabidopsis thaliana, four different dicer-like (DCL) proteins have distinct but partially overlapping functions in the biogenesis of microRNAs (miRNAs) and siRNAs from longer, noncoding precursor RNAs. To analyze the impact of different components of the small RNA biogenesis machinery on the transcriptome, we subjected dcl and other mutants impaired in small RNA biogenesis to whole-genome tiling array analysis. We compared both protein-coding genes and noncoding transcripts, including most pri-miRNAs, in two tissues and several stress conditions. Our analysis revealed a surprising number of common targets in dcl1 and dcl2 dcl3 dcl4 triple mutants. Furthermore, our results suggest that the DCL1 is not only involved in miRNA action but also contributes to silencing of a subset of transposons, apparently through an effect on DNA methylation. PMID:20870966

  16. 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

  17. 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

  18. pY RNA1-s2: A Highly Retina-Enriched Small RNA That Selectively Binds to Matrin 3 (Matr3)

    PubMed Central

    Yamazaki, Fumiyoshi; Kim, Hyun Hee; Lau, Pierre; Hwang, Christopher K.; Iuvone, P. Michael; Klein, David; Clokie, Samuel J. H.

    2014-01-01

    The purpose of this study was to expand our knowledge of small RNAs, which are known to function within protein complexes to modulate the transcriptional output of the cell. Here we describe two previously unrecognized, small RNAs, termed pY RNA1-s1 and pY RNA1-s2 (processed Y RNA1-stem −1 and −2), thereby expanding the list of known small RNAs. pY RNA1-s1 and pY RNA1-s2 were discovered by RNA sequencing and found to be 20-fold more abundant in the retina than in 14 other rat tissues. Retinal expression of pY RNAs is highly conserved, including expression in the human retina, and occurs in all retinal cell layers. Mass spectrometric analysis of pY RNA1-S2 binding proteins in retina indicates that pY RNA1-s2 selectively binds the nuclear matrix protein Matrin 3 (Matr3) and to a lesser degree to hnrpul1 (heterogeneous nuclear ribonucleoprotein U-like protein). In contrast, pY RNA1-s1 does not bind these proteins. Accordingly, the molecular mechanism of action of pY RNA1-s2 is likely be through an action involving Matr3; this 95 kDa protein has two RNA recognition motifs (RRMs) and is implicated in transcription and RNA-editing. The high affinity binding of pY RNA1-s2 to Matr3 is strongly dependent on the sequence of the RNA and both RRMs of Matr3. Related studies also indicate that elements outside of the RRM region contribute to binding specificity and that phosphorylation enhances pY RNA-s2/Matr3 binding. These observations are of significance because they reveal that a previously unrecognized small RNA, pY RNA1-s2, binds selectively to Matr3. Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism. The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision. PMID:24558381

  19. Small Molecule Microarrays of RNA-Focused Peptoids Identifies Inhibitors of a Pathogenic Group I Intron RNA

    PubMed Central

    Labuda, Lucas P.; Pushechnikov, Alexei; Disney, Matthew D.

    2009-01-01

    Peptoids that inhibit the group I intron RNA from Candida albicans, an opportunistic pathogen that kills immunocompromised hosts, have been identified using microarrays. The arrayed peptoid library was constructed using submonomers with moieties similar to ones found in small molecules known to bind RNA. Library members that passed quality control analysis were spotted onto a microarray and screened for binding to the C. albicans group I intron ribozyme. Each ligand binder identified from microarray-based screening inhibited self-splicing in the presence of 1 mM nucleotide concentration of bulk yeast tRNA with IC50’s between 150 and 2200 µM. The binding signals and the corresponding IC50’s were used to identify features in the peptoids that predispose them for RNA binding. After statistical analysis of the peptoids’ structures that bind, a second generation of inhibitors was constructed using these important features; all second generation inhibitors have improved potencies with IC50’s <100 µM. The most potent inhibitor is composed of one phenylguanidine and three tryptamine submonomers and has an IC50 of 31 µM. This compound is 6-fold more potent than pentamidine, a clinically used drug that inhibits self-splicing. These results show that: 1.) modulators of RNA function can be identified by designing RNA-focused chemical libraries and screening them via microarray; 2.) statistical analysis of ligand binders can identify features in leads that predispose them for binding to their targets; and 3.) features can then be programmed into second generation inhibitors to design ligands with improved potencies. PMID:19278238

  20. Unusual RNA plant virus integration in the soybean genome leads to the production of small RNAs.

    PubMed

    da Fonseca, Guilherme Cordenonsi; de Oliveira, Luiz Felipe Valter; de Morais, Guilherme Loss; Abdelnor, Ricardo Vilela; Nepomuceno, Alexandre Lima; Waterhouse, Peter M; Farinelli, Laurent; Margis, Rogerio

    2016-05-01

    Horizontal gene transfer (HGT) is known to be a major force in genome evolution. The acquisition of genes from viruses by eukaryotic genomes is a well-studied example of HGT, including rare cases of non-retroviral RNA virus integration. The present study describes the integration of cucumber mosaic virus RNA-1 into soybean genome. After an initial metatranscriptomic analysis of small RNAs derived from soybean, the de novo assembly resulted a 3029-nt contig homologous to RNA-1. The integration of this sequence in the soybean genome was confirmed by DNA deep sequencing. The locus where the integration occurred harbors the full RNA-1 sequence followed by the partial sequence of an endogenous mRNA and another sequence of RNA-1 as an inverted repeat and allowing the formation of a hairpin structure. This region recombined into a retrotransposon located inside an exon of a soybean gene. The nucleotide similarity of the integrated sequence compared to other Cucumber mosaic virus sequences indicates that the integration event occurred recently. We described a rare event of non-retroviral RNA virus integration in soybean that leads to the production of a double-stranded RNA in a similar fashion to virus resistance RNAi plants. PMID:26993236

  1. Ancient and Novel Small RNA Pathways Compensate for the Loss of piRNAs in Multiple Independent Nematode Lineages

    PubMed Central

    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.

    2015-01-01

    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. PMID:25668728

  2. Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway

    PubMed Central

    Vasale, Jessica J.; Gu, Weifeng; Thivierge, Caroline; Batista, Pedro J; Claycomb, Julie M.; Youngman, Elaine M.; Duchaine, Thomas F.; Mello, Craig C.; Conte, Darryl

    2010-01-01

    Argonaute (AGO) proteins interact with distinct classes of small RNAs to direct multiple regulatory outcomes. In many organisms, including plants, fungi, and nematodes, cellular RNA-dependent RNA polymerases (RdRPs) use AGO targets as templates for amplification of silencing signals. Here, we show that distinct RdRPs function sequentially to produce small RNAs that target endogenous loci in Caenorhabditis elegans. We show that DCR-1, the RdRP RRF-3, and the dsRNA-binding protein RDE-4 are required for the biogenesis of 26-nt small RNAs with a 5′ guanine (26G-RNAs) and that 26G-RNAs engage the Piwi-clade AGO, ERGO-1. Our findings support a model in which targeting by ERGO-1 recruits a second RdRP (RRF-1 or EGO-1), which in turn transcribes 22G-RNAs that interact with worm-specific AGOs (WAGOs) to direct gene silencing. ERGO-1 targets exhibit a nonrandom distribution in the genome and appear to include many gene duplications, suggesting that this pathway may control overexpression resulting from gene expansion. PMID:20133583

  3. 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

  4. 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

  5. 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

  6. MicroRNA: Small RNA mediators of the brains genomic response to environmental stress.

    PubMed

    Hollins, Sharon L; Cairns, Murray J

    2016-08-01

    The developmental processes that establish the synaptic architecture of the brain while retaining capacity for activity-dependent remodeling, are complex and involve a combination of genetic and epigenetic influences. Dysregulation of these processes can lead to problems with neural circuitry which manifest in humans as a range of neurodevelopmental syndromes, such as schizophrenia, bipolar disorder and fragile X mental retardation. Recent studies suggest that prenatal, postnatal and intergenerational environmental factors play an important role in the aetiology of stress-related psychopathology. A number of these disorders have been shown to display epigenetic changes in the postmortem brain that reflect early life experience. These changes affect the regulation of gene expression though chromatin remodeling (transcriptional) and post-transcriptional influences, especially small noncoding microRNA (miRNA). These dynamic and influential molecules appear to play an important function in both brain development and its adaption to stress. In this review, we examine the role of miRNA in mediating the brain's response to both prenatal and postnatal environmental perturbations and explore how stress- induced alterations in miRNA expression can regulate the stress response via modulation of the immune system. Given the close relationship between environmental stress, miRNA, and brain development/function, we assert that miRNA hold a significant position at the molecular crossroads between neural development and adaptations to environmental stress. A greater understanding of the dynamics that mediate an individual's predisposition to stress-induced neuropathology has major human health benefits and is an important area of research. PMID:27317386

  7. Using Small RNA Technology to Efficiently Identify Tomato Viruses and Viroids in Mixed-Infected Field Samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Small interfering RNAs (siRNA) are produced in plants as a defense mechanism against virus or viroid infection. Analysis of a siRNA profile upon virus infection in plants may allow the de novo assembly of the viral genome. In the present study, we were interested in developing an efficient sequenc...

  8. Promoter analysis of influenza virus RNA polymerase.

    PubMed Central

    Parvin, J D; Palese, P; Honda, A; Ishihama, A; Krystal, M

    1989-01-01

    Influenza virus polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicating that the promoter lay solely within the 15-nucleotide 3' terminus. Sequences not specific for the influenza virus termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus-sense cRNA were copied at low levels. The specificity for recognition of the virus sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap endonuclease-primed and primer-free RNA synthesis from these model templates, as well as from genomic-length RNAs. This finding indicates that the reconstituted system has catalytic properties very similar to those of native viral ribonucleoprotein complexes. Images PMID:2585601

  9. Genomic analysis of RNA localization

    PubMed Central

    Taliaferro, J Matthew; Wang, Eric T; Burge, Christopher B

    2014-01-01

    The localization of mRNAs to specific subcellular sites is widespread, allowing cells to spatially restrict and regulate protein production, and playing important roles in development and cellular physiology. This process has been studied in mechanistic detail for several RNAs. However, the generality or specificity of RNA localization systems and mechanisms that impact the many thousands of localized mRNAs has been difficult to assess. In this review, we discuss the current state of the field in determining which RNAs localize, which RNA sequences mediate localization, the protein factors involved, and the biological implications of localization. For each question, we examine prominent systems and techniques that are used to study individual messages, highlight recent genome-wide studies of RNA localization, and discuss the potential for adapting other high-throughput approaches to the study of localization. PMID:25483039

  10. 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

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

    PubMed

    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

  12. Quantitative transcriptome analysis using RNA-seq.

    PubMed

    Külahoglu, Canan; Bräutigam, Andrea

    2014-01-01

    RNA-seq has emerged as the technology of choice to quantify gene expression. This technology is a convenient accurate tool to quantify diurnal changes in gene expression, gene discovery, differential use of promoters, and splice variants for all genes expressed in a single tissue. Thus, RNA-seq experiments provide sequence information and absolute expression values about transcripts in addition to relative quantification available with microarrays or qRT-PCR. The depth of information by sequencing requires careful assessment of RNA intactness and DNA contamination. Although the RNA-seq is comparatively recent, a standard analysis framework has emerged with the packages of Bowtie2, TopHat, and Cufflinks. With rising popularity of RNA-seq tools have become manageable for researchers without much bioinformatical knowledge or programming skills. Here, we present a workflow for a RNA-seq experiment from experimental planning to biological data extraction. PMID:24792045

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

    PubMed

    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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

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

    PubMed

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

    2015-07-01

    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

  1. 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

  2. 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

  3. Using machine learning and high-throughput RNA sequencing to classify the precursors of small non-coding RNAs.

    PubMed

    Ryvkin, Paul; Leung, Yuk Yee; Ungar, Lyle H; Gregory, Brian D; Wang, Li-San

    2014-05-01

    Recent advances in high-throughput sequencing allow researchers to examine the transcriptome in more detail than ever before. Using a method known as high-throughput small RNA-sequencing, we can now profile the expression of small regulatory RNAs such as microRNAs and small interfering RNAs (siRNAs) with a great deal of sensitivity. However, there are many other types of small RNAs (<50nt) present in the cell, including fragments derived from snoRNAs (small nucleolar RNAs), snRNAs (small nuclear RNAs), scRNAs (small cytoplasmic RNAs), tRNAs (transfer RNAs), and transposon-derived RNAs. Here, we present a user's guide for CoRAL (Classification of RNAs by Analysis of Length), a computational method for discriminating between different classes of RNA using high-throughput small RNA-sequencing data. Not only can CoRAL distinguish between RNA classes with high accuracy, but it also uses features that are relevant to small RNA biogenesis pathways. By doing so, CoRAL can give biologists a glimpse into the characteristics of different RNA processing pathways and how these might differ between tissue types, biological conditions, or even different species. CoRAL is available at http://wanglab.pcbi.upenn.edu/coral/. PMID:24145223

  4. 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. PMID:24357407

  5. Intronic regions of plant genes potentially encode RDR (RNA-dependent RNA polymerase)-dependent small RNAs

    PubMed Central

    Qin, Jingping; Ma, Xiaoxia; Yi, Zili; Tang, Zhonghai; Meng, Yijun

    2015-01-01

    Recent research has linked the non-coding intronic regions of plant genes to the production of small RNAs (sRNAs). Certain introns, called ‘mirtrons’ and ‘sirtrons’, could serve as the single-stranded RNA precursors for the generation of microRNA and small interfering RNA, respectively. However, whether the intronic regions could serve as the template for double-stranded RNA synthesis and then for sRNA biogenesis through an RDR (RNA-dependent RNA polymerase)-dependent pathway remains unclear. In this study, a genome-wide search was made for the RDR-dependent sRNA loci within the intronic regions of the Arabidopsis genes. Hundreds of intronic regions encoding three or more RDR-dependent sRNAs were found to be covered by dsRNA-seq (double-stranded RNA sequencing) reads, indicating that the intron-derived sRNAs were indeed generated from long double-stranded RNA precursors. More interestingly, phase-distributed sRNAs were discovered on some of the dsRNA-seq read-covered intronic regions, and those sRNAs were largely 24 nt in length. Based on these results, the opinion is put forward that the intronic regions might serve as the genomic origins for the RDR-dependent sRNAs. This opinion might add a novel layer to the current biogenesis model of the intron-derived sRNAs. PMID:25609829

  6. 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

  7. Toward Reprogramming Bacteria with Small Molecules and RNA

    PubMed Central

    Gallivan, Justin P.

    2007-01-01

    Summary 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. Regulatory mechanisms of exoribonuclease PNPase and regulatory small RNA on T3SS of dickeya dadantii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The type III secretion system (T3SS) is an essential virulence factor for many bacterial pathogens. Polynucleotide phosphorylase (PNPase) is one of the major exoribonucleases in bacteria and plays important roles in mRNA degradation, tRNA processing, and small RNA (sRNA) turnover. In this study, we ...

  9. 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

  10. RNA-seq profiling of small numbers of Drosophila neurons.

    PubMed

    Abruzzi, Katharine; Chen, Xiao; Nagoshi, Emi; Zadina, Abby; Rosbash, Michael

    2015-01-01

    Drosophila melanogaster has a robust circadian clock, which drives a rhythmic behavior pattern: locomotor activity increases in the morning shortly before lights on (M peak) and in the evening shortly before lights off (E peak). This pattern is controlled by ~75 pairs of circadian neurons in the Drosophila brain. One key group of neurons is the M-cells (PDF(+) large and small LNvs), which control the M peak. A second key group is the E-cells, consisting of four LNds and the fifth small LNv, which control the E peak. Recent studies show that the M-cells have a second role in addition to controlling the M peak; they communicate with the E-cells (as well as DN1s) to affect their timing, probably as a function of environmental conditions (Guo, Cerullo, Chen, & Rosbash, 2014). To learn about molecules within the M-cells important for their functional roles, we have adapted methods to manually sort fluorescent protein-expressing neurons of interest from dissociated Drosophila brains. We isolated mRNA and miRNA from sorted M-cells and amplified the resulting DNAs to create deep-sequencing libraries. Visual inspection of the libraries illustrates that they are specific to a particular neuronal subgroup; M-cell libraries contain timeless and dopaminergic cell libraries contain ple/TH. Using these data, it is possible to identify cycling transcripts as well as many mRNAs and miRNAs specific to or enriched in particular groups of neurons. PMID:25662465

  11. Increased Plasma miRNA-30a as a Biomarker for Non-Small Cell Lung Cancer

    PubMed Central

    Sun, Ling; Chen, Yifan; Su, Qiaoli; Tang, Xiaoju; Liang, Yasha; Che, Guowei; Luo, Fengming

    2016-01-01

    Background MicroRNA (miRNA) is a small, non-coding RNA molecule which plays a role in the carcinogenesis and progression of cancers. Abnormal expression of miRNA in plasma has been found in some patients with malignant tumors. Material/Methods This study was conducted to investigate the expression of miRNA-30a in plasma of patients with non-small cell lung cancer (NSCLC). The plasma miRNA-30a in 87 patients with NSCLC, 20 patients with benign lung diseases, and 76 healthy subjects were measured by real-time PCR. The diagnostic value of miRNA-30a in NSCLC was evaluated via the ROC curve method. Results Plasma miRNA-30a level was significantly higher in the NSCLC group compared with benign control and healthy control groups (P<0.01). No statistically significant difference was found in the expression level of miRNA-30a among various clinical pathologic features in NSCLC. ROC curve analysis showed that the specificity and sensitivity cut-off points were at 61.0% and 84.3% for NSCLC. The specificity and sensitivity values were 54.9% and 94.4%, respectively, in the analysis based on in-patients only. Conclusions All these results suggest that plasma miRNA-30a measurement may be a novel and noninvasive method for NSCLC preliminary screening and differential diagnosis. PMID:26918265

  12. 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

  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. 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

  15. 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

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

    PubMed

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

    2016-02-01

    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

  17. Targeted delivery of small interfering RNA to colon cancer cells using chitosan and PEGylated chitosan nanoparticles.

    PubMed

    Rudzinski, Walter E; Palacios, Adriana; Ahmed, Abuzar; Lane, Michelle A; Aminabhavi, Tejraj M

    2016-08-20

    Small interfering RNA (siRNA) molecules specifically target messenger RNA species, decreasing intracellular protein levels. β-Catenin protein concentrations are increased in 70-80% of colon tumors, promoting tumor progression. Chitosan exhibits low levels of toxicity and can be transported across mucosal membranes; therefore, our objective was to develop chitosan and poly(ethylene glycol)-grafted (PEGylated) chitosan nanoparticles, 100-150nm in diameter, encapsulating anti-β-catenin siRNA for transfection into colon cancer cells. Encapsulation efficiencies up to 97% were observed. Confocal microscopy visualized the entry of fluorescently-tagged siRNA into cells. Western blot analysis showed that both chitosan and PEGylated chitosan nanoparticles containing anti-β-catenin siRNA decreased β-catenin protein levels in cultured colon cancer cells. These results indicate that nanoparticles made with chitosan and PEGylated chitosan can successfully enter colon cancer cells and decrease the level of a protein that promotes tumor progression. These or similar nanoparticles may prove beneficial for the treatment of colon cancer in humans. PMID:27178938

  18. 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

  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. 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

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

    PubMed

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

    2016-07-01

    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

  2. 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

  3. From early lessons to new frontiers: the worm as a treasure trove of small RNA biology.

    PubMed

    Youngman, Elaine M; Claycomb, Julie M

    2014-01-01

    In the past 20 years, the tiny soil nematode Caenorhabditis elegans has provided critical insights into our understanding of the breadth of small RNA-mediated gene regulatory activities. The first microRNA was identified in C. elegans in 1993, and the understanding that dsRNA was the driving force behind RNA-mediated gene silencing came from experiments performed in C. elegans in 1998. Likewise, early genetic screens in C. elegans for factors involved in RNA interference pointed to conserved mechanisms for small RNA-mediated gene silencing pathways, placing the worm squarely among the founding fathers of a now extensive field of molecular biology. Today, the worm continues to be at the forefront of ground-breaking insight into small RNA-mediated biology. Recent studies have revealed with increasing mechanistic clarity that C. elegans possesses an extensive nuclear small RNA regulatory network that encompasses not only gene silencing but also gene activating roles. Further, a portrait is emerging whereby small RNA pathways play key roles in integrating responses to environmental stimuli and transmitting epigenetic information about such responses from one generation to the next. Here we discuss endogenous small RNA pathways in C. elegans and the insight worm biology has provided into the mechanisms employed by these pathways. We touch on the increasingly spectacular diversity of small RNA biogenesis and function, and discuss the relevance of lessons learned in the worm for human biology. PMID:25505902

  4. RNA Cytidine Acetyltransferase of Small-Subunit Ribosomal RNA: Identification of Acetylation Sites and the Responsible Acetyltransferase in Fission Yeast, Schizosaccharomyces pombe

    PubMed Central

    Taoka, Masato; Ishikawa, Daisuke; Nobe, Yuko; Ishikawa, Hideaki; Yamauchi, Yoshio; Terukina, Goro; Nakayama, Hiroshi; Hirota, Kouji; Takahashi, Nobuhiro; Isobe, Toshiaki

    2014-01-01

    The eukaryotic small-subunit (SSU) ribosomal RNA (rRNA) has two evolutionarily conserved acetylcytidines. However, the acetylation sites and the acetyltransferase responsible for the acetylation have not been identified. We performed a comprehensive MS-based analysis covering the entire sequence of the fission yeast, Schizosaccharomyces pombe, SSU rRNA and identified two acetylcytidines at positions 1297 and 1815 in the 3′ half of the rRNA. To identify the enzyme responsible for the cytidine acetylation, we searched for an S. pombe gene homologous to TmcA, a bacterial tRNA N-acetyltransferase, and found one potential candidate, Nat10. A temperature-sensitive strain of Nat10 with a mutation in the Walker A type ATP-binding motif abolished the cytidine acetylation in SSU rRNA, and the wild-type Nat10 supplemented to this strain recovered the acetylation, providing evidence that Nat10 is necessary for acetylation of SSU rRNA. The Nat10 mutant strain showed a slow-growth phenotype and was defective in forming the SSU rRNA from the precursor RNA, suggesting that cytidine acetylation is necessary for ribosome assembly. PMID:25402480

  5. 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

  6. Host miRNA degradation by Herpesvirus saimiri small nuclear RNA requires an unstructured interacting region.

    PubMed

    Pawlica, Paulina; Moss, Walter N; Steitz, Joan A

    2016-08-01

    Herpesvirus saimiri, an oncogenic herpesvirus, during latency produces seven small nuclear RNAs, called the Herpesvirus saimiri U RNAs (HSUR1-7). HSUR1 mediates degradation of the host microRNA, miR-27, via a process that requires imperfect base-pairing. The decreased levels of miR-27 lead to prolonged T-cell activation and likely contribute to oncogenesis. To gain insight into HSUR1-mediated degradation of miR-27, we probed the in vivo secondary structure of HSUR1 and coupled this with bioinformatic structural analyses. The results suggest that HSUR1 adopts a conformation different than previously believed and that the region complementary to miR-27 lacks stable structure. To determine whether HSUR1 structural flexibility is important for its ability to mediate miR-27 degradation, we performed structurally informative mutagenic analyses of HSUR1. HSUR1 mutants in which the miR-27 binding site sequence is preserved, but sequestered in predicted helices, lose their ability to decrease miR-27 levels. These results indicate that the HSUR1 miR27-binding region must be available in a conformationally flexible segment for noncoding RNA function. PMID:27335146

  7. 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. PMID:22744314

  8. Myelin Basic Protein synthesis is regulated by small non-coding RNA 715

    PubMed Central

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

    2012-01-01

    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. PMID:22744314

  9. Intrinsic noise in post-transcriptional gene regulation by small non-coding RNA.

    PubMed

    Jia, Ya; Liu, Wangheng; Li, Anbang; Yang, Lijian; Zhan, Xuan

    2009-07-01

    Small non-coding RNA (sRNA) plays very important role in the post transcriptional regulation in various organisms. In complex regulatory networks, highly significant relative fluctuations in RNAs copy numbers can not be neglected due to very small copy number of individual RNA molecules. Here we consider two simple regulation schemes, where one is single target gene regulated by a sRNA and the other is two target mRNAs (mRNA(R) and mRNA(T)) regulated by one sRNA. The Fano factor (a measure of the relative size of the internal fluctuations) formulae of RNA molecules in the post transcriptional regulation are theoretically derived by using of the Langevin theory. For single target gene regulated by a sRNA, it is shown that the intrinsic noise of both mRNA and sRNA approaches the bare Poissonian limit in the regimen of both target RNA silencing and surviving. However, the strong anti-correlation between the fluctuations of two components result in a large intrinsic fluctuations in the level of RNA molecules in the regimen of crossover. For two target mRNAs regulated by one sRNA, in the regimen of crossover, it is found that, with the increasing of transcription rate of target mRNA(T), the maximal intrinsic fluctuation of RNA molecules is shifted from sRNA to target mRNA(R), and then to target mRNA(T). The intrinsic noise intensity of target mRNA(R) is determined by both the transcriptional rate of itself and that of sRNA, and independent of the transcriptional rate of the other target mRNA(T). PMID:19403234

  10. Methods to enable the design of bioactive small molecules targeting RNA

    PubMed Central

    Disney, Matthew D.; Yildirim, Ilyas; Childs-Disney, Jessica L.

    2014-01-01

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including Structure-Activity Relationships Through Sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome. PMID:24357181

  11. Methods to enable the design of bioactive small molecules targeting RNA.

    PubMed

    Disney, Matthew D; Yildirim, Ilyas; Childs-Disney, Jessica L

    2014-02-21

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including structure-activity relationships through sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome. PMID:24357181

  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. Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation

    PubMed Central

    Jensen, Kirsty; Anderson, Jennifer A.; Glass, Elizabeth J.

    2014-01-01

    The manipulation of the RNA interference pathway using small interfering RNA (siRNA) has become the most frequently used gene silencing method. However, siRNA delivery into primary cells, especially primary macrophages, is often considered challenging. Here we report the investigation of the suitability of two methodologies: transient transfection and electroporation, to deliver siRNA targeted against the putative immunomodulatory gene Mediterranean fever (MEFV) into primary bovine monocyte-derived macrophages (bMDM). Eleven commercial transfection reagents were investigated with variable results with respect to siRNA uptake, target gene knock-down, cell toxicity and type I interferon (IFN) response induction. Three transfection reagents: Lipofectamine 2000, Lipofectamine RNAiMAX and DharmaFECT 3, were found to consistently give the best results. However, all the transfection reagents tested induced an IFN response in the absence of siRNA, which could be minimized by reducing the transfection reagent incubation period. In addition, optimized siRNA delivery into bMDM by electroporation achieved comparable levels of target gene knock-down as transient transfection, without a detectable IFN response, but with higher levels of cell toxicity. The optimized transient transfection and electroporation methodologies may provide a starting point for optimizing siRNA delivery into macrophages derived from other species or other cells considered difficult to investigate with siRNA. PMID:24598124

  15. 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

  16. 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

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

    PubMed

    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

  18. Toward reliable biomarker signatures in the age of liquid biopsies - how to standardize the small RNA-Seq workflow.

    PubMed

    Buschmann, Dominik; Haberberger, Anna; Kirchner, Benedikt; Spornraft, Melanie; Riedmaier, Irmgard; Schelling, Gustav; Pfaffl, Michael W

    2016-07-27

    Small RNA-Seq has emerged as a powerful tool in transcriptomics, gene expression profiling and biomarker discovery. Sequencing cell-free nucleic acids, particularly microRNA (miRNA), from liquid biopsies additionally provides exciting possibilities for molecular diagnostics, and might help establish disease-specific biomarker signatures. The complexity of the small RNA-Seq workflow, however, bears challenges and biases that researchers need to be aware of in order to generate high-quality data. Rigorous standardization and extensive validation are required to guarantee reliability, reproducibility and comparability of research findings. Hypotheses based on flawed experimental conditions can be inconsistent and even misleading. Comparable to the well-established MIQE guidelines for qPCR experiments, this work aims at establishing guidelines for experimental design and pre-analytical sample processing, standardization of library preparation and sequencing reactions, as well as facilitating data analysis. We highlight bottlenecks in small RNA-Seq experiments, point out the importance of stringent quality control and validation, and provide a primer for differential expression analysis and biomarker discovery. Following our recommendations will encourage better sequencing practice, increase experimental transparency and lead to more reproducible small RNA-Seq results. This will ultimately enhance the validity of biomarker signatures, and allow reliable and robust clinical predictions. PMID:27317696

  19. 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

  20. 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

  1. Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

    PubMed Central

    Lakatos, Lóránt; Csorba, Tibor; Pantaleo, Vitantonio; Chapman, Elisabeth J; Carrington, James C; Liu, Yu-Ping; Dolja, Valerian V; Calvino, Lourdes Fernández; López-Moya, Juan José; Burgyán, József

    2006-01-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation. PMID:16724105

  2. Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors.

    PubMed

    Lakatos, Lóránt; Csorba, Tibor; Pantaleo, Vitantonio; Chapman, Elisabeth J; Carrington, James C; Liu, Yu-Ping; Dolja, Valerian V; Calvino, Lourdes Fernández; López-Moya, Juan José; Burgyán, József

    2006-06-21

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation. PMID:16724105

  3. Divergent patterns of endogenous small RNA populations from seed and vegetative tissues of Glycine max

    PubMed Central

    2012-01-01

    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 gained mostly through studies with Arabidopsis. In recent years, high throughput sequencing of smRNA populations has enabled extension of knowledge from model systems to plants with larger, more complex genomes. Soybean (Glycine max) now has many genomics resources available including a complete genome sequence and predicted gene models. Relatively little is known, however, about the full complement of its endogenous smRNAs populations and the silenced genes. Results Using Illumina sequencing and computational analysis, we characterized eight smRNA populations from multiple tissues and organs of soybean including developing seed and vegetative tissues. A total of 41 million raw sequence reads collapsed into 135,055 unique reads were mapped to the soybean genome and its predicted cDNA gene models. Bioinformatic analyses were used to distinguish miRNAs and siRNAs and to determine their genomic origins and potential target genes. In addition, we identified two soybean TAS3 gene homologs, the miRNAs that putatively guide cleavage of their transcripts, and the derived tasiRNAs that could target soybean genes annotated as auxin response factors. Tissue-differential expression based on the flux of normalized miRNA and siRNA abundances in the eight smRNA libraries was evident, some of which was confirmed by smRNA blotting. Our global view of these smRNA populations also revealed that the size classes of smRNAs varied amongst different tissues, with the developing seed and seed coat having greater numbers of unique smRNAs of the 24-nt class compared to the vegetative tissues of germinating seedlings. The 24-nt class is known to be derived from repetitive elements including transposons

  4. Transcriptional regulation of human small nuclear RNA genes

    PubMed Central

    Jawdekar, Gauri W.; Henry, R. William

    2009-01-01

    The products of human snRNA genes have been frequently described as performing housekeeping functions and their synthesis refractory to regulation. However, recent studies have emphasized that snRNA and other related non-coding RNA molecules control multiple facets of the central dogma, and their regulated expression is critical to cellular homeostasis during normal growth and in response to stress. Human snRNA genes contain compact and yet powerful promoters that are recognized by increasingly well-characterized transcription factors, thus providing a premier model system to study gene regulation. This review summarizes many recent advances deciphering the mechanism by which the transcription of human snRNA and related genes are regulated. PMID:18442490

  5. 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, ...

  6. 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...

  7. 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

  8. 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. PMID:24138283

  9. 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

  10. 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

  11. 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

  12. 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. PMID:26414664

  13. 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.

  14. Mouse nucleolin binds to 4.5S RNAh, a small noncoding RNA.

    PubMed

    Hirose, Yutaka; Harada, Fumio

    2008-01-01

    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. PMID:17971306

  15. tRid, an enabling method to isolate previously inaccessible small RNA fractions.

    PubMed

    Futai, Kazuki; Terasaka, Naohiro; Katoh, Takayuki; Suga, Hiroaki

    2016-08-15

    Detection of rare small RNA species whose sizes are overlapping with tRNAs often suffers from insufficient sensitivity due to the overwhelming abundance of tRNAs. We here report a method, named tRid (tRNA rid), for removing abundant tRNAs from small RNA fractions regardless of tRNA sequence species. By means of tRid, we are able to selectively enrich small RNAs which have been previously difficult to access due to mass existence of tRNAs in such fractions. A flexible tRNA-acylation ribozyme, known as flexizyme, is a key tool where the total tRNAs are aminoacylated with N-biotinylated phenylalanine regardless of tRNA sequences, and therefore the biotin-tagged tRNAs could be readily removed from the small RNA fractions by the use of streptavidin-immobilized magnetic beads. Next generation sequencing of the isolated small RNA fraction revealed that small RNAs with less than 200nt were effectively enriched, allowing us to identify previously unknown small RNAs in HeLa and E. coli. PMID:27163863

  16. 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

  17. Phylogenetic position of the genus Perkinsus (Protista, Apicomplexa) based on small subunit ribosomal RNA.

    PubMed

    Goggin, C L; Barker, S C

    1993-07-01

    Parasites of the genus Perkinsus destroy marine molluscs worldwide. Their phylogenetic position within the kingdom Protista is controversial. Nucleotide sequence data (1792 bp) from the small subunit rRNA gene of Perkinsus sp. from Anadara trapezia (Mollusca: Bivalvia) from Moreton Bay, Queensland, was used to examine the phylogenetic affinities of this enigmatic genus. These data were aligned with nucleotide sequences from 6 apicomplexans, 3 ciliates, 3 flagellates, a dinoflagellate, 3 fungi, maize and human. Phylogenetic trees were constructed after analysis with maximum parsimony and distance matrix methods. Our analyses indicate that Perkinsus is phylogenetically closer to dinoflagellates and to coccidean and piroplasm apicomplexans than to fungi or flagellates. PMID:8366895

  18. 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

  19. Biogenesis of Y RNA-derived small RNAs is independent of the microRNA pathway.

    PubMed

    Nicolas, Francisco Esteban; Hall, Adam E; Csorba, Tibor; Turnbull, Carly; Dalmay, Tamas

    2012-04-24

    Y RNAs are approximately 100 nucleotide long conserved cytoplasmic non-coding RNAs, which produce smaller RNA fragments during apoptosis. Here we show that these smaller RNA molecules are also produced in non-stressed cells and in a range of human cancerous and non-cancerous cell types. Recent reports have speculated that the cleavage products of Y RNAs enter the microRNA pathway. We tested this hypothesis and found that Y5 and Y3 RNA fragments are Dicer independent, they are in different complexes than microRNAs and that they are not co-immunoprecipitated with Ago2. Therefore we conclude that Y RNA fragments do not enter the microRNA pathway. PMID:22575660

  20. Genome-Wide Analysis of miRNA-mRNA Interactions in Marrow Stromal Cells

    PubMed Central

    Balakrishnan, Ilango; Yang, Xiaodong; Brown, Joseph; Ramakrishnan, Aravind; Torok–Storb, Beverly; Kabos, Peter; Hesselberth, Jay R.; Pillai, Manoj M.

    2014-01-01

    Regulation of hematopoietic stem cell proliferation, lineage commitment, and differentiation in adult vertebrates requires extrinsic signals provided by cells in the marrow microenvironment (ME) located within the bone marrow. Both secreted and cell-surface bound factors critical to this regulation have been identified, yet control of their expression by cells within the ME has not been addressed. Herein we hypothesize that microRNAs (miRNAs) contribute to their controlled expression. MiRNAs are small noncoding RNAs that bind to target mRNAs and downregulate gene expression by either initiating mRNA degradation or preventing peptide translation. Testing the role of miRNAs in downregulating gene expression has been difficult since conventional techniques used to define miRNA-mRNA interactions are indirect and have high false-positive and negative rates. In this report, a genome-wide biochemical technique (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation or HITS-CLIP) was used to generate unbiased genome-wide maps of miRNA-mRNA interactions in two critical cellular components of the marrow ME: marrow stromal cells and bone marrow endothelial cells. Analysis of these datasets identified miRNAs as direct regulators of JAG1, WNT5A, MMP2, and VEGFA; four factors that are important to ME function. Our results show the feasibility and utility of unbiased genome-wide biochemical techniques in dissecting the role of miRNAs in regulation of complex tissues such as the marrow ME. PMID:24038734

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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. PMID:26343506

  7. A single RNA-dependent RNA polymerase assembles with mutually exclusive nucleotidyl transferase subunits to direct different pathways of small RNA biogenesis.

    PubMed

    Lee, Suzanne Rebecca; Talsky, Kristin Benjamin; Collins, Kathleen

    2009-07-01

    Members of the conserved family of eukaryotic RNA-dependent RNA polymerases (Rdrs) synthesize double-stranded RNA (dsRNA) intermediates in diverse pathways of small RNA (sRNA) biogenesis and RNA-mediated silencing. Rdr-dependent pathways of sRNA production are poorly characterized relative to Rdr-independent pathways, and the Rdr enzymes themselves are poorly characterized relative to their viral RNA-dependent RNA polymerase counterparts. We previously described a physical and functional coupling of the Tetrahymena thermophila Rdr, Rdr1, and a Dicer enzyme, Dcr2, in the production of approximately 24-nucleotide (nt) sRNA in vitro. Here we characterize the endogenous complexes that harbor Rdr1, termed RDRCs. Distinct RDRCs assemble to contain Rdr1 and subsets of the total of four tightly Rdr1-associated proteins. Of particular interest are two RDRC subunits, Rdn1 and Rdn2, which possess noncanonical ribonucleotidyl transferase motifs. We show that the two Rdn proteins are uridine-specific polymerases of separate RDRCs. Two additional RDRC subunits, Rdf1 and Rdf2, are present only in RDRCs containing Rdn1. Rdr1 catalytic activity is retained in RDRCs purified from cell extracts lacking any of the nonessential RDRC subunits (Rdn2, Rdf1, Rdf2) or if the RDRC harbors a catalytically inactive Rdn. However, specific disruption of each RDRC imposes distinct loss-of-function consequences at the cellular level and has a differential impact on the accumulation of specific 23-24-nt sRNA sequences in vivo. The biochemical and biological phenotypes of RDRC subunit disruption reveal a previously unanticipated complexity of Rdr-dependent sRNA biogenesis in vivo. PMID:19451546

  8. MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant.

    PubMed

    Lu, Cheng; Kulkarni, Karthik; Souret, Frédéric F; MuthuValliappan, Ramesh; Tej, Shivakundan Singh; Poethig, R Scott; Henderson, Ian R; Jacobsen, Steven E; Wang, Wenzhong; Green, Pamela J; Meyers, Blake C

    2006-10-01

    The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is approximately 155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1-7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs. PMID:16954541

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

    PubMed

    Collier, Aaron M; Lyytinen, Outi L; Guo, Yusong R; Toh, Yukimatsu; Poranen, Minna M; Tao, Yizhi J

    2016-04-01

    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

  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. Specific ligation to double-stranded RNA for analysis of cellular RNA::RNA interactions.

    PubMed

    Faridani, Omid R; McInerney, Gerald M; Gradin, Katarina; Good, Liam

    2008-09-01

    Double-stranded RNA (dsRNA) is formed in cells as intra- and intermolecular RNA interactions and is involved in a range of biological processes including RNA metabolism, RNA interference and translation control mediated by natural antisense RNA and microRNA. Despite this breadth of activities, few molecular tools are available to analyse dsRNA as native hybrids. We describe a two-step ligation method for enzymatic joining of dsRNA adaptors to any dsRNA molecule in its duplex form without a need for prior sequence or termini information. The method is specific for dsRNA and can ligate various adaptors to label, map or amplify dsRNA sequences. When combined with reverse transcription-polymerase chain reaction, the method is sensitive and can detect low nanomolar concentrations of dsRNA in total RNA. As examples, we mapped dsRNA/single-stranded RNA junctions within Escherichia coli hok mRNA and the human immunodeficiency virus TAR element using RNA from bacteria and mammalian cells. PMID:18628292

  12. On-Enzyme Refolding Permits Small RNA and tRNA Surveillance by the CCA-Adding Enzyme

    PubMed Central

    Kuhn, Claus-D.; Wilusz, Jeremy E.; Zheng, Yuxuan; Beal, Peter A.; Joshua-Tor, Leemor

    2015-01-01

    SUMMARY Transcription in eukaryotes produces a number of long noncoding RNAs (lncRNAs). Two of these, MALAT1 and Menβ, generate a tRNA-like small RNA in addition to the mature lncRNA. The stability of these tRNA-like small RNAs and bona fide tRNAs is monitored by the CCA-adding enzyme. Whereas CCA is added to stable tRNAs and tRNA-like transcripts, a second CCA repeat is added to certain unstable transcripts to initiate their degradation. Here, we characterize how these two scenarios are distinguished. Following the first CCA addition cycle, nucleotide binding to the active site triggers a clockwise screw motion, producing torque on the RNA. This ejects stable RNAs, whereas unstable RNAs are refolded while bound to the enzyme and subjected to a second CCA catalytic cycle. Intriguingly, with the CCA-adding enzyme acting as a molecular vise, the RNAs proofread themselves through differential responses to its interrogation between stable and unstable substrates. PMID:25640237

  13. A genome-wide analysis of C/D and H/ACA-like small nucleolar RNAs in Trypanosoma brucei reveals a trypanosome-specific pattern of rRNA modification

    PubMed Central

    LIANG, XUE-HAI; ULIEL, SHAI; HURY, AVRAHAM; BARTH, SARIT; DONIGER, TIRZA; UNGER, RON; MICHAELI, SHULAMIT

    2005-01-01

    Small nucleolar RNAs (snoRNAs) constitute newly discovered noncoding small RNAs, most of which function in guiding modifications such as 2′-O-ribose methylation and pseudouridylation on rRNAs and snRNAs. To investigate the genome organization of Trypanosoma brucei snoRNAs and the pattern of rRNA modifications, we used a whole-genome approach to identify the repertoire of these guide RNAs. Twenty-one clusters encoding for 57 C/D snoRNAs and 34 H/ACA-like RNAs, which have the potential to direct 84 methylations and 32 pseudouridines, respectively, were identified. The number of 2′-O-methyls (Nms) identified on rRNA represent 80% of the expected modifications. The modifications guided by these RNAs suggest that trypanosomes contain many modifications and guide RNAs relative to their genome size. Interestingly, ~40% of the Nms are species-specific modifications that do not exist in yeast, humans, or plants, and 40% of the species-specific predicted modifications are located in unique positions outside the highly conserved domains. Although most of the guide RNAs were found in reiterated clusters, a few single-copy genes were identified. The large repertoire of modifications and guide RNAs in trypanosomes suggests that these modifications possibly play a central role in these parasites. PMID:15840815

  14. U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro.

    PubMed

    Somarelli, Jason A; Mesa, Annia; Rodriguez, Carol E; Sharma, Shalini; Herrera, Rene J

    2014-04-25

    The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5' splice sites at exon/intron boundaries. U1 snRNAs associate with 5' splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the 'Smith antigen', or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs. PMID:24583175

  15. Small-RNA loading licenses Argonaute for assembly into a transcriptional silencing complex

    PubMed Central

    Holoch, Daniel; Moazed, Danesh

    2015-01-01

    Argonautes and their small-RNA cofactors form the core effectors of ancient and diverse gene-silencing mechanisms whose roles include regulation of gene expression and defense against foreign genetic elements. Although Argonautes generally act within multisubunit complexes, what governs their assembly into these machineries is not well defined. Here, we show that loading of small RNAs onto Argonaute is a checkpoint for Argonaute’s association with conserved GW-protein components of silencing complexes. We demonstrate that the Argonaute small interfering RNA chaperone (ARC) complex mediates loading of small RNAs onto Ago1 in Schizosaccharomyces pombe and that deletion of its subunits, or mutations in Ago1 that prevent small-RNA loading, abolish the assembly of the GW protein–containing RNA-induced transcriptional silencing (RITS) complex. Our studies uncover a mechanism that ensures that Argonaute loading precedes RITS assembly and thereby averts the formation of inert and potentially deleterious complexes. PMID:25730778

  16. 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

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

    PubMed Central

    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. PMID:24755616

  18. 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. PMID:24755616

  19. Small RNA and RNA-IP Sequencing Identifies and Validates Novel MicroRNAs in Human Mesenchymal Stem Cells.

    PubMed

    Tsai, Chin-Han; Liao, Ko-Hsun; Shih, Chuan-Chi; Chan, Chia-Hao; Hsieh, Jui-Yu; Tsai, Cheng-Fong; Wang, Hsei-Wei; Chang, Shing-Jyh

    2016-03-01

    Organ regeneration therapies using multipotent mesenchymal stem cells (MSCs) are currently being investigated for a variety of common complex diseases. Understanding the molecular regulation of MSC biology will benefit regenerative medicine. MicroRNAs (miRNAs) act as regulators in MSC stemness. There are approximately 2500 currently known human miRNAs that have been recorded in the miRBase v21 database. In the present study, we identified novel microRNAs involved in MSC stemness and differentiation by obtaining the global microRNA expression profiles (miRNomes) of MSCs from two anatomical locations bone marrow (BM-MSCs) and umbilical cord Wharton's jelly (WJ-MSCs) and from osteogenically and adipogenically differentiated progenies of BM-MSCs. Small RNA sequencing (smRNA-seq) and bioinformatics analyses predicted that 49 uncharacterized miRNA candidates had high cellular expression values in MSCs. Another independent batch of Ago1/2-based RNA immunoprecipitation (RNA-IP) sequencing datasets validated the existence of 40 unreported miRNAs in cells and their associations with the RNA-induced silencing complex (RISC). Nine of these 40 new miRNAs were universally overexpressed in both MSC types; nine others were overexpressed in differentiated cells. A novel miRNA (UNI-118-3p) was specifically expressed in BM-MSCs, as verified using RT-qPCR. Taken together, this report offers comprehensive miRNome profiles for two MSC types, as well as cells differentiated from BM-MSCs. MSC transplantation has the potential to ameliorate degenerative disorders and repair damaged tissues. Interventions involving the above 40 new microRNA members in transplanted MSCs may potentially guide future clinical applications. PMID:26910904

  20. Slicer-independent mechanism drives small-RNA strand separation during human RISC assembly

    PubMed Central

    Park, June Hyun; Shin, Chanseok

    2015-01-01

    Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (AGOs 1–4 in humans). A fundamental step during RISC assembly involves the separation of two strands of a small RNA duplex, whereby only the guide strand is retained to form the mature RISC, a process not well understood. Despite the widely accepted view that ‘slicer-dependent unwinding’ via passenger-strand cleavage is a prerequisite for the assembly of a highly complementary siRNA into the AGO2-RISC, here we show by careful re-examination that ‘slicer-independent unwinding’ plays a more significant role in human RISC maturation than previously appreciated, not only for a miRNA duplex, but, unexpectedly, for a highly complementary siRNA as well. We discovered that ‘slicer-dependency’ for the unwinding was affected primarily by certain parameters such as temperature and Mg2+. We further validate these observations in non-slicer AGOs (1, 3 and 4) that can be programmed with siRNAs at the physiological temperature of humans, suggesting that slicer-independent mechanism is likely a common feature of human AGOs. Our results now clearly explain why both miRNA and siRNA are found in all four human AGOs, which is in striking contrast to the strict small-RNA sorting system in Drosophila. PMID:26384428

  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. 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

  3. 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

  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. 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

  6. 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

  7. 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

  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. Small-Molecule Inhibitors of Staphylococcus aureus RnpA-Mediated RNA Turnover and tRNA Processing

    PubMed Central

    Eidem, Tess M.; Lounsbury, Nicole; Emery, John F.; Bulger, Jeffrey; Smith, Andrew; Abou-Gharbia, Magid

    2015-01-01

    New agents are urgently needed for the therapeutic treatment of Staphylococcus aureus infections. In that regard, S. aureus RNase RnpA may represent a promising novel dual-function antimicrobial target that participates in two essential cellular processes, RNA degradation and tRNA maturation. Accordingly, we previously used a high-throughput screen to identify small-molecule inhibitors of the RNA-degrading activity of the enzyme and showed that the RnpA inhibitor RNPA1000 is an attractive antimicrobial development candidate. In this study, we used a series of in vitro and cellular assays to characterize a second RnpA inhibitor, RNPA2000, which was identified in our initial screening campaign and is structurally distinct from RNPA1000. In doing so, it was found that S. aureus RnpA does indeed participate in 5′-precursor tRNA processing, as was previously hypothesized. Further, we show that RNPA2000 is a bactericidal agent that inhibits both RnpA-associated RNA degradation and tRNA maturation activities both in vitro and within S. aureus. The compound appears to display specificity for RnpA, as it did not significantly affect the in vitro activities of unrelated bacterial or eukaryotic ribonucleases and did not display measurable human cytotoxicity. Finally, we show that RNPA2000 exhibits antimicrobial activity and inhibits tRNA processing in efflux-deficient Gram-negative pathogens. Taken together, these data support the targeting of RnpA for antimicrobial development purposes, establish that small-molecule inhibitors of both of the functions of the enzyme can be identified, and lend evidence that RnpA inhibitors may have broad-spectrum antimicrobial activities. PMID:25605356

  10. Genome-Wide Analysis of C/D and H/ACA-Like Small Nucleolar RNAs in Leishmania major Indicates Conservation among Trypanosomatids in the Repertoire and in Their rRNA Targets▿ †

    PubMed Central

    Liang, Xue-hai; Hury, Avraham; Hoze, Ehud; Uliel, Shai; Myslyuk, Inna; Apatoff, Avihay; Unger, Ron; Michaeli, Shulamit

    2007-01-01

    Small nucleolar RNAs (snoRNAs) are a large group of noncoding RNAs that exist in eukaryotes and archaea and guide modifications such as 2′-O-ribose methylations and pseudouridylation on rRNAs and snRNAs. Recently, we described a genome-wide screening approach with Trypanosoma brucei that revealed over 90 guide RNAs. In this study, we extended this approach to analyze the repertoire of the closely related human pathogen Leishmania major. We describe 23 clusters that encode 62 C/Ds that can potentially guide 79 methylations and 37 H/ACA-like RNAs that can potentially guide 30 pseudouridylation reactions. Like T. brucei, Leishmania also contains many modifications and guide RNAs relative to its genome size. This study describes 10 H/ACAs and 14 C/Ds that were not found in T. brucei. Mapping of 2′-O-methylations in rRNA regions rich in modifications suggests the existence of trypanosomatid-specific modifications conserved in T. brucei and Leishmania. Structural features of C/D snoRNAs, such as copy number, conservation of boxes, K turns, and intragenic and extragenic base pairing, were examined to elucidate the great variation in snoRNA abundance. This study highlights the power of comparative genomics for determining conserved features of noncoding RNAs. PMID:17189491

  11. Polysome Preparation, RNA Isolation and Analysis

    PubMed Central

    Zhang, Hailong; Zhou, Muxiang

    2016-01-01

    During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosome can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be size-fractionated by sucrose density gradient centrifugation. The more specific mRNA in polysomes implies more active translational status of the mRNA.

  12. The plasma lncRNA acting as fingerprint in non-small-cell lung cancer.

    PubMed

    Hu, Xiaodong; Bao, Jitao; Wang, Zhen; Zhang, Zigang; Gu, Peijie; Tao, Feng; Cui, Di; Jiang, Weilong

    2016-03-01

    Recent studies have indicated that long non-coding RNAs (lncRNAs) could act as non-invasive tumor markers in both diagnosis and predicting the prognosis. In this study, we focused to determine the expression of circulating lncRNAs in patients suffering from non-small-cell lung cancer (NSCLC), aiming to found the potential lncRNA as predictor. Twenty-one lncRNAs which previously identified were selected as candidate targets for subsequent circulating lncRNA assay. The candidate lncRNAs were validated by qRT-PCR arranged in the training and validation sets. Circulating SPRY4-IT1, ANRIL, and NEAT1 were significantly increased in plasma samples of NSCLC patients during training set and validation set. Receiver operating characteristic curve (ROC) analysis revealed that plasma ANRIL provided the highest diagnostic performance with an area under ROC curve value (AUC) of 0.798. Further combination with the three factors indicated a higher power (AUC, 0.876; sensitivity, 82.8 %; specificity, 92.3 %). The stableness detection of the three factors indicated that circulating SPRY4-IT1, ANRIL, and NEAT might serve as a predictor for the early warning of non-small-cell lung cancer. PMID:26453113

  13. Structure of the myotonic dystrophy type 2 RNA and designed small molecules that reduce toxicity.

    PubMed

    Childs-Disney, Jessica L; Yildirim, Ilyas; Park, HaJeung; Lohman, Jeremy R; Guan, Lirui; Tran, Tuan; Sarkar, Partha; Schatz, George C; Disney, Matthew D

    2014-02-21

    Myotonic dystrophy type 2 (DM2) is an incurable neuromuscular disorder caused by a r(CCUG) expansion (r(CCUG)(exp)) that folds into an extended hairpin with periodically repeating 2×2 nucleotide internal loops (5'CCUG/3'GUCC). We designed multivalent compounds that improve DM2-associated defects using information about RNA-small molecule interactions. We also report the first crystal structure of r(CCUG) repeats refined to 2.35 Å. Structural analysis of the three 5'CCUG/3'GUCC repeat internal loops (L) reveals that the CU pairs in L1 are each stabilized by one hydrogen bond and a water-mediated hydrogen bond, while CU pairs in L2 and L3 are stabilized by two hydrogen bonds. Molecular dynamics (MD) simulations reveal that the CU pairs are dynamic and stabilized by Na(+) and water molecules. MD simulations of the binding of the small molecule to r(CCUG) repeats reveal that the lowest free energy binding mode occurs via the major groove, in which one C residue is unstacked and the cross-strand nucleotides are displaced. Moreover, we modeled the binding of our dimeric compound to two 5'CCUG/3'GUCC motifs, which shows that the scaffold on which the RNA-binding modules are displayed provides an optimal distance to span two adjacent loops. PMID:24341895

  14. Structure of the Myotonic Dystrophy Type 2 RNA and Designed Small Molecules That Reduce Toxicity

    PubMed Central

    Park, HaJeung; Lohman, Jeremy R.; Guan, Lirui; Tran, Tuan; Sarkar, Partha; Schatz, George C.; Disney, Matthew D.

    2014-01-01

    Myotonic dystrophy type 2 (DM2) is an untreatable neuromuscular disorder caused by a r(CCUG) expansion (r(CCUG)exp) that folds into an extended hairpin with periodically repeating 2×2 nucleotide internal loops (5’CCUG/3’GUCC). We designed multivalent compounds that improve DM2-associated defects using information about RNA-small molecule interactions. We also report the first crystal structure of r(CCUG)exp refined to 2.35 Å. Structural analysis of the three 5’CCUG/3’GUCC repeat internal loops (L) reveals that the CU pairs in L1 are each stabilized by one hydrogen bond and a water-mediated hydrogen bond while CU pairs in L2 and L3 are stabilized by two hydrogen bonds. Molecular dynamics (MD) simulations reveal that the CU pairs are dynamic and stabilized by Na+ and water molecules. MD simulations of the binding of the small molecule to r(CCUG) repeats reveal that the lowest free energy binding mode occurs via the major groove, in which one C residue is unstacked and the cross-strand nucleotides are displaced. Moreover, we modeled the binding of our dimeric compound to two 5’CCUG/3’GUCC motifs, which shows that the scaffold on which the RNA-binding modules are displayed provides an optimal distance to span two adjacent loops. PMID:24341895

  15. Caryotricha minuta (Xu et al., 2008) nov. comb., a unique marine ciliate (Protista, Ciliophora, Spirotrichea), with phylogenetic analysis of the ambiguous genus Caryotricha inferred from the small-subunit rRNA gene sequence.

    PubMed

    Miao, Miao; Shao, Chen; Jiang, Jiamei; Li, Liqiong; Stoeck, Thorsten; Song, Weibo

    2009-02-01

    A population of Kiitricha minuta Xu et al., 2008, a small kiitrichid ciliate, was isolated from a brackish water sample in Jiaozhou Bay, Qingdao, northern China. After comparison of its morphology and infraciliature, it is believed that this morphotype should be assigned to the genus Caryotricha; hence, a new combination is suggested, Caryotricha minuta (Xu et al., 2008) nov. comb. The small-subunit (SSU) rRNA gene sequence was determined in order to elucidate the phylogenetic position of this poorly known, ambiguous genus. The organism can be clearly separated from its congener, Caryotricha convexa Kahl, 1932, by the extremely shortened ventral cirral rows in the posterior ends. Based on the data available, an improved diagnosis is given for the genus: marine Kiitrichidae with prominent buccal field; two highly developed undulating membranes; non-grouped, uniform cirral rows on both ventral and dorsal sides; enlarged transverse cirri present, which are the only differentiated cirri; marginal cirri not present; one short migratory row located posterior to buccal field; structure of dorsal kineties generally in Kiitricha pattern. The sequence of the SSU rRNA gene of C. minuta differs by 13 % from that of Kiitricha marina. Molecular phylogenetic analyses (Bayesian inference, least squares, neighbour joining, maximum parsimony) indicate that Caryotricha, together with Kiitricha, diverges at a deep level from all other spirotrichs. Its branching position is between Phacodiniidia and Licnophoridia. The results strongly support the distinct separation of the Kiitricha-Caryotricha clade, which always branches basal to the Stichotrichia-Hypotrichia-Oligotrichia-Choreotrichia assemblage. These results also confirm the previous hypothesis that the Kiitricha-Caryotricha group, long assumed to be a close relation to the euplotids, represents a taxon at subclass level within the spirotrichs. PMID:19196791

  16. A Bifurcated Proteoglycan Binding Small Molecule Carrier for siRNA Delivery

    PubMed Central

    Gooding, Matt; Adigbli, Derick; Edith Chan, A W; Melander, Roberta J; MacRobert, Alexander J; Selwood, David L

    2014-01-01

    A wider application of siRNA- and miRNA- based therapeutics is restricted by the currently available delivery systems. We have designed a new type of small molecule carrier (SMoC) system for siRNA modeled to interact with cell surface proteoglycans. This bifurcated SMoC has similar affinity for the model proteoglycan heparin to an equivalent polyarginine peptide and exhibits significant mRNA knockdown of protein levels comparable to lipofectamine and the previously reported linear SMoC. PMID:24472581

  17. 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. PMID:25625965

  18. 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

  19. The disturbance of small RNA pathways enhanced abscisic acid response and multiple stress responses in Arabidopsis.

    PubMed

    Zhang, Jian-Feng; Yuan, Li-Jie; Shao, Yi; Du, Wei; Yan, Da-Wei; Lu, Ying-Tang

    2008-04-01

    The phytohormone abscisic acid (ABA) regulates plant growth and development as well as stress tolerance. To gain more insights into ABA signalling, a population of chemical-inducible activation-tagged Arabidopsis mutants was screened on the basis of the ABA effect on the inhibition of seed germination. Two novel ABA supersensitive mutants ABA supersensitive during germination1 (absg1) and absg2 were characterized as alleles of Dicer-like1 (DCL1) and HEN1, respectively, as microRNA biogenesis genes, and accordingly, these two mutants were renamed dcl1-11 and hen1-16. The dcl1-11 mutant was an ABA hypersensitive mutant for seed germination and root growth. Reverse transcriptase polymerase chain reaction assays revealed that the expression of ABA- and stress-responsive genes was increased in dcl1-11, as compared with the wild type (WT). Furthermore, the germination assay showed that dcl1-11 was also more sensitive to salt and osmotic stress. The hen1-16 mutant also showed supersensitive to ABA during seed germination. Further analysis showed that, among the microRNA biogenesis genes, all the other mutants were not only enhanced in sensitivity to ABA, salt and osmotic stress, but also enhanced the expression of ABA-responsive genes. In addition to the mutants in the microRNA biogenesis, the interruption of the production of crucial components of other small RNA pathways such as dcl2, dcl3 and dcl4 also caused ABA supersensitive during germination. PMID:18208512

  20. Design of a bioactive small molecule that targets the myotonic dystrophy type 1 RNA via an RNA motif-ligand database and chemical similarity searching.

    PubMed

    Parkesh, Raman; Childs-Disney, Jessica L; Nakamori, Masayuki; Kumar, Amit; Wang, Eric; Wang, Thomas; Hoskins, Jason; Tran, Tuan; Housman, David; Thornton, Charles A; Disney, Matthew D

    2012-03-14

    Myotonic dystrophy type 1 (DM1) is a triplet repeating disorder caused by expanded CTG repeats in the 3'-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The transcribed repeats fold into an RNA hairpin with multiple copies of a 5'CUG/3'GUC motif that binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Sequestration of MBNL1 by expanded r(CUG) repeats causes splicing defects in a subset of pre-mRNAs including the insulin receptor, the muscle-specific chloride ion channel, sarco(endo)plasmic reticulum Ca(2+) ATPase 1, and cardiac troponin T. Based on these observations, the development of small-molecule ligands that target specifically expanded DM1 repeats could be of use as therapeutics. In the present study, chemical similarity searching was employed to improve the efficacy of pentamidine and Hoechst 33258 ligands that have been shown previously to target the DM1 triplet repeat. A series of in vitro inhibitors of the RNA-protein complex were identified with low micromolar IC(50)'s, which are >20-fold more potent than the query compounds. Importantly, a bis-benzimidazole identified from the Hoechst query improves DM1-associated pre-mRNA splicing defects in cell and mouse models of DM1 (when dosed with 1 mM and 100 mg/kg, respectively). Since Hoechst 33258 was identified as a DM1 binder through analysis of an RNA motif-ligand database, these studies suggest that lead ligands targeting RNA with improved biological activity can be identified by using a synergistic approach that combines analysis of known RNA-ligand interactions with chemical similarity searching. PMID:22300544

  1. Design of a Bioactive Small Molecule that Targets the Myotonic Dystrophy Type 1 RNA Via an RNA Motif-Ligand Database & Chemical Similarity Searching

    PubMed Central

    Parkesh, Raman; Childs-Disney, Jessica L.; Nakamori, Masayuki; Kumar, Amit; Wang, Eric; Wang, Thomas; Hoskins, Jason; Tran, Tuan; Housman, David; Thornton, Charles A.; Disney, Matthew D.

    2012-01-01

    Myotonic dystrophy type 1 (DM1) is a triplet repeating disorder caused by expanded CTG repeats in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The transcribed repeats fold into an RNA hairpin with multiple copies of a 5′CUG/3′GUC motif that binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Sequestration of MBNL1 by expanded r(CUG) repeats causes splicing defects in a subset of pre-mRNAs including the insulin receptor, the muscle-specific chloride ion channel, Sarco(endo)plasmic reticulum Ca2+ ATPase 1 (Serca1/Atp2a1), and cardiac troponin T (cTNT). Based on these observations, the development of small molecule ligands that target specifically expanded DM1 repeats could serve as therapeutics. In the present study, computational screening was employed to improve the efficacy of pentamidine and Hoechst 33258 ligands that have been shown previously to target the DM1 triplet repeat. A series of inhibitors of the RNA-protein complex with low micromolar IC50’s, which are >20-fold more potent than the query compounds, were identified. Importantly, a bis-benzimidazole identified from the Hoechst query improves DM1-associated pre-mRNA splicing defects in cell and mouse models of DM1 (when dosed with 1 mM and 100 mg/kg, respectively). Since Hoechst 33258 was identified as a DM1 binder through analysis of an RNA motif-ligand database, these studies suggest that lead ligands targeting RNA with improved biological activity can be identified by using a synergistic approach that combines analysis of known RNA-ligand interactions with virtual screening. PMID:22300544

  2. Long-circulating siRNA nanoparticles for validating Prohibitin1-targeted non-small cell lung cancer treatment

    PubMed Central

    Zhu, Xi; Xu, Yingjie; Solis, Luisa M.; Tao, Wei; Wang, Liangzhe; Behrens, Carmen; Xu, Xiaoyang; Zhao, Lili; Liu, Danny; Wu, Jun; Zhang, Ning; Wistuba, Ignacio I.; Farokhzad, Omid C.; Zetter, Bruce R.; Shi, Jinjun

    2015-01-01

    RNA interference (RNAi) represents a promising strategy for identification and validation of putative therapeutic targets and for treatment of a myriad of important human diseases including cancer. However, the effective systemic in vivo delivery of small interfering RNA (siRNA) to tumors remains a formidable challenge. Using a robust self-assembly strategy, we develop a unique nanoparticle (NP) platform composed of a solid polymer/cationic lipid hybrid core and a lipid-poly(ethylene glycol) (lipid-PEG) shell for systemic siRNA delivery. The new generation lipid–polymer hybrid NPs are small and uniform, and can efficiently encapsulate siRNA and control its sustained release. They exhibit long blood circulation (t1/2 ∼8 h), high tumor accumulation, effective gene silencing, and negligible in vivo side effects. With this RNAi NP, we delineate and validate the therapeutic role of Prohibitin1 (PHB1), a target protein that has not been systemically evaluated in vivo due to the lack of specific and effective inhibitors, in treating non-small cell lung cancer (NSCLC) as evidenced by the drastic inhibition of tumor growth upon PHB1 silencing. Human tissue microarray analysis also reveals that high PHB1 tumor expression is associated with poorer overall survival in patients with NSCLC, further suggesting PHB1 as a therapeutic target. We expect this long-circulating RNAi NP platform to be of high interest for validating potential cancer targets in vivo and for the development of new cancer therapies. PMID:26056316

  3. Long-circulating siRNA nanoparticles for validating Prohibitin1-targeted non-small cell lung cancer treatment.

    PubMed

    Zhu, Xi; Xu, Yingjie; Solis, Luisa M; Tao, Wei; Wang, Liangzhe; Behrens, Carmen; Xu, Xiaoyang; Zhao, Lili; Liu, Danny; Wu, Jun; Zhang, Ning; Wistuba, Ignacio I; Farokhzad, Omid C; Zetter, Bruce R; Shi, Jinjun

    2015-06-23

    RNA interference (RNAi) represents a promising strategy for identification and validation of putative therapeutic targets and for treatment of a myriad of important human diseases including cancer. However, the effective systemic in vivo delivery of small interfering RNA (siRNA) to tumors remains a formidable challenge. Using a robust self-assembly strategy, we develop a unique nanoparticle (NP) platform composed of a solid polymer/cationic lipid hybrid core and a lipid-poly(ethylene glycol) (lipid-PEG) shell for systemic siRNA delivery. The new generation lipid-polymer hybrid NPs are small and uniform, and can efficiently encapsulate siRNA and control its sustained release. They exhibit long blood circulation (t1/2 ∼ 8 h), high tumor accumulation, effective gene silencing, and negligible in vivo side effects. With this RNAi NP, we delineate and validate the therapeutic role of Prohibitin1 (PHB1), a target protein that has not been systemically evaluated in vivo due to the lack of specific and effective inhibitors, in treating non-small cell lung cancer (NSCLC) as evidenced by the drastic inhibition of tumor growth upon PHB1 silencing. Human tissue microarray analysis also reveals that high PHB1 tumor expression is associated with poorer overall survival in patients with NSCLC, further suggesting PHB1 as a therapeutic target. We expect this long-circulating RNAi NP platform to be of high interest for validating potential cancer targets in vivo and for the development of new cancer therapies. PMID:26056316

  4. 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

  5. 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

  6. 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.

  7. Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection

    PubMed Central

    Wang, Xingyu; Li, Can; Gao, Xiaomeng; Wang, Jing; Liang, Xingguo

    2015-01-01

    A facile and robust RNA preparation protocol was developed by combining rolling circle transcription (RCT) with RNA cleavage by RNase H. Circular DNA with a complementary sequence was used as the template for promoter-free transcription. With the aid of a 2′-O-methylated DNA, the RCT-generated tandem repeats of the desired RNA sequence were disconnected at the exact end-to-end position to harvest the desired RNA oligomers. Compared with the template DNA, more than 4 × 103 times the amount of small RNA products were obtained when modest cleavage was carried out during transcription. Large amounts of RNA oligomers could easily be obtained by simply increasing the reaction volume. PMID:25584899

  8. Complete probabilistic analysis of RNA shapes

    PubMed Central

    Voß, Björn; Giegerich, Robert; Rehmsmeier, Marc

    2006-01-01

    Background Soon after the first algorithms for RNA folding became available, it was recognised that the prediction of only one energetically optimal structure is insufficient to achieve reliable results. An in-depth analysis of the folding space as a whole appeared necessary to deduce the structural properties of a given RNA molecule reliably. Folding space analysis comprises various methods such as suboptimal folding, computation of base pair probabilities, sampling procedures and abstract shape analysis. Common to many approaches is the idea of partitioning the folding space into classes of structures, for which certain properties can be derived. Results In this paper we extend the approach of abstract shape analysis. We show how to compute the accumulated probabilities of all structures that share the same shape. While this implies a complete (non-heuristic) analysis of the folding space, the computational effort depends only on the size of the shape space, which is much smaller. This approach has been integrated into the tool RNAshapes, and we apply it to various RNAs. Conclusion Analyses of conformational switches show the existence of two shapes with probabilities approximately 23 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaadaWcaaqaaiabikdaYaqaaiabiodaZaaaaaa@2EA2@ vs. 13 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaadaWcaaqaaiabigdaXaqaaiabiodaZaaaaaa@2EA0@, whereas the analysis of a microRNA precursor reveals one shape with a probability near to 1.0. Furthermore, it is shown that a shape can outperform an energetically more favourable one by achieving a higher probability

  9. 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

  10. 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. PMID:23933212

  11. Methods for small RNA preparation for digital gene expression profiling by next-generation sequencing.

    PubMed

    Linsen, Sam E V; Cuppen, Edwin

    2012-01-01

    Digital gene expression (DGE) profiling techniques are playing an eminent role in the detection, localization, and differential expression quantification of many small RNA species, including microRNAs (1-3). Procedures in small RNA library preparation techniques typically include adapter ligation by RNA ligase, followed by reverse transcription and amplification by PCR. This chapter describes three protocols that were successfully applied to generate small RNA sequencing SOLiD(TM) libraries. The Ambion SREK(TM)-adopted protocol can be readily used for multiplexing samples; the modban-based protocol is cost-efficient, but biased toward certain microRNAs; the poly(A)-based protocol is less biased, but less precise because of the A-tail that is introduced. In summary, each of these protocols has its advantages and disadvantages with respect to the ease of including barcodes, costs, and outcome. PMID:22144201

  12. Searching for small RNA genes in Xylella fastidiosa genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial non-coding small RNAs (sRNAs) have attracted considerable attention due to their ubiquitous nature and roles in controlling numerous cellular processes including survival, adaptation and pathogenesis. Xylella fastidiosa is a bacterial pathogen causing many economically important diseases s...

  13. 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

    2016-03-01

    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. PMID:26853797

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

    PubMed

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

    2015-11-17

    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

  15. 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

  16. Six RNA viruses and forty-one hosts: viral small RNAs and modulation of small RNA repertoires in vertebrate and invertebrate systems.

    PubMed

    Parameswaran, Poornima; Sklan, Ella; Wilkins, Courtney; Burgon, Trever; Samuel, Melanie A; Lu, Rui; Ansel, K Mark; Heissmeyer, Vigo; Einav, Shirit; Jackson, William; Doukas, Tammy; Paranjape, Suman; Polacek, Charlotta; dos Santos, Flavia Barreto; Jalili, Roxana; Babrzadeh, Farbod; Gharizadeh, Baback; Grimm, Dirk; Kay, Mark; Koike, Satoshi; Sarnow, Peter; Ronaghi, Mostafa; Ding, Shou-Wei; Harris, Eva; Chow, Marie; Diamond, Michael S; Kirkegaard, Karla; Glenn, Jeffrey S; Fire, Andrew Z

    2010-02-01

    We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare" (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs"). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3' overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts. PMID:20169186

  17. Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

    PubMed Central

    Parameswaran, Poornima; Sklan, Ella; Wilkins, Courtney; Burgon, Trever; Samuel, Melanie A.; Lu, Rui; Ansel, K. Mark; Heissmeyer, Vigo; Einav, Shirit; Jackson, William; Doukas, Tammy; Paranjape, Suman; Polacek, Charlotta; dos Santos, Flavia Barreto; Jalili, Roxana; Babrzadeh, Farbod; Gharizadeh, Baback; Grimm, Dirk; Kay, Mark; Koike, Satoshi; Sarnow, Peter; Ronaghi, Mostafa; Ding, Shou-Wei; Harris, Eva; Chow, Marie; Diamond, Michael S.; Kirkegaard, Karla; Glenn, Jeffrey S.; Fire, Andrew Z.

    2010-01-01

    We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts. PMID:20169186

  18. Identification of microRNAs by small RNA deep sequencing for synthetic microRNA mimics to control Spodoptera exigua.

    PubMed

    Zhang, Yu Liang; Huang, Qi Xing; Yin, Guo Hua; Lee, Samantha; Jia, Rui Zong; Liu, Zhi Xin; Yu, Nai Tong; Pennerman, Kayla K; Chen, Xin; Guo, An Ping

    2015-02-25

    Beet armyworm, Spodoptera exigua, is a major pest of cotton around the world. With the increase of resistance to Bacillus thuringiensis (Bt) toxin in transgenic cotton plants, there is a need to develop an alternative control approach that can be used in combination with Bt transgenic crops as part of resistance management strategies. MicroRNAs (miRNAs), a non-coding small RNA family (18-25 nt), play crucial roles in various biological processes and over-expression of miRNAs has been shown to interfere with the normal development of insects. In this study, we identified 127 conserved miRNAs in S. exigua by using small RNA deep sequencing technology. From this, we tested the effects of 11 miRNAs on larval development. We found three miRNAs, Sex-miR-10-1a, Sex-miR-4924, and Sex-miR-9, to be differentially expressed during larval stages of S. exigua. Oral feeding experiments using synthetic miRNA mimics of Sex-miR-10-1a, Sex-miR-4924, and Sex-miR-9 resulted in suppressed growth of S. exigua and mortality. Over-expression of Sex-miR-4924 caused a significant reduction in the expression level of chitinase 1 and caused abortive molting in the insects. Therefore, we demonstrated a novel approach of using miRNA mimics to control S. exigua development. PMID:25528266

  19. Expression of CRISPR/Cas single guide RNAs using small tRNA promoters.

    PubMed

    Mefferd, Adam L; Kornepati, Anand V R; Bogerd, Hal P; Kennedy, Edward M; Cullen, Bryan R

    2015-09-01

    The in vivo application of CRISPR/Cas-based DNA editing technology will require the development of efficient delivery methods that likely will be dependent on adeno-associated virus (AAV)-based viral vectors. However, AAV vectors have only a modest, ∼4.7-kb packaging capacity, which will necessitate the identification and characterization of highly active Cas9 proteins that are substantially smaller than the prototypic Streptococcus pyogenes Cas9 protein, which covers ∼4.2 kb of coding sequence, as well as the development of single guide RNA (sgRNA) expression cassettes substantially smaller than the current ∼360 bp size. Here, we report that small, ∼70-bp tRNA promoters can be used to express high levels of tRNA:sgRNA fusion transcripts that are efficiently and precisely cleaved by endogenous tRNase Z to release fully functional sgRNAs. Importantly, cells stably expressing functional tRNA:sgRNA precursors did not show a detectable change in the level of endogenous tRNA expression. This novel sgRNA expression strategy should greatly facilitate the construction of effective AAV-based Cas9/sgRNA vectors for future in vivo use. PMID:26187160

  20. 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

  1. 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

  2. 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...

  3. 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.

  4. 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

  5. Modeling RNA topological structures using small angle X-ray scattering.

    PubMed

    Bhandari, Yuba R; Jiang, Wei; Stahlberg, Eric A; Stagno, Jason R; Wang, Yun-Xing

    2016-07-01

    Detailed understanding of the structure and function relationship of RNA requires knowledge about RNA three-dimensional (3D) topological folding. However, there are very few unique RNA entries in structure databases. This is due to challenges in determining 3D structures of RNA using conventional methods, such as X-ray crystallography and NMR spectroscopy, despite significant advances in both of these technologies. Computational methods have come a long way in accurately predicting the 3D structures of small (<50nt) RNAs to within a few angstroms compared to their native folds. However, lack of an apparent correlation between an RNA primary sequence and its 3D fold ultimately limits the success of purely computational approaches. In this context, small angle X-ray scattering (SAXS) serves as a valuable tool by providing global shape information of RNA. In this article, we review the progress in determining RNA 3D topological structures, including a new method that combines secondary structural information and SAXS data to sample conformations generated through hierarchical moves of commonly observed RNA motifs. PMID:27090001

  6. An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons

    PubMed Central

    Luo, Miaw-Chyi; Zhang, Dong-Qin; Ma, Shou-Wu; Huang, Yuan-Yuan; Shuster, Sam J; Porreca, Frank; Lai, Josephine

    2005-01-01

    We have developed a highly effective method for in vivo gene silencing in the spinal cord and dorsal root ganglia (DRG) by a cationic lipid facilitated delivery of synthetic, small interfering RNA (siRNA). A siRNA to the delta opioid receptor (DOR), or a mismatch RNA, was mixed with the transfection reagent, i-Fect™ (vehicle), and delivered as repeated daily bolus doses (0.5 μg to 4 μg) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats were tested for antinociception by the DOR selective agonist, [D-Ala2, Glu4]deltorphin II (DELT), or the mu opioid receptor (MOR) selective agonist, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). Pretreatment with the siRNA, but not the mismatch RNA or vehicle alone, blocked DELT antinociception dose-dependently. The latter was concomitant with a reduction in the spinal immunoreactivity and receptor density of DOR, and in DOR transcripts in the lumbar DRG and spinal dorsal horn. Neither siRNA nor mismatch RNA pretreatment altered spinal immunoreactivity of MOR or antinociception by spinal DAMGO, and had no effect on the baseline thermal nociceptive threshold. The inhibition of function and expression of DOR by siRNA was reversed by 72 hr after the last RNA injection. The uptake of fluorescence-tagged siRNA was detected in both DRG and spinal cord. The low effective dose of siRNA/i-Fect™ complex reflects an efficient delivery of the siRNA to peripheral and spinal neurons, produced no behavioral signs of toxicity. This delivery method may be optimized for other gene targets. PMID:16191203

  7. Optimized In Vivo Transfer of Small Interfering RNA Targeting Dermal Tissue Using In Vivo Surface Electroporation

    PubMed Central

    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

  8. 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

  9. 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

  10. 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

  11. H/ACA small nucleolar RNA pseudouridylation pockets bind substrate RNA to form three-way junctions that position the target U for modification

    PubMed Central

    Wu, Haihong; Feigon, Juli

    2007-01-01

    During the biogenesis of eukaryotic ribosomal RNA (rRNA) and spliceosomal small nuclear RNA (snRNA), uridines at specific sites are converted to pseudouridines by H/ACA ribonucleoprotein particles (RNPs). Each H/ACA RNP contains a substrate-specific H/ACA RNA and four common proteins, the pseudouridine synthase Cbf5, Nop10, Gar1, and Nhp2. The H/ACA RNA contains at least one pseudouridylation (ψ) pocket, which is complementary to the sequences flanking the target uridine. In this article, we show structural evidence that the ψ pocket can form the predicted base pairs with substrate RNA in the absence of protein components. We report the solution structure of the complex between an RNA hairpin derived from the 3′ ψ pocket of human U65 H/ACA small nucleolar RNA (snoRNA) and the substrate rRNA. The snoRNA–rRNA substrate complex has a unique structure with two offset parallel pairs of stacked helices and two unusual intermolecular three-way junctions, which together organize the substrate for docking into the active site of Cbf5. The substrate RNA interacts on one face of the snoRNA in the complex, forming a structure that easily could be accommodated in the H/ACA RNP, and explains how successive substrate RNAs could be loaded onto and unloaded from the H/ACA RNA in the RNP. PMID:17412831

  12. 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

  13. 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

  14. RNA-Seq of the Nucleolus Reveals Abundant SNORD44-Derived Small RNAs

    PubMed Central

    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. PMID:25203660

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. Temporal small RNA transcriptome profiling unraveled partitioned miRNA expression in developing maize endosperms between reciprocal crosses.

    PubMed

    Xin, Mingming; Yang, Guanghui; Yao, Yingyin; Peng, Huiru; Hu, Zhaorong; Sun, Qixin; Wang, Xiangfeng; Ni, Zhongfu

    2015-01-01

    In angiosperms, the endosperm nurtures the embryo and provides nutrients for seed germination. To identify the expression pattern of small interfering RNA in the developing maize endosperm, we have performed high-throughput small RNA transcriptome sequencing of kernels at 0, 3, and 5 days after pollination (DAP) and endosperms at 7, 10, and 15 DAP using B73 and Mo17 reciprocal crosses in previous study. Here, we further explored these small RNA-seq data to investigate the potential roles of miRNAs in regulating the gene expression process. In total, 57 conserved miRNAs and 18 novel miRNAs were observed highly expressed in maize endosperm. Temporal expression profiling indicated that these miRNAs exhibited dynamic and partitioned expression patterns at different developmental stages between maize reciprocal crosses, and quantitative RT-PCR results further confirmed our observation. In addition, we found a subset of distinct tandem miRNAs are generated from a single stem-loop structure in maize that might be conserved in monocots. Furthermore, a SNP variation of Zma-miR408-5p at 11th base position was characterized between B73 and Mo17 which might lead to completely different functions in repressing targets. More interestingly, Zma-miR408-5p exhibited B73-biased expression pattern in the B73 and Mo17 reciprocal hybrid endosperms at 7, 10, and 15 DAP according to the reads abundance with SNPs and CAPS experiment. Together, this study suggests that miRNA plays a crucial role in regulating endosperm development, and exhibited distinct expression patterns in developing endosperm between maize reciprocal crosses. PMID:26442057

  4. 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

  5. YM500v2: a small RNA sequencing (smRNA-seq) database for human cancer miRNome research.

    PubMed

    Cheng, Wei-Chung; Chung, I-Fang; Tsai, Cheng-Fong; Huang, Tse-Shun; Chen, Chen-Yang; Wang, Shao-Chuan; Chang, Ting-Yu; Sun, Hsing-Jen; Chao, Jeffrey Yung-Chuan; Cheng, Cheng-Chung; Wu, Cheng-Wen; Wang, Hsei-Wei

    2015-01-01

    We previously presented YM500, which is an integrated database for miRNA quantification, isomiR identification, arm switching discovery and novel miRNA prediction from 468 human smRNA-seq datasets. Here in this updated YM500v2 database (http://ngs.ym.edu.tw/ym500/), we focus on the cancer miRNome to make the database more disease-orientated. New miRNA-related algorithms developed after YM500 were included in YM500v2, and, more significantly, more than 8000 cancer-related smRNA-seq datasets (including those of primary tumors, paired normal tissues, PBMC, recurrent tumors, and metastatic tumors) were incorporated into YM500v2. Novel miRNAs (miRNAs not included in the miRBase R21) were not only predicted by three independent algorithms but also cleaned by a new in silico filtration strategy and validated by wetlab data such as Cross-Linked ImmunoPrecipitation sequencing (CLIP-seq) to reduce the false-positive rate. A new function 'Meta-analysis' is additionally provided for allowing users to identify real-time differentially expressed miRNAs and arm-switching events according to customer-defined sample groups and dozens of clinical criteria tidying up by proficient clinicians. Cancer miRNAs identified hold the potential for both basic research and biotech applications. PMID:25398902

  6. 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

  7. 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

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

    PubMed

    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

  9. Challenges in the analysis of long noncoding RNA functionality.

    PubMed

    Leone, Sergio; Santoro, Raffaella

    2016-08-01

    Long noncoding RNA (lncRNA) are emerging as important regulators of diverse biological functions. Although mechanistic models are starting to emerge, it is also clear that the lncRNA field needs appropriate model systems in order to better elucidate the functions of lncRNA and their roles in both physiological and pathological conditions. The field of lncRNA is new, and the biochemical and genetic methods used to address function and mechanisms of lncRNA have only recently been developed or adapted from techniques used to investigate protein-coding genes. In this review, we discuss the strengths and weaknesses of available techniques for the analysis of chromatin-associated lncRNA and emerging models for the recruitment to specific genomic sites such as triple-helix, RNA-protein-DNA recognition and proximity-guided search models. PMID:27417130

  10. MiRNA expression profile and miRNA-mRNA integrated analysis (MMIA) during podocyte differentiation.

    PubMed

    Li, Zhigui; Wang, Lifeng; Xu, Jing; Yang, Zhuo

    2015-06-01

    The podocyte is a prominent cell type, which encases the capillaries of glomerulus. Podocyte-selective deletion of Dicer or Drosha was reported to induce proteinuria and glomerulosclerosis, suggesting the essential role of microRNA (miRNA) in podocytes for renal function. However, no comprehensive miRNA expression or miRNA-mRNA integrated analysis (MMIA) can be found during podocyte differentiation. Herein, miRNA and mRNA microarrays are presented, which were carried out in differentiated and undifferentiated mouse podocyte cell lines (MPC5). A total of 50 abnormal miRNAs (26 down-regulated and 24 up-regulated) were identified in differentiated and undifferentiated podocytes. Using MMIA, 80 of the 743 mRNAs (>twofold change) were predicted for potential crosstalk with 30 miRNAs of the 50 abnormal miRNAs. In addition, the gene ontology of mRNAs and the pathway analysis of miRNAs revealed a new potential-regulated network during podocyte differentiation. The expressions of three remarkably changed miRNAs (miR-34c, miR-200a and miR-467e) and four mRNAs (Runx1t1, Atp2a2, Glrp1, and Mmp15), were randomly chosen for further validation by the quantitative real-time polymerase chain reaction, and their expression trends were consistent with the microarray data. Reference searching was also conducted to confirm our data and to find potential new molecules and miRNA-target pairs involved in the podocyte differentiation. The dual luciferase reporter assay for miR-200a/GLRX and let-7b/ARL4D confirmed the prediction of MMIA. The results of this study provide a detailed integration of mRNA and miRNA during podocyte differentiation. The molecular integration mode will open up new perspectives for a better understanding of the mechanism during podocyte differentiation. PMID:25433550

  11. Analysis of siRNA specificity on targets with double-nucleotide mismatches

    PubMed Central

    Dahlgren, Cecilia; Zhang, Hong-Yan; Du, Quan; Grahn, Maria; Norstedt, Gunnar; Wahlestedt, Claes

    2008-01-01

    Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that ∼35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site. PMID:18420656

  12. 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

  13. 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.

  14. Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA.

    PubMed

    Costales, Matthew G; Rzuczek, Suzanne G; Disney, Matthew D

    2016-06-01

    Potential RNA targets for chemical probes and therapeutic modalities are pervasive in the transcriptome. Oligonucleotide-based therapeutics are commonly used to target RNA sequence. Small molecules are emerging as a modality to target RNA structures selectively, but their development is still in its infancy. In this work, we compare the activity of oligonucleotides and several classes of small molecules that target the non-coding r(CCUG) repeat expansion (r(CCUG)(exp)) that causes myotonic dystrophy type 2 (DM2), an incurable disease that is the second-most common cause of adult onset muscular dystrophy. Small molecule types investigated include monomers, dimers, and multivalent compounds synthesized on-site by using RNA-templated click chemistry. Oligonucleotides investigated include phosphorothioates that cleave their target and vivo-morpholinos that modulate target RNA activity via binding. We show that compounds assembled on-site that recognize structure have the highest potencies amongst small molecules and are similar in potency to a vivo-morpholino modified oligonucleotide that targets sequence. These studies are likely to impact the design of therapeutic modalities targeting other repeats expansions that cause fragile X syndrome and amyotrophic lateral sclerosis, for example. PMID:27117425

  15. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning.

    PubMed

    Henderson, Ian R; Zhang, Xiaoyu; Lu, Cheng; Johnson, Lianna; Meyers, Blake C; Green, Pamela J; Jacobsen, Steven E

    2006-06-01

    Small RNAs have several important biological functions. MicroRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) regulate mRNA stability and translation, and siRNAs cause post-transcriptional gene silencing of transposons, viruses and transgenes and are important in both the establishment and maintenance of cytosine DNA methylation. Here, we study the role of the four Arabidopsis thaliana DICER-LIKE genes (DCL1-DCL4) in these processes. Sequencing of small RNAs from a dcl2 dcl3 dcl4 triple mutant showed markedly reduced tasiRNA and siRNA production and indicated that DCL1, in addition to its role as the major enzyme for processing miRNAs, has a previously unknown role in the production of small RNAs from endogenous inverted repeats. DCL2, DCL3 and DCL4 showed functional redundancy in siRNA and tasiRNA production and in the establishment and maintenance of DNA methylation. Our studies also suggest that asymmetric DNA methylation can be maintained by pathways that do not require siRNAs. PMID:16699516

  16. Nanocarrier-based co-delivery of small molecules and siRNA/miRNA for treatment of cancer.

    PubMed

    Chitkara, Deepak; Singh, Saurabh; Mittal, Anupama

    2016-04-01

    Aberrant gene expression can trigger several vital molecular events that not only result in carcinogenesis but also cause chemoresistance, metastasis and relapse. Gene-based therapies using siRNA/miRNA have been suggested as new treatment method to improve the current regimen. Although these agents can restore the normal molecular cascade thereby resensitizing the cancer cells, delivering a standard regimen (either subsequently or simultaneously) is necessary to achieve the therapeutic benefit. However, co-delivery using a single carrier could give an additional advantage of similar biodistribution profile of the loaded agents. While much research has been carried out in this field in recent years, challenges involved in designing combination formulations including efficient coloading, stability, appropriate biodistribution and target specificity have hampered their clinical translation. This article highlights current aspects of nano-carriers used for co-delivery of small molecules and genes to treat cancer. PMID:27010986

  17. Small activating RNA binds to the genomic target site in a seed-region-dependent manner

    PubMed Central

    Meng, Xing; Jiang, Qian; Chang, Nannan; Wang, Xiaoxia; Liu, Chujun; Xiong, Jingwei; Cao, Huiqing; Liang, Zicai

    2016-01-01

    RNA activation (RNAa) is the upregulation of gene expression by small activating RNAs (saRNAs). In order to investigate the mechanism by which saRNAs act in RNAa, we used the progesterone receptor (PR) gene as a model, established a panel of effective saRNAs and assessed the involvement of the sense and antisense strands of saRNA in RNAa. All active saRNAs had their antisense strand effectively incorporated into Ago2, whereas such consistency did not occur for the sense strand. Using a distal hotspot for saRNA targeting at 1.6-kb upstream from the PR transcription start site, we further established that gene activation mediated by saRNA depended on the complementarity of the 5′ region of the antisense strand, and that such activity was largely abolished by mutations in this region of the saRNA. We found markedly reduced RNAa effects when we created mutations in the genomic target site of saRNA PR-1611, thus providing evidence that RNAa depends on the integrity of the DNA target. We further demonstrated that this saRNA bound the target site on promoter DNA. These results demonstrated that saRNAs work via an on-site mechanism by binding to target genomic DNA in a seed-region-dependent manner, reminiscent of miRNA-like target recognition. PMID:26873922

  18. A Short Open Reading Frame Encompassing the MicroRNA173 Target Site Plays a Role in trans-Acting Small Interfering RNA Biogenesis.

    PubMed

    Yoshikawa, Manabu; Iki, Taichiro; Numa, Hisataka; Miyashita, Kyoko; Meshi, Tetsuo; Ishikawa, Masayuki

    2016-05-01

    trans-Acting small interfering RNAs (tasiRNAs) participate in the regulation of organ morphogenesis and determination of developmental timing in plants by down-regulating target genes through mRNA cleavage. The production of tasiRNAs is triggered by microRNA173 (miR173) and other specific microRNA-mediated cleavage of 5'-capped and 3'-polyadenylated primary TAS transcripts (pri-TASs). Although pri-TASs are not thought to encode functional proteins, they contain multiple short open reading frames (ORFs). For example, the primary TAS2 transcript (pri-TAS2) contains 11 short ORFs, and the third ORF from the 5' terminus (ORF3) encompasses the miR173 target site. Here, we show that nonsense mutations in ORF3 of pri-TAS2 upstream of the miR173 recognition site suppress tasiRNA accumulation and that ORF3 is translated in vitro. Glycerol gradient centrifugation analysis of Arabidopsis (Arabidopsis thaliana) plant extracts revealed that pri-TAS2 and its miR173-cleaved 5' and 3' fragments are fractionated together in the polysome fractions. These and previous results suggest that the 3' fragment of pri-TAS2, which is a source of tasiRNAs, forms a huge complex containing SGS3, miR173-programmed AGO1 RNA-induced silencing complex, the 5' fragment, and ribosomes. This complex overaccumulated, moderately accumulated, and did not accumulate in rdr6, sde5, and sgs3 mutants, respectively. The sgs3 sde5 and rdr6 sde5 double mutants showed phenotypes similar to those of sgs3 and sde5 single mutants, respectively, with regard to the TAS2-related RNA accumulation, suggesting that the complex is formed in an SGS3-dependent manner, somehow modified and stabilized by SDE5, and becomes competent for RDR6 action. Ribosomes in this complex likely play an important role in this process. PMID:26966170

  19. MicroRNA superfamilies descended from miR390 and their roles in secondary small interfering RNA Biogenesis in Eudicots.

    PubMed

    Xia, Rui; Meyers, Blake C; Liu, Zhongchi; Beers, Eric P; Ye, Songqing; Liu, Zongrang

    2013-05-01

    Trans-acting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway, that of miR173-TAS1/2, produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA (miRNA)-trans-acting small interfering RNA gene (TAS)-pentatricopeptide repeat-containing gene (PPR)-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR phasiRNA production is triggered by different 22-nucleotide miRNAs, including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting or indirect targeting through TAS-like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA-TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families. PMID:23695981

  20. RNA Interference for Wheat Functional Gene Analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference (RNAi) refers to a common mechanism of RNA-based post-transcriptional gene silencing in eukaryotic cells. In model plant species such as Arabidopsis and rice, RNAi has been routinely used to characterize gene function and to engineer novel phenotypes. In polyploid species, this appr...

  1. Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive.

    PubMed

    Childs-Disney, Jessica L; Hoskins, Jason; Rzuczek, Suzanne G; Thornton, Charles A; Disney, Matthew D

    2012-05-18

    RNA is an important drug target, but it is difficult to design or discover small molecules that modulate RNA function. In the present study, we report that rationally designed, modularly assembled small molecules that bind the RNA that causes myotonic dystrophy type 1 (DM1) are potently bioactive in cell culture models. DM1 is caused when an expansion of r(CUG) repeats, or r(CUG)(exp), is present in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. r(CUG)(exp) folds into a hairpin with regularly repeating 5'CUG/3'GUC motifs and sequesters muscleblind-like 1 protein (MBNL1). A variety of defects are associated with DM1, including (i) formation of nuclear foci, (ii) decreased translation of DMPK mRNA due to its nuclear retention, and (iii) pre-mRNA splicing defects due to inactivation of MBNL1, which controls the alternative splicing of various pre-mRNAs. Previously, modularly assembled ligands targeting r(CUG)(exp) were designed using information in an RNA motif-ligand database. These studies showed that a bis-benzimidazole (H) binds the 5'CUG/3'GUC motif in r(CUG)(exp.) Therefore, we designed multivalent ligands to bind simultaneously multiple copies of this motif in r(CUG)(exp). Herein, we report that the designed compounds improve DM1-associated defects including improvement of translational and pre-mRNA splicing defects and the disruption of nuclear foci. These studies may establish a foundation to exploit other RNA targets in genomic sequence. PMID:22332923

  2. Rationally Designed Small Molecules Targeting the RNA That Causes Myotonic Dystrophy Type 1 Are Potently Bioactive

    PubMed Central

    Childs-Disney, Jessica L.; Hoskins, Jason; Rzuczek, Suzanne G.; Thornton, Charles A.; Disney, Matthew D.

    2012-01-01

    RNA is an important drug target, but it is difficult to design or discover small molecules that modulate RNA function. In the present study, we report that rationally designed, modularly assembled small molecules that bind the RNA that causes myotonic dystrophy type 1 (DM1) are potently bioactive in cell culture models. DM1 is caused when an expansion of r(CUG) repeats, or r(CUG)exp, is present in the 3′ untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. r(CUG)exp folds into a hairpin with regularly repeating 5′CUG/3′GUC motifs and sequester muscleblind-like 1 protein (MBNL1). A variety of defects are associated with DM1 including: (i) formation of nuclear foci, (ii) decreased translation of DMPK mRNA due to its nuclear retention, and (iii) pre-mRNA splicing defects due to inactivation of MBNL1, which controls the alternative splicing of various pre-mRNAs. Previously, modularly assembled ligands targeting r(CUG)exp were designed using information in an RNA motif-ligand database. These studies showed that a bis-benzimidazole (H) binds the 5′CUG/3′GUC motif in r(CUG)exp. Therefore, we designed multivalent ligands to bind multiple copies of this motif simultaneously in r(CUG)exp. Herein, we report that the designed compounds improve DM1-associated defects including improvement of translational and pre-mRNA splicing defects and the disruption of nuclear foci. These studies may establish a foundation to exploit other RNA targets in genomic sequence. PMID:22332923

  3. 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

  4. 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

  5. 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

  6. 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

  7. High-Throughput Analysis of RNA Structure and Ribonucleoprotein Assembly

    PubMed Central

    McGinnis, Jennifer L.; Duncan, Caia D. S.; Weeks, Kevin M.

    2016-01-01

    RNA folds to form complex structures vital to many cellular functions. Proteins facilitate RNA folding at both the secondary and tertiary structure levels. An absolute prerequisite for understanding RNA folding and ribonucleoprotein (RNP) assembly reactions is a complete understanding of the RNA structure at each stage of the folding or assembly process. Here we provide a guide for comprehensive and high-throughput analysis of RNA secondary and tertiary structure using SHAPE and hydroxyl radical footprinting. As an example of the strong and sometimes surprising conclusions that can emerge from high-throughput analysis of RNA folding and RNP assembly, we summarize the structure of the bI3 group I intron RNA in four distinct states. Dramatic structural rearrangements occur in both secondary and tertiary structure as the RNA folds from the free state to the active, six-component, RNP complex. As high-throughput and high-resolution approaches are applied broadly to large protein-RNA complexes, other proteins previously viewed as making simple contributions to RNA folding are also likely to be found to exert multifaceted, long-range, cooperative, and non-additive effects on RNA folding. These protein-induced contributions add another level of control, and potential regulatory function, in RNP complexes. PMID:20946765

  8. Mechanism of MicroRNA-Target Interaction: Molecular Dynamics Simulations and Thermodynamics Analysis

    PubMed Central

    Wang, Yonghua; Li, Yan; Ma, Zhi; Yang, Wei; Ai, Chunzhi

    2010-01-01

    MicroRNAs (miRNAs) are endogenously produced ∼21-nt riboregulators that associate with Argonaute (Ago) proteins to direct mRNA cleavage or repress the translation of complementary RNAs. Capturing the molecular mechanisms of miRNA interacting with its target will not only reinforce the understanding of underlying RNA interference but also fuel the design of more effective small-interfering RNA strands. To address this, in the present work the RNA-bound (Ago-miRNA, Ago-miRNA-target) and RNA-free Ago forms were analyzed by performing both molecular dynamics simulations and thermodynamic analysis. Based on the principal component analysis results of the simulation trajectories as well as the correlation analysis in fluctuations of residues, we discover that: 1) three important (PAZ, Mid and PIWI) domains exist in Argonaute which define the global dynamics of the protein; 2) the interdomain correlated movements are so crucial for the interaction of Ago-RNAs that they not only facilitate the relaxation of the interactions between residues surrounding the RNA binding channel but also induce certain conformational changes; and 3) it is just these conformational changes that expand the cavity of the active site and open putative pathways for both the substrate uptake and product release. In addition, by thermodynamic analysis we also discover that for both the guide RNA 5′-end recognition and the facilitated site-specific cleavage of the target, the presence of two metal ions (of Mg2+) plays a predominant role, and this conclusion is consistent with the observed enzyme catalytic cleavage activity in the ternary complex (Ago-miRNA-mRNA). Our results find that it is the set of arginine amino acids concentrated in the nucleotide-binding channel in Ago, instead of the conventionally-deemed seed base-paring, that makes greater contributions in stabilizing the binding of the nucleic acids to Ago. PMID:20686687

  9. 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

  10. Recognition of the small regulatory RNA RydC by the bacterial Hfq protein

    PubMed Central

    Dimastrogiovanni, Daniela; Fröhlich, Kathrin S; Bandyra, Katarzyna J; Bruce, Heather A; Hohensee, Susann; Vogel, Jörg; Luisi, Ben F

    2014-01-01

    Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a ‘seed’ region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at a 3.48 Å resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein–RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target. DOI: http://dx.doi.org/10.7554/eLife.05375.001 PMID:25551292

  11. Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation.

    PubMed

    Zhang, Guoqiang; Estève, Pierre-Olivier; Chin, Hang Gyeong; Terragni, Jolyon; Dai, Nan; Corrêa, Ivan R; Pradhan, Sriharsa

    2015-07-13

    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

  12. Heat shock increases lifetime of a small RNA and induces its accumulation in cells.

    PubMed

    Tatosyan, Karina A; Kramerov, Dmitri A

    2016-08-01

    4.5SH and 4.5SI RNA are two abundant small non-coding RNAs specific for several related rodent families including Muridae. These RNAs have a number of common characteristics such as the short length (about 100nt), transcription by RNA polymerase III, and origin from Short Interspersed Elements (SINEs). However, their stabilities in cells substantially differ: the half-life of 4.5SH RNA is about 20min, while that of 4.5SI RNA is 22h. Here we studied the influence of cell stress such as heat shock or viral infection on these two RNAs. We found that the level of 4.5SI RNA did not change in stressed cells; whereas heat shock increased the abundance of 4.5SH RNA 3.2-10.5 times in different cell lines; and viral infection, 5 times. Due to the significant difference in the turnover rates of these two RNAs, a similar activation of their transcription by heat shock increases the level of the short-lived 4.5SH RNA and has minor effect on the level of the long-lived 4.5SI RNA. In addition, the accumulation of 4.5SH RNA results not only from the induction of its transcription but also from a substantial retardation of its decay. To our knowledge, it is the first example of a short-lived non-coding RNA whose elongated lifetime contributes significantly to its accumulation in stressed cells. PMID:27085482

  13. MicroRNA Superfamilies Descended from miR390 and Their Roles in Secondary Small Interfering RNA Biogenesis in Eudicots[W

    PubMed Central

    Xia, Rui; Meyers, Blake C.; Liu, Zhongchi; Beers, Eric P.; Ye, Songqing; Liu, Zongrang

    2013-01-01

    Trans-acting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway, that of miR173-TAS1/2, produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA (miRNA)-trans-acting small interfering RNA gene (TAS)-pentatricopeptide repeat-containing gene (PPR)-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR phasiRNA production is triggered by different 22-nucleotide miRNAs, including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting or indirect targeting through TAS-like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA–TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families. PMID:23695981

  14. 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

  15. Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection

    PubMed Central

    Qiao, Yongli; Shi, Jinxia; Zhai, Yi; Hou, Yingnan; Ma, Wenbo

    2015-01-01

    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. PMID:25902521

  16. Functional Specialization of the Small Interfering RNA Pathway in Response to Virus Infection

    PubMed Central

    Marques, Joao Trindade; Wang, Ji-Ping; Wang, Xiaohong; de Oliveira, Karla Pollyanna Vieira; Gao, Catherine; Aguiar, Eric Roberto Guimaraes Rocha; Jafari, Nadereh; Carthew, Richard W.

    2013-01-01

    In Drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs-PD). siRNAs are then loaded onto Argonaute-2 (Ago2) by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to siRNAs. Although Dcr-2, R2D2, and Ago2 are essential for innate antiviral defense, the mechanism of virus-derived siRNA (vsiRNA) biogenesis and viral target inhibition remains unclear. Here, we characterize the response mechanism mediated by siRNAs against two different RNA viruses that infect Drosophila. In both cases, we show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and, to a lesser extent, viral transcription. These vsiRNAs seem to preferentially target viral polyadenylated RNA to inhibit viral replication. Loqs-PD is completely dispensable for silencing of the viruses, in contrast to its role in silencing endogenous targets. Biogenesis of vsiRNAs is independent of both Loqs-PD and R2D2. R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression. Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD. This suggests that triggering of the antiviral pathway is not related to viral mode of entry but recognition of intrinsic features of virus RNA. Our results indicate the existence of a vsiRNA pathway that is separate from the endogenous siRNA pathway and is specifically triggered by virus RNA. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response. PMID:24009507

  17. Systematic coarse-grained modeling of complexation between small interfering RNA and polycations

    NASA Astrophysics Data System (ADS)

    Wei, Zonghui; Luijten, Erik

    2015-12-01

    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.

  18. MicroRNA-like small RNAs prediction in the development of Antrodia cinnamomea.

    PubMed

    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

  19. Development of a software tool and criteria evaluation for efficient design of small interfering RNA.

    PubMed

    Chaudhary, Aparna; Srivastava, Sonam; Garg, Sanjeev

    2011-01-01

    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. PMID:21145307

  20. 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

  1. Phylogenetic relationships of the green alga Volvox carteri deduced from small-subunit ribosomal RNA comparisons.

    PubMed

    Rausch, H; Larsen, N; Schmitt, R

    1989-09-01

    The 1788-nucleotide sequence of the small-subunit ribosomal RNA (srRNA) coding region from the chlorophyte Volvox carteri was determined. The secondary structure bears features typical of the universal model of srRNA, including about 40 helices and a division into four domains. Phylogenetic relationships to 17 other eukaryotes, including two other chlorophytes, were explored by comparing srRNA sequences. Similarity values and the inspection of phylogenetic trees derived by distance matrix methods revealed a close relationship between V. carteri and Chlamydomonas reinhardtii. The results are consistent with the view that these Volvocales, and the third green alga, Nanochlorum eucaryotum, are more closely related to higher plants than to any other major eukaryotic group, but constitute a distinct lineage that has long been separated from the line leading to the higher plants. PMID:2506359

  2. 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.

  3. 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

  4. Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

    PubMed

    Mars, Ruben A T; Nicolas, Pierre; Ciccolini, Mariano; Reilman, Ewoud; Reder, Alexander; Schaffer, Marc; Mäder, Ulrike; Völker, Uwe; van Dijl, Jan Maarten; Denham, Emma L

    2015-03-01

    Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs) could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 interaction allows B. subtilis to increase the cell-to-cell variation in AbrB protein levels, despite strong negative autoregulation of the abrB promoter. This behavior is consistent with existing mathematical models of sRNA action, thus suggesting that induction of protein expression noise could be a new general aspect of sRNA regulation. Importantly, we show that the sRNA-induced diversity in AbrB levels generates heterogeneity in growth rates during the exponential growth phase. Based on these findings, we hypothesize that the resulting subpopulations of fast- and slow-growing B. subtilis cells reflect a bet-hedging strategy for enhanced survival of unfavorable conditions. PMID:25790031

  5. 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

  6. A Small RNA-Based Immune System Defends Germ Cells against Mobile Genetic Elements.

    PubMed

    Haase, Astrid D

    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

  7. Evolution of small putative group I introns in the SSU rRNA gene locus of Phialophora species

    PubMed Central

    2011-01-01

    Background Group I introns (specifically subgroup IC1) are common in the nuclear ribosomal RNA genes of fungi. While most range in length from more than 200 to nearly 1800 nucleotides (nt) in length, several small putative (or degenerate) group I introns have been described that are between 56 and 81 nt. Although small, previously we demonstrated that the PaSSU intron in the rRNA small subunit gene of Phialophora americana isolate Wang 1046 is capable of in vitro splicing using a standard group I intron pathway, thus qualifying it as a functional ribozyme. Findings Here, we describe eight short putative group I introns, ranging in length from 63 to 75 nt, in the rRNA small subunit genes of Phialophora isolates, a fungal genus that ranges from saprobic to pathogenic on plants and animals. All contain putative pairing regions P1, P7, and P10, as well as a pairing region formed between the middle of the intron and part of the 3' exon. The other pairing regions common in the core of standard group I introns are absent. However, parts of the 3' exon may aid in the stabilization of these small introns. Although the eight putative group I introns were from at least three species of Phialophora, phylogenetic analysis indicated that the eight are monophyletic. They are also monophyletic with the small introns of two lichen-forming fungi, Porpidia crustulata and Arthonia lapidicola. Conclusions The small putative group I introns in Phialophora have common features that may represent group I introns at their minima. They appear to have a single origin as indicated by their monophyly in phylogenetic analyses. PMID:21781325

  8. Approaches to Validate and Manipulate RNA Targets with Small Molecules in Cells.

    PubMed

    Childs-Disney, Jessica L; Disney, Matthew D

    2016-01-01

    RNA has become an increasingly important target for therapeutic interventions and for chemical probes that dissect and manipulate its cellular function. Emerging targets include human RNAs that have been shown to directly cause cancer, metabolic disorders, and genetic disease. In this review, we describe various routes to obtain bioactive compounds that target RNA, with a particular emphasis on the development of small molecules. We use these cases to describe approaches that are being developed for target validation, which include target-directed cleavage, classic pull-down experiments, and covalent cross-linking. Thus, tools are available to design small molecules to target RNA and to identify the cellular RNAs that are their targets. PMID:26514201

  9. 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

  10. Modification of Small RNAs Associated with Suppression of RNA Silencing by Tobamovirus Replicase Protein▿

    PubMed Central

    Vogler, Hannes; Akbergenov, Rashid; Shivaprasad, Padubidri V.; Dang, Vy; Fasler, Monika; Kwon, Myoung-Ok; Zhanybekova, Saule; Hohn, Thomas; Heinlein, Manfred

    2007-01-01

    Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA. PMID:17634237

  11. 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

  12. 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...

  13. 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

  14. The small regulatory RNA SyR1/PsrR1 controls photosynthetic functions in cyanobacteria.

    PubMed

    Georg, Jens; Dienst, Dennis; Schürgers, Nils; Wallner, Thomas; Kopp, Dominik; Stazic, Damir; Kuchmina, Ekaterina; Klähn, Stephan; Lokstein, Heiko; Hess, Wolfgang R; Wilde, Annegret

    2014-09-01

    Little is known so far about RNA regulators of photosynthesis in plants, algae, or cyanobacteria. The small RNA PsrR1 (formerly SyR1) has been discovered in Synechocystis sp PCC 6803 and appears to be widely conserved within the cyanobacterial phylum. Expression of PsrR1 is induced shortly after a shift from moderate to high-light conditions. Artificial overexpression of PsrR1 led to a bleaching phenotype under moderate light growth conditions. Advanced computational target prediction suggested that several photosynthesis-related mRNAs could be controlled by PsrR1, a finding supported by the results of transcriptome profiling experiments upon pulsed overexpression of this small RNA in Synechocystis sp PCC 6803. We confirmed the interaction between PsrR1 and the ribosome binding regions of the psaL, psaJ, chlN, and cpcA mRNAs by mutational analysis in a heterologous reporter system. Focusing on psaL as a specific target, we show that the psaL mRNA is processed by RNase E only in the presence of PsrR1. Furthermore, we provide evidence for a posttranscriptional regulation of psaL by PsrR1 in the wild type at various environmental conditions and analyzed the consequences of PsrR1-based regulation on photosystem I. In summary, computational and experimental data consistently establish the small RNA PsrR1 as a regulatory factor controlling photosynthetic functions. PMID:25248550

  15. Analysis of hepatitis C virus RNA dimerization and core–RNA interactions

    PubMed Central

    Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

    2006-01-01

    The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus. PMID:16707664

  16. 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

  17. Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing

    PubMed Central

    Hong, Yoonki; Bang, Chi Young; Lee, Jae Cheol; Oh, Yeon-Mok

    2016-01-01

    Background Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. Methods RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. Results RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. Conclusion In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required. PMID:27066085

  18. 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.

  19. 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

  20. miARma-Seq: a comprehensive tool for miRNA, mRNA and circRNA analysis.

    PubMed

    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

  1. 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

  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. miEAA: microRNA enrichment analysis and annotation.

    PubMed

    Backes, Christina; Khaleeq, Qurratulain T; Meese, Eckart; Keller, Andreas

    2016-07-01

    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. 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

  5. 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. PMID:26256260

  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. 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

  8. 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

  9. Identification of More Feasible MicroRNA-mRNA Interactions within Multiple Cancers Using Principal Component Analysis Based Unsupervised Feature Extraction.

    PubMed

    Taguchi, Y-H

    2016-01-01

    MicroRNA(miRNA)-mRNA interactions are important for understanding many biological processes, including development, differentiation and disease progression, but their identification is highly context-dependent. When computationally derived from sequence information alone, the identification should be verified by integrated analyses of mRNA and miRNA expression. The drawback of this strategy is the vast number of identified interactions, which prevents an experimental or detailed investigation of each pair. In this paper, we overcome this difficulty by the recently proposed principal component analysis (PCA)-based unsupervised feature extraction (FE), which reduces the number of identified miRNA-mRNA interactions that properly discriminate between patients and healthy controls without losing biological feasibility. The approach is applied to six cancers: hepatocellular carcinoma, non-small cell lung cancer, esophageal squamous cell carcinoma, prostate cancer, colorectal/colon cancer and breast cancer. In PCA-based unsupervised FE, the significance does not depend on the number of samples (as in the standard case) but on the number of features, which approximates the number of miRNAs/mRNAs. To our knowledge, we have newly identified miRNA-mRNA interactions in multiple cancers based on a single common (universal) criterion. Moreover, the number of identified interactions was sufficiently small to be sequentially curated by literature searches. PMID:27171078

  10. 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.

  11. Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA

    NASA Astrophysics Data System (ADS)

    Woodrow, Kim A.; Cu, Yen; Booth, Carmen J.; Saucier-Sawyer, Jennifer K.; Wood, Monica J.; Mark Saltzman, W.

    2009-06-01

    Vaginal instillation of small-interfering RNA (siRNA) using liposomes has led to silencing of endogenous genes in the genital tract and protection against challenge from infectious disease. Although siRNA lipoplexes are easily formulated, several of the most effective transfection agents available commercially may be toxic to the mucosal epithelia and none are able to provide controlled or sustained release. Here, we demonstrate an alternative approach using nanoparticles composed entirely of FDA-approved materials. To render these materials effective for gene silencing, we developed novel approaches to load them with high amounts of siRNA. A single dose of siRNA-loaded nanoparticles to the mouse female reproductive tract caused efficient and sustained gene silencing. Knockdown of gene expression was observed proximal (in the vaginal lumen) and distal (in the uterine horns) to the site of topical delivery. In addition, nanoparticles penetrated deep into the epithelial tissue. This is the first report demonstrating that biodegradable polymer nanoparticles are effective delivery vehicles for siRNA to the vaginal mucosa.

  12. 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

  13. Aberrant 3' oligoadenylation of spliceosomal U6 small nuclear RNA in poikiloderma with neutropenia.

    PubMed

    Hilcenko, Christine; Simpson, Paul J; Finch, Andrew J; Bowler, Frank R; Churcher, Mark J; Jin, Li; Packman, Len C; Shlien, Adam; Campbell, Peter; Kirwan, Michael; Dokal, Inderjeet; Warren, Alan J

    2013-02-01

    The recessive disorder poikiloderma with neutropenia (PN) is caused by mutations in the C16orf57 gene that encodes the highly conserved USB1 protein. Here, we present the 1.1 Å resolution crystal structure of human USB1, defining it as a member of the LigT-like superfamily of 2H phosphoesterases. We show that human USB1 is a distributive 3'-5' exoribonuclease that posttranscriptionally removes uridine and adenosine nucleosides from the 3' end of spliceosomal U6 small nuclear RNA (snRNA), directly catalyzing terminal 2', 3' cyclic phosphate formation. USB1 measures the appropriate length of the U6 oligo(U) tail by reading the position of a key adenine nucleotide (A102) and pausing 5 uridine residues downstream.We show that the 3' ends of U6 snRNA in PN patient lymphoblasts are elongated and unexpectedly carry nontemplated 3' oligo(A) tails that are characteristic of nuclear RNA surveillance targets. Thus, our study reveals a novel quality control pathway in which posttranscriptional 3'-end processing by USB1 protects U6 snRNA from targeting and destruction by the nuclear exosome. Our data implicate aberrant oligoadenylation of U6 snRNA in the pathogenesis of the leukemia predisposition disorder PN. PMID:23190533

  14. 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.

  15. 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.

  16. Small Players Ruling the Hard Game: siRNA in Bone Regeneration.

    PubMed

    Ghadakzadeh, Saber; Mekhail, Mina; Aoude, Ahmed; Hamdy, Reggie; Tabrizian, Maryam

    2016-03-01

    Silencing gene expression through a sequence-specific manner can be achieved by small interfering RNAs (siRNAs). The discovery of this process has opened the doors to the development of siRNA therapeutics. Although several preclinical and clinical studies have shown great promise in the treatment of neurological disorders, cancers, dominant disorders, and viral infections with siRNA, siRNA therapy is still gaining ground in musculoskeletal tissue repair and bone regeneration. Here we present a comprehensive review of the literature to summarize different siRNA delivery strategies utilized to enhance bone regeneration. With advancement in understanding the targetable biological pathways involved in bone regeneration and also the rapid progress in siRNA technologies, application of siRNA for bone regeneration has great therapeutic potential. High rates of musculoskeletal injuries and diseases, and their inevitable consequences, impose a huge financial burden on individuals and healthcare systems worldwide. © 2016 American Society for Bone and Mineral Research. PMID:26890411

  17. 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

  18. 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

  19. SURVIV for survival analysis of mRNA isoform variation.

    PubMed

    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

  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. 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. Analysis of PKR Structure by Small-Angle Scattering

    SciTech Connect

    VanOudenhove, Jennifer; Anderson, Eric; Krueger, Susan; Cole, James L.

    2009-04-27

    Protein kinase R (PKR) is a key component of the interferon antiviral defense pathway. Upon binding double-stranded RNA, PKR undergoes autophosphorylation reactions that activate the kinase. PKR contains an N-terminal double-stranded RNA binding domain, which consists of two tandem double-stranded RNA binding motifs, and a C-terminal kinase domain. We have used small-angle X-ray scattering and small-angle neutron scattering to define the conformation of latent PKR in solution. Guinier analysis indicates a radius of gyration of about 35 {angstrom}. The p(r) distance distribution function exhibits a peak near 30 {angstrom}, with a broad shoulder extending to longer distances. Good fits to the scattering data require models that incorporate multiple compact and extended conformations of the two interdomain linker regions. Thus, PKR belongs to the growing family of proteins that contain intrinsically unstructured regions. We propose that the flexible linkers may allow PKR to productively dimerize upon interaction with RNA activators that have diverse structures.

  3. 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

  4. 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

  5. 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

  6. Small nucleolar RNA clusters in trypanosomatid Leptomonas collosoma. Genome organization, expression studies, and the potential role of sequences present upstream from the first repeated cluster.

    PubMed

    Liang, Xue-hai; Ochaion, Avivit; Xu, Yu-xin; Liu, Qing; Michaeli, Shulamit

    2004-02-13

    Trypanosomatid small nucleolar RNA (snoRNA) genes are clustered in the genome. snoRNAs are transcribed polycistronically and processed into mature RNAs. In this study, we characterized four snoRNA clusters in Leptomonas collosoma. All of the clusters analyzed carry both C/D and H/ACA RNAs. The H/ACA RNAs are composed of a single hairpin, a structure typical to trypanosome and archaea guide RNAs. Using deletion and mutational analysis of a tagged C/D snoRNA situated within the snoRNA cluster, we identified 10-nucleotide flanking sequences that are essential for processing snoRNA from its precursor. Chromosome walk was performed on a snoRNA cluster, and a sequence of 700 bp was identified between the first repeat and the upstream open reading frame. Cloning of this sequence in an episome vector enhanced the expression of a tagged snoRNA gene in an orientation-dependent manner. However, continuous transcript spanning of this region was detected in steady-state RNA, suggesting that snoRNA transcription also originates from an upstream-long polycistronic transcriptional unit. The 700-bp fragment may therefore represent an example of many more elements to be discovered that enhance transcription along the chromosome, especially when transcription from the upstream gene is reduced or when enhanced transcription is needed. PMID:14645367

  7. sPARTA: a parallelized pipeline for integrated analysis of plant miRNA and cleaved mRNA data sets, including new miRNA target-identification software

    PubMed Central

    Kakrana, Atul; Hammond, Reza; Patel, Parth; Nakano, Mayumi; Meyers, Blake C.

    2014-01-01

    Parallel analysis of RNA ends (PARE) is a technique utilizing high-throughput sequencing to profile uncapped, mRNA cleavage or decay products on a genome-wide basis. Tools currently available to validate miRNA targets using PARE data employ only annotated genes, whereas important targets may be found in unannotated genomic regions. To handle such cases and to scale to the growing availability of PARE data and genomes, we developed a new tool, ‘sPARTA’ (small RNA-PARE target analyzer) that utilizes a built-in, plant-focused target prediction module (aka ‘miRferno’). sPARTA not only exhibits an unprecedented gain in speed but also it shows greater predictive power by validating more targets, compared to a popular alternative. In addition, the novel ‘seed-free’ mode, optimized to find targets irrespective of complementarity in the seed-region, identifies novel intergenic targets. To fully capitalize on the novelty and strengths of sPARTA, we developed a web resource, ‘comPARE’, for plant miRNA target analysis; this facilitates the systematic identification and analysis of miRNA-target interactions across multiple species, integrated with visualization tools. This collation of high-throughput small RNA and PARE datasets from different genomes further facilitates re-evaluation of existing miRNA annotations, resulting in a ‘cleaner’ set of microRNAs. PMID:25120269

  8. Improvement of SMN2 Pre-mRNA Processing Mediated by Exon-Specific U1 Small Nuclear RNA

    PubMed Central

    Dal Mas, Andrea; Rogalska, Malgorzata Ewa; Bussani, Erica; Pagani, Franco

    2015-01-01

    Exon-specific U1 snRNAs (ExSpe U1s) are modified U1 snRNAs that interact with intronic sequences downstream of the 5′ splice site (ss) by complementarity. This process restores exon skipping caused by different types of mutation. We have investigated the molecular mechanism and activity of these molecules in spinal muscular atrophy (SMA), a genetic neuromuscular disease where a silent exonic transition on the survival motor neuron 2 (SMN2) leads to exon 7 (E7) skipping. By using different cellular models, we show that a single chromosome-integrated copy of ExSpe U1 induced a significant correction of endogenous SMN2 E7 splicing and resulted in the restoration of the corresponding SMN protein levels. Interestingly, the analysis of pre-mRNA transcript abundance and decay showed that ExSpe U1s promote E7 inclusion and stabilizes the SMN pre-mRNA intermediate. This selective effect on pre-mRNA stability resulted in higher levels of SMN mRNAs in comparison with those after treatment with an antisense oligonucleotide (AON) that targets corresponding intronic sequences. In mice harboring the SMN2 transgene, AAV-mediated delivery of ExSpe U1 increased E7 inclusion in brain, heart, liver, kidney, and skeletal muscle. The positive effect of ExSpe U1s on SMN pre-mRNA processing highlights their therapeutic potential in SMA and in other pathologies caused by exon-skipping mutations. PMID:25557785

  9. 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.

  10. 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

  11. mRNA and microRNA expression profiles of radioresistant NCI-H520 non-small cell lung cancer cells.

    PubMed

    Guo, Wei; Xie, Li; Zhao, Long; Zhao, Yuehuan

    2015-08-01

    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 x 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

  12. The small RNA Aar in Acinetobacter baylyi: a putative regulator of amino acid metabolism.

    PubMed

    Schilling, Dominik; Findeiss, Sven; Richter, Andreas S; Taylor, Jennifer A; Gerischer, Ulrike

    2010-09-01

    Small non-coding RNAs (sRNAs) are key players in prokaryotic metabolic circuits, allowing the cell to adapt to changing environmental conditions. Regulatory interference by sRNAs in cellular metabolism is often facilitated by the Sm-like protein Hfq. A search for novel sRNAs in A. baylyi intergenic regions was performed by a biocomputational screening. One candidate, Aar, encoded between trpS and sucD showed Hfq dependency in Northern blot analysis. Aar was expressed strongly during stationary growth phase in minimal medium; in contrast, in complex medium, strongest expression was in the exponential growth phase. Whereas over-expression of Aar in trans did not affect bacterial growth, seven mRNA targets predicted by two in silico approaches were upregulated in stationary growth phase. All seven mRNAs are involved in A. baylyi amino acid metabolism. A putative binding site for Lrp, the global regulator of branched-chain amino acids in E. coli, was observed within the aar gene. Both facts imply an Aar participation in amino acid metabolism. PMID:20559624

  13. A Host Small GTP-binding Protein ARL8 Plays Crucial Roles in Tobamovirus RNA Replication

    PubMed Central

    Nishikiori, Masaki; Mori, Masashi; Dohi, Koji; Okamura, Hideyasu; Katoh, Etsuko; Naito, Satoshi; Meshi, Tetsuo; Ishikawa, Masayuki

    2011-01-01

    Tomato mosaic virus (ToMV), like other eukaryotic positive-strand RNA viruses, replicates its genomic RNA in replication complexes formed on intracellular membranes. Previous studies showed that a host seven-pass transmembrane protein TOM1 is necessary for efficient ToMV multiplication. Here, we show that a small GTP-binding protein ARL8, along with TOM1, is co-purified with a FLAG epitope-tagged ToMV 180K replication protein from solubilized membranes of ToMV-infected tobacco (Nicotiana tabacum) cells. When solubilized membranes of ToMV-infected tobacco cells that expressed FLAG-tagged ARL8 were subjected to immunopurification with anti-FLAG antibody, ToMV 130K and 180K replication proteins and TOM1 were co-purified and the purified fraction showed RNA-dependent RNA polymerase activity that transcribed ToMV RNA. From uninfected cells, TOM1 co-purified with FLAG-tagged ARL8 less efficiently, suggesting that a complex containing ToMV replication proteins, TOM1, and ARL8 are formed on membranes in infected cells. In Arabidopsis thaliana, ARL8 consists of four family members. Simultaneous mutations in two specific ARL8 genes completely inhibited tobamovirus multiplication. In an in vitro ToMV RNA translation-replication system, the lack of either TOM1 or ARL8 proteins inhibited the production of replicative-form RNA, indicating that TOM1 and ARL8 are required for efficient negative-strand RNA synthesis. When ToMV 130K protein was co-expressed with TOM1 and ARL8 in yeast, RNA 5′-capping activity was detected in the membrane fraction. This activity was undetectable or very weak when the 130K protein was expressed alone or with either TOM1 or ARL8. Taken together, these results suggest that TOM1 and ARL8 are components of ToMV RNA replication complexes and play crucial roles in a process toward activation of the replication proteins' RNA synthesizing and capping functions. PMID:22174675

  14. A host small GTP-binding protein ARL8 plays crucial roles in tobamovirus RNA replication.

    PubMed

    Nishikiori, Masaki; Mori, Masashi; Dohi, Koji; Okamura, Hideyasu; Katoh, Etsuko; Naito, Satoshi; Meshi, Tetsuo; Ishikawa, Masayuki

    2011-12-01

    Tomato mosaic virus (ToMV), like other eukaryotic positive-strand RNA viruses, replicates its genomic RNA in replication complexes formed on intracellular membranes. Previous studies showed that a host seven-pass transmembrane protein TOM1 is necessary for efficient ToMV multiplication. Here, we show that a small GTP-binding protein ARL8, along with TOM1, is co-purified with a FLAG epitope-tagged ToMV 180K replication protein from solubilized membranes of ToMV-infected tobacco (Nicotiana tabacum) cells. When solubilized membranes of ToMV-infected tobacco cells that expressed FLAG-tagged ARL8 were subjected to immunopurification with anti-FLAG antibody, ToMV 130K and 180K replication proteins and TOM1 were co-purified and the purified fraction showed RNA-dependent RNA polymerase activity that transcribed ToMV RNA. From uninfected cells, TOM1 co-purified with FLAG-tagged ARL8 less efficiently, suggesting that a complex containing ToMV replication proteins, TOM1, and ARL8 are formed on membranes in infected cells. In Arabidopsis thaliana, ARL8 consists of four family members. Simultaneous mutations in two specific ARL8 genes completely inhibited tobamovirus multiplication. In an in vitro ToMV RNA translation-replication system, the lack of either TOM1 or ARL8 proteins inhibited the production of replicative-form RNA, indicating that TOM1 and ARL8 are required for efficient negative-strand RNA synthesis. When ToMV 130K protein was co-expressed with TOM1 and ARL8 in yeast, RNA 5'-capping activity was detected in the membrane fraction. This activity was undetectable or very weak when the 130K protein was expressed alone or with either TOM1 or ARL8. Taken together, these results suggest that TOM1 and ARL8 are components of ToMV RNA replication complexes and play crucial roles in a process toward activation of the replication proteins' RNA synthesizing and capping functions. PMID:22174675

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase.

    PubMed

    Xu, Hong-Tao; Colby-Germinario, Susan P; Hassounah, Said; Quashie, Peter K; Han, Yingshan; Oliveira, Maureen; Stranix, Brent R; Wainberg, Mark A

    2016-01-01

    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

  2. Deep sequencing reveals unique small RNA repertoire that is regulated during head regeneration in Hydra magnipapillata.

    PubMed

    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

  3. 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

  4. 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.

  5. New insight into RNase P RNA structure from comparative analysis of the archaeal RNA.

    PubMed Central

    Harris, J K; Haas, E S; Williams, D; Frank, D N; Brown, J W

    2001-01-01

    A detailed comparative analysis of archaeal RNase P RNA structure and a comparison of the resulting structural information with that of the bacterial RNA reveals that the archaeal RNase P RNAs are strikingly similar to those of Bacteria. The differences between the secondary structure models of archaeal and bacterial RNase P RNA have largely disappeared, and even variation in the sequence and structure of the RNAs are similar in extent and type. The structure of the cruciform (P7-11) has been reevaluated on the basis of a total of 321 bacterial and archaeal sequences, leading to a model for the structure of this region of the RNA that includes an extension to P11 that consistently organizes the cruciform and adjacent highly-conserved sequences. PMID:11233979

  6. Complex Intra-Operonic Dynamics Mediated by a Small RNA in Streptomyces coelicolor

    PubMed Central

    Hindra; Moody, Matthew J.; Jones, Stephanie E.; Elliot, Marie A.

    2014-01-01

    Streptomyces are predominantly soil-dwelling bacteria that are best known for their multicellular life cycle and their prodigious metabolic capabilities. They are also renowned for their regulatory capacity and flexibility, with each species encoding >60 sigma factors, a multitude of transcription factors, and an increasing number of small regulatory RNAs. Here, we describe our characterization of a conserved small RNA (sRNA), scr4677. In the model species Streptomyces coelicolor, this sRNA is located in the intergenic region separating SCO4677 (an anti-sigma factor-encoding gene) and SCO4676 (a putative regulatory protein-encoding gene), close to the SCO4676 translation start site in an antisense orientation. There appears to be considerable genetic interplay between these different gene products, with wild type expression of scr4677 requiring function of the anti-sigma factor SCO4677, and scr4677 in turn influencing the abundance of SCO4676-associated transcripts. The scr4677-mediated effects were independent of RNase III (a double stranded RNA-specific nuclease), with RNase III having an unexpectedly positive influence on the level of SCO4676-associated transcripts. We have shown that both SCO4676 and SCO4677 affect the production of the blue-pigmented antibiotic actinorhodin under specific growth conditions, and that this activity appears to be independent of scr4677. PMID:24465751

  7. 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

  8. Inhibition of Non-ATG Translational Events in Cells via Covalent Small Molecules Targeting RNA

    PubMed Central

    Yang, Wang-Yong; Wilson, Henry D.; Velagapudi, Sai Pradeep

    2016-01-01

    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. PMID:25825793

  9. 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. PMID:25825793

  10. Identification of Small Molecule Inhibitors of Pre-mRNA Splicing*

    PubMed Central

    Pawellek, Andrea; McElroy, Stuart; Samatov, Timur; Mitchell, Lee; Woodland, Andrew; Ryder, Ursula; Gray, David; Lührmann, Reinhard; Lamond, Angus I.

    2014-01-01

    Eukaryotic pre-mRNA splicing is an essential step in gene expression for all genes that contain introns. In contrast to transcription and translation, few well characterized chemical inhibitors are available with which to dissect the splicing process, particularly in cells. Therefore, the identification of specific small molecules that either inhibit or modify pre-mRNA splicing would be valuable for research and potentially also for therapeutic applications. We have screened a highly curated library of 71,504 drug-like small molecules using a high throughput in vitro splicing assay. This identified 10 new compounds that both inhibit pre-mRNA splicing in vitro and modify splicing of endogenous pre-mRNA in cells. One of these splicing modulators, DDD00107587 (termed “madrasin,” i.e. 2-((7methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one RNAsplicing inhibitor), was studied in more detail. Madrasin interferes with the early stages of spliceosome assembly and stalls spliceosome assembly at the A complex. Madrasin is cytotoxic at higher concentrations, although at lower concentrations it induces cell cycle arrest, promotes a specific reorganization of subnuclear protein localization, and modulates splicing of multiple pre-mRNAs in both HeLa and HEK293 cells. PMID:25281741

  11. Delivery of antiviral small interfering RNA with gold nanoparticles inhibits dengue virus infection in vitro

    PubMed Central

    Paul, Amber M.; Shi, Yongliang; Acharya, Dhiraj; Douglas, Jessica R.; Cooley, Amanda; Anderson, John F.; Huang, Faqing

    2014-01-01

    Dengue virus (DENV) infection in humans can cause flu-like illness, life-threatening haemorrhagic fever or even death. There is no specific anti-DENV therapeutic or approved vaccine currently available, partially due to the possibility of antibody-dependent enhancement reaction. Small interfering RNAs (siRNAs) that target specific viral genes are considered a promising therapeutic alternative against DENV infection. However, in vivo, siRNAs are vulnerable to degradation by serum nucleases and rapid renal excretion due to their small size and anionic character. To enhance siRNA delivery and stability, we complexed anti-DENV siRNAs with biocompatible gold nanoparticles (AuNPs) and tested them in vitro. We found that cationic AuNP–siRNA complexes could enter Vero cells and significantly reduce DENV serotype 2 (DENV-2) replication and infectious virion release under both pre- and post-infection conditions. In addition, RNase-treated AuNP–siRNA complexes could still inhibit DENV-2 replication, suggesting that AuNPs maintained siRNA stability. Collectively, these results demonstrated that AuNPs were able to efficiently deliver siRNAs and control infection in vitro, indicating a novel anti-DENV strategy. PMID:24828333

  12. 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.

  13. 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

  14. Small interfering RNA suppression of polyamine analog-induced spermidine/spermine n1-acetyltransferase.

    PubMed

    Chen, Ying; Kramer, Debora L; Jell, Jason; Vujcic, Slavoljub; Porter, Carl W

    2003-11-01

    N1,N11-diethylnorspermine (DENSPM) is a polyamine analog that down-regulates polyamine biosynthesis and potently upregulates the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT). In certain cells, such as SKMEL-28 human melanoma cells, induction of SSAT is associated with rapid apoptosis. In this study, we used small interfering RNA (siRNA) to examine the role of SSAT induction in mediating polyamine pool depletion and apoptosis. siRNA duplexes were designed to target three independent sites in the SSAT mRNA coding region (siSSAT). When transfected under nontoxic conditions, two of the duplexes selectively reduced basal SSAT mRNA in HEK-293 cells by >80% and prevented DENSPM-induced SSAT mRNA by 95% in SK-MEL-28 cells. Treatment of SK-MEL-28 cells with 10 muM DENSPM in the presence of 83 nM siSSAT selectively prevented the 1400-fold induction of SSAT activity by approximately 90% and, in turn, prevented analog depletion of spermine (Spm) pools by approximately 35%. siSSAT also prevented DENSPM-induced cytochrome c release and caspase-3 cleavage at 36 h and apoptosis at 48 h as measured by annexin V staining. Overall, the data directly link analog induction of SSAT to Spm pool depletion and to caspase-dependent apoptosis in DENSPM-treated SK-MEL-28 cells. This represents the first use of siRNA technology directed toward a polyamine gene and the first unequivocal demonstration that SSAT induction initiates events leading to polyamine analog-induced apoptosis. PMID:14573765

  15. 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

  16. 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

  17. Safe and effective delivery of small interfering RNA with polymer- and liposomes-based complexes.

    PubMed

    Kodama, Yukinobu; Harauchi, Satoe; Kawanabe, Saki; Ichikawa, Nobuhiro; Nakagawa, Hiroo; Muro, Takahiro; Higuchi, Norihide; Nakamura, Tadahiro; Kitahara, Takashi; Sasaki, Hitoshi

    2013-01-01

    We developed binary and ternary complexes based on polymers and liposomes for safe and effective delivery of small interfering RNA (siRNA). Anti-luciferase siRNA was used as a model of nucleic acid medicine. The binary complexes of siRNA were prepared with cationic polymers and cationic liposomes such as polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly-L-arginine (PLA), trimethyl[2,3-(dioleoxy)-propyl]ammonium chloride (DOTMA), and cholesteryl 3β-N-(dimetylaminnoethyl)carbamate hydrochloride (DC-Chol). The ternary complexes were constructed by the addition of γ-polyglutamic acid (γ-PGA) to the binary complexes. The complexes were approximately 54-153 nm in particle size. The binary complexes showed a cationic surface charge although an anionic surface charge was observed in the ternary complexes. The polymer-based complexes did not show a silencing effect in the mouse colon carcinoma cell line expressing luciferase regularly (Colon26/Luc cells). The binary complexes based on liposomes and their ternary complexes coated by γ-PGA showed a significant silencing effect. The binary complexes showed significant cytotoxicity although the ternary complexes coated by γ-PGA did not show significant cytotoxicity. The ternary complexes coated by γ-PGA suppressed luciferase activity in the tumor after their direct injection into the tumors of mice bearing Colon26/Luc cells. Thus, we have newly identified safe and efficient ternary complexes of siRNA for clinical use. PMID:23727920

  18. 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

  19. 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.

  20. Structure and Genome Organization of Cherry Virus A (Capillovirus, Betaflexiviridae) from China Using Small RNA Sequencing

    PubMed Central

    Wang, Jiawei; Zhai, Ying; Liu, Weizhen; Dhingra, Amit

    2016-01-01

    Cherry virus A (CVA) (Capillovirus, Betaflexiviridae) is widely present in cherry-growing areas. We obtained the complete genome of a CVA isolate (CVA-TA) using small RNA deep sequencing, followed by overlapping reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The newly identified 5′-untranslated region (5′-UTR) from CVA-TA may form additional hairpin and loop structures to stabilize the CVA genome. PMID:27174277

  1. Structure and Genome Organization of Cherry Virus A (Capillovirus, Betaflexiviridae) from China Using Small RNA Sequencing.

    PubMed

    Wang, Jiawei; Zhai, Ying; Liu, Weizhen; Dhingra, Amit; Pappu, Hanu R; Liu, Qingzhong

    2016-01-01

    Cherry virus A (CVA) (Capillovirus, Betaflexiviridae) is widely present in cherry-growing areas. We obtained the complete genome of a CVA isolate (CVA-TA) using small RNA deep sequencing, followed by overlapping reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The newly identified 5'-untranslated region (5'-UTR) from CVA-TA may form additional hairpin and loop structures to stabilize the CVA genome. PMID:27174277

  2. 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

  3. Analysis options for high-throughput sequencing in miRNA expression profiling

    PubMed Central

    2014-01-01

    Background Recently high-throughput sequencing (HTS) using next generation sequencing techniques became useful in digital gene expression profiling. Our study introduces analysis options for HTS data based on mapping to miRBase or counting and grouping of identical sequence reads. Those approaches allow a hypothesis free detection of miRNA differential expression. Methods We compare our results to microarray and qPCR data from one set of RNA samples. We use Illumina platforms for microarray analysis and miRNA sequencing of 20 samples from benign follicular thyroid adenoma and malignant follicular thyroid carcinoma. Furthermore, we use three strategies for HTS data analysis to evaluate miRNA biomarkers for malignant versus benign follicular thyroid tumors. Results High correlation of qPCR and HTS data was observed for the proposed analysis methods. However, qPCR is limited in the differential detection of miRNA isoforms. Moreover, we illustrate a much broader dynamic range of HTS compared to microarrays for small RNA studies. Finally, our data confirm hsa-miR-197-3p, hsa-miR-221-3p, hsa-miR-222-3p and both hsa-miR-144-3p and hsa-miR-144-5p as potential follicular thyroid cancer biomarkers. Conclusions Compared to microarrays HTS provides a global profile of miRNA expression with higher specificity and in more detail. Summarizing of HTS reads as isoform groups (analysis pipeline B) or according to functional criteria (seed analysis pipeline C), which better correlates to results of qPCR are promising new options for HTS analysis. Finally, data opens future miRNA research perspectives for HTS and indicates that qPCR might be limited in validating HTS data in detail. PMID:24625073

  4. 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…

  5. Massive-Scale RNA-Seq Analysis of Non Ribosomal Transcriptome in Human Trisomy 21

    PubMed Central

    Costa, Valerio; Angelini, Claudia; D'Apice, Luciana; Mutarelli, Margherita; Casamassimi, Amelia; Sommese, Linda; Gallo, Maria Assunta; Aprile, Marianna; Esposito, Roberta; Leone, Luigi; Donizetti, Aldo; Crispi, Stefania; Rienzo, Monica; Sarubbi, Berardo; Calabrò, Raffaele; Picardi, Marco; Salvatore, Paola; Infante, Teresa; De Berardinis, Piergiuseppe; Napoli, Claudio; Ciccodicola, Alfredo

    2011-01-01

    Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high-throughput RNA sequencing have revealed the complexity of transcriptomes, most of them rely on polyA enrichment protocols, able to detect only a small fraction of total RNA content. On the opposite end, massive-scale RNA sequencing on rRNA-depleted samples allows the survey of the complete set of coding and non-coding RNA species, now emerging as novel contributors to pathogenic mechanisms. Hence, in this work we analysed for the first time the complete transcriptome of human trisomic endothelial progenitor cells to an unprecedented level of resolution and sensitivity by RNA-sequencing. Our analysis allowed us to detect differential expression of even low expressed genes crucial for the pathogenesis, to disclose novel regions of active transcription outside yet annotated loci, and to investigate a plethora of non-polyadenilated long as well as short non coding RNAs. Novel splice isoforms for a large subset of crucial genes, and novel extended untranslated regions for known genes—possibly novel miRNA targets or regulatory sites for gene transcription—were also identified in this study. Coupling the rRNA depletion of samples, followed by high-throughput RNA-sequencing, to the easy availability of these cells renders this approach very feasible for transcriptome studies, offering the possibility of investigating in-depth blood-related pathological features of Down syndrome, as well as other genetic disorders. PMID:21533138

  6. 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

  7. The CRISPR system: small RNA-guided defense in bacteria and archaea

    PubMed Central

    Karginov, Fedor V.; Hannon, Gregory J.

    2010-01-01

    All cellular systems evolve ways to combat predators and genomic parasites. In bacteria and archaea, numerous resistance mechanisms have developed against phage. Our understanding of this defensive repertoire has recently been expanded to include the CRISPR system of Clustered, Regularly Interspaced Short Palindromic Repeats. In this remarkable pathway, short sequence tags from invading genetic elements are actively incorporated into the host's CRISPR locus, to be transcribed and processed into a set of small RNAs that guide the destruction of foreign genetic material. Here, we review the inner workings of this adaptable and heritable immune system and draw comparisons to small RNA-guided defense mechanisms in eukaryotic cells. PMID:20129051

  8. Small RNA profile in moso bamboo root and leaf obtained by high definition adapters.

    PubMed

    Xu, Ping; Mohorianu, Irina; 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

  9. 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

  10. The p122 Subunit of Tobacco Mosaic Virus Replicase Is a Potent Silencing Suppressor and Compromises both Small Interfering RNA- and MicroRNA-Mediated Pathways▿

    PubMed Central

    Csorba, Tibor; Bovi, Aurelie; Dalmay, Tamás; Burgyán, József

    2007-01-01

    One of the functions of RNA silencing in plants is to defend against molecular parasites, such as viruses, retrotransposons, and transgenes. Plant viruses are inducers, as well as targets, of RNA silencing-based antiviral defense. Replication intermediates or folded viral RNAs activate RNA silencing, generating small interfering RNAs (siRNAs), which are the key players in the antiviral response. Viruses are able to counteract RNA silencing by expressing silencing-suppressor proteins. It has been shown that many of the identified silencing-suppressor proteins bind long double-stranded RNA or siRNAs and thereby prevent assembly of the silencing effector complexes. In this study, we show that the 122-kDa replicase subunit (p122) of crucifer-infecting Tobacco mosaic virus (cr-TMV) is a potent silencing-suppressor protein. We found that the p122 protein preferentially binds to double-stranded 21-nucleotide (nt) siRNA and microRNA (miRNA) intermediates with 2-nt 3′ overhangs inhibiting the incorporation of siRNA and miRNA into silencing-related complexes (e.g., RNA-induced silencing complex [RISC]) both in vitro and in planta but cannot interfere with previously programmed RISCs. In addition, our results also suggest that the virus infection and/or sequestration of the siRNA and miRNA molecules by p122 enhances miRNA accumulation despite preventing its methylation. However, the p122 silencing suppressor does not prevent the methylation of certain miRNAs in hst-15 mutants, in which the nuclear export of miRNAs is compromised. PMID:17715232

  11. The p122 subunit of Tobacco Mosaic Virus replicase is a potent silencing suppressor and compromises both small interfering RNA- and microRNA-mediated pathways.

    PubMed

    Csorba, Tibor; Bovi, Aurelie; Dalmay, Tamás; Burgyán, József

    2007-11-01

    One of the functions of RNA silencing in plants is to defend against molecular parasites, such as viruses, retrotransposons, and transgenes. Plant viruses are inducers, as well as targets, of RNA silencing-based antiviral defense. Replication intermediates or folded viral RNAs activate RNA silencing, generating small interfering RNAs (siRNAs), which are the key players in the antiviral response. Viruses are able to counteract RNA silencing by expressing silencing-suppressor proteins. It has been shown that many of the identified silencing-suppressor proteins bind long double-stranded RNA or siRNAs and thereby prevent assembly of the silencing effector complexes. In this study, we show that the 122-kDa replicase subunit (p122) of crucifer-infecting Tobacco mosaic virus (cr-TMV) is a potent silencing-suppressor protein. We found that the p122 protein preferentially binds to double-stranded 21-nucleotide (nt) siRNA and microRNA (miRNA) intermediates with 2-nt 3' overhangs inhibiting the incorporation of siRNA and miRNA into silencing-related complexes (e.g., RNA-induced silencing complex [RISC]) both in vitro and in planta but cannot interfere with previously programmed RISCs. In addition, our results also suggest that the virus infection and/or sequestration of the siRNA and miRNA molecules by p122 enhances miRNA accumulation despite preventing its methylation. However, the p122 silencing suppressor does not prevent the methylation of certain miRNAs in hst-15 mutants, in which the nuclear export of miRNAs is compromised. PMID:17715232

  12. REH2C Helicase and GRBC Subcomplexes May Base Pair through mRNA and Small Guide RNA in Kinetoplastid Editosomes.

    PubMed

    Kumar, Vikas; Madina, Bhaskara R; Gulati, Shelly; Vashisht, Ajay A; Kanyumbu, Chiedza; Pieters, Brittany; Shakir, Afzal; Wohlschlegel, James A; Read, Laurie K; Mooers, Blaine H M; Cruz-Reyes, Jorge

    2016-03-11

    Mitochondrial mRNAs in Trypanosoma brucei undergo extensive insertion and deletion of uridylates that are catalyzed by the RNA editing core complex (RECC) and directed by hundreds of small guide RNAs (gRNAs) that base pair with mRNA. RECC is largely RNA-free, and accessory mitochondrial RNA-binding complex 1 (MRB1) variants serve as scaffolds for the assembly of mRNA-gRNA hybrids and RECC. However, the molecular steps that create higher-order holoenzymes ("editosomes") are unknown. Previously, we identified an RNA editing helicase 2-associated subcomplex (REH2C) and showed that REH2 binds RNA. Here we showed that REH2C is an mRNA-associated ribonucleoprotein (mRNP) subcomplex with editing substrates, intermediates, and products. We isolated this mRNP from mitochondria lacking gRNA-bound RNP (gRNP) subcomplexes and identified REH2-associated cofactors 1 and 2 ((H2)F1 and (H2)F2). (H2)F1 is an octa-zinc finger protein required for mRNP-gRNP docking, pre-mRNA and RECC loading, and RNP formation with a short synthetic RNA duplex. REH2 and other eukaryotic DEAH/RHA-type helicases share a conserved regulatory C-terminal domain cluster that includes an oligonucleotide-binding fold. Recombinant REH2 and (H2)F1 constructs associate in a purified complex in vitro. We propose a model of stepwise editosome assembly that entails controlled docking of mRNP and gRNP modules via specific base pairing between their respective mRNA and gRNA cargo and regulatory REH2 and (H2)F1 subunits of the novel mRNP that may control specificity checkpoints in the editing pathway. PMID:26769962

  13. RADIA: RNA and DNA Integrated Analysis for Somatic Mutation Detection

    PubMed Central

    Radenbaugh, Amie J.; Ma, Singer; Ewing, Adam; Stuart, Joshua M.; Collisson, Eric A.; Zhu, Jingchun; Haussler, David

    2014-01-01

    The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual’s DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84%) and very high precision (98% and 99%) for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA. PMID:25405470

  14. RADIA: RNA and DNA integrated analysis for somatic mutation detection.

    PubMed

    Radenbaugh, Amie J; Ma, Singer; Ewing, Adam; Stuart, Joshua M; Collisson, Eric A; Zhu, Jingchun; Haussler, David

    2014-01-01

    The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84%) and very high precision (98% and 99%) for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA. PMID:25405470

  15. 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

  16. MicroRNA-dependent regulation of transcription in non-small cell lung cancer.

    PubMed

    Molina-Pinelo, Sonia; Gutiérrez, Gabriel; Pastor, Maria Dolores; Hergueta, Marta; Moreno-Bueno, Gema; García-Carbonero, Rocío; Nogal, Ana; Suárez, Rocío; Salinas, Ana; Pozo-Rodríguez, Francisco; Lopez-Rios, Fernando; Agulló-Ortuño, Maria Teresa; Ferrer, Irene; Perpiñá, Asunción; Palacios, José; Carnero, Amancio; Paz-Ares, Luis

    2014-01-01

    Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies. PMID:24625834