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Sample records for abundant small rnas

  1. Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs

    PubMed Central

    Cole, Christian; Sobala, Andrew; Lu, Cheng; Thatcher, Shawn R.; Bowman, Andrew; Brown, John W.S.; Green, Pamela J.; Barton, Geoffrey J.; Hutvagner, Gyorgy

    2009-01-01

    Deep sequencing technologies such as Illumina, SOLiD, and 454 platforms have become very powerful tools in discovering and quantifying small RNAs in diverse organisms. Sequencing small RNA fractions always identifies RNAs derived from abundant RNA species such as rRNAs, tRNAs, snRNA, and snoRNA, and they are widely considered to be random degradation products. We carried out bioinformatic analysis of deep sequenced HeLa RNA and after quality filtering, identified highly abundant small RNA fragments, derived from mature tRNAs that are likely produced by specific processing rather than from random degradation. Moreover, we showed that the processing of small RNAs derived from tRNAGln is dependent on Dicer in vivo and that Dicer cleaves the tRNA in vitro. PMID:19850906

  2. Tailored enrichment strategy detects low abundant small noncoding RNAs in HIV-1 infected cells

    PubMed Central

    2012-01-01

    Background The various classes of small noncoding RNAs (sncRNAs) are important regulators of gene expression across divergent types of organisms. While a rapidly increasing number of sncRNAs has been identified over recent years, the isolation of sncRNAs of low abundance remains challenging. Virally encoded sncRNAs, particularly those of RNA viruses, can be expressed at very low levels. This is best illustrated by HIV-1 where virus encoded sncRNAs represent approximately 0.1-1.0% of all sncRNAs in HIV-1 infected cells or were found to be undetected. Thus, we applied a novel, sequence targeted enrichment strategy to capture HIV-1 derived sncRNAs in HIV-1 infected primary CD4+ T-lymphocytes and macrophages that allows a greater than 100-fold enrichment of low abundant sncRNAs. Results Eight hundred and ninety-two individual HIV-1 sncRNAs were cloned and sequenced from nine different sncRNA libraries derived from five independent experiments. These clones represent up to 90% of all sncRNA clones in the generated libraries. Two hundred and sixteen HIV-1 sncRNAs were distinguishable as unique clones. They are spread throughout the HIV-1 genome, however, forming certain clusters, and almost 10% show an antisense orientation. The length of HIV-1 sncRNAs varies between 16 and 89 nucleotides with an unexpected peak at 31 to 50 nucleotides, thus, longer than cellular microRNAs or short-interfering RNAs (siRNAs). Exemplary HIV-1 sncRNAs were also generated in cells infected with different primary HIV-1 isolates and can inhibit HIV-1 replication. Conclusions HIV-1 infected cells generate virally encoded sncRNAs, which might play a role in the HIV-1 life cycle. Furthermore, the enormous capacity to enrich low abundance sncRNAs in a sequence specific manner highly recommends our selection strategy for any type of investigation where origin or target sequences of the sought-after sncRNAs are known. PMID:22458358

  3. Small RNAs in spermatogenesis.

    PubMed

    Yadav, Ram Prakash; Kotaja, Noora

    2014-01-25

    Spermatogenesis is characterized by meiotic divisions and major morphological changes to produce spermatozoa that are capable of independent movement and fertilization of an egg. Male germ cell differentiation is governed by orchestrated, phase-specific gene expression patterns that are tightly controlled at transcriptional and post-transcriptional level. Post-transcriptional regulation of protein-coding mRNAs becomes prominent during the late steps of spermatogenesis when the compacting sperm nucleus becomes transcriptionally inhibited. Small non-coding RNAs are important regulators of gene expression that mainly function post-transcriptionally to control the properties of their target mRNAs. Male germ cells express several classes of small RNAs, including Dicer-dependent microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), as well as Dicer-independent piwi-interacting RNAs (piRNAs). Increasing evidence supports the essential role of small RNA-mediated RNA regulation in normal spermatogenesis and male fertility.

  4. Abundant pseudogenes for small nuclear RNAs are dispersed in the human genome.

    PubMed Central

    Denison, R A; Van Arsdell, S W; Bernstein, L B; Weiner, A M

    1981-01-01

    We have cloned and partially characterized 24 loci from the human genome which are complementary to U1, U2, or U3, the three major species of small nuclear RNA (snRNA) in HeLa cells. When compared to the known U1 (human) and U2 (rat) snRNA sequences, the DNA sequences we report here for the complementary regions from two of the clones, U1.11 and U2.7, reveal the presence of truncated and divergent gene copies. Furthermore, most if not all of the 24 cloned loci contain gene copies that are significantly divergent from the homologous HeLa snRNA species because DNA from every recombinant phage except U1.7 and U1.15 proved unable to form snRNA.DNA hybrids which protect full-length HeLa snRNA from ild digestion with ribonuclease T1. Hence, we refer to these loci as snRNA pseudogenes. In both clones U1.11 and U2.7, an element of the dominant middle repetitive DNA sequence family in the human genome, the Alu family, is located upstream from the snRNA pseudogene and in the same orientation. Alu elements in the same location and orientation relative to bona fide genes have previously been found in the human beta-globin gene cluster [Duncan, C. H., Biro, P. A., Choudary, P. V., Elder, J. T., Wang, R. C., Forget, G. B., deRiel, J. K. & Weissman, S. M. (1979) Proc. Natl. Acad. Sci. USA 76, 5095-5099]. We discuss the significance of these findings in relation to the nature of snRNA multigene families and other reported examples of pseudogenes. Images PMID:6165010

  5. DASHR: database of small human noncoding RNAs

    PubMed Central

    Leung, Yuk Yee; Kuksa, Pavel P.; Amlie-Wolf, Alexandre; Valladares, Otto; Ungar, Lyle H.; Kannan, Sampath; Gregory, Brian D.; Wang, Li-San

    2016-01-01

    Small non-coding RNAs (sncRNAs) are highly abundant RNAs, typically <100 nucleotides long, that act as key regulators of diverse cellular processes. Although thousands of sncRNA genes are known to exist in the human genome, no single database provides searchable, unified annotation, and expression information for full sncRNA transcripts and mature RNA products derived from these larger RNAs. Here, we present the Database of small human noncoding RNAs (DASHR). DASHR contains the most comprehensive information to date on human sncRNA genes and mature sncRNA products. DASHR provides a simple user interface for researchers to view sequence and secondary structure, compare expression levels, and evidence of specific processing across all sncRNA genes and mature sncRNA products in various human tissues. DASHR annotation and expression data covers all major classes of sncRNAs including microRNAs (miRNAs), Piwi-interacting (piRNAs), small nuclear, nucleolar, cytoplasmic (sn-, sno-, scRNAs, respectively), transfer (tRNAs), and ribosomal RNAs (rRNAs). Currently, DASHR (v1.0) integrates 187 smRNA high-throughput sequencing (smRNA-seq) datasets with over 2.5 billion reads and annotation data from multiple public sources. DASHR contains annotations for ∼48 000 human sncRNA genes and mature sncRNA products, 82% of which are expressed in one or more of the curated tissues. DASHR is available at http://lisanwanglab.org/DASHR. PMID:26553799

  6. Competition between small RNAs: a quantitative view.

    PubMed

    Loinger, Adiel; Shemla, Yael; Simon, Itamar; Margalit, Hanah; Biham, Ofer

    2012-04-18

    Two major classes of small regulatory RNAs--small interfering RNAs (siRNAs) and microRNA (miRNAs)--are involved in a common RNA interference processing pathway. Small RNAs within each of these families were found to compete for limiting amounts of shared components, required for their biogenesis and processing. Association with Argonaute (Ago), the catalytic component of the RNA silencing complex, was suggested as the central mechanistic point in RNA interference machinery competition. Aiming to better understand the competition between small RNAs in the cell, we present a mathematical model and characterize a range of specific cell and experimental parameters affecting the competition. We apply the model to competition between miRNAs and study the change in the expression level of their target genes under a variety of conditions. We show quantitatively that the amount of Ago and miRNAs in the cell are dominant factors contributing greatly to the competition. Interestingly, we observe what to our knowledge is a novel type of competition that takes place when Ago is abundant, by which miRNAs with shared targets compete over them. Furthermore, we use the model to examine different interaction mechanisms that might operate in establishing the miRNA-Ago complexes, mainly those related to their stability and recycling. Our model provides a mathematical framework for future studies of competition effects in regulation mechanisms involving small RNAs.

  7. Small nuclear RNAs in the ciliate Tetrahymena.

    PubMed Central

    Pedersen, N; Hellung-Larsen, P; Engberg, J

    1985-01-01

    We have isolated and partially characterized a family of small nuclear RNAs (snRNAs) from three different species of the protozoan Tetrahymena. We find six distinct snRNAs ranging in size from 100 to 250 nucleotides. The two largest snRNAs, as well as an abundant, heterogenous group of smaller snRNAs are found in the nucleolar RNA fraction. None of the snRNAs are transcription products of the ribosomal RNA gene or its flanking regions, as shown by hybridization tests. The snRNAs are metabolically stable as determined by pulse/chase experiments and several of them contain a number of modified nuclotides. The snRNAs from Tetrahymena all have slightly different sizes from mammalian snRNAs. The cap structure of the snRNAs from Tetrahymena differs from that of the snRNAs from mammalian cells, but has not yet been fully characterized. The relative amount of snRNAs to total RNA is less in Tetrahymena (greater than 0.1%) than in mammalian cells (2%). Images PMID:2409533

  8. Small regulatory RNAs in Archaea.

    PubMed

    Babski, Julia; Maier, Lisa-Katharina; Heyer, Ruth; Jaschinski, Katharina; Prasse, Daniela; Jäger, Dominik; Randau, Lennart; Schmitz, Ruth A; Marchfelder, Anita; Soppa, Jörg

    2014-01-01

    Small regulatory RNAs (sRNAs) are universally distributed in all three domains of life, Archaea, Bacteria, and Eukaryotes. In bacteria, sRNAs typically function by binding near the translation start site of their target mRNAs and thereby inhibit or activate translation. In eukaryotes, miRNAs and siRNAs typically bind to the 3'-untranslated region (3'-UTR) of their target mRNAs and influence translation efficiency and/or mRNA stability. In archaea, sRNAs have been identified in all species investigated using bioinformatic approaches, RNomics, and RNA-Seq. Their size can vary significantly between less than 50 to more than 500 nucleotides. Differential expression of sRNA genes has been studied using northern blot analysis, microarrays, and RNA-Seq. In addition, biological functions have been unraveled by genetic approaches, i.e., by characterization of designed mutants. As in bacteria, it was revealed that archaeal sRNAs are involved in many biological processes, including metabolic regulation, adaptation to extreme conditions, stress responses, and even in regulation of morphology and cellular behavior. Recently, the first target mRNAs were identified in archaea, including one sRNA that binds to the 5'-region of two mRNAs in Methanosarcina mazei Gö1 and a few sRNAs that bind to 3'-UTRs in Sulfolobus solfataricus, three Pyrobaculum species, and Haloferax volcanii, indicating that archaeal sRNAs appear to be able to target both the 5'-UTR or the 3'-UTRs of their respective target mRNAs. In addition, archaea contain tRNA-derived fragments (tRFs), and one tRF has been identified as a major ribosome-binding sRNA in H. volcanii, which downregulates translation in response to stress. Besides regulatory sRNAs, archaea contain further classes of sRNAs, e.g., CRISPR RNAs (crRNAs) and snoRNAs.

  9. Abundant primary piRNAs, endo-siRNAs, and microRNAs in a Drosophila ovary cell line

    PubMed Central

    Lau, Nelson C.; Robine, Nicolas; Martin, Raquel; Chung, Wei-Jen; Niki, Yuzo; Berezikov, Eugene; Lai, Eric C.

    2009-01-01

    Piwi proteins, a subclass of Argonaute-family proteins, carry ∼24–30-nt Piwi-interacting RNAs (piRNAs) that mediate gonadal defense against transposable elements (TEs). We analyzed the Drosophila ovary somatic sheet (OSS) cell line and found that it expresses miRNAs, endogenous small interfering RNAs (endo-siRNAs), and piRNAs in abundance. In contrast to intact gonads, which contain mixtures of germline and somatic cell types that express different Piwi-class proteins, OSS cells are a homogenous somatic cell population that expresses only PIWI and primary piRNAs. Detailed examination of its TE-derived piRNAs and endo-siRNAs revealed aspects of TE defense that do not rely upon ping-pong amplification. In particular, we provide evidence that a subset of piRNA master clusters, including flamenco, are specifically expressed in OSS and ovarian follicle cells. These data indicate that the restriction of certain TEs in somatic gonadal cells is largely mediated by a primary piRNA pathway. PMID:19541914

  10. Small silencing RNAs: an expanding universe

    PubMed Central

    Ghildiyal, Megha; Zamore, Phillip D.

    2009-01-01

    Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNA classes have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, modes of target regulation and in the biological pathways they regulate. There is a growing realization that, despite their differences, these distinct small RNA pathways are interconnected and that small RNA pathways compete and collaborate as they regulate genes and protect the genome from external and internal threats. PMID:19148191

  11. Small silencing RNAs: an expanding universe.

    PubMed

    Ghildiyal, Megha; Zamore, Phillip D

    2009-02-01

    Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNA classes have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, their modes of target regulation and in the biological pathways they regulate. There is a growing realization that, despite their differences, these distinct small RNA pathways are interconnected, and that small RNA pathways compete and collaborate as they regulate genes and protect the genome from external and internal threats.

  12. Small RNAs of Sequoia sempervirens during rejuvenation and phase change.

    PubMed

    Chen, Y-T; Shen, C-H; Lin, W-D; Chu, H-A; Huang, B-L; Kuo, C-I; Yeh, K-W; Huang, L-C; Chang, I-F

    2013-01-01

    In this work, the population of small RNAs (sRNAs) was studied in the gymnosperm Sequoia sempervirens during phase changes, specifically in the juvenile, adult and rejuvenated plants obtained in vitro. The potential target genes of Sequoia sRNAs were predicted through bioinformatics. Rejuvenation is a pivotal process in woody plants that enables them to regain their growth potential, which results in the recovery of physiologic and molecular characteristics that were lost when the juveniles mature into adult plants. The results from the five repeated graftings of juvenile, adult and rejuvenated plants in vitro showed that sRNAs could be classified into structural RNAs (Group I), small interfering RNAs (Group II), annotated microRNAs (Group III, and unannotated sRNAs (Group IV). The results indicate that only 573 among 15,485,415 sRNAs (Groups III and IV) had significantly different expression patterns associated with rejuvenation and phase change. A total of 215 sRNAs exhibited up-regulated expression patterns in adult shoots, and 358 sRNAs were down-regulated. Expression profiling and prediction of possible target genes of these unique small RNAs indicate possible functions in the control of photosynthetic efficiency and rooting competence abundance during plant rejuvenation. Moreover, the increase in SsmiR156 and decrease in SsmiR172 during plant rejuvenation suggested that these two microRNAs extensively affect phase transition.

  13. The expanding world of small RNAs in plants

    PubMed Central

    Borges, Filipe; Martienssen, Robert A.

    2016-01-01

    Plant genomes produce a variety of small RNAs that function in distinct, yet overlapping, genetic and epigenetic silencing pathways. However, the abundance and diversity of small RNA classes varies in different plant species, suggesting co-evolution between environmental adaptations and gene silencing mechanisms. Small RNA biogenesis in plants is well understood, but we are just beginning to uncover their intricate regulation and activity. Here, we discuss the biogenesis of plant small RNAs, such as microRNAs, secondary small-interfering RNAs and heterochromatic small-interfering RNAs, and their diverse cellular and developmental functions, including reproductive transitions, genomic imprinting and paramutation. We also discuss the diversification of small RNA-directed silencing pathways through the expansion of RNA-dependent RNA polymerases, Dicer and Argonaute proteins. PMID:26530390

  14. Endogenous small RNAs in grain: semi-quantification and sequence homology to human and animal genes.

    PubMed

    Ivashuta, Sergey I; Petrick, Jay S; Heisel, Sara E; Zhang, Yuanji; Guo, Liang; Reynolds, Tracey L; Rice, James F; Allen, Edwards; Roberts, James K

    2009-02-01

    Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effector molecules of RNA interference (RNAi), a highly conserved RNA-based gene suppression mechanism in plants, mammals and other eukaryotes. Endogenous RNAi-based gene suppression has been harnessed naturally and through conventional breeding to achieve desired plant phenotypes. The present study demonstrates that endogenous small RNAs, such as siRNAs and miRNAs, are abundant in soybean seeds, corn kernels, and rice grain, plant tissues that are traditionally used for food and feed. Numerous endogenous plant small RNAs were found to have perfect complementarity to human genes as well as those of other mammals. The abundance of endogenous small RNA molecules in grain from safely consumed food and feed crops such as soybean, corn, and rice and the homology of a number of these dietary small RNAs to human and animal genomes and transcriptomes establishes a history of safe consumption for dietary small RNAs.

  15. Novel microRNAs and microsatellite-like small RNAs in sexual and apomictic Boechera species.

    PubMed

    Amiteye, Samuel; Corral, Jose M; Vogel, Heiko; Kuhlmann, Markus; Mette, Michael F; Sharbel, Timothy F

    2013-01-01

    Apomixis refers to plant asexual reproduction through seeds that give rise to progeny which are genotypically identical to the maternal parent. It has evolved from many different sexual taxa although the underlying genetic factors remain unknown. Previous analyses of the over-representation of transcription factors, in a comparison of microdissected ovules from apomictic and sexual Boechera, showed that many transcription factor mRNAs possessed microRNA (miRNAs) binding sites, thus pointing to miRNAs as potentially important factors that may be involved in the regulatory switch from sexual to apomictic reproduction. A microarray-based approach was used to identify (1) 673 microsatellitelike small RNAs (misRNAs) containing predominantly 2-7 repeats of (GAA)n/(CUU)n, (GCA)n/(CGU)n, (GGA)n/(CCU)n, (GGU)n/(CCA)n and (UGA)n/(ACU)n, and (2) 166 more typical non-repeat small RNAs. In total, 87 small RNAs were found to be located in cDNAs that could fold into stem-loop structures and thus represent miRNA molecules. In addition, 109 Boechera small RNAs including both misRNAs and non-repeat small RNAs, showed significant homology to 407 Arabidopsis thaliana small RNAs including the A. thaliana pollen-specific ath-miR5021. This indicates that only a fraction of the identified small RNAs are unique to Boechera. Ten small RNAs were validated using a Northern blot assay on flower and leaf tissues, eight of which showed flower-specific expression with varying abundance. The potential binding sites of many of the misRNAs and non-repeat small RNAs occur predominantly in exonic regions. This feature coupled with their flower-specific pattern of expression is suggestive of their probable role in post-transcriptional gene regulation. We propose that quantitative variation for misRNA target binding (and hence post-transcriptional gene regulation) could arise via microsatellite length polymorphisms occurring either in misRNA precursors or in their gene targets.

  16. MicroRNAs: Small RNAs with Big Effects

    PubMed Central

    Anglicheau, Dany; Muthukumar, Thangamani; Suthanthiran, Manikkam

    2010-01-01

    MicroRNAs are evolutionarily conserved, small (~20–25 nucleotides), single-stranded molecules that suppress the expression of protein-coding genes by translational repression, messenger RNA degradation, or both. More than 700 microRNAs have been identified in the human genome. Amazingly, a single miRNA can regulate the expression of hundreds of mRNAs/proteins within a cell. The small RNAs are fast emerging as master regulators of innate and adaptive immunity, and very likely to play a pivotal role in transplantation. The clinical application of RNA sequencing (“next – generation sequencing”) should facilitate transcriptome profiling at an unprecedented resolution. We provide an overview of microRNA biology and their hypothesized roles in transplantation. PMID:20574417

  17. Abundances of microRNAs in human cells can be estimated as a function of the abundances of YRHB and RHHK tetranucleotides in these microRNAs as an ill-posed inverse problem solution.

    PubMed

    Ponomarenko, Mikhail P; Suslov, Valentin V; Ponomarenko, Petr M; Gunbin, Konstantin V; Stepanenko, Irina L; Vishnevsky, Oleg V; Kolchanov, Nikolay A

    2013-01-01

    Mature microRNAs (miRNAs) are small endogenous non-coding RNAs 18-25 nt in length. They program the RNA Induced Silencing Complex (RISC) to make it inhibit either messenger RNAs or promoter DNAs. We have found that the mean abundance of miRNAs in Arabidopsis is correlated with the abundance of DRYD tetranucleotides near the 3'-end and the abundance of WRHB tetranucleotides in the center of the miRNA sequence. Based on this correlation, we have estimated miRNA abundances in seven organs of this plant, namely: inflorescences, stems, siliques, seedlings, roots, cauline, and rosette leaves. We have also found that the mean affinity of miRNAs for two proteins in the Argonaute family (Ago2 and Ago3) in man is correlated with the abundance of YRHB tetranucleotides near the 3'-end and that the preference of miRNAs for Ago2 is correlated with the abundance of RHHK tetranucleotides in the center of the miRNA sequence. This allowed us to obtain statistically significant estimates of miRNA abundances in human embryonic kidney cells, HEK293T. These findings in relation to two taxonomically distant entities (man and Arabidopsis) fit one another like pieces of a jigsaw puzzle, which allowed us to heuristically generalize them and state that the miRNA abundance in the human brain may be determined by the abundance of YRHB and RHHK tetranucleotides in these miRNAs.

  18. Small RNAs in the genus Clostridium.

    PubMed

    Chen, Yili; Indurthi, Dinesh C; Jones, Shawn W; Papoutsakis, Eleftherios T

    2011-01-25

    The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny.

  19. Designing small multiple-target artificial RNAs

    PubMed Central

    De Guire, Vincent; Caron, Maxime; Scott, Nicolas; Ménard, Catherine; Gaumont-Leclerc, Marie-France; Chartrand, Pascal; Major, François; Ferbeyre, Gerardo

    2010-01-01

    MicroRNAs (miRNAs) are naturally occurring small RNAs that regulate the expression of several genes. MiRNAs’ targeting rules are based on sequence complementarity between their mature products and targeted genes’ mRNAs. Based on our present understanding of those rules, we developed an algorithm to design artificial miRNAs to target simultaneously a set of predetermined genes. To validate in silico our algorithm, we tested different sets of genes known to be targeted by a single miRNA. The algorithm finds the seed of the corresponding miRNA among the solutions, which also include the seeds of new artificial miRNA sequences potentially capable of targeting these genes as well. We also validated the functionality of some artificial miRNAs designed to target simultaneously members of the E2F family. These artificial miRNAs reproduced the effects of E2Fs inhibition in both normal human fibroblasts and prostate cancer cells where they inhibited cell proliferation and induced cellular senescence. We conclude that the current miRNA targeting rules based on the seed sequence work to design multiple-target artificial miRNAs. This approach may find applications in both research and therapeutics. PMID:20453028

  20. Bombyx small RNAs: genomic defense system against transposons in the silkworm, Bombyx mori.

    PubMed

    Kawaoka, Shinpei; Hayashi, Nobumitsu; Katsuma, Susumu; Kishino, Hirohisa; Kohara, Yuji; Mita, Kazuei; Shimada, Toru

    2008-12-01

    Selfish genetic elements called transposons can insert themselves at new locations in host genomes to modify gene structure and alter gene expression. Expansion of transposons can occur when novel transposition events are transmitted to subsequent generations after germline hopping. Therefore, organisms seem likely to have evolved defense mechanisms to silence transposons in the germline. Recently, small RNAs interacting with Piwi proteins (piwi-interacting RNAs: piRNAs) have been demonstrated to be involved in genomic defense mechanism against transposons. Here, we show that piRNA-like small RNAs are present abundantly in the Bombyx ovary. We cloned 38,493 kinds of Bombyx small RNA from the ovary and performed functional characterization. Bombyx small RNAs showed a unimodal length distribution with a peak at 28nt and a strong bias for U at the 5' end. We found that 12,869 kinds of Bombyx small RNAs were associated with transposons or repetitive sequences. We classified them as repeat-associated small interfering RNAs (rasiRNAs), a subclass of piRNAs. Notably, antisense rasiRNAs have a strong bias toward U at 5' ends; in contrast, sense rasiRNAs have a strong bias toward A at nucleotide position 10, indicating that the piRNA amplification loop proposed in Drosophila is evolutionarily conserved in Bombyx. These results suggest that Bombyx small RNAs regulate transposon activity.

  1. Small noncoding RNAs: biogenesis, function, and emerging significance in toxicology.

    PubMed

    Choudhuri, Supratim

    2010-01-01

    In recent years, the discovery of small ncRNAs (noncoding RNAs) has unveiled a slew of powerful riboregulators of gene expression. So far, many different types of small ncRNAs have been described. Of these, miRNAs (microRNAs), siRNAs (small interfering RNAs), and piRNAs (Piwi-interacting RNAs) have been studied in more detail. A significant fraction of genes in most organisms and tissues is targets of these small ncRNAs. Because these tiny RNAs are turning out to be important regulators of gene and genome expression, their aberrant expression profiles are expected to be associated with cellular dysfunction and disease. In fact, an ever-increasing number of studies have implicated miRNAs and siRNAs in human health and disease ranging from metabolic disorders to diseases of various organ systems as well as various forms of cancer. Nevertheless, despite the flurry of research on these small ncRNAs, many aspects of their biology still remain to be understood. The following discussion focuses on some aspects of the biogenesis and function of small ncRNAs with major emphasis on miRNAs since these are the most widespread endogenous small ncRNAs that have been called "micromanagers" of gene expression. Their emerging significance in toxicology is also discussed.

  2. Structure and Gene-Silencing Mechanisms of Small Noncoding RNAs

    NASA Astrophysics Data System (ADS)

    Chu, Chia-Ying; Rana, Tariq M.

    Small (19-31-nucleotides) noncoding RNAs were identified in the past 10 years for their distinct function in gene silencing. The best known gene-silencing phenomenon, RNA interference (RNAi), is triggered in a sequence-specific manner by endogenously produced or exogenously introduced small doubled-stranded RNAs. As knowledge of the structure and function of the RNAi machinery has expanded, this phenomenon has become a powerful tool for biochemical research; it has enormous potential for therapeutics. This chapter summarizes significant aspects of three major classes of small noncoding, regulatory RNAs: small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). Here, we focus on the biogenesis of these small RNAs, their structural features and coupled effectors as well as the mechanisms of each small regulatory RNA pathway which reveal fascinating ways by which gene silencing is controlled and fine-tuned at an epigenetic level.

  3. High throughput sequencing of small RNAs in Potato Leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding the role of small RNAs (sRNAs) in gene regulation, function and development is a rapidly evolving field. sRNAs result from transcript degradation and to regulatory micro RNA (miRNA) and small inhibitory RNA (siRNA) classes involved in gene regulation. The sRNAs from potato leaves were...

  4. Artificial microRNAs and synthetic trans-acting small interfering RNAs interfere with viroid infection.

    PubMed

    Carbonell, Alberto; Daròs, José-Antonio

    2016-12-27

    Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of artificial small RNAs (sRNAs) engineered to silence endogenous transcripts as well as viral RNAs in plants. Here, we explore the possibility of using amiRNAs and syn-tasiRNAs to specifically interfere with infections by viroids, small (250-400 nt) non-coding circular RNAs with compact secondary structure infecting a wide range of plant species. The combined use of recent high-throughput methods for artificial sRNA construct generation and of the Potato spindle tuber viroid (PSTVd)/Nicotiana benthamiana pathosystem allowed for the simple and time-effective screening of multiple artificial sRNAs targeting sites distributed along PSTVd RNAs of (+) or (-) polarity. The majority of amiRNAs were highly active in agroinfiltrated leaves when co-expressed with an infectious PSTVd transcript, as were syn-tasiRNAs derived from a construct including the five most effective amiRNA sequences. A comparative analysis showed that the effects of the most effective amiRNA and of the syn-tasiRNAs were similar in agroinfiltrated leaves, as well as in upper non-agroinfiltrated leaves where PSTVd accumulation was significantly delayed. These results suggest that amiRNAs and syn-tasiRNAs can be used effectively to control viroid infections in plants. This article is protected by copyright. All rights reserved.

  5. Rpl13a small nucleolar RNAs regulate systemic glucose metabolism

    PubMed Central

    Lee, Jiyeon; Harris, Alexis N.; Holley, Christopher L.; Mahadevan, Jana; Pyles, Kelly D.; Lavagnino, Zeno; Scherrer, David E.; Fujiwara, Hideji; Sidhu, Rohini; Zhang, Jessie; Huang, Stanley Ching-Cheng; Piston, David W.; Remedi, Maria S.; Urano, Fumihiko; Ory, Daniel S.

    2016-01-01

    Small nucleolar RNAs (snoRNAs) are non-coding RNAs that form ribonucleoproteins to guide covalent modifications of ribosomal and small nuclear RNAs in the nucleus. Recent studies have also uncovered additional non-canonical roles for snoRNAs. However, the physiological contributions of these small RNAs are largely unknown. Here, we selectively deleted four snoRNAs encoded within the introns of the ribosomal protein L13a (Rpl13a) locus in a mouse model. Loss of Rpl13a snoRNAs altered mitochondrial metabolism and lowered reactive oxygen species tone, leading to increased glucose-stimulated insulin secretion from pancreatic islets and enhanced systemic glucose tolerance. Islets from mice lacking Rpl13a snoRNAs demonstrated blunted oxidative stress responses. Furthermore, these mice were protected against diabetogenic stimuli that cause oxidative stress damage to islets. Our study illuminates a previously unrecognized role for snoRNAs in metabolic regulation. PMID:27820699

  6. The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

    PubMed Central

    Pantano, Lorena; Jodar, Meritxell; Bak, Mads; Ballescà, Josep Lluís; Tommerup, Niels; Oliva, Rafael; Vavouri, Tanya

    2015-01-01

    At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline. PMID:25904136

  7. Small RNAs, DNA methylation and transposable elements in wheat

    PubMed Central

    2010-01-01

    Background More than 80% of the wheat genome is composed of transposable elements (TEs). Since active TEs can move to different locations and potentially impose a significant mutational load, their expression is suppressed in the genome via small non-coding RNAs (sRNAs). sRNAs guide silencing of TEs at the transcriptional (mainly 24-nt sRNAs) and post-transcriptional (mainly 21-nt sRNAs) levels. In this study, we report the distribution of these two types of sRNAs among the different classes of wheat TEs, the regions targeted within the TEs, and their impact on the methylation patterns of the targeted regions. Results We constructed an sRNA library from hexaploid wheat and developed a database that included our library and three other publicly available sRNA libraries from wheat. For five completely-sequenced wheat BAC contigs, most perfectly matching sRNAs represented TE sequences, suggesting that a large fraction of the wheat sRNAs originated from TEs. An analysis of all wheat TEs present in the Triticeae Repeat Sequence database showed that sRNA abundance was correlated with the estimated number of TEs within each class. Most of the sRNAs perfectly matching miniature inverted repeat transposable elements (MITEs) belonged to the 21-nt class and were mainly targeted to the terminal inverted repeats (TIRs). In contrast, most of the sRNAs matching class I and class II TEs belonged to the 24-nt class and were mainly targeted to the long terminal repeats (LTRs) in the class I TEs and to the terminal repeats in CACTA transposons. An analysis of the mutation frequency in potentially methylated sites revealed a three-fold increase in TE mutation frequency relative to intron and untranslated genic regions. This increase is consistent with wheat TEs being preferentially methylated, likely by sRNA targeting. Conclusions Our study examines the wheat epigenome in relation to known TEs. sRNA-directed transcriptional and post-transcriptional silencing plays important roles in

  8. Small Regulatory RNAs of Rickettsia conorii

    PubMed Central

    Narra, Hema P.; Schroeder, Casey L. C.; Sahni, Abha; Rojas, Mark; Khanipov, Kamil; Fofanov, Yuriy; Sahni, Sanjeev K.

    2016-01-01

    Small regulatory RNAs comprise critically important modulators of gene expression in bacteria, yet very little is known about their prevalence and functions in Rickettsia species. R. conorii, the causative agent of Mediterranean spotted fever, is a tick-borne pathogen that primarily infects microvascular endothelium in humans. We have determined the transcriptional landscape of R. conorii during infection of Human Microvascular Endothelial Cells (HMECs) by strand-specific RNA sequencing to identify 4 riboswitches, 13 trans-acting (intergenic), and 22 cis-acting (antisense) small RNAs (termed ‘Rc_sR’s). Independent expression of four novel trans-acting sRNAs (Rc_sR31, Rc_sR33, Rc_sR35, and Rc_sR42) and known bacterial sRNAs (6S, RNaseP_bact_a, ffs, and α-tmRNA) was next confirmed by Northern hybridization. Comparative analysis during infection of HMECs vis-à-vis tick AAE2 cells revealed significantly higher expression of Rc_sR35 and Rc_sR42 in HMECs, whereas Rc_sR31 and Rc_sR33 were expressed at similar levels in both cell types. We further predicted a total of 502 genes involved in all important biological processes as potential targets of Rc_sRs and validated the interaction of Rc_sR42 with cydA (cytochrome d ubiquinol oxidase subunit I). Our findings constitute the first evidence of the existence of post-transcriptional riboregulatory mechanisms in R. conorii and interactions between a novel Rc_sR and its target mRNA. PMID:27834404

  9. Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes

    PubMed Central

    Watanabe, Toshiaki; Takeda, Atsushi; Tsukiyama, Tomoyuki; Mise, Kazuyuki; Okuno, Tetsuro; Sasaki, Hiroyuki; Minami, Naojiro; Imai, Hiroshi

    2006-01-01

    Small RNAs ranging in size between 18 and 30 nucleotides (nt) are found in many organisms including yeasts, plants, and animals. Small RNAs are involved in the regulation of gene expression through translational repression, mRNA degradation, and chromatin modification. In mammals, microRNAs (miRNAs) are the only small RNAs that have been well characterized. Here, we have identified two novel classes of small RNAs in the mouse germline. One class consists of ∼20- to 24-nt small interfering RNAs (siRNAs) from mouse oocytes, which are derived from retroelements including LINE, SINE, and LTR retrotransposons. Addition of retrotransposon-derived sequences to the 3′ untranslated region (UTR) of a reporter mRNA destabilizes the mRNA significantly when injected into full-grown oocytes. These results suggest that retrotransposons are suppressed through the RNAi pathway in mouse oocytes. The other novel class of small RNAs is 26- to 30-nt germline small RNAs (gsRNAs) from testes. gsRNAs are expressed during spermatogenesis in a developmentally regulated manner, are mapped to the genome in clusters, and have strong strand bias. These features are reminiscent of Tetrahymena ∼23- to 24-nt small RNAs and Caenorhabditis elegans X-cluster small RNAs. A conserved novel small RNA pathway may be present in diverse animals. PMID:16766679

  10. Horizontal Transfer of Small RNAs to and from Plants

    PubMed Central

    Han, Lu; Luan, Yu-Shi

    2015-01-01

    Genetic information is traditionally thought to be transferred from parents to offspring. However, there is evidence indicating that gene transfer can also occur from microbes to higher species, such as plants, invertebrates, and vertebrates. This horizontal transfer can be carried out by small RNAs (sRNAs). sRNAs have been recently reported to move across kingdoms as mobile signals, spreading silencing information toward targeted genes. sRNAs, especially microRNAs (miRNAs) and small interfering RNAs (siRNAs), are non-coding molecules that control gene expression at the transcriptional or post-transcriptional level. Some sRNAs act in a cross-kingdom manner between animals and their parasites, but little is known about such sRNAs associated with plants. In this report, we provide a brief introduction to miRNAs that are transferred from plants to mammals/viruses and siRNAs that are transferred from microbes to plants. Both miRNAs and siRNAs can exert corresponding functions in the target organisms. Additionally, we provide information concerning a host-induced gene silencing system as a potential application that utilizes the transgenic trafficking of RNA molecules to silence the genes of interacting organisms. Moreover, we lay out the controversial views regarding cross-kingdom miRNAs and call for better methodology and experimental design to confirm this unique function of miRNAs. PMID:26697056

  11. Characterization of Sus scrofa small non-coding RNAs present in both female and male gonads.

    PubMed

    Kowalczykiewicz, Dorota; Świercz, Aleksandra; Handschuh, Luiza; Leśniak, Katarzyna; Figlerowicz, Marek; Wrzesinski, Jan

    2014-01-01

    Small non-coding RNAs (sncRNAs) are indispensable for proper germ cell development, emphasizing the need for greater elucidation of the mechanisms of germline development and regulation of this process by sncRNAs. We used deep sequencing to characterize three families of small non-coding RNAs (piRNAs, miRNAs, and tRFs) present in Sus scrofa gonads and focused on the small RNA fraction present in both male and female gonads. Although similar numbers of reads were obtained from both types of gonads, the number of unique RNA sequences in the ovaries was several times lower. Of the sequences detected in the testes, 2.6% of piRNAs, 9% of miRNAs, and 10% of tRFs were also present in the ovaries. Notably, the majority of the shared piRNAs mapped to ribosomal RNAs and were derived from clustered loci. In addition, the most abundant miRNAs present in the ovaries and testes are conserved and are involved in many biological processes such as the regulation of homeobox genes, the control of cell proliferation, and carcinogenesis. Unexpectedly, we detected a novel sncRNA type, the tRFs, which are 30-36-nt RNA fragments derived from tRNA molecules, in gonads. Analysis of S. scrofa piRNAs show that testes specific piRNAs are biased for 5' uracil but both testes and ovaries specific piRNAs are not biased for adenine at the 10th nucleotide position. These observations indicate that adult porcine piRNAs are predominantly produced by a primary processing pathway or other mechanisms and secondary piRNAs generated by ping-pong mechanism are absent.

  12. Small RNAs as Guardians of the Genome

    PubMed Central

    Malone, Colin D.; Hannon, Gregory J.

    2009-01-01

    Transposons populate the landscape of all eukaryotic genomes. Often considered purely genomic parasites, transposons can also benefit their hosts, playing roles in gene regulation and in genome organization and evolution. Peaceful coexistence with mobile elements depends upon adaptive control mechanisms, since unchecked transposon activity can impact long-term fitness and acutely reduce the fertility of progeny. Here, we review the conserved roles played by small RNAs in the adaptation of eukaryotes to coexist with their genomic colonists. An understanding of transposon-defense pathways has uncovered recurring themes in the mechanisms by which genomes distinguish “self” from “non-self” and selectively silence the latter. PMID:19239887

  13. A Comprehensive Expression Profile of MicroRNAs and Other Classes of Non-Coding Small RNAs in Barley Under Phosphorous-Deficient and -Sufficient Conditions

    PubMed Central

    Hackenberg, Michael; Huang, Po-Jung; Huang, Chun-Yuan; Shi, Bu-Jun; Gustafson, Perry; Langridge, Peter

    2013-01-01

    Phosphorus (P) is essential for plant growth. MicroRNAs (miRNAs) play a key role in phosphate homeostasis. However, little is known about P effect on miRNA expression in barley (Hordeum vulgare L.). In this study, we used Illumina's next-generation sequencing technology to sequence small RNAs (sRNAs) in barley grown under P-deficient and P-sufficient conditions. We identified 221 conserved miRNAs and 12 novel miRNAs, of which 55 were only present in P-deficient treatment while 32 only existed in P-sufficient treatment. Total 47 miRNAs were significantly differentially expressed between the two P treatments (|log2| > 1). We also identified many other classes of sRNAs, including sense and antisense sRNAs, repeat-associated sRNAs, transfer RNA (tRNA)-derived sRNAs and chloroplast-derived sRNAs, and some of which were also significantly differentially expressed between the two P treatments. Of all the sRNAs identified, antisense sRNAs were the most abundant sRNA class in both P treatments. Surprisingly, about one-fourth of sRNAs were derived from the chloroplast genome, and a chloroplast-encoded tRNA-derived sRNA was the most abundant sRNA of all the sRNAs sequenced. Our data provide valuable clues for understanding the properties of sRNAs and new insights into the potential roles of miRNAs and other classes of sRNAs in the control of phosphate homeostasis. PMID:23266877

  14. Endogenous small RNAs and antibacterial immunity in plants.

    PubMed

    Jin, Hailing

    2008-08-06

    Small RNAs are non-coding regulatory RNA molecules that control gene expression by mediating mRNA degradation, translational inhibition, or chromatin modification. Virus-derived small RNAs induce silencing of viral RNAs and are essential for antiviral defense in both animal and plant systems. The role of host endogenous small RNAs on antibacterial immunity has only recently been recognized. Host disease resistance and defense responses are achieved by activation and repression of a large array of genes. Certain endogenous small RNAs in plants, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are induced or repressed in response to pathogen attack and subsequently regulate the expression of genes involved in disease resistance and defense responses by mediating transcriptional or post-transcriptional gene silencing. Thus, these small RNAs play an important role in gene expression reprogramming in plant disease resistance and defense responses. This review focuses on the recent findings of plant endogenous small RNAs in antibacterial immunity.

  15. Differential expression of small non-coding RNAs in serum from cattle challenged with viruses causing bovine respiratory disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    MicroRNAs and tRNA-derived RNA fragments (tRFs) are the two most abundant groups of small non-coding RNAs. The potential for microRNAs and tRFs to be used as pathogen exposure indicators is yet to be fully explored. Our objective was to identify microRNAs and tRFs in cattle challenged with a non-cy...

  16. Dicer independent small RNAs associate with telomeric heterochromatin

    PubMed Central

    Cao, Fang; Li, Xiangzhi; Hiew, Samantha; Brady, Hugh; Liu, Yifan; Dou, Yali

    2009-01-01

    Small RNAs play important roles in the establishment and maintenance of heterochromatin structures. We show the presence of telomere specific small RNAs (tel-sRNAs) in mouse embryonic stem cells that are ∼24 nucleotides in length, Dicer-independent, and 2′-O-methylated at the 3′ terminus. The tel-sRNAs are asymmetric with specificity toward telomere G-rich strand, and evolutionarily conserved from protozoan to mammalian cells. Furthermore, tel-sRNAs are up-regulated in cells that carry null mutation of H3K4 methyltransferase MLL (Mll(−/−)) and down-regulated in cells that carry null mutations of histone H3K9 methyltransferase SUV39H (Suv39h1/h2(−/−)), suggesting that they are subject to epigenetic regulation. These results support that tel-sRNAs are heterochromatin associated pi-like small RNAs. PMID:19460867

  17. A small nucleolar RNP protein is required for pseudouridylation of eukaryotic ribosomal RNAs.

    PubMed Central

    Bousquet-Antonelli, C; Henry, Y; G'elugne, J P; Caizergues-Ferrer, M; Kiss, T

    1997-01-01

    Eukaryotic rRNAs possess numerous post-transcriptionally modified nucleotides. The most abundant modifications, 2'-O-ribose methylation and pseudouridylation, occur in the nucleolus during rRNA processing. The nucleolus contains a large number of small nucleolar RNAs (snoRNAs) most of which can be classified into two distinct families defined by conserved sequence boxes and common associated proteins. The C and D box-containing snoRNAs are associated with fibrillarin, and most of them function as guide RNAs in site-specific ribose methylation of rRNAs. The nucleolar function of the other class of snoRNAs, which share box H and ACA elements and are associated with a glycine- and arginine-rich nucleolar protein, Gar1p, remains elusive. Here we demonstrate that the yeast Saccharomyces cerevisiae Gar1 snoRNP protein plays an essential and specific role in the overall pseudouridylation of yeast rRNAs. These results establish a novel function for Gar1 protein and indicate that the box H/ACA snoRNAs, or at least a subset of these snoRNAs, function in the site-specific pseudouridylation of rRNAs. PMID:9303321

  18. Profiling of Small Nucleolar RNAs by Next Generation Sequencing: Potential New Players for Breast Cancer Prognosis

    PubMed Central

    Krishnan, Preethi; Ghosh, Sunita; Wang, Bo; Heyns, Mieke; Graham, Kathryn; Mackey, John R.; Kovalchuk, Olga; Damaraju, Sambasivarao

    2016-01-01

    One of the most abundant, yet least explored, classes of RNA is the small nucleolar RNAs (snoRNAs), which are well known for their involvement in post-transcriptional modifications of other RNAs. Although snoRNAs were only considered to perform housekeeping functions for a long time, recent studies have highlighted their importance as regulators of gene expression and as diagnostic/prognostic markers. However, the prognostic potential of these RNAs has not been interrogated for breast cancer (BC). The objective of the current study was to identify snoRNAs as prognostic markers for BC. Small RNA sequencing (Illumina Genome Analyzer IIx) was performed for 104 BC cases and 11 normal breast tissues. Partek Genomics Suite was used for analyzing the sequencing files. Two independent and proven approaches were used to identify prognostic markers: case-control (CC) and case-only (CO). For both approaches, snoRNAs significant in the permutation test, following univariate Cox proportional hazards regression model were used for constructing risk scores. Risk scores were subsequently adjusted for potential confounders in a multivariate Cox model. For both approaches, thirteen snoRNAs were associated with overall survival and/or recurrence free survival. Patients belonging to the high-risk group were associated with poor outcomes, and the risk score was significant after adjusting for confounders. Validation of representative snoRNAs (SNORD46 and SNORD89) using qRT-PCR confirmed the observations from sequencing experiments. We also observed 64 snoRNAs harboring piwi-interacting RNAs and/or microRNAs that were predicted to target genes (mRNAs) involved in tumorigenesis. Our results demonstrate the potential of snoRNAs to serve (i) as novel prognostic markers for BC and (ii) as indirect regulators of gene expression. PMID:27631501

  19. Biocomputational prediction of small non-coding RNAs in Streptomyces

    PubMed Central

    Pánek, Josef; Bobek, Jan; Mikulík, Karel; Basler, Marek; Vohradský, Jiří

    2008-01-01

    Background The first systematic study of small non-coding RNAs (sRNA, ncRNA) in Streptomyces is presented. Except for a few exceptions, the Streptomyces sRNAs, as well as the sRNAs in other genera of the Actinomyces group, have remained unstudied. This study was based on sequence conservation in intergenic regions of Streptomyces, localization of transcription termination factors, and genomic arrangement of genes flanking the predicted sRNAs. Results Thirty-two potential sRNAs in Streptomyces were predicted. Of these, expression of 20 was detected by microarrays and RT-PCR. The prediction was validated by a structure based computational approach. Two predicted sRNAs were found to be terminated by transcription termination factors different from the Rho-independent terminators. One predicted sRNA was identified computationally with high probability as a Streptomyces 6S RNA. Out of the 32 predicted sRNAs, 24 were found to be structurally dissimilar from known sRNAs. Conclusion Streptomyces is the largest genus of Actinomyces, whose sRNAs have not been studied. The Actinomyces is a group of bacterial species with unique genomes and phenotypes. Therefore, in Actinomyces, new unique bacterial sRNAs may be identified. The sequence and structural dissimilarity of the predicted Streptomyces sRNAs demonstrated by this study serve as the first evidence of the uniqueness of Actinomyces sRNAs. PMID:18477385

  20. Small RNAs Originated from Pseudogenes: cis- or trans-Acting?

    PubMed Central

    Guo, Xingyi; Zhang, Zhaolei; Gerstein, Mark B.; Zheng, Deyou

    2009-01-01

    Pseudogenes are significant components of eukaryotic genomes, and some have acquired novel regulatory roles. To date, no study has characterized rice pseudogenes systematically or addressed their impact on the structure and function of the rice genome. In this genome-wide study, we have identified 11,956 non-transposon-related rice pseudogenes, most of which are from gene duplications. About 12% of the rice protein-coding genes, half of which are in singleton families, have a pseudogene paralog. Interestingly, we found that 145 of these pseudogenes potentially gave rise to antisense small RNAs after examining ∼1.5 million small RNAs from developing rice grains. The majority (>50%) of these antisense RNAs are 24-nucleotides long, a feature often seen in plant repeat-associated small interfering RNAs (siRNAs) produced by RNA-dependent RNA polymerase (RDR2) and Dicer-like protein 3 (DCL3), suggesting that some pseudogene-derived siRNAs may be implicated in repressing pseudogene transcription (i.e., cis-acting). Multiple lines of evidence, however, indicate that small RNAs from rice pseudogenes might also function as natural antisense siRNAs either by interacting with the complementary sense RNAs from functional parental genes (38 cases) or by forming double-strand RNAs with transcripts of adjacent paralogous pseudogenes (2 cases) (i.e., trans-acting). Further examinations of five additional small RNA libraries revealed that pseudogene-derived antisense siRNAs could be produced in specific rice developmental stages or physiological growth conditions, suggesting their potentially important roles in normal rice development. In summary, our results show that pseudogenes derived from protein-coding genes are prevalent in the rice genome, and a subset of them are strong candidates for producing small RNAs with novel regulatory roles. Our findings suggest that pseudogenes of exapted functions may be a phenomenon ubiquitous in eukaryotic organisms. PMID:19649160

  1. High-throughput sequencing of small RNAs and anatomical characteristics associated with leaf development in celery.

    PubMed

    Jia, Xiao-Ling; Li, Meng-Yao; Jiang, Qian; Xu, Zhi-Sheng; Wang, Feng; Xiong, Ai-Sheng

    2015-06-09

    MicroRNAs (miRNAs) exhibit diverse and important roles in plant growth, development, and stress responses and regulate gene expression at the post-transcriptional level. Knowledge about the diversity of miRNAs and their roles in leaf development in celery remains unknown. To elucidate the roles of miRNAs in celery leaf development, we identified leaf development-related miRNAs through high-throughput sequencing. Small RNA libraries were constructed using leaves from three stages (10, 20, and 30 cm) of celery cv.'Ventura' and then subjected to high-throughput sequencing and bioinformatics analysis. At Stage 1, Stage 2, and Stage 3 of 'Ventura', a total of 333, 329, and 344 conserved miRNAs (belonging to 35, 35, and 32 families, respectively) were identified. A total of 131 miRNAs were identified as novel in 'Ventura'. Potential miRNA target genes were predicted and annotated using the eggNOG, GO, and KEGG databases to explore gene functions. The abundance of five conserved miRNAs and their corresponding potential target genes were validated. Expression profiles of novel potential miRNAs were also detected. Anatomical characteristics of the leaf blades and petioles at three leaf stages were further analyzed. This study contributes to our understanding on the functions and molecular regulatory mechanisms of miRNAs in celery leaf development.

  2. Design and Characterization of Topological Small RNAs.

    PubMed

    Hassall, Jack; MacDonald, Paul; Cordero, Teresa; Rostain, William; Jaramillo, Alfonso

    2015-01-01

    RNA can self-assemble into complex structures through base pairing, as well as encode information and bind with proteins to induce enzymatic activity. Furthermore, RNA can possess intrinsic enzymatic-like (ribozymatic) activity, a property that, if necessary, can be activated only upon the binding of a small molecule or another RNA (as is the case in aptazymes). As such, RNA could be of use in nanotechnology as a programmable polymer capable of self-assembling into complex topological structures. In this chapter we describe a method for designing advanced topological structures using self-circulating RNA, exemplified by three tiers of topologically manipulated self-assembling synthetic RNA systems. The first tier of topological manipulation, the RNA knot is a physically locked structure, formed by circularizing one monomer of knotted single-stranded RNA left with loose ends (an "open" knot). The second tier, a two interlocking ring system, is made by interlocking two circular RNA components: a circular RNA target, and an RNA lasso designed to intercalate the target before circularizing. The third tier naturally extends this system into a string of topologically locked circular RNA molecules (an RNA chain). We detail the methodology used for designing such topologically complex RNAs, including computational predictions of secondary structure, and where appropriate, RNA-RNA interactions, illustrated by examples. We then describe the experimental methods used for characterizing such structures, and provide sequences of building blocks that can be used for topological manipulation of RNA.

  3. The role of RNases in the regulation of small RNAs.

    PubMed

    Saramago, Margarida; Bárria, Cátia; Dos Santos, Ricardo F; Silva, Inês J; Pobre, Vânia; Domingues, Susana; Andrade, José M; Viegas, Sandra C; Arraiano, Cecília M

    2014-04-01

    Ribonucleases (RNases) are key factors in the control of biological processes, since they modulate the processing, degradation and quality control of RNAs. This review gives many illustrative examples of the role of RNases in the regulation of small RNAs (sRNAs). RNase E and PNPase have been shown to degrade the free pool of sRNAs. RNase E can also be recruited to cleave mRNAs when they are interacting with sRNAs. RNase III cleaves double-stranded structures, and can cut both the sRNA and its RNA target when they are hybridized. Overall, ribonucleases act as conductors in the control of sRNAs. Therefore, it is very important to further understand their role in the post-transcriptional control of gene expression.

  4. Small non-coding RNAs as novel therapeutics.

    PubMed

    Rossbach, M

    2010-06-01

    RNA interference (RNAi), an evolutionarily conserved sequence-specific post-transcriptional gene silencing mechanism, is triggered by double-stranded RNA (dsRNA) that results in the degradation of homologous mRNA or in the inhibition of mRNA translation. The naturally occurring triggers for the RNAi pathway are small regulatory RNAs, including small interfering RNAs (siRNAs), processed from longer dsRNAs by the RNAse III enzyme Dicer, and microRNAs (miRNAs), generated in a regulated multistep process from endogenous primary transcripts (pri-miRNA). These primary transcripts are capped, polyadenylated and spliced, thus resembling conventional mRNAs. It is estimated that miRNAs regulate more than one third of all cellular mRNAs, and bioinformatic data indicate that each miRNA can control hundreds of gene targets. Thus, there are likely to be few biological processes not regulated by miRNAs. Although the biological functions of miRNAs are not completely revealed, there is growing evidence that miRNA pathways are a new mechanism of gene regulation in both normal and diseased conditions. Recent evidence has shown that miRNA mutations or aberrant expression patterns correlate with various diseases, such as cancer, viral infections, cardiovascular or neurodegenerative diseases and indicates that miRNAs can function as tumor suppressors and oncogenes. MiRNAs have not only emerged as a powerful tool for gene regulation studies but also for the development of novel drugs. Since they do not encode proteins, they are not traditional therapeutic targets of small-molecule inhibitors and thus comprise a novel class of therapeutics. This article will focus on the current progress in drug discovery using the miRNA strategy.

  5. Small RNAs regulate plant responses to filamentous pathogens.

    PubMed

    Kuan, Tung; Zhai, Yi; Ma, Wenbo

    2016-08-01

    Small RNAs are central players of RNA silencing in eukaryotes. These short RNA molecules (20-25 nucleotides in length) repress target gene expression based on sequence complementarity. While small RNAs are well-known for their essential function in regulating growth and development, recent research has revealed that they also influence plant immunity. Extensive changes in small RNA accumulation have been observed during infection. This review focuses on specific small RNA changes that are involved in plant responses to filamentous eukaryotic pathogens including fungi and oomycetes. We describe how changes in small RNA accumulation influence plant immunity and summarize the cellular processes affected by these small RNAs. In particular, we discuss secondary small interfering RNAs that directly modulate the expression of defense-related genes.

  6. Argonaute and Argonaute-Bound Small RNAs in Stem Cells

    PubMed Central

    Zhai, Lihong; Wang, Lin; Teng, Feng; Zhou, Lanting; Zhang, Wenjing; Xiao, Juan; Liu, Ying; Deng, Wenbin

    2016-01-01

    Small RNAs are essential for a variety of cellular functions. Argonaute (AGO) proteins are associated with all of the different classes of small RNAs, and are indispensable in small RNA-mediated regulatory pathways. AGO proteins have been identified in various types of stem cells in diverse species from plants and animals. This review article highlights recent progress on how AGO proteins and AGO-bound small RNAs regulate the self-renewal and differentiation of distinct stem cell types, including pluripotent, germline, somatic, and cancer stem cells. PMID:26861290

  7. Deep sequencing of small RNAs in plants: applied bioinformatics.

    PubMed

    Studholme, David J

    2012-01-01

    Small RNAs, including microRNA and short-interfering RNAs, play important roles in plants. In recent years, developments in sequencing technology have enabled the large-scale discovery of sRNAs in various cells, tissues and developmental stages and in response to various stresses. This review describes the bioinformatics challenges to analysing these large datasets of short-RNA sequences and some of the solutions to those challenges.

  8. High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants

    PubMed Central

    Klahre, Ulrich; Crété, Patrice; Leuenberger, Sabrina A.; Iglesias, Victor A.; Meins, Frederick

    2002-01-01

    Posttranscriptional gene silencing (PTGS) in transgenic plants is an epigenetic form of RNA degradation related to PTGS and RNA interference (RNAi) in fungi and animals. Evidence suggests that transgene loci and RNA viruses can generate double-stranded RNAs similar in sequence to the transcribed region of target genes, which then undergo endonucleolytic cleavage to generate small interfering RNAs (siRNA) that promote degradation of cognate RNAs. The silent state in transgenic plants and in Caenorhabditis elegans can spread systemically, implying that mobile silencing signals exist. Neither the chemical nature of these signals nor their exact source in the PTGS pathway is known. Here, we use a positive marker system and real-time monitoring of green fluorescent protein expression to show that large sense, antisense, and double-stranded RNAs as well as double-stranded siRNAs delivered biolistically into plant cells trigger silencing capable of spreading locally and systemically. Systemically silenced leaves show greatly reduced levels of target RNA and accumulate siRNAs, confirming that RNA can induce systemic PTGS. The induced siRNAs represent parts of the target RNA that are outside of the region of homology with the triggering siRNA. Our results imply that siRNAs themselves or intermediates induced by siRNAs could comprise silencing signals and that these signals induce self-amplifying production of siRNAs. PMID:12181491

  9. Small RNAs – secrets and surprises of the genome

    PubMed Central

    Chen, Xuemei

    2011-01-01

    SUMMARY Small RNAs associated with post-transcriptional gene silencing were first discovered in plants in 1999. Although this study marked the beginning of small RNA biology in plants, the sequence of the Arabidopsis genome and related genomic resources that were soon to become available to the Arabidopsis community launched the research on small RNAs at a remarkable pace. In 2000, when the genetic blueprint of the first plant species was revealed, the tens of thousands of endogenous small RNA species as we know today remained hidden features of the genome. However, the subsequent 10 years have witnessed an explosion of our knowledge of endogenous small RNAs: their widespread existence, diversity, biogenesis, mode of action and biological functions. As key sequence-specific regulators of gene expression in the nucleus and the cytoplasm, small RNAs influence almost all aspects of plant biology. Because of the extensive conservation of mechanisms concerning the biogenesis and molecular actions of small RNAs, research in the model plant Arabidopsis has contributed vital knowledge to the small RNA field in general. Our knowledge of small RNAs gained primarily from Arabidopsis has also led to the invention of effective gene knock-down technologies that are applicable to diverse plant species, including crop plants. Here, I attempt to recount the developments of the small RNA field in the pre- and post-genomic era, in celebration of the 10th anniversary of the completion of the first plant genome. PMID:20409269

  10. Diversity of small RNAs expressed in Pseudomonas species.

    PubMed

    Gómez-Lozano, María; Marvig, Rasmus L; Molina-Santiago, Carlos; Tribelli, Paula M; Ramos, Juan-Luis; Molin, Søren

    2015-04-01

    RNA sequencing (RNA-seq) has revealed several hundreds of previously undetected small RNAs (sRNAs) in all bacterial species investigated, including strains of Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas syringae. Nonetheless, only little is known about the extent of conservation of expressed sRNAs across strains and species. In this study, we have used RNA-seq to identify sRNAs in P. putida DOT-T1E and Pseudomonas extremaustralis 14-3b. This is the first strain of P. extremaustralis and the second strain of P. putida to have their transcriptomes analysed for sRNAs, and we identify the presence of around 150 novel sRNAs in each strain. Furthermore, we provide a comparison based on sequence conservation of all the sRNAs detected by RNA-seq in the Pseudomonas species investigated so far. Our results show that the extent of sRNA conservation across different species is very limited. In addition, when comparing the sRNAs expressed in different strains of the same species, we observe that numerous sRNAs exhibit a strain-specific expression pattern. These results support the idea that the evolution of most bacterial sRNAs is rapid, which limits the extent of both interspecies and intraspecies conservation.

  11. Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA).

    PubMed

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S; Harlow, Mark L

    2015-10-08

    Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5' ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission.

  12. Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA)

    PubMed Central

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S.; Harlow, Mark L.

    2015-01-01

    Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5′ ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission. PMID:26446566

  13. 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

  14. Delivery of Small Interfering RNAs to Cells via Exosomes.

    PubMed

    Wahlgren, Jessica; Statello, Luisa; Skogberg, Gabriel; Telemo, Esbjörn; Valadi, Hadi

    2016-01-01

    Exosomes are small membrane bound vesicles between 30 and 100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. Exosomes play a role in intercellular communication by transferring proteins, lipids, and RNAs to recipient cells.Exosomes from human cells could be used as vectors to provide cells with therapeutic RNAs. Here we describe how exogenous small interfering RNAs may successfully be introduced into various kinds of human exosomes using electroporation and subsequently delivered to recipient cells. Methods used to confirm the presence of siRNA inside exosomes and cells are presented, such as flow cytometry, confocal microscopy, and Northern blot.

  15. Wolbachia small noncoding RNAs and their role in cross-kingdom communications.

    PubMed

    Mayoral, Jaime G; Hussain, Mazhar; Joubert, D Albert; Iturbe-Ormaetxe, Iñaki; O'Neill, Scott L; Asgari, Sassan

    2014-12-30

    In prokaryotes, small noncoding RNAs (snRNAs) of 50-500 nt are produced that are important in bacterial virulence and response to environmental stimuli. Here, we identified and characterized snRNAs from the endosymbiotic bacteria, Wolbachia, which are widespread in invertebrates and cause reproductive manipulations. Most importantly, some strains of Wolbachia inhibit replication of several vector-borne pathogens in insects. We demonstrate that two abundant snRNAs, WsnRNA-46 and WsnRNA-49, are expressed in Wolbachia from noncoding RNA transcripts that contain precursors with stem-loop structures. WsnRNAs were detected in Aedes aegypti mosquitoes infected with the wMelPop-CLA strain of Wolbachia and in Drosophila melanogaster and Drosophila simulans infected with wMelPop and wAu strains, respectively, indicating that the WsnRNAs are conserved across species and strains. In addition, we show that the WsnRNAs may potentially regulate host genes and Wolbachia genes. Our findings provide evidence for the production of functional snRNAs by Wolbachia that play roles in cross-kingdom communication between the endosymbiont and the host.

  16. Big impacts by small RNAs in plant development.

    PubMed

    Chuck, George; Candela, Héctor; Hake, Sarah

    2009-02-01

    The identification and study of small RNAs, including microRNAs and trans-acting small interfering RNAs, have added a layer of complexity to the many pathways that regulate plant development. These molecules, which function as negative regulators of gene expression, are now known to have greatly expanded roles in a variety of developmental processes affecting all major plant structures, including meristems, leaves, roots, and inflorescences. Mutants with specific developmental phenotypes have also advanced our knowledge of the biogenesis and mode of action of these diverse small RNAs. In addition, previous models on the cell autonomy of microRNAs may have to be revised as more data accumulate supporting their long distance transport. As many of these small RNAs appear to be conserved across different species, knowledge gained from one species is expected to have general application. However, a few surprising differences in small RNA function seem to exist between monocots and dicots regarding meristem initiation and sex determination. Integrating these unique functions into the overall scheme for plant growth will give a more complete picture of how they have evolved as unique developmental systems.

  17. The role of small RNAs in vegetative shoot development

    PubMed Central

    Fouracre, Jim P.; Poethig, R. Scott

    2016-01-01

    Shoot development consists of the production of lateral organs in predictable spatial and temporal patterns at the shoot apex. To properly integrate such programs of growth across different cell and tissue types, plants require highly complex and robust genetic networks. Over the last twenty years, the roles of small, non-coding RNAs (sRNAs) in these networks have become increasingly apparent, not least in vegetative shoot growth. In this review, we describe recent progress in understanding the contribution of sRNAs to the regulation of vegetative shoot growth, and outline persisting experimental limitations in the field. PMID:26745378

  18. Identification and Characterization of Novel Small RNAs in Rickettsia prowazekii

    PubMed Central

    Schroeder, Casey L. C.; Narra, Hema P.; Sahni, Abha; Rojas, Mark; Khanipov, Kamil; Patel, Jignesh; Shah, Riya; Fofanov, Yuriy; Sahni, Sanjeev K.

    2016-01-01

    Emerging evidence implicates a critically important role for bacterial small RNAs (sRNAs) as post-transcriptional regulators of physiology, metabolism, stress/adaptive responses, and virulence, but the roles of sRNAs in pathogenic Rickettsia species remain poorly understood. Here, we report on the identification of both novel and well-known bacterial sRNAs in Rickettsia prowazekii, known to cause epidemic typhus in humans. RNA sequencing of human microvascular endothelial cells (HMECs), the preferred targets during human rickettsioses, infected with R. prowazekii revealed the presence of 35 trans-acting and 23 cis-acting sRNAs, respectively. Of these, expression of two trans-acting (Rp_sR17 and Rp_sR60) and one cis-acting (Rp_sR47) novel sRNAs and four well-characterized bacterial sRNAs (RNaseP_bact_a, α-tmRNA, 4.5S RNA, 6S RNA) was further confirmed by Northern blot or RT-PCR analyses. The transcriptional start sites of five novel rickettsial sRNAs and 6S RNA were next determined using 5′ RLM-RACE yielding evidence for their independent biogenesis in R. prowazekii. Finally, computational approaches were employed to determine the secondary structures and potential mRNA targets of novel sRNAs. Together, these results establish the presence and expression of sRNAs in R. prowazekii during host cell infection and suggest potential functional roles for these important post-transcriptional regulators in rickettsial biology and pathogenesis. PMID:27375581

  19. Small RNAs and the competing endogenous RNA network in high grade serous ovarian cancer tumor spread

    PubMed Central

    Bachmayr-Heyda, Anna; Auer, Katharina; Sukhbaatar, Nyamdelger; Aust, Stefanie; Deycmar, Simon; Reiner, Agnes T.; Polterauer, Stephan; Dekan, Sabine; Pils, Dietmar

    2016-01-01

    High grade serous ovarian cancer (HGSOC) is among the most deadly malignancies in women, frequently involving peritoneal tumor spread. Understanding molecular mechanisms of peritoneal metastasis is essential to develop urgently needed targeted therapies. We described two peritoneal tumor spread types in HGSOC apparent during surgery: miliary (numerous millet-sized implants) and non-miliary (few big, bulky implants). The former one is defined by a more epithelial-like tumor cell characteristic with less immune cell reactivity and with significant worse prognosis, even if corrected for typical clinicopathologic factors. 23 HGSOC patients were enrolled in this study. Isolated tumor cells from fresh tumor tissues of ovarian and peritoneal origin and from ascites were used for ribosomal RNA depleted RNA and small RNA sequencing. RT-qPCR was used to validate results and an independent cohort of 32 patients to validate the impact on survival. Large and small RNA sequencing data were integrated and a new gene-miRNA set analysis method was developed. Thousands of new small RNAs (miRNAs and piwi-interacting RNAs) were predicted and a 13 small RNA signature was developed to predict spread type from formalin-fixed paraffin-embedded tissues. Furthermore, integrative analyses of RNA sequencing and small RNA sequencing data revealed a global upregulation of the competing endogenous RNA network in tumor tissues of non-miliary compared to miliary spread, i.e. higher expression of circular RNAs and long non-coding RNAs compared to coding RNAs but unchanged abundance of small RNAs. This global deregulated expression pattern could be co-responsible for the spread characteristic, miliary or non-miliary, in ovarian cancer. PMID:27172797

  20. High-Resolution Profiling and Analysis of Viral and Host Small RNAs during Human Cytomegalovirus Infection

    PubMed Central

    Stark, Thomas J.; Arnold, Justin D.; Spector, Deborah H.

    2012-01-01

    Human cytomegalovirus (HCMV) contributes its own set of microRNAs (miRNAs) during lytic infection of cells, likely fine-tuning conditions important for viral replication. To enhance our understanding of this component of the HCMV-host transcriptome, we have conducted deep-sequencing analysis of small RNAs (smRNA-seq) from infected human fibroblast cells. We found that HCMV-encoded miRNAs accumulate to ∼20% of the total smRNA population at late stages of infection, and our analysis led to improvements in viral miRNA annotations and identification of two novel HCMV miRNAs, miR-US22 and miR-US33as. Both of these miRNAs were capable of functionally repressing synthetic targets in transient transfection experiments. Additionally, through cross-linking and immunoprecipitation (CLIP) of Argonaute (Ago)-bound RNAs from infected cells, followed by high-throughput sequencing, we have obtained direct evidence for incorporation of all HCMV miRNAs into the endogenous host silencing machinery. Surprisingly, three HCMV miRNA precursors exhibited differential incorporation of their mature miRNA arms between Ago2 and Ago1 complexes. Host miRNA abundances were also affected by HCMV infection, with significant upregulation observed for an miRNA cluster containing miR-96, miR-182, and miR-183. In addition to miRNAs, we also identified novel forms of virus-derived smRNAs, revealing greater complexity within the smRNA population during HCMV infection. PMID:22013051

  1. Influence of small RNAs on biofilm formation process in bacteria.

    PubMed

    Ghaz-Jahanian, Mohammad Ali; Khodaparastan, Fatemeh; Berenjian, Aydin; Jafarizadeh-Malmiri, Hoda

    2013-11-01

    Small non-coding RNAs (sRNAs) play a significant role in regulation of bacterial physiological behaviors. After sensing any environmental cue such as fluctuation of nutrient concentration, temperature, pH, and osmolarity, these sRNAs interfere to transmit these signals to target regulators and genes. sRNAs have key role in biofilm formation process by base pairing with target mRNAs or interaction with modulating proteins to both positive and negative regulation mechanisms. There are various regulatory systems to characterize the initiation and formation of special bacterial biofilms that are mostly described as two component systems based on sRNAs functions. In this study, regulatory pathways that are important for biofilm formation and genetic responses to environmental stimuli in mature biofilms were evaluated. Some of the regulatory systems that produce common types of biofilms such as curli, PGA, cellulose and polysaccharides such as alginate, colonic acid, Psl and their involved sRNAs functions were also discussed.

  2. Small RNAs and regulation of transposons in plants.

    PubMed

    Ito, Hidetaka

    2013-01-01

    RNA interference is now a well-recognized post-transcriptional mechanism for regulation of gene expression in both animals and plants. In this process, microRNAs (miRNAs) direct silencing complexes to complementary RNA sequences, leading to either degradation or repression of translation. Plants also contain another type of small RNA, small interfering RNAs (siRNAs), that play a role in gene silencing by directing cytosine methylation activities of complementary DNA sequences and thus, differ from miRNAs. This nuclear regulation system is referred to as RNA-directed DNA methylation (RdDM). In plant genomes, transposable elements were initially thought to be regulated by DNA methylation alone. However, several recent reports have revealed that siRNAs and RdDM also play crucial roles in silencing of transposons and endogenous repeats. It is also becoming apparent that transposons are subjected to different levels of regulation in response to developmental and environmental cues. Transposons are tightly regulated in germ cells to protect the host genome from transgenerational mutagenic activity. In plants, transposons are also activated by biotic and abiotic stress. The regulation of transposons in these different situations has been associated with both the DNA methylation and siRNA-mediated regulation systems, suggesting that plants likely evolved "multi-lock" systems for transposon regulation to ensure tight control during the developmental phase and environmental changes.

  3. Comparative analysis of virus-derived small RNAs within cassava (Manihot esculenta Crantz) infected with cassava brown streak viruses.

    PubMed

    Ogwok, Emmanuel; Ilyas, Muhammad; Alicai, Titus; Rey, Marie E C; Taylor, Nigel J

    2016-04-02

    Infection of plant cells by viral pathogens triggers RNA silencing, an innate antiviral defense mechanism. In response to infection, small RNAs (sRNAs) are produced that associate with Argonaute (AGO)-containing silencing complexes which act to inactivate viral genomes by posttranscriptional gene silencing (PTGS). Deep sequencing was used to compare virus-derived small RNAs (vsRNAs) in cassava genotypes NASE 3, TME 204 and 60444 infected with the positive sense single-stranded RNA (+ssRNA) viruses cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), the causal agents of cassava brown streak disease (CBSD). An abundance of 21-24nt vsRNAs was detected and mapped, covering the entire CBSV and UCBSV genomes. The 21nt vsRNAs were most predominant, followed by the 22 nt class with a slight bias toward sense compared to antisense polarity, and a bias for adenine and uracil bases present at the 5'-terminus. Distribution and frequency of vsRNAs differed between cassava genotypes and viral genomes. In susceptible genotypes TME 204 and 60444, CBSV-derived sRNAs were seen in greater abundance than UCBSV-derived sRNAs. NASE 3, known to be resistant to UCBSV, accumulated negligible UCBSV-derived sRNAs but high populations of CBSV-derived sRNAs. Transcript levels of cassava homologues of AGO2, DCL2 and DCL4, which are central to the gene-silencing complex, were found to be differentially regulated in CBSV- and UCBSV-infected plants across genotypes, suggesting these proteins play a role in antiviral defense. Irrespective of genotype or viral pathogen, maximum populations of vsRNAs mapped to the cytoplasmic inclusion, P1 and P3 protein-encoding regions. Our results indicate disparity between CBSV and UCBSV host-virus interaction mechanisms, and provide insight into the role of virus-induced gene silencing as a mechanism of resistance to CBSD.

  4. Deep sequencing of Brachypodium small RNAs at the global genome level identifies microRNAs involved in cold stress response

    PubMed Central

    Zhang, Jingyu; Xu, Yunyuan; Huan, Qing; Chong, Kang

    2009-01-01

    Background MicroRNAs (miRNAs) are endogenous small RNAs having large-scale regulatory effects on plant development and stress responses. Extensive studies of miRNAs have only been performed in a few model plants. Although miRNAs are proved to be involved in plant cold stress responses, little is known for winter-habit monocots. Brachypodium distachyon, with close evolutionary relationship to cool-season cereals, has recently emerged as a novel model plant. There are few reports of Brachypodium miRNAs. Results High-throughput sequencing and whole-genome-wide data mining led to the identification of 27 conserved miRNAs, as well as 129 predicted miRNAs in Brachypodium. For multiple-member conserved miRNA families, their sizes in Brachypodium were much smaller than those in rice and Populus. The genome organization of miR395 family in Brachypodium was quite different from that in rice. The expression of 3 conserved miRNAs and 25 predicted miRNAs showed significant changes in response to cold stress. Among these miRNAs, some were cold-induced and some were cold-suppressed, but all the conserved miRNAs were up-regulated under cold stress condition. Conclusion Our results suggest that Brachypodium miRNAs are composed of a set of conserved miRNAs and a large proportion of non-conserved miRNAs with low expression levels. Both kinds of miRNAs were involved in cold stress response, but all the conserved miRNAs were up-regulated, implying an important role for cold-induced miRNAs. The different size and genome organization of miRNA families in Brachypodium and rice suggest that the frequency of duplication events or the selection pressure on duplicated miRNAs are different between these two closely related plant species. PMID:19772667

  5. 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

  6. MicroRNAs and the heart: small things do matter.

    PubMed

    Papoutsidakis, Nikolaos; Deftereos, Spyridon; Kaoukis, Andreas; Bouras, Georgios; Giannopoulos, Georgios; Theodorakis, Andreas; Angelidis, Christos; Hatzis, Georgios; Stefanadis, Christodoulos

    2013-01-01

    MicroRNAs are small RNA molecules and constitute a relatively novel class of gene expression regulators, found in the great majority of eukaryotic cells. Their role in human physiology and pathology is actively being researched with new exciting discoveries continuously coming to the forefront. MicroRNAs play a crucial role in the biogenesis and function of the cardiovascular system and act as important regulators of various metabolic and signaling pathways in cardiovascular disease. In this review there will be a summary on current knowledge about the expression, regulation and function of microRNAs in the most common diseases of the cardiovascular system as well as a presentation of and discussion about their promising future role as new biomarkers and therapeutic targets.

  7. Efficient degradation and expression prioritization with small RNAs

    NASA Astrophysics Data System (ADS)

    Mitarai, Namiko; Andersson, Anna M. C.; Krishna, Sandeep; Semsey, Szabolcs; Sneppen, Kim

    2007-09-01

    We build a simple model for feedback systems involving small RNA (sRNA) molecules based on the iron metabolism system in the bacterium E. coli, and compare it with the corresponding system in H. pylori which uses purely transcriptional regulation. This reveals several unique features of sRNA-based regulation that could be exploited by cells. Firstly, we show that sRNA regulation can maintain a smaller turnover of target mRNAs than transcriptional regulation, without sacrificing the speed of response to external shocks. Secondly, we propose that a single sRNA can prioritize the usage of different target mRNAs. This suggests that sRNA regulation would be more common in more complex systems which need to co-regulate many mRNAs efficiently.

  8. Engineering small interfering RNAs by strategic chemical modification.

    PubMed

    Bramsen, Jesper B; Kjems, Jørgen

    2013-01-01

    Synthetic small interfering RNAs (siRNAs) have revolutionized functional genomics in mammalian cell cultures due to their reliability, efficiency, and ease of use. This success, however, has not fully translated into siRNA applications in vivo and in siRNA therapeutics where initial optimism has been dampened by a lack of efficient delivery strategies and reports of siRNA off-target effects and immunogenicity. Encouragingly, most aspects of siRNA behavior can be addressed by careful engineering of siRNAs incorporating beneficial chemical modifications into discrete nucleotide positions during siRNA synthesis. Here, we review the literature (Subheadings 1 -3) and provide a quick guide (Subheading 4) to how the performance of siRNA can be improved by chemical modification to suit specific applications in vitro and in vivo.

  9. Evidence for Small RNAs Homologous to Effector-Encoding Genes and Transposable Elements in the Oomycete Phytophthora infestans

    PubMed Central

    Vetukuri, Ramesh R.; Åsman, Anna K. M.; Tellgren-Roth, Christian; Jahan, Sultana N.; Reimegård, Johan; Fogelqvist, Johan; Savenkov, Eugene; Söderbom, Fredrik; Avrova, Anna O.; Whisson, Stephen C.; Dixelius, Christina

    2012-01-01

    Phytophthora infestans is the oomycete pathogen responsible for the devastating late blight disease on potato and tomato. There is presently an intense research focus on the role(s) of effectors in promoting late blight disease development. However, little is known about how they are regulated, or how diversity in their expression may be generated among different isolates. Here we present data from investigation of RNA silencing processes, characterized by non-coding small RNA molecules (sRNA) of 19–40 nt. From deep sequencing of sRNAs we have identified sRNAs matching numerous RxLR and Crinkler (CRN) effector protein genes in two isolates differing in pathogenicity. Effector gene-derived sRNAs were present in both isolates, but exhibited marked differences in abundance, especially for CRN effectors. Small RNAs in P. infestans grouped into three clear size classes of 21, 25/26 and 32 nt. Small RNAs from all size classes mapped to RxLR effector genes, but notably 21 nt sRNAs were the predominant size class mapping to CRN effector genes. Some effector genes, such as PiAvr3a, to which sRNAs were found, also exhibited differences in transcript accumulation between the two isolates. The P. infestans genome is rich in transposable elements, and the majority of sRNAs of all size classes mapped to these sequences, predominantly to long terminal repeat (LTR) retrotransposons. RNA silencing of Dicer and Argonaute genes provided evidence that generation of 21 nt sRNAs is Dicer-dependent, while accumulation of longer sRNAs was impacted by silencing of Argonaute genes. Additionally, we identified six microRNA (miRNA) candidates from our sequencing data, their precursor sequences from the genome sequence, and target mRNAs. These miRNA candidates have features characteristic of both plant and metazoan miRNAs. PMID:23272103

  10. Small-interfering RNAs (siRNAs) as a promising tool for ocular therapy

    PubMed Central

    Guzman-Aranguez, A; Loma, P; Pintor, J

    2013-01-01

    RNA interference (RNAi) can be used to inhibit the expression of specific genes in vitro and in vivo, thereby providing an extremely useful tool for investigating gene function. Progress in the understanding of RNAi-based mechanisms has opened up new perspectives in therapeutics for the treatment of several diseases including ocular disorders. The eye is currently considered a good target for RNAi therapy mainly because it is a confined compartment and, therefore, enables local delivery of small-interfering RNAs (siRNAs) by topical instillation or direct injection. However, delivery strategies that protect the siRNAs from degradation and are suitable for long-term treatment would be help to improve the efficacy of RNAi-based therapies for ocular pathologies. siRNAs targeting critical molecules involved in the pathogenesis of glaucoma, retinitis pigmentosa and neovascular eye diseases (age-related macular degeneration, diabetic retinopathy and corneal neovascularization) have been tested in experimental animal models, and clinical trials have been conducted with some of them. This review provides an update on the progress of RNAi in ocular therapeutics, discussing the advantages and drawbacks of RNAi-based therapeutics compared to previous treatments. PMID:23937539

  11. Characterisation of the Small RNAs in the Biomedically Important Green-Bottle Blowfly Lucilia sericata

    PubMed Central

    Blenkiron, Cherie; Tsai, Peter; Brown, Lisa A.; Askelund, Kathryn J.; Windsor, John A.; Phillips, Anthony R.

    2015-01-01

    Background The green bottle fly maggot, Lucilia sericata, is a species with importance in medicine, agriculture and forensics. Improved understanding of this species’ biology is of great potential benefit to many research communities. MicroRNAs (miRNA) are a short non-protein coding regulatory RNA, which directly regulate a host of protein coding genes at the translational level. They have been shown to have developmental and tissue specific distributions where they impact directly on gene regulation. In order to improve understanding of the biology of L. sericata maggots we have performed small RNA-sequencing of their secretions and tissue at different developmental stages. Results We have successfully isolated RNA from the secretions of L. sericata maggots. Illumina small RNA-sequencing of these secretions and the three tissues (crop, salivary gland, gut) revealed that the most common small RNA fragments were derived from ribosomal RNA and transfer RNAs of both insect and bacterial origins. These RNA fragments were highly specific, with the most common tRNAs, such as GlyGCC, predominantly represented by reads derived from the 5’ end of the mature maggot tRNA. Each library also had a unique profile of miRNAs with a high abundance of miR-10-5p in the maggot secretions and gut and miR-8 in the food storage organ the crop and salivary glands. The pattern of small RNAs in the bioactive maggot secretions suggests they originate from a combination of saliva, foregut and hindgut tissues. Droplet digital RT-PCR validation of the RNA-sequencing data shows that not only are there differences in the tissue profiles for miRNAs and small RNA fragments but that these are also modulated through developmental stages of the insect. Conclusions We have identified the small-RNAome of the medicinal maggots L. sericata and shown that there are distinct subsets of miRNAs expressed in specific tissues that also alter during the development of the insect. Furthermore there are very

  12. A Tale of Two RNAs during Viral Infection: How Viruses Antagonize mRNAs and Small Non-Coding RNAs in The Host Cell

    PubMed Central

    Herbert, Kristina M.; Nag, Anita

    2016-01-01

    Viral infection initiates an array of changes in host gene expression. Many viruses dampen host protein expression and attempt to evade the host anti-viral defense machinery. Host gene expression is suppressed at several stages of host messenger RNA (mRNA) formation including selective degradation of translationally competent messenger RNAs. Besides mRNAs, host cells also express a variety of noncoding RNAs, including small RNAs, that may also be subject to inhibition upon viral infection. In this review we focused on different ways viruses antagonize coding and noncoding RNAs in the host cell to its advantage. PMID:27271653

  13. Dynamic changes of small RNAs in rice spikelet development reveal specialized reproductive phasiRNA pathways

    PubMed Central

    Fei, Qili; Yang, Li; Liang, Wanqi; Zhang, Dabing; Meyers, Blake C.

    2016-01-01

    Dissection of the genetic pathways and mechanisms by which anther development occurs in grasses is crucial for both a basic understanding of plant development and for examining traits of agronomic importance such as male sterility. In rice, MULTIPLE SPOROCYTES1 (MSP1), a leucine-rich-repeat receptor kinase, plays an important role in anther development by limiting the number of sporocytes. OsTDL1a (a TPD1-like gene in rice) encodes a small protein that acts as a cofactor of MSP1 in the same regulatory pathway. In this study, we analyzed small RNA and mRNA changes in different stages of spikelets from wild-type rice, and from msp1 and ostdl1a mutants. Analysis of the small RNA data identified miRNAs demonstrating differential abundances. miR2275 was depleted in the two rice mutants; this miRNA is specifically enriched in anthers and functions to trigger the production of 24-nt phased secondary siRNAs (phasiRNAs) from PHAS loci. We observed that the 24-nt phasiRNAs as well as their precursor PHAS mRNAs were also depleted in the two mutants. An analysis of co-expression identified three Argonaute-encoding genes (OsAGO1d, OsAGO2b, and OsAGO18) that accumulate transcripts coordinately with phasiRNAs, suggesting a functional relationship. By mRNA in situ analysis, we demonstrated a strong correlation between the spatiotemporal pattern of these OsAGO transcripts and phasiRNA accumulations. PMID:27702997

  14. Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism

    PubMed Central

    van der Meulen, Sjoerd B.; de Jong, Anne; Kok, Jan

    2016-01-01

    ABSTRACT RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species. PMID:26950529

  15. Age-associated changes in expression of small, noncoding RNAs, including microRNAs, in C. elegans.

    PubMed

    Kato, Masaomi; Chen, Xiaowei; Inukai, Sachi; Zhao, Hongyu; Slack, Frank J

    2011-10-01

    Small, noncoding RNAs (sncRNAs), including microRNAs (miRNAs), impact diverse biological events through the control of gene expression and genome stability. However, the role of these sncRNAs in aging remains largely unknown. To understand the contribution of sncRNAs to the aging process, we performed small RNA profiling by deep-sequencing over the course of Caenorhabditis elegans (C. elegans) aging. Many small RNAs, including a significant number of miRNAs, change their expression during aging in C. elegans. Further studies of miRNA expression changes under conditions that modify lifespan demonstrate the tight control of their expression during aging. Adult-specific loss of argonaute-like gene-1 (alg-1) activity, which is necessary for miRNA maturation and function, resulted in an abnormal lifespan, suggesting that miRNAs are, indeed, required in adulthood for normal aging. miRNA target prediction algorithms combined with transcriptome data and pathway enrichment analysis revealed likely targets of these age-associated miRNAs with known roles in aging, such as mitochondrial metabolism. Furthermore, a computational analysis of our deep-sequencing data identified additional age-associated sncRNAs, including miRNA star strands, novel miRNA candidates, and endo-siRNA sequences. We also show an increase of specific transfer RNA (tRNA) fragments during aging, which are known to be induced in response to stress in several organisms. This study suggests that sncRNAs including miRNAs contribute to lifespan regulation in C. elegans, and indicates new connections between aging, stress responses, and the small RNA world.

  16. Plant-Generated Artificial Small RNAs Mediated Aphid Resistance

    PubMed Central

    Wang, Guiling; Yang, Kun; Wang, Yu; Niu, Libo; Chen, Xiaoying; Fang, Rongxiang

    2014-01-01

    Background RNA silencing is an important mechanism for regulation of endogenous gene expression and defense against genomic intruders in plants. This natural defense system was adopted to generate virus-resistant plants even before the mechanism of RNA silencing was unveiled. With the clarification of that mechanism, transgenic antiviral plants were developed that expressed artificial virus-specific hairpin RNAs (hpRNAs) or microRNAs (amiRNAs) in host plants. Previous works also showed that plant-mediated RNA silencing technology could be a practical method for constructing insect-resistant plants by expressing hpRNAs targeting essential genes of insects. Methodology/Principal findings In this study, we chose aphid Myzus persicae of order Hemiptera as a target insect. To screen for aphid genes vulnerable to attack by plant-mediated RNA silencing to establish plant aphid resistance, we selected nine genes of M. persicae as silencing targets, and constructed their hpRNA-expressing vectors. For the acetylcholinesterase 2 coding gene (MpAChE2), two amiRNA-expressing vectors were also constructed. The vectors were transformed into tobacco plants (Nicotiana tabacum cv. Xanti). Insect challenge assays showed that most of the transgenic plants gained aphid resistance, among which those expressing hpRNAs targeting V-type proton ATPase subunit E-like (V-ATPaseE) or tubulin folding cofactor D (TBCD) genes displayed stronger aphicidal activity. The transgenic plants expressing amiRNAs targeting two different sites in the MpAChE2 gene exhibited better aphid resistance than the plants expressing MpAChE2-specific hpRNA. Conclusions/Significance Our results indicated that plant-mediated insect-RNA silencing might be an effective way to develop plants resistant to insects with piercing-sucking mouthparts, and both the selection of vulnerable target genes and the biogenetic type of the small RNAs were crucial for the effectiveness of aphid control. The expression of insect

  17. Genome-Wide Identification of Small RNAs in the Opportunistic Pathogen Enterococcus faecalis V583

    PubMed Central

    Shioya, Kouki; Michaux, Charlotte; Kuenne, Carsten; Hain, Torsten; Verneuil, Nicolas; Budin-Verneuil, Aurélie; Hartsch, Thomas; Hartke, Axel; Giard, Jean-Christophe

    2011-01-01

    Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the Gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5′ and 3′ RACE-PCR, and Northern blot analysis. Six sRNAs were specifically expressed at exponential phase, two sRNAs were observed at stationary phase, and three were detected during both phases. Searches of putative functions revealed that three of them (EFA0080_EFA0081 and EFB0062_EFB0063 on pTF1 and pTF2 plasmids, respectively, and EF0408_EF04092 located on the chromosome) are similar to antisense RNA involved in plasmid addiction modules. Moreover, EF1097_EF1098 shares strong homologies with tmRNA (bi-functional RNA acting as both a tRNA and an mRNA) and EF2205_EF2206 appears homologous to 4.5S RNA member of the Signal Recognition Particle (SRP) ribonucleoprotein complex. In addition, proteomic analysis of the ΔEF3314_EF3315 sRNA mutant suggests that it may be involved in the turnover of some abundant proteins. The expression patterns of these transcripts were evaluated by tiling array hybridizations performed with samples from cells grown under eleven different conditions some of which may be encountered during infection. Finally, distribution of these sRNAs among genome sequences of 54 E. faecalis strains was assessed. This is the first experimental genome-wide identification of sRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen. PMID:21912655

  18. Genome-wide identification of small RNAs in the opportunistic pathogen Enterococcus faecalis V583.

    PubMed

    Shioya, Kouki; Michaux, Charlotte; Kuenne, Carsten; Hain, Torsten; Verneuil, Nicolas; Budin-Verneuil, Aurélie; Hartsch, Thomas; Hartke, Axel; Giard, Jean-Christophe

    2011-01-01

    Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5' and 3' RACE-PCR, and Northern blot analysis. Six sRNAs were specifically expressed at exponential phase, two sRNAs were observed at stationary phase, and three were detected during both phases. Searches of putative functions revealed that three of them (EFA0080_EFA0081 and EFB0062_EFB0063 on pTF1 and pTF2 plasmids, respectively, and EF0408_EF04092 located on the chromosome) are similar to antisense RNA involved in plasmid addiction modules. Moreover, EF1097_EF1098 shares strong homologies with tmRNA (bi-functional RNA acting as both a tRNA and an mRNA) and EF2205_EF2206 appears homologous to 4.5S RNA member of the Signal Recognition Particle (SRP) ribonucleoprotein complex. In addition, proteomic analysis of the ΔEF3314_EF3315 sRNA mutant suggests that it may be involved in the turnover of some abundant proteins. The expression patterns of these transcripts were evaluated by tiling array hybridizations performed with samples from cells grown under eleven different conditions some of which may be encountered during infection. Finally, distribution of these sRNAs among genome sequences of 54 E. faecalis strains was assessed. This is the first experimental genome-wide identification of sRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen.

  19. Roles of small RNAs in the immune defense mechanisms of crustaceans.

    PubMed

    He, Yaodong; Ju, Chenyu; Zhang, Xiaobo

    2015-12-01

    Small RNAs, 21-24 nucleotides in length, are non-coding RNAs found in most multicellular organisms, as well as in some viruses. There are three main types of small RNAs including microRNA (miRNA), small-interfering RNA (siRNA), and piwi-interacting RNA (piRNA). Small RNAs play key roles in the genetic regulation of eukaryotes; at least 50% of all eukaryote genes are the targets of small RNAs. In recent years, studies have shown that some unique small RNAs are involved in the immune response of crustaceans, leading to lower or higher immune responses to infections and diseases. SiRNAs could be used as therapy for virus infection. In this review, we provide an overview of the diverse roles of small RNAs in the immune defense mechanisms of crustaceans.

  20. The sRNAome mining revealed existence of unique signature small RNAs derived from 5.8SrRNA from Piper nigrum and other plant lineages.

    PubMed

    Asha, Srinivasan; Soniya, E V

    2017-02-01

    Small RNAs derived from ribosomal RNAs (srRNAs) are rarely explored in the high-throughput data of plant systems. Here, we analyzed srRNAs from the deep-sequenced small RNA libraries of Piper nigrum, a unique magnoliid plant. The 5' end of the putative long form of 5.8S rRNA (5.8SLrRNA) was identified as the site for biogenesis of highly abundant srRNAs that are unique among the Piperaceae family of plants. A subsequent comparative analysis of the ninety-seven sRNAomes of diverse plants successfully uncovered the abundant existence and precise cleavage of unique rRF signature small RNAs upstream of a novel 5' consensus sequence of the 5.8S rRNA. The major cleavage process mapped identically among the different tissues of the same plant. The differential expression and cleavage of 5'5.8S srRNAs in Phytophthora capsici infected P. nigrum tissues indicated the critical biological functions of these srRNAs during stress response. The non-canonical short hairpin precursor structure, the association with Argonaute proteins, and the potential targets of 5'5.8S srRNAs reinforced their regulatory role in the RNAi pathway in plants. In addition, this novel lineage specific small RNAs may have tremendous biological potential in the taxonomic profiling of plants.

  1. The sRNAome mining revealed existence of unique signature small RNAs derived from 5.8SrRNA from Piper nigrum and other plant lineages

    PubMed Central

    Asha, Srinivasan; Soniya, E. V.

    2017-01-01

    Small RNAs derived from ribosomal RNAs (srRNAs) are rarely explored in the high-throughput data of plant systems. Here, we analyzed srRNAs from the deep-sequenced small RNA libraries of Piper nigrum, a unique magnoliid plant. The 5′ end of the putative long form of 5.8S rRNA (5.8SLrRNA) was identified as the site for biogenesis of highly abundant srRNAs that are unique among the Piperaceae family of plants. A subsequent comparative analysis of the ninety-seven sRNAomes of diverse plants successfully uncovered the abundant existence and precise cleavage of unique rRF signature small RNAs upstream of a novel 5′ consensus sequence of the 5.8S rRNA. The major cleavage process mapped identically among the different tissues of the same plant. The differential expression and cleavage of 5′5.8S srRNAs in Phytophthora capsici infected P. nigrum tissues indicated the critical biological functions of these srRNAs during stress response. The non-canonical short hairpin precursor structure, the association with Argonaute proteins, and the potential targets of 5′5.8S srRNAs reinforced their regulatory role in the RNAi pathway in plants. In addition, this novel lineage specific small RNAs may have tremendous biological potential in the taxonomic profiling of plants. PMID:28145468

  2. Identification of Small RNAs in Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    Burns, Andrew; Joachimiak, Marcin; Deutschbauer, Adam; Arkin, Adam; Bender, Kelly

    2010-05-17

    Desulfovibrio vulgaris is an anaerobic sulfate-reducing bacterium capable of facilitating the removal of toxic metals such as uranium from contaminated sites via reduction. As such, it is essential to understand the intricate regulatory cascades involved in how D. vulgaris and its relatives respond to stressors in such sites. One approach is the identification and analysis of small non-coding RNAs (sRNAs); molecules ranging in size from 20-200 nucleotides that predominantly affect gene regulation by binding to complementary mRNA in an anti-sense fashion and therefore provide an immediate regulatory response. To identify sRNAs in D. vulgaris, a bacterium that does not possess an annotated hfq gene, RNA was pooled from stationary and exponential phases, nitrate exposure, and biofilm conditions. The subsequent RNA was size fractionated, modified, and converted to cDNA for high throughput transcriptomic deep sequencing. A computational approach to identify sRNAs via the alignment of seven separate Desulfovibrio genomes was also performed. From the deep sequencing analysis, 2,296 reads between 20 and 250 nt were identified with expression above genome background. Analysis of those reads limited the number of candidates to ~;;87 intergenic, while ~;;140 appeared to be antisense to annotated open reading frames (ORFs). Further BLAST analysis of the intergenic candidates and other Desulfovibrio genomes indicated that eight candidates were likely portions of ORFs not previously annotated in the D. vulgaris genome. Comparison of the intergenic and antisense data sets to the bioinformatical predicted candidates, resulted in ~;;54 common candidates. Current approaches using Northern analysis and qRT-PCR are being used toverify expression of the candidates and to further develop the role these sRNAs play in D. vulgaris regulation.

  3. Profiling of small RNAs involved in plant-pathogen interactions.

    PubMed

    Niu, Dongdong; Wang, Zhaoyun; Wang, Shune; Qiao, Lulu; Zhao, Hongwei

    2015-01-01

    Small RNA (sRNA)-mediated gene silencing is an important gene expression regulatory mechanism conserved in eukaryotes. Such sRNAs, first discovered in plants, are involved in diverse biological processes. In plants, sRNAs participate in many growth and developmental processes, such as embryo development, seed germination, flowering, hormone synthesis and distribution, and nutrient assimilation. However, the significance of sRNA in shaping the relationship between plants and their symbiotic microbes or pathogens has been underestimated. Recent progress in profiling sRNA, especially advances in next-generation sequencing technology, has revealed its extensive and complicated involvement in interactions between plants and viruses, bacteria, and fungi. In this review, we will summarize recent findings regarding sRNA in plant-pathogen interactions.

  4. Modular synthetic inverters from zinc finger proteins and small RNAs

    DOE PAGES

    Hsia, Justin; Holtz, William J.; Maharbiz, Michel M.; ...

    2016-02-17

    Synthetic zinc finger proteins (ZFPs) can be created to target promoter DNA sequences, repressing transcription. The binding of small RNA (sRNA) to ZFP mRNA creates an ultrasensitive response to generate higher effective Hill coefficients. Here we combined three “off the shelf” ZFPs and three sRNAs to create new modular inverters in E. coli and quantify their behavior using induction fold. We found a general ordering of the effects of the ZFPs and sRNAs on induction fold that mostly held true when combining these parts. We then attempted to construct a ring oscillator using our new inverters. In conclusion, our chosenmore » parts performed insufficiently to create oscillations, but we include future directions for improvement upon our work presented here.« less

  5. Modular synthetic inverters from zinc finger proteins and small RNAs

    SciTech Connect

    Hsia, Justin; Holtz, William J.; Maharbiz, Michel M.; Arcak, Murat; Keasling, Jay D.; Rao, Christopher V.

    2016-02-17

    Synthetic zinc finger proteins (ZFPs) can be created to target promoter DNA sequences, repressing transcription. The binding of small RNA (sRNA) to ZFP mRNA creates an ultrasensitive response to generate higher effective Hill coefficients. Here we combined three “off the shelf” ZFPs and three sRNAs to create new modular inverters in E. coli and quantify their behavior using induction fold. We found a general ordering of the effects of the ZFPs and sRNAs on induction fold that mostly held true when combining these parts. We then attempted to construct a ring oscillator using our new inverters. In conclusion, our chosen parts performed insufficiently to create oscillations, but we include future directions for improvement upon our work presented here.

  6. Transposable elements and small RNAs: Genomic fuel for species diversity

    PubMed Central

    Hoffmann, Federico G; McGuire, Liam P; Counterman, Brian A; Ray, David A

    2015-01-01

    While transposable elements (TE) have long been suspected of involvement in species diversification, identifying specific roles has been difficult. We recently found evidence of TE-derived regulatory RNAs in a species-rich family of bats. The TE-derived small RNAs are temporally associated with the burst of species diversification, suggesting that they may have been involved in the processes that led to the diversification. In this commentary, we expand on the ideas that were briefly touched upon in that manuscript. Specifically, we suggest avenues of research that may help to identify the roles that TEs may play in perturbing regulatory pathways. Such research endeavors may serve to inform evolutionary biologists of the ways that TEs have influenced the genomic and taxonomic diversity around us. PMID:26904375

  7. Transposable elements and small RNAs: Genomic fuel for species diversity.

    PubMed

    Hoffmann, Federico G; McGuire, Liam P; Counterman, Brian A; Ray, David A

    2015-01-01

    While transposable elements (TE) have long been suspected of involvement in species diversification, identifying specific roles has been difficult. We recently found evidence of TE-derived regulatory RNAs in a species-rich family of bats. The TE-derived small RNAs are temporally associated with the burst of species diversification, suggesting that they may have been involved in the processes that led to the diversification. In this commentary, we expand on the ideas that were briefly touched upon in that manuscript. Specifically, we suggest avenues of research that may help to identify the roles that TEs may play in perturbing regulatory pathways. Such research endeavors may serve to inform evolutionary biologists of the ways that TEs have influenced the genomic and taxonomic diversity around us.

  8. Multiple roles for small RNAs during plant reproduction.

    PubMed

    Van Ex, Frédéric; Jacob, Yannick; Martienssen, Robert A

    2011-10-01

    Germline development and early embryogenesis in eukaryotes are characterized by large-scale genome reprogramming events. In companion cells of the Arabidopsis male gametophyte, epigenome reorganization leads to loss of heterochromatin and production of a distinct small RNA (sRNA) population. A specific class of sRNA derived from transposons appears to be mobile and can accumulate in germ cells. In the germline of maize, rice, and Arabidopsis, specific ARGONAUTE-sRNA silencing complexes appear to play key roles in reproductive development, including meiosis and regulation of germ cell fate. These results reveal new roles for sRNAs during plant reproduction and suggest that mobility of sRNAs could be critical for some of these functions.

  9. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond.

    PubMed

    Castel, Stephane E; Martienssen, Robert A

    2013-02-01

    A growing number of functions are emerging for RNA interference (RNAi) in the nucleus, in addition to well-characterized roles in post-transcriptional gene silencing in the cytoplasm. Epigenetic modifications directed by small RNAs have been shown to cause transcriptional repression in plants, fungi and animals. Additionally, increasing evidence indicates that RNAi regulates transcription through interaction with transcriptional machinery. Nuclear small RNAs include small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) and are implicated in nuclear processes such as transposon regulation, heterochromatin formation, developmental gene regulation and genome stability.

  10. A rapid, quantitative assay for direct detection of microRNAs and other small RNAs using splinted ligation

    PubMed Central

    Maroney, Patricia A.; Chamnongpol, Sangpen; Souret, Frédéric; Nilsen, Timothy W.

    2007-01-01

    The discovery and characterization of microRNAs (miRNAs) and other families of short RNAs has led to a rapid expansion of research directed at elucidating their expression patterns and regulatory functions. Here, we describe a convenient, sensitive, and straightforward method to detect and quantitate specific miRNA levels in unfractionated total RNA samples. The method, based on splinted ligation, does not require specialized equipment or any amplification step, and is significantly faster and more sensitive than Northern blotting. We demonstrate that the method can be used to detect various classes of small regulatory RNAs from different organisms. PMID:17456563

  11. In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs.

    PubMed

    Martínez de Alba, Angel Emilio; Moreno, Ana Beatriz; Gabriel, Marc; Mallory, Allison C; Christ, Aurélie; Bounon, Rémi; Balzergue, Sandrine; Aubourg, Sebastien; Gautheret, Daniel; Crespi, Martin D; Vaucheret, Hervé; Maizel, Alexis

    2015-03-11

    Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5' cap structure and their subsequent 5' to 3' degradation in cytoplasmic processing (P-)bodies. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to double-stranded RNAs by the cellular RNA-dependent RNA polymerase 6 (RDR6) in cytoplasmic siRNA-bodies. PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts. We show that the lethality of decapping mutants is suppressed by impairing RDR6 activity. We establish that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of rqc-siRNAs, that over-accumulate when RQC processes are impaired, a subset of which depending on RDR6 for their production. We observe that P- and siRNA-bodies often are dynamically juxtaposed, potentially allowing for cross-talk of the two machineries. Our results suggest that the decapping of endogenous RNA limits their entry into the PTGS pathway. We anticipate that the rqc-siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs.

  12. In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs

    PubMed Central

    Martínez de Alba, Angel Emilio; Moreno, Ana Beatriz; Gabriel, Marc; Mallory, Allison C.; Christ, Aurélie; Bounon, Rémi; Balzergue, Sandrine; Aubourg, Sebastien; Gautheret, Daniel; Crespi, Martin D.; Vaucheret, Hervé; Maizel, Alexis

    2015-01-01

    Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5′ cap structure and their subsequent 5′ to 3′ degradation in cytoplasmic processing (P-)bodies. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to double-stranded RNAs by the cellular RNA-dependent RNA polymerase 6 (RDR6) in cytoplasmic siRNA-bodies. PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts. We show that the lethality of decapping mutants is suppressed by impairing RDR6 activity. We establish that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of rqc-siRNAs, that over-accumulate when RQC processes are impaired, a subset of which depending on RDR6 for their production. We observe that P- and siRNA-bodies often are dynamically juxtaposed, potentially allowing for cross-talk of the two machineries. Our results suggest that the decapping of endogenous RNA limits their entry into the PTGS pathway. We anticipate that the rqc-siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs. PMID:25694514

  13. Small RNAs Derived from the T-DNA of Agrobacterium rhizogenes in Hairy Roots of Phaseolus vulgaris

    PubMed Central

    Peláez, Pablo; Hernández-López, Alejandrina; Estrada-Navarrete, Georgina; Sanchez, Federico

    2017-01-01

    Agrobacterium rhizogenes is a pathogenic bacteria that causes hairy root disease by transferring bacterial DNA into the plant genome. It is an essential tool for industry and research due to its capacity to produce genetically modified roots and whole organisms. Here, we identified and characterized small RNAs generated from the transfer DNA (T-DNA) of A. rhizogenes in hairy roots of common bean (Phaseolus vulgaris). Distinct abundant A. rhizogenes T-DNA-derived small RNAs (ArT-sRNAs) belonging to several oncogenes were detected in hairy roots using high-throughput sequencing. The most abundant and diverse species of ArT-sRNAs were those of 21- and 22-nucleotides in length. Many T-DNA encoded genes constituted phasiRNA producing loci (PHAS loci). Interestingly, degradome analysis revealed that ArT-sRNAs potentially target genes of P. vulgaris. In addition, we detected low levels of ArT-sRNAs in the A. rhizogenes-induced calli generated at the wound site before hairy root emergence. These results suggest that RNA silencing targets several genes from T-DNA of A. rhizogenes in hairy roots of common bean. Therefore, the role of RNA silencing observed in this study has implications in our understanding and usage of this unique plant-bacteria interaction. PMID:28203245

  14. Small RNAs Derived from the T-DNA of Agrobacterium rhizogenes in Hairy Roots of Phaseolus vulgaris.

    PubMed

    Peláez, Pablo; Hernández-López, Alejandrina; Estrada-Navarrete, Georgina; Sanchez, Federico

    2017-01-01

    Agrobacterium rhizogenes is a pathogenic bacteria that causes hairy root disease by transferring bacterial DNA into the plant genome. It is an essential tool for industry and research due to its capacity to produce genetically modified roots and whole organisms. Here, we identified and characterized small RNAs generated from the transfer DNA (T-DNA) of A. rhizogenes in hairy roots of common bean (Phaseolus vulgaris). Distinct abundant A. rhizogenes T-DNA-derived small RNAs (ArT-sRNAs) belonging to several oncogenes were detected in hairy roots using high-throughput sequencing. The most abundant and diverse species of ArT-sRNAs were those of 21- and 22-nucleotides in length. Many T-DNA encoded genes constituted phasiRNA producing loci (PHAS loci). Interestingly, degradome analysis revealed that ArT-sRNAs potentially target genes of P. vulgaris. In addition, we detected low levels of ArT-sRNAs in the A. rhizogenes-induced calli generated at the wound site before hairy root emergence. These results suggest that RNA silencing targets several genes from T-DNA of A. rhizogenes in hairy roots of common bean. Therefore, the role of RNA silencing observed in this study has implications in our understanding and usage of this unique plant-bacteria interaction.

  15. Identification and Characterization of Small Noncoding RNAs in Genome Sequences of the Edible Fungus Pleurotus ostreatus

    PubMed Central

    Zhao, Mengran; Hsiang, Tom; Feng, Xiaoxing

    2016-01-01

    Noncoding RNAs (ncRNAs) have been identified in many fungi. However, no genome-scale identification of ncRNAs has been inventoried for basidiomycetes. In this research, we detected 254 small noncoding RNAs (sncRNAs) in a genome assembly of an isolate (CCEF00389) of Pleurotus ostreatus, which is a widely cultivated edible basidiomycetous fungus worldwide. The identified sncRNAs include snRNAs, snoRNAs, tRNAs, and miRNAs. SnRNA U1 was not found in CCEF00389 genome assembly and some other basidiomycetous genomes by BLASTn. This implies that if snRNA U1 of basidiomycetes exists, it has a sequence that varies significantly from other organisms. By analyzing the distribution of sncRNA loci, we found that snRNAs and most tRNAs (88.6%) were located in pseudo-UTR regions, while miRNAs are commonly found in introns. To analyze the evolutionary conservation of the sncRNAs in P. ostreatus, we aligned all 254 sncRNAs to the genome assemblies of some other Agaricomycotina fungi. The results suggest that most sncRNAs (77.56%) were highly conserved in P. ostreatus, and 20% were conserved in Agaricomycotina fungi. These findings indicate that most sncRNAs of P. ostreatus were not conserved across Agaricomycotina fungi. PMID:27703969

  16. Small Comet Abundance and Solar System Location

    NASA Astrophysics Data System (ADS)

    Phillips, C. B.; Moore, J.; Zahnle, K.

    2001-12-01

    We present geological, historical, and observational evidence which implies that the proportion of small comets is related to where in the solar system the population is counted. A detailed count of craters in the 1-5 km size range on Jupiter's moon Europa yields a slope of -1.45 for the cumulative size-frequency crater distribution. This is strongly depleted in small objects relative to the far outer solar system. We compare the Europan distribution to crater counts on Neptune's moon Triton and extrapolated observational evidence of Kuiper Belt Objects. We find that small comets appear to be rarer at Triton than in the Kuiper Belt, and that small comets are five times rarer at Europa than they appear to be at Triton. We have extended our study into the inner solar system by considering the discovery histories of long and short period comets. We find that, while large comets have been discovered at ever-increasing rates over the last few centuries, small comets have not paralleled this increase. In one example, the known number of small Jupiter-family comets in Earth-crossing orbits has not changed significantly in the two and a half centuries since comet detection has been intensely pursued. In another example, we focus on the sungrazing comets as found by the SOHO satellite and otherwise. Many of the sungrazers are undoubtedly small comets. But 94 of the 95 known sungrazers follow the same orbit - i.e., they are fragments of one great comet disrupted by close passage to the Sun long ago. Thus SOHO could see unique small comets, if they were present, and so the number of unique small objects in the inner solar system is few to none. Small comets are therefore found relatively frequently in the outer solar system, but their numbers decrease by the orbit of Jupiter, and they are very rare in the inner solar system. One explanation for this decrease is the preferential sublimation and coupled sublimation-enhancing disaggregation of small icy bodies as they approach

  17. Exploration of small RNA-seq data for small non-coding RNAs in Human Colorectal Cancer

    PubMed Central

    Koduru, Srinivas V; Tiwari, Amit K; Hazard, Sprague W; Mahajan, Milind; Ravnic, Dino J

    2017-01-01

    Background: Improved healthcare and recent breakthroughs in technology have substantially reduced cancer mortality rates worldwide. Recent advancements in next-generation sequencing (NGS) have allowed genomic analysis of the human transcriptome. Now, using NGS we can further look into small non-coding regions of RNAs (sncRNAs) such as microRNAs (miRNAs), Piwi-interacting-RNAs (piRNAs), long non-coding RNAs (lncRNAs), and small nuclear/nucleolar RNAs (sn/snoRNAs) among others. Recent studies looking at sncRNAs indicate their role in important biological processes such as cancer progression and predict their role as biomarkers for disease diagnosis, prognosis, and therapy. Results: In the present study, we data mined publically available small RNA sequencing data from colorectal tissue samples of eight matched patients (benign, tumor, and metastasis) and remapped the data for various small RNA annotations. We identified aberrant expression of 13 miRNAs in tumor and metastasis specimens [tumor vs benign group (19 miRNAs) and metastasis vs benign group (38 miRNAs)] of which five were upregulated, and eight were downregulated, during disease progression. Pathway analysis of aberrantly expressed miRNAs showed that the majority of miRNAs involved in colon cancer were also involved in other cancers. Analysis of piRNAs revealed six to be over-expressed in the tumor vs benign cohort and 24 in the metastasis vs benign group. Only two piRNAs were shared between the two cohorts. Examining other types of small RNAs [sn/snoRNAs, mt_rRNA, miscRNA, nonsense mediated decay (NMD), and rRNAs] identified 15 sncRNAs in the tumor vs benign group and 104 in the metastasis vs benign group, with only four others being commonly expressed. Conclusion: In summary, our comprehensive analysis on publicly available small RNA-seq data identified multiple differentially expressed sncRNAs during colorectal cancer progression at different stages compared to normal colon tissue. We speculate that

  18. Isolation and detection of small RNAs from plant tissues.

    PubMed

    Smith, Neil A; Eamens, Andrew L

    2012-01-01

    In plants, several classes of non-coding small RNA (sRNA) have been shown to be important regulators of gene expression in a wide variety of biological processes. The two main classes of sRNA, the small-interfering RNA (siRNA) and microRNA (miRNA) classes, are well documented and several experimental approaches have been developed to allow for their routine isolation and detection from plant tissues. Here, we describe the current methods used for the isolation of total RNA and the subsequent enrichment of low-molecular-weight (LMW) RNA species, as well as to outline how sRNAs are detected from such nucleic acid preparations.

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

    PubMed

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

    2014-01-01

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

  20. Small Cajal body-specific RNAs of Drosophila function in the absence of Cajal bodies.

    PubMed

    Deryusheva, Svetlana; Gall, Joseph G

    2009-12-01

    During their biogenesis small nuclear RNAs (snRNAs) undergo multiple covalent modifications that require guide RNAs to direct methylase and pseudouridylase enzymes to the appropriate nucleotides. Because of their localization in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific RNAs (scaRNAs). Using a fluorescent primer extension technique, we mapped the modified nucleotides in Drosophila U1, U2, U4, and U5 snRNAs. By fluorescent in situ hybridization (FISH) we showed that seven Drosophila scaRNAs are concentrated in easily detectable CBs. We used two assays based on Xenopus oocyte nuclei to demonstrate that three of these Drosophila scaRNAs do, in fact, function as guide RNAs. In flies null for the CB marker protein coilin, CBs are absent and there are no localized FISH signals for the scaRNAs. Nevertheless, biochemical experiments show that scaRNAs are present at normal levels and snRNAs are properly modified. Our experiments demonstrate that several scaRNAs are concentrated as expected in the CBs of wild-type Drosophila, but they function equally well in the nucleoplasm of mutant flies that lack CBs. We propose that the snRNA modification machinery is not limited to CBs, but is dispersed throughout the nucleoplasm of cells in general.

  1. 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

  2. RNA sequencing reveals small RNAs differentially expressed between incipient Japanese threespine sticklebacks

    PubMed Central

    2013-01-01

    Background Non-coding small RNAs, ranging from 20 to 30 nucleotides in length, mediate the regulation of gene expression and play important roles in many biological processes. One class of small RNAs, microRNAs (miRNAs), are highly conserved across taxa and mediate the regulation of the chromatin state and the post-transcriptional regulation of messenger RNA (mRNA). Another class of small RNAs is the Piwi-interacting RNAs, which play important roles in the silencing of transposons and other functional genes. Although the biological functions of the different small RNAs have been elucidated in several laboratory animals, little is known regarding naturally occurring variation in small RNA transcriptomes among closely related species. Results We employed next-generation sequencing technology to compare the expression profiles of brain small RNAs between sympatric species of the Japanese threespine stickleback (Gasterosteus aculeatus). We identified several small RNAs that were differentially expressed between sympatric Pacific Ocean and Japan Sea sticklebacks. Potential targets of several small RNAs were identified as repetitive sequences. Female-biased miRNA expression from the old X chromosome was also observed, and it was attributed to the degeneration of the Y chromosome. Conclusions Our results suggest that expression patterns of small RNA can differ between incipient species and may be a potential mechanism underlying differential mRNA expression and transposon activity. PMID:23547919

  3. 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

  4. Roles of small RNAs in soybean defense against Phytophthora sojae infection.

    PubMed

    Wong, James; Gao, Lei; Yang, Yang; Zhai, Jixian; Arikit, Siwaret; Yu, Yu; Duan, Shuyi; Chan, Vicky; Xiong, Qin; Yan, Jun; Li, Shengben; Liu, Renyi; Wang, Yuanchao; Tang, Guiliang; Meyers, Blake C; Chen, Xuemei; Ma, Wenbo

    2014-09-01

    The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat-inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding-leucine rich repeat proteins and genes encoding pentatricopeptide repeat-containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense-associated genes in soybean during Phytophthora infection.

  5. Small RNAs and heritable epigenetic variation in plants.

    PubMed

    Bond, Donna M; Baulcombe, David C

    2014-02-01

    Recent studies suggest that inheritance of phenotypes in plants is more likely to involve epigenetics than in mammals. There are two reasons for this difference. First, there is a RNA-based system in plants involving small (s)RNAs that influences de novo establishment and maintenance of DNA methylation at many sites in plant genomes. These regions of methylated DNA are epigenetic marks with the potential to affect gene expression that are transmitted between dividing cells of the same generation. Second, unlike mammals, DNA methyltransferases in plants are active during gametogenesis and embryogenesis so that patterns of DNA methylation can persist from parent to progeny and do not need to be reset. We discuss how the effects of stress and genome interactions in hybrid plants are two systems that illustrate how RNA-based mechanisms can influence heritable phenotypes in plants.

  6. Big roles for small RNAs in polyploidy, hybrid vigor, and hybrid incompatibility.

    PubMed

    Ng, Danny W-K; Lu, Jie; Chen, Z Jeffrey

    2012-04-01

    Small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and trans-acting siRNAs (ta-siRNAs), mediate gene expression and epigenetic regulation. While siRNAs are highly diverged, miRNAs and ta-siRNAs are generally conserved but many are differentially expressed between related species and in interspecific hybrids and allopolyploids. On one hand, combination of diverged maternal and paternal siRNAs in the same nucleus may exert cis-acting and trans-acting effects on transposable elements (TEs) and TE-associated genes, leading to genomic instability and endosperm and embryo failures, constituting a bottleneck for the evolution of hybrids and polyploids. On the other hand, cis and trans-acting small RNAs induce quantitative and qualitative changes in epigenetic regulation, leading to morphological variation and hybrid vigor in F1 hybrids and stable allopolyploids as well as transgressive phenotypes in the progeny, increasing a potential for adaptive evolution.

  7. Biogenic mechanisms and utilization of small RNAs derived from human protein-coding genes.

    PubMed

    Valen, Eivind; Preker, Pascal; Andersen, Peter Refsing; Zhao, Xiaobei; Chen, Yun; Ender, Christine; Dueck, Anne; Meister, Gunter; Sandelin, Albin; Jensen, Torben Heick

    2011-08-07

    Efforts to catalog eukaryotic transcripts have uncovered many small RNAs (sRNAs) derived from gene termini and splice sites. Their biogenesis pathways are largely unknown, but a mechanism based on backtracking of RNA polymerase II (RNAPII) has been suggested. By sequencing transcripts 12-100 nucleotides in length from cells depleted of major RNA degradation enzymes and RNAs associated with Argonaute (AGO1/2) effector proteins, we provide mechanistic models for sRNA production. We suggest that neither splice site-associated (SSa) nor transcription start site-associated (TSSa) RNAs arise from RNAPII backtracking. Instead, SSa RNAs are largely degradation products of splicing intermediates, whereas TSSa RNAs probably derive from nascent RNAs protected by stalled RNAPII against nucleolysis. We also reveal new AGO1/2-associated RNAs derived from 3' ends of introns and from mRNA 3' UTRs that appear to draw from noncanonical microRNA biogenesis pathways.

  8. Endogenous Small-Noncoding RNAs and Potential Functions in Desiccation Tolerance in Physcomitrella Patens

    PubMed Central

    Xia, Jing; Wang, Xiaoqin; Perroud, Pierre-François; He, Yikun; Quatrano, Ralph; Zhang, Weixiong

    2016-01-01

    Early land plants like moss Physcomitrella patens have developed remarkable drought tolerance. Phytohormone abscisic acid (ABA) protects seeds during water stress by activating genes through transcription factors such as ABSCISIC ACID INSENSITIVE (ABI3). Small noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs), are key gene regulators in eukaryotes, playing critical roles in stress tolerance in plants. Combining next-generation sequencing and computational analysis, we profiled and characterized sncRNA species from two ABI3 deletion mutants and the wild type P. patens that were subject to ABA treatment in dehydration and rehydration stages. Small RNA profiling using deep sequencing helped identify 22 novel miRNAs and 6 genomic loci producing trans-acting siRNAs (ta-siRNAs) including TAS3a to TAS3e and TAS6. Data from degradome profiling showed that ABI3 genes (ABI3a/b/c) are potentially regulated by the plant-specific miR536 and that other ABA-relevant genes are regulated by miRNAs and ta-siRNAs. We also observed broad variations of miRNAs and ta-siRNAs expression across different stages, suggesting that they could potentially influence desiccation tolerance. This study provided evidence on the potential roles of sncRNA in mediating desiccation-responsive pathways in early land plants. PMID:27443635

  9. Novel microRNA-like viral small regulatory RNAs arising during human hepatitis A virus infection.

    PubMed

    Shi, Jiandong; Sun, Jing; Wang, Bin; Wu, Meini; Zhang, Jing; Duan, Zhiqing; Wang, Haixuan; Hu, Ningzhu; Hu, Yunzhang

    2014-10-01

    MicroRNAs (miRNAs), including host miRNAs and viral miRNAs, play vital roles in regulating host-virus interactions. DNA viruses encode miRNAs that regulate the viral life cycle. However, it is generally believed that cytoplasmic RNA viruses do not encode miRNAs, owing to inaccessible cellular miRNA processing machinery. Here, we provide a comprehensive genome-wide analysis and identification of miRNAs that were derived from hepatitis A virus (HAV; Hu/China/H2/1982), which is a typical cytoplasmic RNA virus. Using deep-sequencing and in silico approaches, we identified 2 novel virally encoded miRNAs, named hav-miR-1-5p and hav-miR-2-5p. Both of the novel virally encoded miRNAs were clearly detected in infected cells. Analysis of Dicer enzyme silencing demonstrated that HAV-derived miRNA biogenesis is Dicer dependent. Furthermore, we confirmed that HAV mature miRNAs were generated from viral miRNA precursors (pre-miRNAs) in host cells. Notably, naturally derived HAV miRNAs were biologically and functionally active and induced post-transcriptional gene silencing (PTGS). Genomic location analysis revealed novel miRNAs located in the coding region of the viral genome. Overall, our results show that HAV naturally generates functional miRNA-like small regulatory RNAs during infection. This is the first report of miRNAs derived from the coding region of genomic RNA of a cytoplasmic RNA virus. These observations demonstrate that a cytoplasmic RNA virus can naturally generate functional miRNAs, as DNA viruses do. These findings also contribute to improved understanding of host-RNA virus interactions mediated by RNA virus-derived miRNAs.

  10. Small nucleolar RNAs that guide modification in trypanosomatids: repertoire, targets, genome organisation, and unique functions.

    PubMed

    Uliel, Shai; Liang, Xue-hai; Unger, Ron; Michaeli, Shulamit

    2004-03-29

    Small nucleolar RNAs constitute a family of newly discovered non-coding small RNAs, most of which function in guiding RNA modifications. Two prevalent types of modifications are 2'-O-methylation and pseudouridylation. The modification is directed by the formation of a canonical small nucleolar RNA-target duplex. Initially, RNA-guided modification was shown to take place on rRNA, but recent studies suggest that small nuclear RNA, mRNA, tRNA, and the trypanosome spliced leader RNA also undergo guided modifications. Trypanosomes contain more modifications and potentially more small nucleolar RNAs than yeast, and the increased number of modifications may help to preserve ribosome function under adverse environmental conditions during the cycling between the insect and mammalian host. The genome organisation in clusters carrying the two types of small nucleolar RNAs, C/D and H/ACA-like RNAs, resembles that in plants. However, the trypanosomatid H/ACA RNAs are similar to those found in Archaea and are composed of a single hairpin that may represent the primordial H/ACA RNA. In this review we summarise this new field of trypanosome small nucleolar RNAs, emphasising the open questions regarding the number of small nucleolar RNAs, the repertoire, genome organisation, and the unique function of guided modifications in these protozoan parasites.

  11. Sensitive whole mount in situ localization of small RNAs in plants.

    PubMed

    Ghosh Dastidar, Mouli; Mosiolek, Magdalena; Bleckmann, Andrea; Dresselhaus, Thomas; Nodine, Michael D; Maizel, Alexis

    2016-11-01

    Small RNAs, such as microRNAs (miRNAs), regulate gene expression and play important roles in many plant processes. Although our knowledge of their biogenesis and mode of action has significantly progressed, we still have comparatively little information about their biological functions. In particular, knowledge about their spatio-temporal expression patterns rely on either indirect detection by use of reporter constructs or labor-intensive direct detection by in situ hybridization on sectioned material. None of the current approaches allows a systematic investigation of small RNA expression patterns. Here, we present a sensitive method for in situ detection of miRNAs and siRNAs in intact plant tissues that utilizes both double-labeled probes and a specific cross-linker. We determined the expression patterns of several small RNAs in diverse plant tissues.

  12. High throughput sequencing of small RNAs transcriptomes in two Crassostrea oysters identifies microRNAs involved in osmotic stress response

    PubMed Central

    Zhao, Xuelin; Yu, Hong; Kong, Lingfeng; Liu, Shikai; Li, Qi

    2016-01-01

    Increasing evidence suggests that microRNAs post-transcriptionally regulate gene expression and are involved in responses to biotic and abiotic stress. However, the role of miRNAs involved in osmotic plasticity remains largely unknown in marine bivalves. In the present study, we performed low salinity challenge with two Crassostrea species (C. gigas and C. hongkongensis), and conducted high-throughput sequencing of four small RNA libraries constructed from the gill tissues. A total of 202 and 87 miRNAs were identified from C. gigas and C. hongkongensis, respectively. Six miRNAs in C. gigas and two in C. hongkongensis were differentially expressed in response to osmotic stress. The expression profiles of these eight miRNAs were validated by qRT-PCR. Based on GO enrichment and KEGG pathway analysis, genes associated with microtubule-based process and cellular component movement were enriched in both species. In addition, five miRNA-mRNA interaction pairs that showed opposite expression patterns were identified in the C. hongkongensis, Differential expression analysis identified the miRNAs that play important regulatory roles in response to low salinity stress, providing insights into molecular mechanisms that are essential for salinity tolerance in marine bivalves. PMID:26940974

  13. Biogenesis, Function, and Applications of Virus-Derived Small RNAs in Plants

    PubMed Central

    Zhang, Chao; Wu, Zujian; Li, Yi; Wu, Jianguo

    2015-01-01

    RNA silencing, an evolutionarily conserved and sequence-specific gene-inactivation system, has a pivotal role in antiviral defense in most eukaryotic organisms. In plants, a class of exogenous small RNAs (sRNAs) originating from the infecting virus called virus-derived small interfering RNAs (vsiRNAs) are predominantly responsible for RNA silencing-mediated antiviral immunity. Nowadays, the process of vsiRNA formation and the role of vsiRNAs in plant viral defense have been revealed through deep sequencing of sRNAs and diverse genetic analysis. The biogenesis of vsiRNAs is analogous to that of endogenous sRNAs, which require diverse essential components including dicer-like (DCL), argonaute (AGO), and RNA-dependent RNA polymerase (RDR) proteins. vsiRNAs trigger antiviral defense through post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) of viral RNA, and they hijack the host RNA silencing system to target complementary host transcripts. Additionally, several applications that take advantage of the current knowledge of vsiRNAs research are being used, such as breeding antiviral plants through genetic engineering technology, reconstructing of viral genomes, and surveying viral ecology and populations. Here, we will provide an overview of vsiRNA pathways, with a primary focus on the advances in vsiRNA biogenesis and function, and discuss their potential applications as well as the future challenges in vsiRNAs research. PMID:26617580

  14. New and emerging roles of small RNAs in neurodegeneration, muscle, cardiovascular and inflammatory diseases.

    PubMed

    Hruska-Plochan, Marian; Li, Bei; Kyburz, Diego; Krützfeld, Jan; Landmesser, Ulf; Aguzzi, Adriano; Polymenidou, Magdalini

    2015-01-01

    Small noncoding RNAs (snRNAs) were discovered more than two decades ago, yet it was not until relatively recently that their important role in genome regulation was recognised. With such a substantial role in genome regulation, it is not surprising that snRNAs are crucial contributors to an ever-increasing number of diseases, as evidenced by the long list of published studies. Currently, microRNAs (miRNAs) represent the most intensively studied snRNAs. Dysregulation of miRNAs has been confirmed in numerous diseases, and changes in their levels could play an essential role in disease onset and progression and could be used for prognosis and potential therapy. Indeed, disease-altered miRNAs may either signify a direct trigger or a consequence of the disease. Therefore, miRNAs represent unique targets for disease intervention through their down- or up-regulation. Importantly, miRNAs may facilitate disease monitoring by detection of disease-altered miRNAs in easily accessible bodily fluids, such as blood or cerebrospinal fluid. Therefore, study of these events is of utmost importance for understanding the molecular mechanisms that drive disease, as well as for diagnosis and therapy. Here we attempted to synthesise a large number of studies to highlight the crucial role of miRNAs in the pathogenesis of neurodegenerative, muscle, cardiovascular and inflammatory diseases.

  15. Small RNAs: the qualified candidates for gene manipulation in diverse clinical pathologies.

    PubMed

    Kaur, Indu Pal; Chopra, Kanwaljit; Rishi, Parveen; Puri, Sanjeev; Sharma, Gaurav

    2014-01-01

    In recent years, RNA interference (RNAi) has become a tool of choice to analyze and target the in vitro and in vivo function of mammalian genes. RNAi down-regulates gene expression by inducing enzyme-dependent degradation of targeted mRNA. This can be achieved using small double-stranded RNAs (dsRNAs), including small interfering RNAs (siRNAs), micro RNAs, and piwi RNAs. These active, small dsRNAs can regulate endogenous genes in both somatic and germ cells such that they can defend the genome from invasive nucleic acids. Extension of the concept of RNAi to preclinical studies indicates its probable application in the treatment of cancers, viral infections, arterial stenosis, and genetic disorders. Exciting results from ongoing clinical trials have raised the expectations of the scientific community for the use of RNAi in "real cures". Rational pharmaceutical design of these molecules can further open a Pandora's box of newer therapeutic options. However, efficient delivery of these small dsRNAs to target tissues or cells and their unanticipated nonspecific effects comprise a few important issues that still need to be addressed. In this review, we offer an overview of different small dsRNAs, their biogenesis, and their applicability in therapeutics. Current delivery strategies and formulation techniques to achieve the desired transfection capability and RNAi products in clinical trials are also discussed.

  16. Phytophthora infestans Argonaute 1 binds microRNA and small RNAs from effector genes and transposable elements.

    PubMed

    Åsman, Anna K M; Fogelqvist, Johan; Vetukuri, Ramesh R; Dixelius, Christina

    2016-08-01

    Phytophthora spp. encode large sets of effector proteins and distinct populations of small RNAs (sRNAs). Recent evidence has suggested that pathogen-derived sRNAs can modulate the expression of plant defense genes. Here, we studied the sRNA classes and functions associated with Phytophthora infestans Argonaute (Ago) proteins. sRNAs were co-immunoprecipitated with three PiAgo proteins and deep sequenced. Twenty- to twenty-two-nucleotide (nt) sRNAs were identified as the main interaction partners of PiAgo1 and high enrichment of 24-26-nt sRNAs was seen in the PiAgo4-bound sample. The frequencies and sizes of transposable element (TE)-derived sRNAs in the different PiAgo libraries suggested diversified roles of the PiAgo proteins in the control of different TE classes. We further provide evidence for the involvement of PiAgo1 in the P. infestans microRNA (miRNA) pathway. Protein-coding genes are probably regulated by the shared action of PiAgo1 and PiAgo5, as demonstrated by analysis of differential expression. An abundance of sRNAs from genes encoding host cell death-inducing Crinkler (CRN) effectors was bound to PiAgo1, implicating this protein in the regulation of the expanded CRN gene family. The data suggest that PiAgo1 plays an essential role in gene regulation and that at least two RNA silencing pathways regulate TEs in the plant-pathogenic oomycete P. infestans.

  17. MicroRNAs and PIWI-interacting RNAs in oncology

    PubMed Central

    Liu, Yong

    2016-01-01

    RNA molecules that are unable to translate into proteins are classified as non-coding RNA. Non-coding RNA (ncRNA) genes include highly abundant and functionally important RNAs such as transfer RNAs, microRNAs (miRNAs), siRNAs, snRNAs, exRNAs and piRNAs. The number of ncRNAs encoded within the human genome is unknown; however, recent transcriptomic and bioinformatic studies suggest the existence of thousands of ncRNAs. Furthermore, small ncRNAs, including miRNAs and PIWI-interacting RNAs (piRNAs), play an imperative role in the regulation of gene expression of numerous biological and pathological processes. Investigation into the expression and function of small RNA in cancer cells has contributed to gaining a greater understanding of the roles of small RNAs in carcinogenesis. The present review is aimed primarily to discuss the importance of the expression and functions of these small RNAs in carcinogenesis. These studies may provide useful information for future therapies in cancer. PMID:27698791

  18. Identification of associations between small molecule drugs and miRNAs based on functional similarity

    PubMed Central

    Dai, EnYu; Yang, Feng; Wang, Shuyuan; Chen, Xiaowen; Yang, Lei; Wang, Yuwen; Jiang, Wei

    2016-01-01

    MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate gene expression at post-transcriptional level. Increasing evidences show aberrant expression of miRNAs in varieties of diseases. Targeting the dysregulated miRNAs with small molecule drugs has become a novel therapy for many human diseases, especially cancer. Here, we proposed a novel computational approach to identify associations between small molecules and miRNAs based on functional similarity of differentially expressed genes. At the significance level of p < 0.01, we constructed the small molecule and miRNA functional similarity network involving 111 small molecules and 20 miRNAs. Moreover, we also predicted associations between drugs and diseases through integrating our identified small molecule-miRNA associations with experimentally validated disease related miRNAs. As a result, we identified 2265 associations between FDA approved drugs and diseases, in which ~35% associations have been validated by comprehensive literature reviews. For breast cancer, we identified 19 potential drugs, in which 12 drugs were supported by previous studies. In addition, we performed survival analysis for the patients from TCGA and GEO database, which indicated that the associated miRNAs of 4 drugs might be good prognosis markers in breast cancer. Collectively, this study proposed a novel approach to predict small molecule and miRNA associations based on functional similarity, which may pave a new way for miRNA-targeted therapy and drug repositioning. PMID:27232942

  19. The tRNA-Derived Small RNAs Regulate Gene Expression through Triggering Sequence-Specific Degradation of Target Transcripts in the Oomycete Pathogen Phytophthora sojae

    PubMed Central

    Wang, Qinhu; Li, Tingting; Xu, Ke; Zhang, Wei; Wang, Xiaolong; Quan, Junli; Jin, Weibo; Zhang, Meixiang; Fan, Guangjin; Wang, Ming-Bo; Shan, Weixing

    2016-01-01

    Along with the well-studied microRNA (miRNA) and small interfering RNA (siRNA) is a new class of transfer RNA-derived small RNA (tsRNA), which has recently been detected in multiple organisms and is implicated in gene regulation. However, while miRNAs and siRNAs are known to repress gene expression through sequence-specific RNA cleavage or translational repression, how tsRNAs regulate gene expression remains unclear. Here we report the identification and functional characterization of tsRNAs in the oomycete pathogen Phytophthora sojae. We show that multiple tRNAs are processed into abundant tsRNAs, which accumulate in a similar developmental stage-specific manner and are negatively correlated with the expression of predicted target genes. Degradome sequencing and 5′ RLM RACE experiments indicate tsRNAs can trigger degradation of target transcripts. Transient expression assays using GUS sensor constructs confirmed the requirement of sequence complementarity in tsRNA-mediated RNA degradation in P. sojae. Our results show that the tsRNA are a class of functional endogenous sRNAs and suggest that tsRNA regulate gene expression through inducing sequence-specific degradation of target RNAs in oomycetes. PMID:28066490

  20. Small regulatory RNAs from low-GC Gram-positive bacteria

    PubMed Central

    Brantl, Sabine; Brückner, Reinhold

    2014-01-01

    Small regulatory RNAs (sRNAs) that act by base-pairing were first discovered in so-called accessory DNA elements—plasmids, phages, and transposons—where they control replication, maintenance, and transposition. Since 2001, a huge body of work has been performed to predict and identify sRNAs in a multitude of bacterial genomes. The majority of chromosome-encoded sRNAs have been investigated in E. coli and other Gram-negative bacteria. However, during the past five years an increasing number of sRNAs were found in Gram-positive bacteria. Here, we outline our current knowledge on chromosome-encoded sRNAs from low-GC Gram-positive species that act by base-pairing, i.e., an antisense mechanism. We will focus on sRNAs with known targets and defined regulatory mechanisms with special emphasis on Bacillus subtilis. PMID:24576839

  1. 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/.

  2. Small RNA deep sequencing identifies novel and salt-stress-regulated microRNAs from roots of Medicago sativa and Medicago truncatula.

    PubMed

    Long, Rui-Cai; Li, Ming-Na; Kang, Jun-Mei; Zhang, Tie-Jun; Sun, Yan; Yang, Qing-Chuan

    2015-05-01

    Small 21- to 24-nucleotide (nt) ribonucleic acids (RNAs), notably the microRNA (miRNA), are emerging as a posttranscriptional regulation mechanism. Salt stress is one of the primary abiotic stresses that cause the crop losses worldwide. In saline lands, root growth and function of plant are determined by the action of environmental salt stress through specific genes that adapt root development to the restrictive condition. To elucidate the role of miRNAs in salt stress regulation in Medicago, we used a high-throughput sequencing approach to analyze four small RNA libraries from roots of Zhongmu-1 (Medicago sativa) and Jemalong A17 (Medicago truncatula), which were treated with 300 mM NaCl for 0 and 8 h. Each library generated about 20 million short sequences and contained predominantly small RNAs of 24-nt length, followed by 21-nt and 22-nt small RNAs. Using sequence analysis, we identified 385 conserved miRNAs from 96 families, along with 68 novel candidate miRNAs. Of all the 68 predicted novel miRNAs, 15 miRNAs were identified to have miRNA*. Statistical analysis on abundance of sequencing read revealed specific miRNA showing contrasting expression patterns between M. sativa and M. truncatula roots, as well as between roots treated for 0 and 8 h. The expression of 10 conserved and novel miRNAs was also quantified by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The miRNA precursor and target genes were predicted by bioinformatics analysis. We concluded that the salt stress related conserved and novel miRNAs may have a large variety of target mRNAs, some of which might play key roles in salt stress regulation of Medicago.

  3. LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance.

    PubMed

    Wilbert, Melissa L; Huelga, Stephanie C; Kapeli, Katannya; Stark, Thomas J; Liang, Tiffany Y; Chen, Stella X; Yan, Bernice Y; Nathanson, Jason L; Hutt, Kasey R; Lovci, Michael T; Kazan, Hilal; Vu, Anthony Q; Massirer, Katlin B; Morris, Quaid; Hoon, Shawn; Yeo, Gene W

    2012-10-26

    LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.

  4. LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance

    PubMed Central

    Wilbert, Melissa L.; Huelga, Stephanie C.; Kapeli, Katannya; Stark, Thomas J.; Liang, Tiffany Y.; Chen, Stella X.; Yan, Bernice Y.; Nathanson, Jason L.; Hutt, Kasey R.; Lovci, Michael T.; Kazan, Hilal; Vu, Anthony Q; Massirer, Katlin B.; Morris, Quaid; Hoon, Shawn; Yeo, Gene W.

    2012-01-01

    SUMMARY LIN28 is a conserved RNA binding protein implicated in pluripotency, reprogramming and oncogenesis. Previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through cross-linking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28 binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions. PMID:22959275

  5. Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles

    PubMed Central

    Caswell, Clayton C.; Oglesby-Sherrouse, Amanda G.; Murphy, Erin R.

    2014-01-01

    Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators. PMID:25389522

  6. Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs

    PubMed Central

    Zheng, Qiupeng; Bao, Chunyang; Guo, Weijie; Li, Shuyi; Chen, Jie; Chen, Bing; Luo, Yanting; Lyu, Dongbin; Li, Yan; Shi, Guohai; Liang, Linhui; Gu, Jianren; He, Xianghuo; Huang, Shenglin

    2016-01-01

    Circular RNAs (circRNAs) represent a class of widespread and diverse endogenous RNAs that may regulate gene expression in eukaryotes. However, the regulation and function of human circRNAs remain largely unknown. Here we generate ribosomal-depleted RNA sequencing data from six normal tissues and seven cancers, and detect at least 27,000 circRNA candidates. Many of these circRNAs are differently expressed between the normal and cancerous tissues. We further characterize one abundant circRNA derived from Exon2 of the HIPK3 gene, termed circHIPK3. The silencing of circHIPK3 but not HIPK3 mRNA significantly inhibits human cell growth. Via a luciferase screening assay, circHIPK3 is observed to sponge to 9 miRNAs with 18 potential binding sites. Specifically, we show that circHIPK3 directly binds to miR-124 and inhibits miR-124 activity. Our results provide evidence that circular RNA produced from precursor mRNA may have a regulatory role in human cells. PMID:27050392

  7. Novel small noncoding RNAs in mouse spermatozoa, zygotes and early embryos.

    PubMed

    Kawano, Mitsuoki; Kawaji, Hideya; Grandjean, Valérie; Kiani, Jafar; Rassoulzadegan, Minoo

    2012-01-01

    The recent discovery of a significant amount of RNA in spermatozoa contradicted the previously held belief that paternal contribution was limited to one copy of the genome. Furthermore, detection of RNA in sperm raised the intriguing question of its possible role in embryonic development. The possibility that RNAs may serve as epigenetic determinants was supported by experiments showing inheritance of epigenetic traits in mice mediated by RNA. We used high-throughput, large-scale sequencing technology to analyze sperm RNA. The RNA sequences generated were diverse in terms of length and included mRNAs, rRNAs, piRNAs, and miRNAs. We studied two small noncoding RNAs enriched in mature sperm, designated sperm RNAs (spR) -12 and -13. They are both encoded in a piRNA locus on chromosome 17, but neither their length (20-21 nt), nor their sequences correspond to known piRNAs or miRNAs. They are resistant to periodate-oxidation-mediated reaction, implying that they undergo terminal post-transcriptional modification. Both were detected in sperm and ovulated unfertilized oocytes, present in one-cell embryos and maintained in preimplantation stages, but not at later differentiation stages. These findings offer a new perspective regarding a possibly important role for gamete-specific small RNAs in early embryogenesis.

  8. Novel Small Noncoding RNAs in Mouse Spermatozoa, Zygotes and Early Embryos

    PubMed Central

    Kawano, Mitsuoki; Kawaji, Hideya; Grandjean, Valérie; Kiani, Jafar; Rassoulzadegan, Minoo

    2012-01-01

    The recent discovery of a significant amount of RNA in spermatozoa contradicted the previously held belief that paternal contribution was limited to one copy of the genome. Furthermore, detection of RNA in sperm raised the intriguing question of its possible role in embryonic development. The possibility that RNAs may serve as epigenetic determinants was supported by experiments showing inheritance of epigenetic traits in mice mediated by RNA. We used high-throughput, large-scale sequencing technology to analyze sperm RNA. The RNA sequences generated were diverse in terms of length and included mRNAs, rRNAs, piRNAs, and miRNAs. We studied two small noncoding RNAs enriched in mature sperm, designated sperm RNAs (spR) −12 and −13. They are both encoded in a piRNA locus on chromosome 17, but neither their length (20–21 nt), nor their sequences correspond to known piRNAs or miRNAs. They are resistant to periodate-oxidation-mediated reaction, implying that they undergo terminal post-transcriptional modification. Both were detected in sperm and ovulated unfertilized oocytes, present in one-cell embryos and maintained in preimplantation stages, but not at later differentiation stages. These findings offer a new perspective regarding a possibly important role for gamete-specific small RNAs in early embryogenesis. PMID:22984523

  9. Small non-coding RNAs in plant-pathogenic Xanthomonas spp.

    PubMed

    Abendroth, Ulrike; Schmidtke, Cornelius; Bonas, Ulla

    2014-01-01

    The genus Xanthomonas comprises a large group of plant-pathogenic bacteria. The infection and bacterial multiplication in the plant tissue depends on the type III secretion system and other virulence determinants. Recent studies revealed that bacterial virulence is also controlled at the post-transcriptional level by small non-coding RNAs (sRNAs). In this review, we highlight our current knowledge about sRNAs and RNA-binding proteins in Xanthomonas species.

  10. Introduction: MicroRNAs in human reproduction: small molecules with crucial regulatory roles.

    PubMed

    Imbar, Tal; Galliano, Daniela; Pellicer, Antonio; Laufer, Neri

    2014-06-01

    MicroRNAs constitute a large family of approximately 21-nucleotide-long, noncoding RNAs. They emerged more than 20 years ago as key posttranscriptional regulators of gene expression. The regulatory role of these small RNA molecules has recently begun to be explored in the human reproductive system. In this issue's Views and Reviews, the authors present the current knowledge regarding the involvement of microRNAs in several aspects of human reproduction and discuss its future implications for clinical practice.

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

    PubMed

    Usme-Ciro, Jose A; Campillo-Pedroza, Natalia; Almazán, Fernando; Gallego-Gomez, Juan C

    2013-06-07

    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.

  12. Both endo-siRNAs and tRNA-derived small RNAs are involved in the differentiation of primitive eukaryote Giardia lamblia.

    PubMed

    Liao, Jian-You; Guo, Yan-Hua; Zheng, Ling-Ling; Li, Yan; Xu, Wen-Li; Zhang, Yu-Chan; Zhou, Hui; Lun, Zhao-Rong; Ayala, Francisco J; Qu, Liang-Hu

    2014-09-30

    Small RNAs (sRNAs), including microRNAs and endogenous siRNAs (endo-siRNAs), regulate most important biologic processes in eukaryotes, such as cell division and differentiation. Although sRNAs have been extensively studied in various eukaryotes, the role of sRNAs in the early emergence of eukaryotes is unclear. To address these questions, we deep sequenced the sRNA transcriptome of four different stages in the differentiation of Giardia lamblia, one of the most primitive eukaryotes. We identified a large number of endo-siRNAs in this fascinating parasitic protozoan and found that they were produced from live telomeric retrotransposons and three genomic regions (i.e., endo-siRNA generating regions [eSGRs]). eSGR-derived endo-siRNAs were proven to target mRNAs in trans. Gradual up-regulation of endo-siRNAs in the differentiation of Giardia suggested that they might be involved in the regulation of this process. This hypothesis was supported by the impairment of the differentiation ability of Giardia when GLDICER, essential for the biogenesis of endo-siRNAs, was knocked down. Endo-siRNAs are not the only sRNA regulators in Giardia differentiation, because a great number of tRNAs-derived sRNAs showed more dramatic expression changes than endo-siRNAs in this process. We totally identified five novel kinds of tRNAs-derived sRNAs and found that the biogenesis in four of them might be correlated with that of stress-induced tRNA-derived RNA (sitRNA), which was discovered in our previous studies. Our studies reveal an unexpected complex panorama of sRNA in G. lamblia and shed light on the origin and functional evolution of eukaryotic sRNAs.

  13. Viral small RNAs reveal the genomic variations of three grapevine vein clearing virus quasispecies populations.

    PubMed

    Howard, Susanne; Qiu, Wenping

    2017-02-02

    Viral small RNAs (vsRNAs) include viral small interfering RNAs (vsiRNAs) that are initiators and products of RNA silencing, and small RNAs that are derived from viral RNAs with function still unknown. Sequencing of vsRNAs allows assembling of viral genomes and revelation of viral population variations at genomic levels. Grapevine vein clearing virus (GVCV) is a new member of the family Caulimoviridae whose DNA genome is replicated by reverse transcription of pre-genomic RNA molecules. In this short report, three genomic sequences of GVCV were assembled from vsRNAs that were isolated and sequenced from three individual grapevines in commercial vineyards and compared to the GVCV-CHA reference genome. Profiles of single nucleotide polymorphism among three viral populations indicated a closer relatedness between two populations in different grape cultivars at the same location than those in the same grape cultivar at different locations, suggesting the spread of GVCV populations among vineyards of close proximity. Classic types of vsiRNAs (21-nt, 22-nt, and 24-nt) were found in the three GVCV vsiRNA populations, but these did not produce alignment hotspots on the GVCV-CHA reference genome. The number of 36-nt reads is the highest among vsRNAs, the role of these vsRNAs remains unclear. The analysis of vsRNAs provides a first holistic picture of genomic variations among GVCV viral quasispecies populations that help monitor epidemics and evolution of GVCV populations, an emerging virus that is becoming a threat to grape production in the Midwest region of the USA.

  14. The coilin interactome identifies hundreds of small noncoding RNAs that traffic through Cajal bodies.

    PubMed

    Machyna, Martin; Kehr, Stephanie; Straube, Korinna; Kappei, Dennis; Buchholz, Frank; Butter, Falk; Ule, Jernej; Hertel, Jana; Stadler, Peter F; Neugebauer, Karla M

    2014-11-06

    Coilin protein scaffolds Cajal bodies (CBs)-subnuclear compartments enriched in small nuclear RNAs (snRNAs)-and promotes efficient spliceosomal snRNP assembly. The molecular function of coilin, which is intrinsically disordered with no defined motifs, is poorly understood. We use UV crosslinking and immunoprecipitation (iCLIP) to determine whether mammalian coilin binds RNA in vivo and to identify targets. Robust detection of snRNA transcripts correlated with coilin ChIP-seq peaks on snRNA genes, indicating that coilin binding to nascent snRNAs is a site-specific CB nucleator. Surprisingly, several hundred small nucleolar RNAs (snoRNAs) were identified as coilin interactors, including numerous unannotated mouse and human snoRNAs. We show that all classes of snoRNAs concentrate in CBs. Moreover, snoRNAs lacking specific CB retention signals traffic through CBs en route to nucleoli, consistent with the role of CBs in small RNP assembly. Thus, coilin couples snRNA and snoRNA biogenesis, making CBs the cellular hub of small ncRNA metabolism.

  15. Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs.

    PubMed

    Yan, Ming; Wen, Jing; Liang, Min; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S Y

    2015-01-01

    Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30 nm) polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture.

  16. Identification of MicroRNA-like small RNAs from fungus parasite Nosema ceranae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously found transcripts encoding Dicer and Argonaute in the honey bee parasite Nosema ceranae. Since these proteins are involved in the production of regulatory microRNAs we carried out controlled infections and genetic screens in order to identify microRNAs in Nosema . We sequenced small R...

  17. Tailor: a computational framework for detecting non-templated tailing of small silencing RNAs.

    PubMed

    Chou, Min-Te; Han, Bo W; Hsiao, Chiung-Po; Zamore, Phillip D; Weng, Zhiping; Hung, Jui-Hung

    2015-09-30

    Small silencing RNAs, including microRNAs, endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs), have been shown to play important roles in fine-tuning gene expression, defending virus and controlling transposons. Loss of small silencing RNAs or components in their pathways often leads to severe developmental defects, including lethality and sterility. Recently, non-templated addition of nucleotides to the 3' end, namely tailing, was found to associate with the processing and stability of small silencing RNAs. Next Generation Sequencing has made it possible to detect such modifications at nucleotide resolution in an unprecedented throughput. Unfortunately, detecting such events from millions of short reads confounded by sequencing errors and RNA editing is still a tricky problem. Here, we developed a computational framework, Tailor, driven by an efficient and accurate aligner specifically designed for capturing the tailing events directly from the alignments without extensive post-processing. The performance of Tailor was fully tested and compared favorably with other general-purpose aligners using both simulated and real datasets for tailing analysis. Moreover, to show the broad utility of Tailor, we used Tailor to reanalyze published datasets and revealed novel findings worth further experimental validation. The source code and the executable binaries are freely available at https://github.com/jhhung/Tailor.

  18. 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.

  19. A simple and efficient method for isolating small RNAs from different plant species

    PubMed Central

    2011-01-01

    Background Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species. Results We developed a simple and efficient method to isolate small RNAs from different plant species by first comparing different total RNA extraction protocols, followed by streamlining the best one, finally resulting in a small RNA extraction method that has no need of first total RNA extraction and is not based on the commercially available TRIzol® Reagent or columns. This small RNA extraction method not only works well for plant tissues with high polysaccharide content, like cactus, agave, banana, and tomato, but also for plant species like Arabidopsis or tobacco. Furthermore, the obtained small RNA samples were successfully used in northern blot assays. Conclusion Here we provide a simple and efficient method to isolate small RNAs from different plant species, such as cactus, agave, banana, tomato, Arabidopsis, and tobacco, and the small RNAs from this simplified and low cost method is suitable for downstream handling like northern blot assays. PMID:21349188

  20. Expression of microRNAs and other small RNAs in prefrontal cortex in schizophrenia, bipolar disorder and depressed subjects.

    PubMed

    Smalheiser, Neil R; Lugli, Giovanni; Zhang, Hui; Rizavi, Hooriyah; Cook, Edwin H; Dwivedi, Yogesh

    2014-01-01

    Because of the role played by miRNAs in post-transcriptional regulation of an array of genes, their impact in neuropsychiatric disease pathophysiology has increasingly been evident. In the present study, we assessed microRNA expression in prefrontal cortex (Brodmann area 10) of a well-characterized cohort of major depressed, bipolar, and schizophrenia subjects (obtained from Stanley Neuropathology Consortium; n = 15 in each group), using high throughput RT-PCR plates. Discrete miRNA alterations were observed in all disorders, as well as in suicide subjects (pooled across diagnostic categories) compared to all non-suicide subjects. The changes in the schizophrenia group were partially similar to those in the bipolar group, but distinct from changes in depression and suicide. Intriguingly, those miRNAs which were down-regulated in the schizophrenia group tended to be synaptically enriched, whereas up-regulated miRNAs tended not to be. To follow this up, we purified synaptosomes from pooled samples of the schizophrenia vs. control groups and subjected them to Illumina deep sequencing. There was a significant loss of small RNA expression in schizophrenia synaptosomes only for certain sequence lengths within the miRNA range. Moreover, 73 miRNAs were significantly down-regulated whereas only one was up-regulated. Strikingly, across all expressed miRNAs in synaptosomes, there was a significant inverse correlation between the fold-change of a given miRNA seen in schizophrenia and its synaptic enrichment ratio observed in controls. Thus, synaptic miRNAs tended to be down-regulated in schizophrenia, and the more highly synaptically enriched miRNAs tended to show greater down-regulation. These findings point to some deficit in miRNA biogenesis, transport, processing or turnover in schizophrenia that is selective for the synaptic compartment. A novel class of ncRNA-derived small RNAs, shown to be strongly induced during an early phase of learning in mouse, is also

  1. Therapeutic potential of small interfering RNAs/micro interfering RNA in hepatocellular carcinoma.

    PubMed

    Farra, Rossella; Grassi, Mario; Grassi, Gabriele; Dapas, Barbara

    2015-08-14

    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.

  2. Retinal expression of small non-coding RNAs in a murine model of proliferative retinopathy

    PubMed Central

    Liu, Chi-Hsiu; Wang, Zhongxiao; Sun, Ye; SanGiovanni, John Paul; Chen, Jing

    2016-01-01

    Ocular neovascularization is a leading cause of blindness in proliferative retinopathy. Small non-coding RNAs (sncRNAs) play critical roles in both vascular and neuronal development of the retina through post-transcriptional regulation of target gene expression. To identify the function and therapeutic potential of sncRNAs in retinopathy, we assessed the expression profile of retinal sncRNAs in a mouse model of oxygen-induced retinopathy (OIR) with pathologic proliferation of neovessels. Approximately 2% of all analyzed sncRNAs were significantly altered in OIR retinas compared with normoxic controls. Twenty three microRNAs with substantial up- or down-regulation were identified, including miR-351, -762, -210, 145, -155, -129-5p, -150, -203, and -375, which were further analyzed for their potential target genes in angiogenic, hypoxic, and immune response-related pathways. In addition, nineteen small nucleolar RNAs also revealed differential expression in OIR retinas compared with control retinas. A decrease of overall microRNA expression in OIR retinas was consistent with reduced microRNA processing enzyme Dicer, and increased expression of Alu element in OIR. Together, our findings elucidated a group of differentially expressed sncRNAs in a murine model of proliferative retinopathy. These sncRNAs may exert critical post-transcriptional regulatory roles in regulating pathological neovascularization in eye diseases. PMID:27653551

  3. Genetic analysis of small nuclear RNAs in Saccharomyces cerevisiae: viable sextuple mutant.

    PubMed Central

    Parker, R; Simmons, T; Shuster, E O; Siliciano, P G; Guthrie, C

    1988-01-01

    Saccharomyces cerevisiae contains at least 24 distinct small nuclear RNAs (snRNAs), several of which are known to be essential for viability and to participate in the splicing of pre-mRNAs; the RNAs in this subset contain binding sites for the Sm antigen, a hallmark of metazoan snRNAs involved in mRNA processing. In contrast, we showed previously that the single-copy genes for three other snRNAs (snR3, snR4, and snR10) are not required for viability, although cells lacking snR10 are growth impaired at low temperature. None of these RNAs associates with the Sm antigen. To assess this apparent correlation, we cloned and sequenced the genes encoding three additional non-Sm snRNAs. Comparison of these genes with nine additional yeast snRNA genes revealed a highly conserved TATA box located 92 +/- 8 nucleotides 5' of the transcriptional start site. By using the technique of gene replacement with null alleles, each of these three single copy genes was shown to be completely dispensable. We constructed multiple mutants to test the hypothesis that, individually, each of these snRNAs is nonessential because the snRNAs play functionally overlapping roles. A mutant lacking five snRNAs (snR3, snR4, snR5, snR8, snR9) was indistinguishable from the wild type, and growth of the sextuple mutant was no more impaired than that in strains lacking only snR10. This widespread dispensability of snRNAs was completely unexpected and forces us to reconsider the possible roles of these ubiquitous RNAs. Images PMID:2905424

  4. 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

  5. RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse

    PubMed Central

    Hüttenhofer, Alexander; Kiefmann, Martin; Meier-Ewert, Sebastian; O’Brien, John; Lehrach, Hans; Bachellerie, Jean-Pierre; Brosius, Jürgen

    2001-01-01

    In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non-messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAs. Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAs. Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAs. Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINEs. The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAs. PMID:11387227

  6. A big role for small RNAs in HDL homeostasis

    PubMed Central

    Ouimet, Mireille; Moore, Kathryn J.

    2013-01-01

    High-density lipoproteins play a central role in systemic cholesterol homeostasis by stimulating the efflux of excess cellular cholesterol and transporting it to the liver for biliary excretion. HDL has long been touted as the “good cholesterol” because of the strong inverse correlation of plasma HDL cholesterol levels with coronary heart disease. However, the disappointing outcomes of recent clinical trials involving therapeutic elevations of HDL cholesterol have called this moniker into question and revealed our lack of understanding of this complex lipoprotein. At the same time, the discovery of microRNAs (miRNAs) that regulate HDL biogenesis and function have led to a surge in our understanding of the posttranscriptional mechanisms regulating plasma levels of HDL. Furthermore, HDL has recently been shown to selectively transport miRNAs and thereby facilitate cellular communication by shuttling these potent gene regulators to distal tissues. Finally, that miRNA cargo carried by HDL may be altered during disease states further broadened our perspective of how this lipoprotein can have complex effects on target cells and tissues. The unraveling of how these tiny RNAs govern HDL metabolism and contribute to its actions promises to reveal new therapeutic strategies to optimize cardiovascular health. PMID:23509405

  7. Engineering artificial small RNAs for conditional gene silencing in Escherichia coli.

    PubMed

    Sharma, Vandana; Yamamura, Asami; Yokobayashi, Yohei

    2012-01-20

    It has become increasingly evident that noncoding small RNAs (sRNAs) play a significant and global role in bacterial gene regulation. A majority of the trans-acting sRNAs in bacteria interact with the 5' untranslated region (UTR) and/or the translation initiation region of the targeted mRNAs via imperfect base pairing, resulting in reduced translation efficiency and/or mRNA stability. Additionally, bacterial sRNAs often contain distinct scaffolds that recruit RNA chaperones such as Hfq to facilitate gene regulation. In this study, we describe a strategy to engineer artificial sRNAs that can regulate desired endogenous genes in Escherichia coli. Using a fluorescent reporter gene that was translationally fused to a native 5' mRNA leader sequence, active artificial sRNAs were screened from libraries in which natural sRNA scaffolds were fused to a randomized antisense domain. Artificial sRNAs that posttranscriptionally repress two endogenous genes ompF and fliC were isolated and characterized. We anticipate that the artificial sRNAs will be useful for dynamic control and fine-tuning of endogenous gene expression in bacteria for applications in synthetic biology.

  8. Roles for small noncoding RNAs in silencing of retrotransposons in the mammalian brain.

    PubMed

    Nandi, Sayan; Chandramohan, Dhruva; Fioriti, Luana; Melnick, Ari M; Hébert, Jean M; Mason, Christopher E; Rajasethupathy, Priyamvada; Kandel, Eric R

    2016-10-24

    Piwi-interacting RNAs (piRNAs), long thought to be restricted to germline, have recently been discovered in neurons of Aplysia, with a role in the epigenetic regulation of gene expression underlying long-term memory. We here ask whether piwi/piRNAs are also expressed and have functional roles in the mammalian brain. Large-scale RNA sequencing and subsequent analysis of protein expression revealed the presence in brain of several piRNA biogenesis factors including a mouse piwi (Mili), as well as small RNAs, albeit at low levels, resembling conserved piRNAs in mouse testes [primarily LINE1 (long interspersed nuclear element1) retrotransposon-derived]. Despite the seeming low expression of these putative piRNAs, single-base pair CpG methylation analyses across the genome of Mili/piRNA-deficient (Mili(-/-)) mice demonstrate that brain genomic DNA is preferentially hypomethylated within intergenic areas and LINE1 promoter areas of the genome. Furthermore, Mili mutant mice exhibit behavioral deficits such as hyperactivity and reduced anxiety. These results suggest that putative piRNAs exist in mammalian brain, and similar to the role of piRNAs in testes, they may be involved in the silencing of retrotransposons, which in brain have critical roles in contributing to genomic heterogeneity underlying adaptation, stress response, and brain pathology. We also describe the presence of another class of small RNAs in the brain, with features of endogenous siRNAs, which may have taken over the role of invertebrate piRNAs in their capacity to target both transposons, as well as protein-coding genes. Thus, RNA interference through gene and retrotransposon silencing previously encountered in Aplysia may also have potential roles in the mammalian brain.

  9. A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae

    PubMed Central

    Rossi, Ciro C.; Bossé, Janine T.; Li, Yanwen; Witney, Adam A.; Gould, Kate A.; Langford, Paul R.; Bazzolli, Denise M.S.

    2016-01-01

    Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are frequently connected to the expression of virulence factors in diverse bacteria. Only a few sRNAs have been described for Pasteurellaceae pathogens and no in-depth analysis of sRNAs has been described for Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, responsible for considerable losses in the swine industry. To search for sRNAs in A. pleuropneumoniae, we developed a strategy for the computational analysis of the bacterial genome by using four algorithms with different approaches, followed by experimental validation. The coding strand and expression of 17 out of 23 RNA candidates were confirmed by Northern blotting, RT-PCR, and RNA sequencing. Among them, two are likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied GcvB and 6S sRNAs, and 10 are putative novel trans-acting sRNAs, never before described for any bacteria. The latter group has several potential mRNA targets, many of which are involved with virulence, stress resistance, or metabolism, and connect the sRNAs in a complex gene regulatory network. The sRNAs identified are well conserved among the Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same host. Our results show that the combination of newly developed computational programs can be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus providing clues for novel aspects of virulence that will be explored in further studies. PMID:27402897

  10. Argonaute-bound small RNAs from promoter-proximal RNA polymerase II.

    PubMed

    Zamudio, Jesse R; Kelly, Timothy J; Sharp, Phillip A

    2014-02-27

    Argonaute (Ago) proteins mediate posttranscriptional gene repression by binding guide miRNAs to regulate targeted RNAs. To confidently assess Ago-bound small RNAs, we adapted a mouse embryonic stem cell system to express a single epitope-tagged Ago protein family member in an inducible manner. Here, we report the small RNA profile of Ago-deficient cells and show that Ago-dependent stability is a common feature of mammalian miRNAs. Using this criteria and immunopurification, we identified an Ago-dependent class of noncanonical miRNAs derived from protein-coding gene promoters, which we name transcriptional start site miRNAs (TSS-miRNAs). A subset of promoter-proximal RNA polymerase II (RNAPII) complexes produces hairpin RNAs that are processed in a DiGeorge syndrome critical region gene 8 (Dgcr8)/Drosha-independent but Dicer-dependent manner. TSS-miRNA activity is detectable from endogenous levels and following overexpression of mRNA constructs. Finally, we present evidence of differential expression and conservation in humans, suggesting important roles in gene regulation.

  11. Involvement of Small RNAs in Phosphorus and Sulfur Sensing, Signaling and Stress: Current Update

    PubMed Central

    Kumar, Smita; Verma, Saurabh; Trivedi, Prabodh K.

    2017-01-01

    Plants require several essential mineral nutrients for their growth and development. These nutrients are required to maintain physiological processes and structural integrity in plants. The root architecture has evolved to absorb nutrients from soil and transport them to other parts of the plant. Nutrient deficiency affects several physiological and biological processes in plants and leads to reduction in crop productivity and yield. To compensate this adversity, plants have developed adaptive mechanisms to enhance the acquisition, conservation, and mobilization of these nutrients under deficient or adverse conditions. In addition, plants have evolved an intricate nexus of complex signaling cascades, which help in nutrient sensing and uptake as well as to maintain nutrient homeostasis. In recent years, small non-coding RNAs such as micro RNAs (miRNAs) and endogenous small interfering RNAs have emerged as important component in regulating plant stress responses. A set of these small RNAs (sRNAs) have been implicated in regulating various processes involved in nutrient uptake, assimilation, and deficiency. In response to phosphorus (P) and sulphur (S) deficiencies, role of sRNAs, miR395 and miR399, have been identified to be instrumental; however, many more miRNAs might be involved in regulating the plant response to these nutrient stresses. These sRNAs modulate expression of target genes in response to P and S deficiencies and regulate their uptake and utilization for proper growth and development of the plant. This review summarizes the current understanding of uptake, sensing, and signaling of P and S and highlights the regulatory role of sRNAs in adaptive responses to these nutrient stresses in plants. PMID:28344582

  12. Virus-derived small RNAs in the penaeid shrimp Fenneropenaeus chinensis during acute infection of the DNA virus WSSV

    PubMed Central

    Liu, Chengzhang; Li, Fuhua; Sun, Yumiao; Zhang, Xiaojun; Yuan, Jianbo; Yang, Hui; Xiang, Jianhai

    2016-01-01

    Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are two classes of small RNAs (sRNAs) that are critical for virus-host interplay via the RNA interference (RNAi) pathway. One virus-derived siRNA and numerous miRNAs has been reported for the double-stranded DNA virus white spot syndrome virus (WSSV), however, the expression profiles of these different types of sRNAs have not been assessed. Here, by sequencing the sRNAs and mRNAs of WSSV-infected Chinese shrimp (Fenneropenaeus chinensis), we found that the viral transcripts were universally targeted by WSSV-derived siRNAs, supporting a pivotal role for RNAi in the anti-viral immunity of shrimp. The genesis of WSSV-derived siRNAs was associated with long RNA structures. Moreover, by separating miRNAs from siRNAs, 12 WSSV miRNAs were identified. Investigation of conserved viral miRNA targets in different host species indicated the involvement of viral miRNAs in host immune responses. Collectively, our data provide new insights into the role of the RNAi pathway in the interplay between DNA viruses and crustaceans. PMID:27349643

  13. Cell contact-dependent acquisition of cellular and viral nonautonomously encoded small RNAs

    PubMed Central

    Rechavi, Oded; Erlich, Yaniv; Amram, Hila; Flomenblit, Lena; Karginov, Fedor V.; Goldstein, Itamar; Hannon, Gregory J.; Kloog, Yoel

    2009-01-01

    In some organisms, small RNA pathways can act nonautonomously, with responses spreading from cell to cell. Dedicated intercellular RNA delivery pathways have not yet been characterized in mammals, although secretory compartments have been found to contain RNA. Here we show that, upon cell contact, T cells acquire from B cells small RNAs that can impact the expression of target genes in the recipient T cells. Synthetic microRNA (miRNA) mimetics, viral miRNAs expressed by infected B cells, and endogenous miRNAs could all be transferred into T cells. These mechanisms may allow small RNA-mediated communication between immune cells. The documented transfer of viral miRNAs raises the possible exploitation of these pathways for viral manipulation of the host immune response. PMID:19684116

  14. 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

  15. 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.

  16. Cotton Leaf Curl Multan Virus-Derived Viral Small RNAs Can Target Cotton Genes to Promote Viral Infection

    PubMed Central

    Wang, Jinyan; Tang, Yafei; Yang, Yuwen; Ma, Na; Ling, Xitie; Kan, Jialiang; He, Zifu; Zhang, Baolong

    2016-01-01

    RNA silencing is a conserved mechanism in plants that targets viruses. Viral small RNAs (vsiRNAs) can be generated from viral double-stranded RNA replicative intermediates within the infected host, or from host RNA-dependent RNA polymerases activity on viral templates. The abundance and profile of vsiRNAs in viral infections have been reported previously. However, the involvement of vsiRNAs during infection of the Geminiviridae family member cotton leaf curl virus (CLCuD), which causes significant economic losses in cotton growing regions, remains largely uncharacterized. Cotton leaf curl Multan virus (CLCuMuV) associated with a betasatellite called Cotton leaf curl Multan betasatellite (CLCuMuB) is a major constraint to cotton production in South Asia and is now established in Southern China. In this study, we obtained the profiles of vsiRNAs from CLCuMV and CLCuMB in infected upland cotton (Gossypium hirsutum) plants by deep sequencing. Our data showed that vsiRNA that were derived almost equally from sense and antisense CLCuD DNA strands accumulated preferentially as 21- and 22-nucleotide (nt) small RNA population and had a cytosine bias at the 5′-terminus. Polarity distribution revealed that vsiRNAs were almost continuously present along the CLCuD genome and hotspots of sense and antisense strands were mainly distributed in the Rep proteins region of CLCuMuV and in the C1 protein of CLCuMuB. In addition, hundreds of host transcripts targeted by vsiRNAs were predicted, many of which encode transcription factors associated with biotic and abiotic stresses. Quantitative real-time polymerase chain reaction analysis of selected potential vsiRNA targets showed that some targets were significantly down-regulated in CLCuD-infected cotton plants. We also verified the potential function of vsiRNA targets that may be involved in CLCuD infection by virus-induced gene silencing (VIGS) and 5′-rapid amplification of cDNA end (5′-RACE). Here, we provide the first report

  17. Identification of Conserved and Potentially Regulatory Small RNAs in Heterocystous Cyanobacteria.

    PubMed

    Brenes-Álvarez, Manuel; Olmedo-Verd, Elvira; Vioque, Agustín; Muro-Pastor, Alicia M

    2016-01-01

    Small RNAs (sRNAs) are a growing class of non-protein-coding transcripts that participate in the regulation of virtually every aspect of bacterial physiology. Heterocystous cyanobacteria are a group of photosynthetic organisms that exhibit multicellular behavior and developmental alternatives involving specific transcriptomes exclusive of a given physiological condition or even a cell type. In the context of our ongoing effort to understand developmental decisions in these organisms we have undertaken an approach to the global identification of sRNAs. Using differential RNA-Seq we have previously identified transcriptional start sites for the model heterocystous cyanobacterium Nostoc sp. PCC 7120. Here we combine this dataset with a prediction of Rho-independent transcriptional terminators and an analysis of phylogenetic conservation of potential sRNAs among 89 available cyanobacterial genomes. In contrast to predictive genome-wide approaches, the use of an experimental dataset comprising all active transcriptional start sites (differential RNA-Seq) facilitates the identification of bona fide sRNAs. The output of our approach is a dataset of predicted potential sRNAs in Nostoc sp. PCC 7120, with different degrees of phylogenetic conservation across the 89 cyanobacterial genomes analyzed. Previously described sRNAs appear among the predicted sRNAs, demonstrating the performance of the algorithm. In addition, new predicted sRNAs are now identified that can be involved in regulation of different aspects of cyanobacterial physiology, including adaptation to nitrogen stress, the condition that triggers differentiation of heterocysts (specialized nitrogen-fixing cells). Transcription of several predicted sRNAs that appear exclusively in the genomes of heterocystous cyanobacteria is experimentally verified by Northern blot. Cell-specific transcription of one of these sRNAs, NsiR8 (nitrogen stress-induced RNA 8), in developing heterocysts is also demonstrated.

  18. Identification of Conserved and Potentially Regulatory Small RNAs in Heterocystous Cyanobacteria

    PubMed Central

    Brenes-Álvarez, Manuel; Olmedo-Verd, Elvira; Vioque, Agustín; Muro-Pastor, Alicia M.

    2016-01-01

    Small RNAs (sRNAs) are a growing class of non-protein-coding transcripts that participate in the regulation of virtually every aspect of bacterial physiology. Heterocystous cyanobacteria are a group of photosynthetic organisms that exhibit multicellular behavior and developmental alternatives involving specific transcriptomes exclusive of a given physiological condition or even a cell type. In the context of our ongoing effort to understand developmental decisions in these organisms we have undertaken an approach to the global identification of sRNAs. Using differential RNA-Seq we have previously identified transcriptional start sites for the model heterocystous cyanobacterium Nostoc sp. PCC 7120. Here we combine this dataset with a prediction of Rho-independent transcriptional terminators and an analysis of phylogenetic conservation of potential sRNAs among 89 available cyanobacterial genomes. In contrast to predictive genome-wide approaches, the use of an experimental dataset comprising all active transcriptional start sites (differential RNA-Seq) facilitates the identification of bona fide sRNAs. The output of our approach is a dataset of predicted potential sRNAs in Nostoc sp. PCC 7120, with different degrees of phylogenetic conservation across the 89 cyanobacterial genomes analyzed. Previously described sRNAs appear among the predicted sRNAs, demonstrating the performance of the algorithm. In addition, new predicted sRNAs are now identified that can be involved in regulation of different aspects of cyanobacterial physiology, including adaptation to nitrogen stress, the condition that triggers differentiation of heterocysts (specialized nitrogen-fixing cells). Transcription of several predicted sRNAs that appear exclusively in the genomes of heterocystous cyanobacteria is experimentally verified by Northern blot. Cell-specific transcription of one of these sRNAs, NsiR8 (nitrogen stress-induced RNA 8), in developing heterocysts is also demonstrated. PMID

  19. Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena

    PubMed Central

    Schoeberl, Ursula E.; Kurth, Henriette M.; Noto, Tomoko; Mochizuki, Kazufumi

    2012-01-01

    The ciliated protozoan Tetrahymena undergoes extensive programmed DNA elimination when the germline micronucleus produces the new macronucleus during sexual reproduction. DNA elimination is epigenetically controlled by DNA sequences of the parental macronuclear genome, and this epigenetic regulation is mediated by small RNAs (scan RNAs [scnRNAs]) of ∼28–30 nucleotides that are produced and function by an RNAi-related mechanism. Here, we examine scnRNA production and turnover by deep sequencing. scnRNAs are produced exclusively from the micronucleus and nonhomogeneously from a variety of chromosomal locations. scnRNAs are preferentially derived from the eliminated sequences, and this preference is mainly determined at the level of transcription. Despite this bias, a significant fraction of scnRNAs is also derived from the macronuclear-destined sequences, and these scnRNAs are degraded during the course of sexual reproduction. These results indicate that the pattern of DNA elimination in the new macronucleus is shaped by the biased transcription in the micronucleus and the selective degradation of scnRNAs in the parental macronucleus. PMID:22855833

  20. Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia.

    PubMed

    Marcial-Quino, Jaime; Gómez-Manzo, Saúl; Fierro, Francisco; Vanoye-Carlo, America; Rufino-González, Yadira; Sierra-Palacios, Edgar; Castillo-Villanueva, Adriana; Castillo-Rodríguez, Rosa Angélica; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto; Reyes-Vivas, Horacio

    2016-12-20

    Stem-loop quantitative reverse transcription PCR (RT-qPCR) is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL)-based design to detect-in a rapid, sensitive, specific, and reproducible way-the small nucleolar RNA (snoRNA) GlsR17 and its derived miRNA (miR2) of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G. lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G. lamblia. This methodology will be a powerful tool for studying gene regulation in G. lamblia, and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi) pathway in G. lamblia.

  1. Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes.

    PubMed

    Gaspin, C; Cavaillé, J; Erauso, G; Bachellerie, J P

    2000-04-07

    Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into

  2. SARS-CoV-Encoded Small RNAs Contribute to Infection-Associated Lung Pathology.

    PubMed

    Morales, Lucía; Oliveros, Juan Carlos; Fernandez-Delgado, Raúl; tenOever, Benjamin Robert; Enjuanes, Luis; Sola, Isabel

    2017-03-08

    Severe acute respiratory syndrome coronavirus (SARS-CoV) causes lethal disease in humans, which is characterized by exacerbated inflammatory response and extensive lung pathology. To address the relevance of small non-coding RNAs in SARS-CoV pathology, we deep sequenced RNAs from the lungs of infected mice and discovered three 18-22 nt small viral RNAs (svRNAs). The three svRNAs were derived from the nsp3 (svRNA-nsp3.1 and -nsp3.2) and N (svRNA-N) genomic regions of SARS-CoV. Biogenesis of CoV svRNAs was RNase III, cell type, and host species independent, but it was dependent on the extent of viral replication. Antagomir-mediated inhibition of svRNA-N significantly reduced in vivo lung pathology and pro-inflammatory cytokine expression. Taken together, these data indicate that svRNAs contribute to SARS-CoV pathogenesis and highlight the potential of svRNA-N antagomirs as antivirals.

  3. Small interfering RNAs as a tool to assign Rho GTPase exchange-factor function in vivo.

    PubMed Central

    Gampel, Alexandra; Mellor, Harry

    2002-01-01

    Rho GTPases control a complex network of intracellular signalling pathways. Whereas progress has been made in identifying downstream signalling partners for these proteins, the characterization of Rho upstream regulatory guanine-nucleotide exchange factors (GEFs) has been hampered by a lack of suitable research tools. Here we use small interfering RNAs (siRNAs) to examine the cellular regulation of the RhoB GTPase, and show that RhoB is activated downstream of the epidermal-growth-factor receptor through the Vav2 exchange factor. These studies demonstrate that siRNAs are an ideal research tool for the assignment of Rho GEF function in vivo. PMID:12113653

  4. Structure based approaches for targeting non-coding RNAs with small molecules

    PubMed Central

    Shortridge, Matthew D.; Varani, Gabriele

    2015-01-01

    The increasing appreciation of the central role of non-coding RNAs (miRNAs and long non coding RNAs) in chronic and degenerative human disease makes them attractive therapeutic targets. This would not be unprecedented: the bacterial ribosomal RNA is a mainstay for antibacterial treatment, while the conservation and functional importance of viral RNA regulatory elements has long suggested they would constitute attractive targets for new antivirals. Oligonucleotide-based chemistry has obvious appeals but also considerable pharmacological limitations that are yet to be addressed satisfactorily. Recent studies identifying small molecules targeting non-coding RNAs may provide an alternative approach to oligonucleotide methods. Here we review recent work investigating new structural and chemical principles for targeting RNA with small molecules. PMID:25687935

  5. Dicer-Dependent Biogenesis of Small RNAs and Evidence for MicroRNA-Like RNAs in the Penicillin Producing Fungus Penicillium chrysogenum.

    PubMed

    Dahlmann, Tim A; Kück, Ulrich

    2015-01-01

    MicroRNAs (miRNAs) are non-coding small RNAs (sRNAs) that regulate gene expression in a wide range of eukaryotes. In this study, we analyzed regulatory sRNAs in Penicillium chrysogenum, the industrial producer of the β-lactam antibiotic penicillin. To identify sRNAs and microRNA-like RNAs (milRNAs) on a global approach, two sRNA sequencing libraries were constructed. One library was created with pooled total RNA, obtained from twelve differently grown cultures (RNA Mix), and the other with total RNA from a single submerged cultivation (∆ku70FRT2). Illumina sequencing of both RNA libraries produced 84,322,825 mapped reads. To distinguish between Dicer-dependent and independent sRNA formation, we further constructed two single dicer gene mutants (∆dcl2 and ∆dcl1) and a dicer double mutant (∆dcl2∆dcl1) and analyzed an sRNA library from the Dicer-deficient double-mutant. We identified 661 Dicer-dependent loci and in silico prediction revealed 34 milRNAs. Northern blot hybridization of two milRNAs provided evidence for mature milRNAs that are processed either in a complete or partial Dicer-dependent manner from an RNA precursor. Identified milRNAs share typical characteristics of previously discovered fungal milRNAs, like a strong preference for a 5' uracil and the typical length distribution. The detection of potential milRNA target sites in the genome suggests that milRNAs might play a role in posttranscriptional gene regulation. Our data will further increase our knowledge of sRNA dependent gene regulation processes, which is an important prerequisite to develop more effective strategies for improving industrial fermentations with P. chrysogenum.

  6. Dicer-Dependent Biogenesis of Small RNAs and Evidence for MicroRNA-Like RNAs in the Penicillin Producing Fungus Penicillium chrysogenum

    PubMed Central

    Dahlmann, Tim A.; Kück, Ulrich

    2015-01-01

    MicroRNAs (miRNAs) are non-coding small RNAs (sRNAs) that regulate gene expression in a wide range of eukaryotes. In this study, we analyzed regulatory sRNAs in Penicillium chrysogenum, the industrial producer of the β-lactam antibiotic penicillin. To identify sRNAs and microRNA-like RNAs (milRNAs) on a global approach, two sRNA sequencing libraries were constructed. One library was created with pooled total RNA, obtained from twelve differently grown cultures (RNA Mix), and the other with total RNA from a single submerged cultivation (∆ku70FRT2). Illumina sequencing of both RNA libraries produced 84,322,825 mapped reads. To distinguish between Dicer-dependent and independent sRNA formation, we further constructed two single dicer gene mutants (∆dcl2 and ∆dcl1) and a dicer double mutant (∆dcl2∆dcl1) and analyzed an sRNA library from the Dicer-deficient double-mutant. We identified 661 Dicer-dependent loci and in silico prediction revealed 34 milRNAs. Northern blot hybridization of two milRNAs provided evidence for mature milRNAs that are processed either in a complete or partial Dicer-dependent manner from an RNA precursor. Identified milRNAs share typical characteristics of previously discovered fungal milRNAs, like a strong preference for a 5' uracil and the typical length distribution. The detection of potential milRNA target sites in the genome suggests that milRNAs might play a role in posttranscriptional gene regulation. Our data will further increase our knowledge of sRNA dependent gene regulation processes, which is an important prerequisite to develop more effective strategies for improving industrial fermentations with P. chrysogenum. PMID:25955857

  7. Small RNAs in plant defense responses during viral and bacterial interactions: similarities and differences

    PubMed Central

    Peláez, Pablo; Sanchez, Federico

    2013-01-01

    Small non-coding RNAs constitute an important class of gene expression regulators that control different biological processes in most eukaryotes. In plants, several small RNA (sRNA) silencing pathways have evolved to produce a wide range of small RNAs with specialized functions. Evidence for the diverse mode of action of the small RNA pathways has been highlighted during plant–microbe interactions. Host sRNAs and small RNA silencing pathways have been recognized as essential components of plant immunity. One way plants respond and defend against pathogen infections is through the small RNA silencing immune system. To deal with plant defense responses, pathogens have evolved sophisticated mechanisms to avoid and counterattack this defense strategy. The relevance of the small RNA-mediated plant defense responses during viral infections has been well-established. Recent evidence points out its importance also during plant–bacteria interactions. Herein, this review discusses recent findings, similarities and differences about the small RNA-mediated arms race between plants and these two groups of microbes, including the small RNA silencing pathway components that contribute to plant immune responses, the pathogen-responsive endogenous sRNAs and the pathogen-delivered effector proteins. PMID:24046772

  8. Small RNAs in plant defense responses during viral and bacterial interactions: similarities and differences.

    PubMed

    Peláez, Pablo; Sanchez, Federico

    2013-09-05

    Small non-coding RNAs constitute an important class of gene expression regulators that control different biological processes in most eukaryotes. In plants, several small RNA (sRNA) silencing pathways have evolved to produce a wide range of small RNAs with specialized functions. Evidence for the diverse mode of action of the small RNA pathways has been highlighted during plant-microbe interactions. Host sRNAs and small RNA silencing pathways have been recognized as essential components of plant immunity. One way plants respond and defend against pathogen infections is through the small RNA silencing immune system. To deal with plant defense responses, pathogens have evolved sophisticated mechanisms to avoid and counterattack this defense strategy. The relevance of the small RNA-mediated plant defense responses during viral infections has been well-established. Recent evidence points out its importance also during plant-bacteria interactions. Herein, this review discusses recent findings, similarities and differences about the small RNA-mediated arms race between plants and these two groups of microbes, including the small RNA silencing pathway components that contribute to plant immune responses, the pathogen-responsive endogenous sRNAs and the pathogen-delivered effector proteins.

  9. β-Cell MicroRNAs: Small but Powerful

    PubMed Central

    Filios, Stephen R.

    2015-01-01

    Noncoding RNA and especially microRNAs (miRs) have emerged as important regulators of key processes in cell biology, including development, differentiation, and survival. Currently, over 2,500 mature miRs have been reported in humans, and considering that each miR has multiple targets, the number of genes and pathways potentially affected is huge. Not surprisingly, many miRs have also been implicated in diabetes, and more recently, some have been discovered to play important roles in the pancreatic islet, including β-cell function, proliferation, and survival. The goal of this Perspective is to offer an overview of this rapidly evolving field and the miRs involved, reveal novel networks of β-cell miR signaling, and provide an outlook of the opportunities and challenges ahead. PMID:26494215

  10. Prediction of non-small cell lung cancer metastasis-associated microRNAs using bioinformatics

    PubMed Central

    Wang, Rong; Chen, Xiao-Feng; Shu, Yong-Qian

    2015-01-01

    Distant metastasis is one of the most common causes for failure in treatment of advanced NSCLC, and it is a key factor to determine the patients’ prognosis. This study aims to screen the microRNAs associated with non-small cell lung cancer metastasis, so as to provide theoretical basis for investigating their roles in non-small cell lung cancer metastasis. In this study, the fluorescent transfected human non-small cell lung cancer cell lines H460 developed tumors subcutaneously, which were then in situ transplanted into the left lung of nude mice to obtain the tissue specimens of primary tumor and metastatic tumor. The differentially expressed microRNAs associated with non-small cell lung cancer metastasis were identified using the microRNA microarray and real-time quantitative polymerase chain reaction (RT-PCR) analysis, and bioinformatics analysis of the microRNAs was performed. The microarray analysis results revealed that 17 microRNAs with up-regulated expression and 7 with down-regulated expression between the non-small cell lung cancer metastatic primary loci and the non-metastatic primary loci (Group A), while 20 microRNAs with up-regulated expression (ratio > 1.5 times, P < 0.05) and 16 with down-regulated expression (ratio < 0.65 times, P < 0.05) between the non-small cell lung cancer metastatic loci and the metastatic primary loci (Group B). RT-PCR validation and bioinformatics analysis of some microRNAs identified 2 microRNAs with up-regulated expression, miR-10b and miR-144, and 3 microRNAs with down-regulated expression, miR-9, miR-31 and miR-34b in Group A; and 4 microRNAs with down-regulated expression, miR-25, miR-92a, miR-202 and miR-326 in Group B, which may be mediated by transcription factors activator protein 1 (AP-1), p53, STATs and NF-κB, regulate cell development, proliferation and cycle, DNA and RNA metabolism and signal transduction pathway, and promote tumor growth and metastasis through the effects on target genes like RARβ, RASSF1

  11. miRNAs: small genes with big potential in metazoan phylogenetics.

    PubMed

    Tarver, James E; Sperling, Erik A; Nailor, Audrey; Heimberg, Alysha M; Robinson, Jeffrey M; King, Benjamin L; Pisani, Davide; Donoghue, Philip C J; Peterson, Kevin J

    2013-11-01

    microRNAs (miRNAs) are a key component of gene regulatory networks and have been implicated in the regulation of virtually every biological process found in multicellular eukaryotes. What makes them interesting from a phylogenetic perspective is the high conservation of primary sequence between taxa, their accrual in metazoan genomes through evolutionary time, and the rarity of secondary loss in most metazoan taxa. Despite these properties, the use of miRNAs as phylogenetic markers has not yet been discussed within a clear conceptual framework. Here we highlight five properties of miRNAs that underlie their utility in phylogenetics: 1) The processes of miRNA biogenesis enable the identification of novel miRNAs without prior knowledge of sequence; 2) The continuous addition of miRNA families to metazoan genomes through evolutionary time; 3) The low level of secondary gene loss in most metazoan taxa; 4) The low substitution rate in the mature miRNA sequence; and 5) The small probability of convergent evolution of two miRNAs. Phylogenetic analyses using both Bayesian and parsimony methods on a eumetazoan miRNA data set highlight the potential of miRNAs to become an invaluable new tool, especially when used as an additional line of evidence, to resolve previously intractable nodes within the tree of life.

  12. Ancestral vinclozolin exposure alters the epigenetic transgenerational inheritance of sperm small noncoding RNAs.

    PubMed

    Schuster, Andrew; Skinner, Michael K; Yan, Wei

    Exposure to the agricultural fungicide vinclozolin during gestation promotes a higher incidence of various diseases in the subsequent unexposed F3 and F4 generations. This phenomenon is termed epigenetic transgenerational inheritance and has been shown to in part involve alterations in DNA methylation, but the role of other epigenetic mechanisms remains unknown. The current study investigated the alterations in small noncoding RNA (sncRNA) in the sperm from F3 generation control and vinclozolin lineage rats. Over 200 differentially expressed sncRNAs were identified and the tRNA-derived sncRNAs, namely 5' halves of mature tRNAs (5' halves), displayed the most dramatic changes. Gene targets of the altered miRNAs and tRNA 5' halves revealed associations between the altered sncRNAs and differentially DNA methylated regions. Dysregulated sncRNAs appear to correlate with mRNA profiles associated with the previously observed vinclozolin-induced disease phenotypes. Data suggest potential connections between sperm-borne RNAs and the vinclozolin-induced epigenetic transgenerational inheritance phenomenon.

  13. Ancestral vinclozolin exposure alters the epigenetic transgenerational inheritance of sperm small noncoding RNAs

    PubMed Central

    Schuster, Andrew; Skinner, Michael K.; Yan, Wei

    2016-01-01

    Exposure to the agricultural fungicide vinclozolin during gestation promotes a higher incidence of various diseases in the subsequent unexposed F3 and F4 generations. This phenomenon is termed epigenetic transgenerational inheritance and has been shown to in part involve alterations in DNA methylation, but the role of other epigenetic mechanisms remains unknown. The current study investigated the alterations in small noncoding RNA (sncRNA) in the sperm from F3 generation control and vinclozolin lineage rats. Over 200 differentially expressed sncRNAs were identified and the tRNA-derived sncRNAs, namely 5′ halves of mature tRNAs (5′ halves), displayed the most dramatic changes. Gene targets of the altered miRNAs and tRNA 5′ halves revealed associations between the altered sncRNAs and differentially DNA methylated regions. Dysregulated sncRNAs appear to correlate with mRNA profiles associated with the previously observed vinclozolin-induced disease phenotypes. Data suggest potential connections between sperm-borne RNAs and the vinclozolin-induced epigenetic transgenerational inheritance phenomenon. PMID:27390623

  14. Stochastic Modeling of Regulation of Gene Expression by Multiple Competing Small RNAs

    NASA Astrophysics Data System (ADS)

    Baker, Charles; Jia, Tao; Kulkarni, Rahul

    2011-03-01

    A wealth of new research has highlighted the critical roles of small RNAs (sRNAs) in diverse processes such as quorum sensing and cellular responses to stress. The pathways controlling these processes often have a central motif comprised of a key protein regulated by multiple sRNAs. However, the regulation of stochastic gene expression of a single target gene by multiple sRNAs is currently not well understood. To address this issue, we analyze a stochastic model of regulation of gene expression by multiple sRNAs. For this model, we derive exact analytic results for the regulated protein distribution including compact expressions for its mean and variance. The derived results provide novel insights into the roles of multiple sRNAs in fine-tuning the noise in gene expression. In particular, we show that, in contrast to regulation by a single sRNA, multiple sRNAs provide a mechanism for independently controlling the mean and variance of the regulated protein distribution.

  15. Stochastic modeling of regulation of gene expression by multiple small RNAs

    NASA Astrophysics Data System (ADS)

    Baker, Charles; Jia, Tao; Kulkarni, Rahul V.

    2012-06-01

    A wealth of new research has highlighted the critical roles of small noncoding RNAs (sRNAs) in diverse processes, such as quorum sensing and cellular responses to stress. The pathways controlling these processes often have a central motif composed of a master regulator protein whose expression is controlled by multiple sRNAs. However, the stochastic gene expression of a single target gene regulated by multiple sRNAs is currently not well understood. To address this issue, we analyze a stochastic model of regulation of gene expression by multiple sRNAs. For this model, we derive exact analytic results for the regulated protein distribution, including compact expressions for its mean and variance. The derived results provide insights into the roles of multiple sRNAs in fine-tuning the noise in gene expression. In particular, we show that, in contrast to regulation by a single sRNA, multiple sRNAs provide a mechanism for independently controlling the mean and variance of the regulated protein distribution.

  16. The ribonuclease polynucleotide phosphorylase can interact with small regulatory RNAs in both protective and degradative modes

    PubMed Central

    Bandyra, Katarzyna J.; Sinha, Dhriti; Syrjanen, Johanna; Luisi, Ben F.; De Lay, Nicholas R.

    2016-01-01

    In all bacterial species examined thus far, small regulatory RNAs (sRNAs) contribute to intricate patterns of dynamic genetic regulation. Many of the actions of these nucleic acids are mediated by well-characterized chaperones such as the Hfq protein, but genetic screens have also recently identified the 3′-to-5′ exoribonuclease polynucleotide phosphorylase (PNPase) as an unexpected stabilizer and facilitator of sRNAs in vivo. To understand how a ribonuclease might mediate these effects, we tested the interactions of PNPase with sRNAs and found that the enzyme can readily degrade these nucleic acids in vitro but, nonetheless, copurifies from cell extracts with the same sRNAs without discernible degradation or modification to their 3′ ends, suggesting that the associated RNA is protected against the destructive activity of the ribonuclease. In vitro, PNPase, Hfq, and sRNA can form a ternary complex in which the ribonuclease plays a nondestructive, structural role. Such ternary complexes might be formed transiently in vivo, but could help to stabilize particular sRNAs and remodel their population on Hfq. Taken together, our results indicate that PNPase can be programmed to act on RNA in either destructive or stabilizing modes in vivo and may form complex, protective ribonucleoprotein assemblies that shape the landscape of sRNAs available for action. PMID:26759452

  17. Discovery of putative small non-coding RNAs from the obligate intracellular bacterium Wolbachia pipientis.

    PubMed

    Woolfit, Megan; Algama, Manjula; Keith, Jonathan M; McGraw, Elizabeth A; Popovici, Jean

    2015-01-01

    Wolbachia pipientis is an endosymbiotic bacterium that induces a wide range of effects in its insect hosts, including manipulation of reproduction and protection against pathogens. Little is known of the molecular mechanisms underlying the insect-Wolbachia interaction, though it is likely to be mediated via the secretion of proteins or other factors. There is an increasing amount of evidence that bacteria regulate many cellular processes, including secretion of virulence factors, using small non-coding RNAs (sRNAs), but sRNAs have not previously been described from Wolbachia. We have used two independent approaches, one based on comparative genomics and the other using RNA-Seq data generated for gene expression studies, to identify candidate sRNAs in Wolbachia. We experimentally characterized the expression of one of these candidates in four Wolbachia strains, and showed that it is differentially regulated in different host tissues and sexes. Given the roles played by sRNAs in other host-associated bacteria, the conservation of the candidate sRNAs between different Wolbachia strains, and the sex- and tissue-specific differential regulation we have identified, we hypothesise that sRNAs may play a significant role in the biology of Wolbachia, and in particular in its interactions with its host.

  18. Monitoring the Spatiotemporal Activities of miRNAs in Small Animal Models Using Molecular Imaging Modalities

    PubMed Central

    Baril, Patrick; Ezzine, Safia; Pichon, Chantal

    2015-01-01

    MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy. PMID:25749473

  19. MicroRNAs in Amoebozoa: Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs

    PubMed Central

    Avesson, Lotta; Reimegård, Johan; Wagner, E. Gerhart H.; Söderbom, Fredrik

    2012-01-01

    The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs. PMID:22875808

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

    PubMed

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

    2015-01-01

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

  1. Fast-forward generation of effective artificial small RNAs for enhanced antiviral defense in plants.

    PubMed

    Carbonell, Alberto; Carrington, James C; Daròs, José-Antonio

    Artificial small RNAs (sRNAs) are short ≈21-nt non-coding RNAs engineered to inactivate sequence complementary RNAs. In plants, they have been extensively used to silence cellular transcripts in gene function analyses and to target invading RNA viruses to induce resistance. Current artificial sRNA-based antiviral resistance in plants is mainly limited to a single virus, and is jeopardized by the emergence of mutations in the artificial sRNA target site or by the presence of co-infecting viruses. Hence, there is a need to further develop the artificial sRNA approach to generate more broad and durable antiviral resistance in plants. A recently developed toolbox allows for the time and cost-effective large-scale production of artificial sRNA constructs in plants. The toolbox includes the P-SAMS web tool for the automated design of artificial sRNAs, and a new generation of artificial microRNA and synthetic trans-acting small interfering RNA (syn-tasiRNA) vectors for direct cloning and high expression of artificial sRNAs. Here we describe how the simplicity and high-throughput capability of these new technologies should accelerate the study of artificial sRNA-based antiviral resistance in plants. In particular, we discuss the potential of the syn-tasiRNA approach as a promising strategy for developing more effective, durable and broad antiviral resistance in plants.

  2. MicroRNA-Like Small RNAs Prediction in the Development of Antrodia cinnamomea

    PubMed Central

    Lin, Yan-Liang; Ma, Li-Ting; Lee, Yi-Ru; Lin, Shih-Shun; Wang, Sheng-Yang; Chang, Tun-Tschu; Shaw, Jei-Fu; Li, Wen-Hsiung; Chu, Fang-Hua

    2015-01-01

    Antrodia cinnamomea, a precious, host-specific brown-rot fungus that has been used as a folk medicine in Taiwan for centuries is known to have diverse bioactive compounds with potent pharmaceutical activity. In this study, different fermentation states of A. cinnamomea (wild-type fruiting bodies and liquid cultured mycelium) were sequenced using the next-generation sequencing (NGS) technique. A 45.58 Mb genome encoding 6,522 predicted genes was obtained. High quality reads were assembled into a total of 13,109 unigenes. Using a previously constructed pipeline to search for microRNAs (miRNAs), we then identified 4 predicted conserved miRNA and 63 novel predicted miRNA-like small RNA (milRNA) candidates. Target prediction revealed several interesting proteins involved in tri-terpenoid synthesis, mating type recognition, chemical or physical sensory protein and transporters predicted to be regulated by the miRNAs and milRNAs. PMID:25860872

  3. Development of RNA interference-based therapeutics and application of multi-target small interfering RNAs.

    PubMed

    Li, Tiejun; Wu, Meihua; Zhu, York Yuanyuan; Chen, Jianxin; Chen, Li

    2014-08-01

    RNA interference (RNAi) has been proven in recent years to be a newly advanced and powerful tool for development of therapeutic agents toward various unmet medical needs such as cancer, in particular, a great attention has been paid to the development of antineoplastic agents. Recent success in clinical trials related to RNAi-based therapeutics on cancer and ocular disease has validated that small interfering RNAs (siRNAs) constitute a new promising class of therapeutics. Currently, a great wealth of multi-target based siRNA structural modifications is available for promoting siRNA-mediated gene silencing with low side effects. Here, the latest developments in RNAi-based therapeutics and novel structural modifications described for siRNAs--in particular multi-target siRNAs--are reviewed.

  4. Metatranscriptomic analysis of small RNAs present in soybean deep sequencing libraries

    PubMed Central

    Molina, Lorrayne Gomes; da Fonseca, Guilherme Cordenonsi; de Morais, Guilherme Loss; de Oliveira, Luiz Felipe Valter; de Carvalho, Joseane Biso; Kulcheski, Franceli Rodrigues; Margis, Rogerio

    2012-01-01

    A large number of small RNAs unrelated to the soybean genome were identified after deep sequencing of soybean small RNA libraries. A metatranscriptomic analysis was carried out to identify the origin of these sequences. Comparative analyses of small interference RNAs (siRNAs) present in samples collected in open areas corresponding to soybean field plantations and samples from soybean cultivated in greenhouses under a controlled environment were made. Different pathogenic, symbiotic and free-living organisms were identified from samples of both growth systems. They included viruses, bacteria and different groups of fungi. This approach can be useful not only to identify potentially unknown pathogens and pests, but also to understand the relations that soybean plants establish with microorganisms that may affect, directly or indirectly, plant health and crop production. PMID:22802714

  5. Metatranscriptomic analysis of small RNAs present in soybean deep sequencing libraries.

    PubMed

    Molina, Lorrayne Gomes; da Fonseca, Guilherme Cordenonsi; de Morais, Guilherme Loss; de Oliveira, Luiz Felipe Valter; de Carvalho, Joseane Biso; Kulcheski, Franceli Rodrigues; Margis, Rogerio

    2012-06-01

    A large number of small RNAs unrelated to the soybean genome were identified after deep sequencing of soybean small RNA libraries. A metatranscriptomic analysis was carried out to identify the origin of these sequences. Comparative analyses of small interference RNAs (siRNAs) present in samples collected in open areas corresponding to soybean field plantations and samples from soybean cultivated in greenhouses under a controlled environment were made. Different pathogenic, symbiotic and free-living organisms were identified from samples of both growth systems. They included viruses, bacteria and different groups of fungi. This approach can be useful not only to identify potentially unknown pathogens and pests, but also to understand the relations that soybean plants establish with microorganisms that may affect, directly or indirectly, plant health and crop production.

  6. Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs

    PubMed Central

    Coffman, Stephanie R.; Lu, Jinfeng; Guo, Xunyang; Zhong, Jing; Broitman-Maduro, Gina; Li, Wan-Xiang; Lu, Rui; Maduro, Morris

    2017-01-01

    ABSTRACT Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans. Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans. Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors. PMID:28325765

  7. Identification of novel microRNAs in primates by using the synteny information and small RNA deep sequencing data.

    PubMed

    Yuan, Zhidong; Liu, Hongde; Nie, Yumin; Ding, Suping; Yan, Mingli; Tan, Shuhua; Jin, Yuanchang; Sun, Xiao

    2013-10-16

    Current technologies that are used for genome-wide microRNA (miRNA) prediction are mainly based on BLAST tool. They often produce a large number of false positives. Here, we describe an effective approach for identifying orthologous pre-miRNAs in several primates based on syntenic information. Some of them have been validated by small RNA high throughput sequencing data. This approach uses the synteny information and experimentally validated miRNAs of human, and incorporates currently available algorithms and tools to identify the pre-miRNAs in five other primates. First, we identified 929 potential pre-miRNAs in the marmoset in which miRNAs have not yet been reported. Then, we predicted the miRNAs in other primates, and we successfully re-identified most of the published miRNAs and found 721, 979, 650 and 639 new potential pre-miRNAs in chimpanzee, gorilla, orangutan and rhesus macaque, respectively. Furthermore, the miRNA transcriptome in the four primates have been re-analyzed and some novel predicted miRNAs have been supported by the small RNA sequencing data. Finally, we analyzed the potential functions of those validated miRNAs and explored the regulatory elements and transcription factors of some validated miRNA genes of interest. The results show that our approach can effectively identify novel miRNAs and some miRNAs that supported by small RNA sequencing data maybe play roles in the nervous system.

  8. Bovine Leukemia Virus Small Noncoding RNAs Are Functional Elements That Regulate Replication and Contribute to Oncogenesis In Vivo

    PubMed Central

    Hamaidia, Malik; de Brogniez, Alix; Gutiérrez, Gerónimo; Renotte, Nathalie; Reichert, Michal; Trono, Karina; Willems, Luc

    2016-01-01

    Retroviruses are not expected to encode miRNAs because of the potential problem of self-cleavage of their genomic RNAs. This assumption has recently been challenged by experiments showing that bovine leukemia virus (BLV) encodes miRNAs from intragenomic Pol III promoters. The BLV miRNAs are abundantly expressed in B-cell tumors in the absence of significant levels of genomic and subgenomic viral RNAs. Using deep RNA sequencing and functional reporter assays, we show that miRNAs mediate the expression of genes involved in cell signaling, cancer and immunity. We further demonstrate that BLV miRNAs are essential to induce B-cell tumors in an experimental model and to promote efficient viral replication in the natural host. PMID:27123579

  9. The small noncoding RNAs (sncRNAs) of murine gammaherpesvirus 68 (MHV-68) are involved in regulating the latent-to-lytic switch in vivo

    PubMed Central

    Steer, Beatrix; Strehle, Martin; Sattler, Christine; Bund, Dagmar; Flach, Britta; Stoeger, Tobias; Haas, Jürgen G.; Adler, Heiko

    2016-01-01

    The human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), which are associated with a variety of diseases including tumors, produce various small noncoding RNAs (sncRNAs) such as microRNAs (miRNAs). Like all herpesviruses, they show two stages in their life cycle: lytic replication and latency. During latency, hardly any viral proteins are expressed to avoid recognition by the immune system. Thus, sncRNAs might be exploited since they are less likely to be recognized. Specifically, it has been proposed that sncRNAs might contribute to the maintenance of latency. This has already been shown in vitro, but the respective evidence in vivo is very limited. A natural model system to explore this question in vivo is infection of mice with murine gammaherpesvirus 68 (MHV-68). We used this model to analyze a MHV-68 mutant lacking the expression of all miRNAs. In the absence of the miRNAs, we observed a higher viral genomic load during late latency in the spleens of mice. We propose that this is due to a disturbed regulation of the latent-to-lytic switch, altering the balance between latent and lytic infection. Hence, we provide for the first time evidence that gammaherpesvirus sncRNAs contribute to the maintenance of latency in vivo. PMID:27561205

  10. High Throughput Sequencing of Small RNAs in the Two Cucurbita Germplasm with Different Sodium Accumulation Patterns Identifies Novel MicroRNAs Involved in Salt Stress Response

    PubMed Central

    Xie, Junjun; Lei, Bo; Niu, Mengliang; Huang, Yuan; Kong, Qiusheng; Bie, Zhilong

    2015-01-01

    MicroRNAs (miRNAs), a class of small non-coding RNAs, recognize their mRNA targets based on perfect sequence complementarity. MiRNAs lead to broader changes in gene expression after plants are exposed to stress. High-throughput sequencing is an effective method to identify and profile small RNA populations in non-model plants under salt stresses, significantly improving our knowledge regarding miRNA functions in salt tolerance. Cucurbits are sensitive to soil salinity, and the Cucurbita genus is used as the rootstock of other cucurbits to enhance salt tolerance. Several cucurbit crops have been used for miRNA sequencing but salt stress-related miRNAs in cucurbit species have not been reported. In this study, we subjected two Cucurbita germplasm, namely, N12 (Cucurbita. maxima Duch.) and N15 (Cucurbita. moschata Duch.), with different sodium accumulation patterns, to Illumina sequencing to determine small RNA populations in root tissues after 4 h of salt treatment and control. A total of 21,548,326 and 19,394,108 reads were generated from the control and salt-treated N12 root tissues, respectively. By contrast, 19,108,240 and 20,546,052 reads were obtained from the control and salt-treated N15 root tissues, respectively. Fifty-eight conserved miRNA families and 33 novel miRNAs were identified in the two Cucurbita germplasm. Seven miRNAs (six conserved miRNAs and one novel miRNAs) were up-regulated in salt-treated N12 and N15 samples. Most target genes of differentially expressed novel miRNAs were transcription factors and salt stress-responsive proteins, including dehydration-induced protein, cation/H+ antiporter 18, and CBL-interacting serine/threonine-protein kinase. The differential expression of miRNAs between the two Cucurbita germplasm under salt stress conditions and their target genes demonstrated that novel miRNAs play an important role in the response of the two Cucurbita germplasm to salt stress. The present study initially explored small RNAs in the

  11. High Throughput Sequencing of Small RNAs in the Two Cucurbita Germplasm with Different Sodium Accumulation Patterns Identifies Novel MicroRNAs Involved in Salt Stress Response.

    PubMed

    Xie, Junjun; Lei, Bo; Niu, Mengliang; Huang, Yuan; Kong, Qiusheng; Bie, Zhilong

    2015-01-01

    MicroRNAs (miRNAs), a class of small non-coding RNAs, recognize their mRNA targets based on perfect sequence complementarity. MiRNAs lead to broader changes in gene expression after plants are exposed to stress. High-throughput sequencing is an effective method to identify and profile small RNA populations in non-model plants under salt stresses, significantly improving our knowledge regarding miRNA functions in salt tolerance. Cucurbits are sensitive to soil salinity, and the Cucurbita genus is used as the rootstock of other cucurbits to enhance salt tolerance. Several cucurbit crops have been used for miRNA sequencing but salt stress-related miRNAs in cucurbit species have not been reported. In this study, we subjected two Cucurbita germplasm, namely, N12 (Cucurbita. maxima Duch.) and N15 (Cucurbita. moschata Duch.), with different sodium accumulation patterns, to Illumina sequencing to determine small RNA populations in root tissues after 4 h of salt treatment and control. A total of 21,548,326 and 19,394,108 reads were generated from the control and salt-treated N12 root tissues, respectively. By contrast, 19,108,240 and 20,546,052 reads were obtained from the control and salt-treated N15 root tissues, respectively. Fifty-eight conserved miRNA families and 33 novel miRNAs were identified in the two Cucurbita germplasm. Seven miRNAs (six conserved miRNAs and one novel miRNAs) were up-regulated in salt-treated N12 and N15 samples. Most target genes of differentially expressed novel miRNAs were transcription factors and salt stress-responsive proteins, including dehydration-induced protein, cation/H+ antiporter 18, and CBL-interacting serine/threonine-protein kinase. The differential expression of miRNAs between the two Cucurbita germplasm under salt stress conditions and their target genes demonstrated that novel miRNAs play an important role in the response of the two Cucurbita germplasm to salt stress. The present study initially explored small RNAs in the

  12. Mating of the Stichotrichous Ciliate Oxytricha trifallax Induces Production of a Class of 27 nt Small RNAs Derived from the Parental Macronucleus

    PubMed Central

    Zahler, Alan M.; Neeb, Zachary T.; Lin, Athena; Katzman, Sol

    2012-01-01

    Ciliated protozoans possess two types of nuclei; a transcriptionally silent micronucleus, which serves as the germ line nucleus, and a transcriptionally active macronucleus, which serves as the somatic nucleus. The macronucleus is derived from a new diploid micronucleus after mating, with epigenetic information contributed by the parental macronucleus serving to guide the formation of the new macronucleus. In the stichotrichous ciliate Oxytricha trifallax, the macronuclear DNA is highly processed to yield gene-sized nanochromosomes with telomeres at each end. Here we report that soon after mating of Oxytricha trifallax, abundant 27 nt small RNAs are produced that are not present prior to mating. We performed next generation sequencing of Oxytricha small RNAs from vegetative and mating cells. Using sequence comparisons between macronuclear and micronuclear versions of genes, we found that the 27 nt RNA class derives from the parental macronucleus, not the developing macronucleus. These small RNAs are produced equally from both strands of macronuclear nanochromosomes, but in a highly non-uniform distribution along the length of the nanochromosome, and with a particular depletion in the 30 nt telomere-proximal positions. This production of small RNAs from the parental macronucleus during macronuclear development stands in contrast to the mechanism of epigenetic control in the distantly related ciliate Tetrahymena. In that species, 28–29 nt scanRNAs are produced from the micronucleus and these micronuclear-derived RNAs serve as epigenetic controllers of macronuclear development. Unlike the Tetrahymena scanRNAs, the Oxytricha macronuclear-derived 27 mers are not modified by 2′O-methylation at their 3′ ends. We propose models for the role of these “27macRNAs” in macronuclear development. PMID:22900016

  13. Genome-wide analyses of small non-coding RNAs in streptococci

    PubMed Central

    Patenge, Nadja; Pappesch, Roberto; Khani, Afsaneh; Kreikemeyer, Bernd

    2015-01-01

    Streptococci represent a diverse group of Gram-positive bacteria, which colonize a wide range of hosts among animals and humans. Streptococcal species occur as commensal as well as pathogenic organisms. Many of the pathogenic species can cause severe, invasive infections in their hosts leading to a high morbidity and mortality. The consequence is a tremendous suffering on the part of men and livestock besides the significant financial burden in the agricultural and healthcare sectors. An environmentally stimulated and tightly controlled expression of virulence factor genes is of fundamental importance for streptococcal pathogenicity. Bacterial small non-coding RNAs (sRNAs) modulate the expression of genes involved in stress response, sugar metabolism, surface composition, and other properties that are related to bacterial virulence. Even though the regulatory character is shared by this class of RNAs, variation on the molecular level results in a high diversity of functional mechanisms. The knowledge about the role of sRNAs in streptococci is still limited, but in recent years, genome-wide screens for sRNAs have been conducted in an increasing number of species. Bioinformatics prediction approaches have been employed as well as expression analyses by classical array techniques or next generation sequencing. This review will give an overview of whole genome screens for sRNAs in streptococci with a focus on describing the different methods and comparing their outcome considering sRNA conservation among species, functional similarities, and relevance for streptococcal infection. PMID:26042151

  14. Small Non-coding RNAs Associated with Viral Infectious Diseases of Veterinary Importance: Potential Clinical Applications

    PubMed Central

    Samir, Mohamed; Pessler, Frank

    2016-01-01

    MicroRNAs (miRNAs) represent a class of small non-coding RNA (sncRNA) molecules that can regulate mRNAs by inducing their degradation or by blocking translation. Considering that miRNAs are ubiquitous, stable, and conserved across animal species, it seems feasible to exploit them for clinical applications. Unlike in human viral diseases, where some miRNA-based molecules have progressed to clinical application, in veterinary medicine, this concept is just starting to come into view. Clinically, miRNAs could represent powerful diagnostic tools to pinpoint animal viral diseases and/or prognostic tools to follow up disease progression or remission. Additionally, the possible consequences of miRNA dysregulation make them potential therapeutic targets and open the possibilities to use them as tools to generate viral disease-resistant livestock. This review presents an update of preclinical studies on using sncRNAs to combat viral diseases that affect pet and farm animals. Moreover, we discuss the possibilities and challenges of bringing these bench-based discoveries to the veterinary clinic. PMID:27092305

  15. Characterization and expression patterns of small RNAs in synthesized Brassica hexaploids.

    PubMed

    Shen, Yanyue; Zhao, Qin; Zou, Jun; Wang, Wenliang; Gao, Yi; Meng, Jinling; Wang, Jianbo

    2014-06-01

    Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. We used high-throughput sequencing to compare miRNA expression profiles between Brassica hexaploid and its parents. A total of 613, 784 and 742 known miRNAs were identified in Brassica rapa, Brassica carinata, and Brassica hexaploid, respectively. We detected 618 miRNAs were differentially expressed (log(2)Ratio ≥ 1, P ≤ 0.05) between Brassica hexaploid and its parents, and 425 miRNAs were non-additively expressed in Brassica hexaploid, which suggest a trend of non-additive miRNA regulation following hybridization and polyploidization. Remarkably, majority of the non-additively expressed miRNAs in the Brassica hexaploid are repressed, and there was a bias toward repression of B. rapa miRNAs, which is consistent with the progenitor-biased gene repression in the synthetic allopolyploids. In addition, we identified 653 novel mature miRNAs in Brassica hexaploid and its parents. Finally, we found that almost all the non-additive accumulation of siRNA clusters exhibited a low-parent pattern in Brassica hexaploid. Non-additive small RNA regulation is involved in a range of biological pathways, probably providing a driving force for variation and adaptation in allopolyploids.

  16. Involvement of Enterococcus faecalis Small RNAs in Stress Response and Virulence

    PubMed Central

    Michaux, Charlotte; Hartke, Axel; Martini, Cecilia; Reiss, Swantje; Albrecht, Dirk; Budin-Verneuil, Aurélie; Sanguinetti, Maurizio; Engelmann, Susanne; Hain, Torsten; Verneuil, Nicolas

    2014-01-01

    Candidate small RNAs (sRNAs) have recently been identified in Enterococcus faecalis, a Gram-positive opportunistic pathogen, and six of these candidate sRNAs with unknown functions were selected for a functional study. Deletion mutants and complemented strains were constructed, and their virulence was tested. We were unable to obtain the ef0869-0870 mutant, likely due to an essential role, and the ef0820-0821 sRNA seemed not to be involved in virulence. In contrast, the mutant lacking ef0408-0409 sRNA, homologous to the RNAII component of the toxin-antitoxin system, appeared more virulent and more able to colonize mouse organs. The three other mutants showed reduced virulence. In addition, we checked the responses of these mutant strains to several stresses encountered in the gastrointestinal tract or during the infection process. In parallel, the activities of the sRNA promoters were measured using transcriptional fusion constructions. To attempt to identify the regulons of these candidate sRNAs, proteomics profiles of the mutant strains were compared with that of the wild type. This showed that the selected sRNAs controlled the expression of proteins involved in diverse cellular processes and the stress response. The combined data highlight the roles of certain candidate sRNAs in the adaptation of E. faecalis to environmental changes and in the complex transition process from a commensal to a pathogen. PMID:24914223

  17. Involvement of Enterococcus faecalis small RNAs in stress response and virulence.

    PubMed

    Michaux, Charlotte; Hartke, Axel; Martini, Cecilia; Reiss, Swantje; Albrecht, Dirk; Budin-Verneuil, Aurélie; Sanguinetti, Maurizio; Engelmann, Susanne; Hain, Torsten; Verneuil, Nicolas; Giard, Jean-Christophe

    2014-09-01

    Candidate small RNAs (sRNAs) have recently been identified in Enterococcus faecalis, a Gram-positive opportunistic pathogen, and six of these candidate sRNAs with unknown functions were selected for a functional study. Deletion mutants and complemented strains were constructed, and their virulence was tested. We were unable to obtain the ef0869-0870 mutant, likely due to an essential role, and the ef0820-0821 sRNA seemed not to be involved in virulence. In contrast, the mutant lacking ef0408-0409 sRNA, homologous to the RNAII component of the toxin-antitoxin system, appeared more virulent and more able to colonize mouse organs. The three other mutants showed reduced virulence. In addition, we checked the responses of these mutant strains to several stresses encountered in the gastrointestinal tract or during the infection process. In parallel, the activities of the sRNA promoters were measured using transcriptional fusion constructions. To attempt to identify the regulons of these candidate sRNAs, proteomics profiles of the mutant strains were compared with that of the wild type. This showed that the selected sRNAs controlled the expression of proteins involved in diverse cellular processes and the stress response. The combined data highlight the roles of certain candidate sRNAs in the adaptation of E. faecalis to environmental changes and in the complex transition process from a commensal to a pathogen.

  18. Small regulatory RNAs controlled by genomic imprinting and their contribution to human disease.

    PubMed

    Girardot, Michael; Cavaillé, Jérôme; Feil, Robert

    2012-12-01

    More than a hundred protein-coding genes are controlled by genomic imprinting in humans. These atypical genes are organized in chromosomal domains, each of which is controlled by a differentially methylated "imprinting control region" (ICR). How ICRs mediate the parental allele-specific expression of close-by genes is now becoming understood. At several imprinted domains, this epigenetic mechanism involves the action of long non-coding RNAs. It is less well appreciated that imprinted gene domains also transcribe hundreds of microRNA and small nucleolar RNA genes and that these represent the densest clusters of small RNA genes in mammalian genomes. The evolutionary reasons for this remarkable enrichment of small regulatory RNAs at imprinted domains remain unclear. However, recent studies show that imprinted small RNAs modulate specific functions in development and metabolism and also are frequently perturbed in cancer. Here, we review our current understanding of imprinted small RNAs in the human genome and discuss how perturbation of their expression contributes to disease.

  19. Small regulatory RNAs controlled by genomic imprinting and their contribution to human disease

    PubMed Central

    Girardot, Michael; Cavaillé, Jérôme; Feil, Robert

    2012-01-01

    More than a hundred protein-coding genes are controlled by genomic imprinting in humans. These atypical genes are organized in chromosomal domains, each of which is controlled by a differentially methylated "imprinting control region" (ICR). How ICRs mediate the parental allele-specific expression of close-by genes is now becoming understood. At several imprinted domains, this epigenetic mechanism involves the action of long non-coding RNAs. It is less well appreciated that imprinted gene domains also transcribe hundreds of microRNA and small nucleolar RNA genes and that these represent the densest clusters of small RNA genes in mammalian genomes. The evolutionary reasons for this remarkable enrichment of small regulatory RNAs at imprinted domains remain unclear. However, recent studies show that imprinted small RNAs modulate specific functions in development and metabolism and also are frequently perturbed in cancer. Here, we review our current understanding of imprinted small RNAs in the human genome and discuss how perturbation of their expression contributes to disease. PMID:23154539

  20. Small RNAs and Gene Network in a Durable Disease Resistance Gene—Mediated Defense Responses in Rice

    PubMed Central

    Zhang, Haitao; Xiao, Jinghua; Li, Xianghua; Wang, Shiping

    2015-01-01

    Accumulating data have suggested that small RNAs (sRNAs) have important functions in plant responses to pathogen invasion. However, it is largely unknown whether and how sRNAs are involved in the regulation of rice responses to the invasion of Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight, the most devastating bacterial disease of rice worldwide. We performed simultaneous genome-wide analyses of the expression of sRNAs and genes during early defense responses of rice to Xoo mediated by a major disease resistance gene, Xa3/Xa26, which confers durable and race-specific qualitative resistance. A large number of sRNAs and genes showed differential expression in Xa3/Xa26-mediated resistance. These differentially expressed sRNAs include known microRNAs (miRNAs), unreported miRNAs, and small interfering RNAs. The candidate genes, with expression that was negatively correlated with the expression of sRNAs, were identified, indicating that these genes may be regulated by sRNAs in disease resistance in rice. These results provide a new perspective regarding the putative roles of sRNA candidates and their putative target genes in durable disease resistance in rice. PMID:26335702

  1. The Role of Ctk1 Kinase in Termination of Small Non-Coding RNAs

    PubMed Central

    Lenstra, Tineke L.; Tudek, Agnieszka; Clauder, Sandra; Xu, Zhenyu; Pachis, Spyridon T.; van Leenen, Dik; Kemmeren, Patrick; Steinmetz, Lars M.; Libri, Domenico; Holstege, Frank C. P.

    2013-01-01

    Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is influenced by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex, the recruitment of which is dependent on CTD-Ser2 phosphorylation (Ser2P). Early termination of shorter cryptic unstable transcripts (CUTs) and small nucleolar/nuclear RNAs (sno/snRNAs) is performed by the Nrd1-Nab3-Sen1 (NNS) complex that binds phosphorylated CTD-Ser5 (Ser5P) via the CTD-interacting domain (CID) of Nrd1p. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1p are more similar to those derived from alterations in the Ser5P-dependent NNS pathway, than from loss of CTD-Ser2P binding factors. Tiling array analysis of ctk1Δ cells reveals readthrough at snoRNAs, at many cryptic unstable transcripts (CUTs) and stable uncharacterized transcripts (SUTs), but only at some mRNAs. Despite the suggested predominant role in termination of mRNAs, we observed that a CTK1 deletion or a Pol II CTD mutant lacking all Ser2 positions does not result in a global mRNA termination defect. Rather, termination defects in these strains are widely observed at NNS-dependent genes. These results indicate that Ctk1p and Ser2 CTD phosphorylation have a wide impact in termination of small non-coding RNAs but only affect a subset of mRNA coding genes. PMID:24324601

  2. Small RNA sequencing revealed dysregulated piRNAs in Alzheimer's disease and their probable role in pathogenesis.

    PubMed

    Roy, Jyoti; Sarkar, Arijita; Parida, Sibun; Ghosh, Zhumur; Mallick, Bibekanand

    2017-02-28

    PIWI-interacting RNAs (piRNAs), ∼23-36 nucleotide-long small non-coding RNAs, earlier believed to be germline-specific, have now been identified in somatic cells including neural cells. However, piRNAs have not yet been studied in the human brain (HB) and Alzheimer's disease (AD)-affected brain. In this study, by next-generation small RNA sequencing, 564 and 451 piRNAs were identified in the HB and AD-affected brain respectively. The majority of the neuronal piRNAs have intronic origin wherein primary piRNAs are mostly from the negative strand. piRNAs originating from the coding sequence of mRNAs and tRNAs are highly conserved compared to other genomic contexts. We found 1923 mRNAs significantly down-regulated in AD as the predicted targets of 125 up-regulated piRNAs. The filtering of targets based on our criteria coupled with pathway enrichment analysis of all the predicted targets resulted in five most significant AD-associated pathways enriched with four genes (CYCS, LIN7C, KPNA6, and RAB11A) found to be regulated by four piRNAs. The qRT-PCR study verified the reciprocal expression of piRNAs and their targets. This study provides the first evidence of piRNAs in the HB and AD which will provide the foundation for future studies to unravel the regulatory role of piRNAs in the human brain and associated diseases. The sequencing data have been submitted to the GEO database (Accession no. GSE85075).

  3. The Phloem-Delivered RNA Pool Contains Small Noncoding RNAs and Interferes with Translation1[W][OA

    PubMed Central

    Zhang, Shoudong; Sun, Li; Kragler, Friedrich

    2009-01-01

    In plants, the vascular tissue contains the enucleated sieve tubes facilitating long-distance transport of nutrients, hormones, and proteins. In addition, several mRNAs and small interfering RNAs/microRNAs were shown to be delivered via sieve tubes whose content is embodied by the phloem sap (PS). A number of these phloem transcripts are transported from source to sink tissues and function at targeted tissues. To gain additional insights into phloem-delivered RNAs and their potential role in signaling, we isolated and characterized PS RNA molecules distinct from microRNAs/small interfering RNAs with a size ranging from 30 to 90 bases. We detected a high number of full-length and phloem-specific fragments of noncoding RNAs such as tRNAs, ribosomal RNAs, and spliceosomal RNAs in the PS of pumpkin (Cucurbita maxima). In vitro assays show that small quantities of PS RNA molecules efficiently inhibit translation in an unspecific manner. Proof of concept that PS-specific tRNA fragments may interfere with ribosomal activity was obtained with artificially produced tRNA fragments. The results are discussed in terms of a functional role for long distance delivered noncoding PS RNAs. PMID:19261735

  4. Phylogeny of organisms investigated by the base-pair changes in the stem regions of small and large ribosomal subunit RNAs.

    PubMed

    Otsuka, J; Terai, G; Nakano, T

    1999-02-01

    In order to obtain the evolutionary distance data that are as purely additive as possible, we have developed a novel method for evaluating the evolutionary distances from the base-pair changes in stem regions of ribosomal RNAs (rRNAs). The application of this method to small-subunit (SSU) and large-subunit (LSU) rRNAs provides the distance data, with which both the unweighted pair group method of analysis and the neighbor-joining method give almost the same tree topology of most organisms except for some Protoctista, thermophilic bacteria, parasitic organisms, and endosymbionts. Although the evolutionary distances calculated with LSU rRNAs are somewhat longer than those with SSU rRNAs, the difference, probably due to a slight difference in functional constraint, is substantially decreased when the distances are converted into the divergence times of organisms by the measure of the time scale estimated in each type of rRNAs. The divergence times of main branches agree fairly well with the geological record of organisms, at least after the appearance of oxygen-releasing photosynthesis, although the divergence times of Eukaryota, Archaebacteria, and Eubacteria are somewhat overestimated in comparison with the geological record of Earth formation. This result is explained by considering that the mutation rate is determined by the accumulation of misrepairs for DNA damage caused by radiation and that the effect of radiation had been stronger before the oxygen molecules became abundant in the atmosphere of the Earth.

  5. Genomic prediction and validation of Pospiviroidae-derived small RNAs and their targets during tomato infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Viroids are small, covalently-closed, circular non-coding RNAs that act as pathogens in flowering plants. It is proposed that the symptoms of viroid pathogenesis result from a direct interaction between the viroid genomic RNA and unknown host plant factors. Using a comparative genomic approach we t...

  6. Psmir: a database of potential associations between small molecules and miRNAs

    PubMed Central

    Meng, Fanlin; Wang, Jing; Dai, Enyu; Yang, Feng; Chen, Xiaowen; Wang, Shuyuan; Yu, Xuexin; Liu, Dianming; Jiang, Wei

    2016-01-01

    miRNAs are key post-transcriptional regulators of many essential biological processes, and their dysregulation has been validated in almost all human cancers. Restoring aberrantly expressed miRNAs might be a novel therapeutics. Recently, many studies have demonstrated that small molecular compounds can affect miRNA expression. Thus, prediction of associations between small molecules and miRNAs is important for investigation of miRNA-targeted drugs. Here, we analyzed 39 miRNA-perturbed gene expression profiles, and then calculated the similarity of transcription responses between miRNA perturbation and drug treatment to predict drug-miRNA associations. At the significance level of 0.05, we obtained 6501 candidate associations between 1295 small molecules and 25 miRNAs, which included 624 FDA approved drugs. Finally, we constructed the Psmir database to store all potential associations and the related materials. In a word, Psmir served as a valuable resource for dissecting the biological significance in small molecules’ effects on miRNA expression, which will facilitate developing novel potential therapeutic targets or treatments for human cancers. Psmir is supported by all major browsers, and is freely available at http://www.bio-bigdata.com/Psmir/. PMID:26759061

  7. Psmir: a database of potential associations between small molecules and miRNAs.

    PubMed

    Meng, Fanlin; Wang, Jing; Dai, Enyu; Yang, Feng; Chen, Xiaowen; Wang, Shuyuan; Yu, Xuexin; Liu, Dianming; Jiang, Wei

    2016-01-13

    miRNAs are key post-transcriptional regulators of many essential biological processes, and their dysregulation has been validated in almost all human cancers. Restoring aberrantly expressed miRNAs might be a novel therapeutics. Recently, many studies have demonstrated that small molecular compounds can affect miRNA expression. Thus, prediction of associations between small molecules and miRNAs is important for investigation of miRNA-targeted drugs. Here, we analyzed 39 miRNA-perturbed gene expression profiles, and then calculated the similarity of transcription responses between miRNA perturbation and drug treatment to predict drug-miRNA associations. At the significance level of 0.05, we obtained 6501 candidate associations between 1295 small molecules and 25 miRNAs, which included 624 FDA approved drugs. Finally, we constructed the Psmir database to store all potential associations and the related materials. In a word, Psmir served as a valuable resource for dissecting the biological significance in small molecules' effects on miRNA expression, which will facilitate developing novel potential therapeutic targets or treatments for human cancers. Psmir is supported by all major browsers, and is freely available at http://www.bio-bigdata.com/Psmir/.

  8. 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...

  9. Small RNAs as important regulators for the hybrid vigour of super-hybrid rice.

    PubMed

    Zhang, Lei; Peng, Yonggang; Wei, Xiaoli; Dai, Yan; Yuan, Dawei; Lu, Yufei; Pan, Yangyang; Zhu, Zhen

    2014-11-01

    Heterosis is an important biological phenomenon; however, the role of small RNA (sRNA) in heterosis of hybrid rice remains poorly described. Here, we performed sRNA profiling of F1 super-hybrid rice LYP9 and its parents using high-throughput sequencing technology, and identified 355 distinct mature microRNAs and trans-acting small interfering RNAs, 69 of which were differentially expressed sRNAs (DES) between the hybrid and the mid-parental value. Among these, 34 DES were predicted to target 176 transcripts, of which 112 encoded 94 transcription factors. Further analysis showed that 67.6% of DES expression levels were negatively correlated with their target mRNAs either in flag leaves or panicles. The target genes of DES were significantly enriched in some important biological processes, including the auxin signalling pathway, in which existed a regulatory network mediated by DES and their targets, closely associated with plant growth and development. Overall, 20.8% of DES and their target genes were significantly enriched in quantitative trait loci of small intervals related to important rice agronomic traits including growth vigour, grain yield, and plant architecture, suggesting that the interaction between sRNAs and their targets contributes to the heterotic phenotypes of hybrid rice. Our findings revealed that sRNAs might play important roles in hybrid vigour of super-hybrid rice by regulating their target genes, especially in controlling the auxin signalling pathway. The above finding provides a novel insight into the molecular mechanism of heterosis.

  10. Genome-wide identification of endogenous RNA-directed DNA methylation loci associated with abundant 21-nucleotide siRNAs in Arabidopsis

    PubMed Central

    Zhao, Jian-Hua; Fang, Yuan-Yuan; Duan, Cheng-Guo; Fang, Rong-Xiang; Ding, Shou-Wei; Guo, Hui-Shan

    2016-01-01

    In Arabidopsis, the 24-nucleotide (nt) small interfering RNAs (siRNAs) mediates RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of transposable elements (TEs). In the present study, we examined genome-wide changes in DNA methylation and siRNA accumulation in Arabidopsis induced by expression of the Cucumber mosaic virus silencing suppressor protein 2b known to directly bind to both the 21/24-nt siRNAs as well as their associated Argonaute proteins. We demonstrated a genome-wide reduction of CHH and CHG methylation in the 2b-transgenic plants. We found that 2b suppressed RdDM not only at the previously annotated loci directed by 24-nt siRNAs but also a new set of loci associated with 21/22-nt siRNAs. Further analysis showed that the reduced methylation of TEs and coding genes targeted by 21/22-nt siRNAs was associated with sequestration of the duplex siRNAs by the 2b protein but not with changes in either siRNA production or transcription. Notably, we detected both the deletion and/or the transposition of multicopy TEs associated with 2b-induced hypomethylation, suggesting potential TE reactivation. We propose that the silencing of many TEs in Arabidopsis is controlled by the 24- and 21-nt endogenous siRNAs analogous to Drosophila TE silencing by PIWI-interacting RNAs and siRNAs. PMID:27786269

  11. Silencing of antibiotic resistance in E. coli with engineered phage bearing small regulatory RNAs.

    PubMed

    Libis, Vincent K; Bernheim, Aude G; Basier, Clovis; Jaramillo-Riveri, Sebastián; Deyell, Matthew; Aghoghogbe, Idonnya; Atanaskovic, Iva; Bencherif, Amel Camélia; Benony, Marguerite; Koutsoubelis, Nicolas; Löchner, Anne C; Marinkovic, Zoran S; Zahra, Sarah; Zegman, Yonatan; Lindner, Ariel B; Wintermute, Edwin H

    2014-12-19

    In response to emergent antibiotic resistance, new strategies are needed to enhance the effectiveness of existing antibiotics. Here, we describe a phagemid-delivered, RNA-mediated system capable of directly knocking down antibiotic resistance phenotypes. Small regulatory RNAs (sRNAs) were designed to specifically inhibit translation of chloramphenicol acetyltransferase and kanamycin phosphotransferase. Nonlytic phagemids coding for sRNA expression were able to infect and restore chloramphenicol and kanamycin sensitivity to populations of otherwise resistant E. coli. This modular system could easily be extended to other bacteria with resistance profiles that depend on specific transcripts.

  12. Small RNAs in Plant Responses to Abiotic Stresses: Regulatory Roles and Study Methods

    PubMed Central

    Ku, Yee-Shan; Wong, Johanna Wing-Hang; Mui, Zeta; Liu, Xuan; Hui, Jerome Ho-Lam; Chan, Ting-Fung; Lam, Hon-Ming

    2015-01-01

    To survive under abiotic stresses in the environment, plants trigger a reprogramming of gene expression, by transcriptional regulation or translational regulation, to turn on protective mechanisms. The current focus of research on how plants cope with abiotic stresses has transitioned from transcriptomic analyses to small RNA investigations. In this review, we have summarized and evaluated the current methodologies used in the identification and validation of small RNAs and their targets, in the context of plant responses to abiotic stresses. PMID:26501263

  13. Identification of small RNAs in extracellular vesicles from the commensal yeast Malassezia sympodialis

    PubMed Central

    Rayner, Simon; Bruhn, Sören; Vallhov, Helen; Andersson, Anna; Billmyre, R. Blake; Scheynius, Annika

    2017-01-01

    Malassezia is the dominant fungus in the human skin mycobiome and is associated with common skin disorders including atopic eczema (AE)/dermatitis. Recently, it was found that Malassezia sympodialis secretes nanosized exosome-like vesicles, designated MalaEx, that carry allergens and can induce inflammatory cytokine responses. Extracellular vesicles from different cell-types including fungi have been found to deliver functional RNAs to recipient cells. In this study we assessed the presence of small RNAs in MalaEx and addressed if the levels of these RNAs differ when M. sympodialis is cultured at normal human skin pH versus the elevated pH present on the skin of patients with AE. The total number and the protein concentration of the released MalaEx harvested after 48 h culture did not differ significantly between the two pH conditions nor did the size of the vesicles. From small RNA sequence data, we identified a set of reads with well-defined start and stop positions, in a length range of 16 to 22 nucleotides consistently present in the MalaEx. The levels of small RNAs were not significantly differentially expressed between the two different pH conditions indicating that they are not influenced by the elevated pH level observed on the AE skin. PMID:28051166

  14. Regulation of Small RNAs and Corresponding Targets in Nod Factor-Induced Phaseolus vulgaris Root Hair Cells

    PubMed Central

    Formey, Damien; Martín-Rodríguez, José Ángel; Leija, Alfonso; Santana, Olivia; Quinto, Carmen; Cárdenas, Luis; Hernández, Georgina

    2016-01-01

    A genome-wide analysis identified the set of small RNAs (sRNAs) from the agronomical important legume Phaseolus vulgaris (common bean), including novel P. vulgaris-specific microRNAs (miRNAs) potentially important for the regulation of the rhizobia-symbiotic process. Generally, novel miRNAs are difficult to identify and study because they are very lowly expressed in a tissue- or cell-specific manner. In this work, we aimed to analyze sRNAs from common bean root hairs (RH), a single-cell model, induced with pure Rhizobium etli nodulation factors (NF), a unique type of signal molecule. The sequence analysis of samples from NF-induced and control libraries led to the identity of 132 mature miRNAs, including 63 novel miRNAs and 1984 phasiRNAs. From these, six miRNAs were significantly differentially expressed during NF induction, including one novel miRNA: miR-RH82. A parallel degradome analysis of the same samples revealed 29 targets potentially cleaved by novel miRNAs specifically in NF-induced RH samples; however, these novel miRNAs were not differentially accumulated in this tissue. This study reveals Phaseolus vulgaris-specific novel miRNA candidates and their corresponding targets that meet all criteria to be involved in the regulation of the early nodulation events, thus setting the basis for exploring miRNA-mediated improvement of the common bean–rhizobia symbiosis. PMID:27271618

  15. Small RNAs tackle large viruses: RNA interference-based antiviral defense against DNA viruses in insects.

    PubMed

    Bronkhorst, Alfred W; Miesen, Pascal; van Rij, Ronald P

    2013-01-01

    The antiviral RNA interference (RNAi) pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNA) that guide the recognition and cleavage of complementary viral target RNAs. In RNA virus infections, viral replication intermediates, dsRNA genomes or viral structured RNAs have been implicated as Dicer-2 substrates. In a recent publication, we demonstrated that a double-stranded DNA virus, Invertebrate iridescent virus 6, is a target of the Drosophila RNAi machinery, and we proposed that overlapping converging transcripts base pair to form the dsRNA substrates for vsiRNA biogenesis. Here, we discuss the role of RNAi in antiviral defense to DNA viruses in Drosophila and other invertebrate model systems.

  16. Plasma and EBC microRNAs as early biomarkers of non-small-cell lung cancer.

    PubMed

    Mozzoni, Paola; Banda, Iris; Goldoni, Matteo; Corradi, Massimo; Tiseo, Marcello; Acampa, Olga; Balestra, Valeria; Ampollini, Luca; Casalini, Angelo; Carbognani, Paolo; Mutti, Antonio

    2013-12-01

    Lung cancer is a major cause of death in Western countries. Current screening methods are invasive and still lead to a high percentage of false positives. There is, therefore, a need to find biomarkers that increase the probability of detecting lung cancer early. MicroRNAs (miRNAs) are stable molecules in blood plasma and exhaled breath condensate (EBC). We quantified miRNA-21 and miRNA-486 expression from plasma and EBC samples from patients with a diagnosis of non-small-cell lung cancer (NSCLC) and controls. miRNA-21 was significantly higher in plasma and in EBC of the NSCLC patients and miRNA-486 was significantly lower. This difference indicates a significantly improved diagnostic value, and suggests that these miRNAs could be clinically used as a first-line screening test in high-risk subjects.

  17. Identification and profiling of novel microRNAs in the Brassica rapa genome based on small RNA deep sequencing

    PubMed Central

    2012-01-01

    Background MicroRNAs (miRNAs) are one of the functional non-coding small RNAs involved in the epigenetic control of the plant genome. Although plants contain both evolutionary conserved miRNAs and species-specific miRNAs within their genomes, computational methods often only identify evolutionary conserved miRNAs. The recent sequencing of the Brassica rapa genome enables us to identify miRNAs and their putative target genes. In this study, we sought to provide a more comprehensive prediction of B. rapa miRNAs based on high throughput small RNA deep sequencing. Results We sequenced small RNAs from five types of tissue: seedlings, roots, petioles, leaves, and flowers. By analyzing 2.75 million unique reads that mapped to the B. rapa genome, we identified 216 novel and 196 conserved miRNAs that were predicted to target approximately 20% of the genome’s protein coding genes. Quantitative analysis of miRNAs from the five types of tissue revealed that novel miRNAs were expressed in diverse tissues but their expression levels were lower than those of the conserved miRNAs. Comparative analysis of the miRNAs between the B. rapa and Arabidopsis thaliana genomes demonstrated that redundant copies of conserved miRNAs in the B. rapa genome may have been deleted after whole genome triplication. Novel miRNA members seemed to have spontaneously arisen from the B. rapa and A. thaliana genomes, suggesting the species-specific expansion of miRNAs. We have made this data publicly available in a miRNA database of B. rapa called BraMRs. The database allows the user to retrieve miRNA sequences, their expression profiles, and a description of their target genes from the five tissue types investigated here. Conclusions This is the first report to identify novel miRNAs from Brassica crops using genome-wide high throughput techniques. The combination of computational methods and small RNA deep sequencing provides robust predictions of miRNAs in the genome. The finding of numerous novel miRNAs

  18. Diverse Functions of Small RNAs in Different Plant–Pathogen Communications

    PubMed Central

    Huang, Juan; Yang, Meiling; Lu, Lu; Zhang, Xiaoming

    2016-01-01

    RNA silencing is a conserved mechanism that utilizes small RNAs (sRNAs) to direct the regulation of gene expression at the transcriptional or post-transcriptional level. Plants utilizing RNA silencing machinery to defend pathogen infection was first identified in plant–virus interaction and later was observed in distinct plant–pathogen interactions. RNA silencing is not only responsible for suppressing RNA accumulation and movement of virus and viroid, but also facilitates plant immune responses against bacterial, oomycete, and fungal infection. Interestingly, even the same plant sRNA can perform different roles when encounters with different pathogens. On the other side, pathogens counteract by generating sRNAs that directly regulate pathogen gene expression to increase virulence or target host genes to facilitate pathogen infection. Here, we summarize the current knowledge of the characterization and biogenesis of host- and pathogen-derived sRNAs, as well as the different RNA silencing machineries that plants utilize to defend against different pathogens. The functions of these sRNAs in defense and counter-defense and their mechanisms for regulation during different plant–pathogen interactions are also discussed. PMID:27757103

  19. Regulation of spermatogenesis by small non-coding RNAs: role of the germ granule.

    PubMed

    de Mateo, Sara; Sassone-Corsi, Paolo

    2014-05-01

    The spermatogenic process relays in highly regulated gene expression mechanisms at the transcriptional and post-transcriptional levels to generate the male gamete that is needed for the perpetuation of the species. Small non-coding RNA pathways have been determined to participate in the post-transcriptional regulatory processes of germ cells. The most important sncRNA molecules that are critically involved in spermatogenesis belong to the miRNA and piRNAs pathways as illustrated by animal models where ablation of specific protein components displays male infertility. Several elements of these regulatory pathways have been found in the nuage or germ granule, a non-membranous cytoplasmatic structure that can be seen in spermatocytes and spermatids. This notion suggests that germ granules may act as organizer centers for silencing pathways in the germline. In general, miRNAs regulate spermatogenesis through targeting and down-regulation of specific transcripts to eventually promote sperm development. However, piRNAs are powerful repressors of transposon elements expression in the spermatogenic process. Here we describe the suggested functions that miRNA and piRNAs pathways execute in the regulation of spermatogenesis and include some recent studies in the field. Despite major strides on the detailed molecular mechanisms of sncRNAs in relation to spermatogenesis, there is plenty to discover on this fascinating regulatory program.

  20. Molecular characterization of geminivirus-derived small RNAs in different plant species

    PubMed Central

    Akbergenov, Rashid; Si-Ammour, Azeddine; Blevins, Todd; Amin, Imran; Kutter, Claudia; Vanderschuren, Herve; Zhang, Peng; Gruissem, Wilhelm; Meins, Frederick; Hohn, Thomas; Pooggin, Mikhail M.

    2006-01-01

    DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs—the hallmarks of silencing—associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5′ end phosphorylated, as shown by phosphatase treatments, and methylated at the 3′-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to β-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions. PMID:16421273

  1. Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.).

    PubMed

    Asha, Srinivasan; Soniya, Eppurath V

    2016-01-01

    Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5'tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5'tRFs in the infected leaf and root. The abundance of 5'tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5'Ala(CGC) tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5'Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper.

  2. Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.)

    PubMed Central

    Asha, Srinivasan; Soniya, Eppurath V.

    2016-01-01

    Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5′tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5′tRFs in the infected leaf and root. The abundance of 5′tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5′AlaCGC tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5′Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper. PMID:27313593

  3. High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.)

    PubMed Central

    Donaire, Livia; Pedrola, Laia; de la Rosa, Raúl; Llave, César

    2011-01-01

    Small RNAs (sRNAs) of 20 to 25 nucleotides (nt) in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the sRNA component of more than 40 plant species. Here, we used deep sequencing and molecular methods to report the first inventory of sRNAs in olive (Olea europaea L.). sRNA libraries prepared from juvenile and adult shoots revealed that the 24-nt class dominates the sRNA transcriptome and atypically accumulates to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. A total of 18 known miRNA families were identified in the libraries. Also, 5 other sRNAs derived from potential hairpin-like precursors remain as plausible miRNA candidates. RNA blots confirmed miRNA expression and suggested tissue- and/or developmental-specific expression patterns. Target mRNAs of conserved miRNAs were computationally predicted among the olive cDNA collection and experimentally validated through endonucleolytic cleavage assays. Finally, we use expression data to uncover genetic components of the miR156, miR172 and miR390/TAS3-derived trans-acting small interfering RNA (tasiRNA) regulatory nodes, suggesting that these interactive networks controlling developmental transitions are fully operational in olive. PMID:22140484

  4. The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative argonautes in Arabidopsis thaliana.

    PubMed

    Wang, Xian-Bing; Jovel, Juan; Udomporn, Petchthai; Wang, Ying; Wu, Qingfa; Li, Wan-Xiang; Gasciolli, Virginie; Vaucheret, Herve; Ding, Shou-Wei

    2011-04-01

    Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-Δ2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-Δ2b requires SUPPRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-Δ2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-Δ2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21- and 22-nucleotide secondary siRNAs are discussed.

  5. Next-generation sequencing of small RNAs from HIV-infected cells identifies phased microrna expression patterns and candidate novel microRNAs differentially expressed upon infection.

    PubMed

    Chang, Stewart T; Thomas, Matthew J; Sova, Pavel; Green, Richard R; Palermo, Robert E; Katze, Michael G

    2013-02-05

    HIV infection of CD4(+) T cells induces a range of host transcriptional changes in mRNAs as well as microRNAs that may coordinate changes in mRNAs. To survey these dynamic changes, we applied next-generation sequencing, analyzing the small RNA fraction of HIV-infected cells at 5, 12, and 24 h postinfection (RNA-Seq). These time points afforded a view of the transcriptomic changes occurring both before and during viral replication. In the resulting small RNA-Seq data set, we detected a phased pattern of microRNA expression. Largely distinct sets of microRNAs were found to be suppressed at 5 and 12 h postinfection, and both sets of changes rebounded later in infection. A larger set of microRNA changes was observed at 24 h postinfection. When integrated with mRNA expression data, the small RNA-Seq data indicated a role for microRNAs in transcriptional regulation, T cell activation, and cell cycle during HIV infection. As a unique benefit of next-generation sequencing, we also detected candidate novel host microRNAs differentially expressed during infection, including one whose downregulation at 24 h postinfection may allow full replication of HIV to proceed. Collectively, our data provide a uniquely comprehensive view of the changes in host microRNAs induced by HIV during cellular infection. IMPORTANCE New sequencing technologies allow unprecedented views into changes occurring in virus-infected cells, including comprehensive and largely unbiased measurements of different types of RNA. In this study, we used next-generation sequencing to profile dynamic changes in cellular microRNAs occurring in HIV-infected cells. The sensitivity afforded by sequencing allowed us to detect changes in microRNA expression early in infection, before the onset of viral replication. A phased pattern of expression was evident among these microRNAs, and many that were initially suppressed were later overexpressed at the height of infection, providing unique signatures of infection. By

  6. Dynamics and Mechanism of A Quorum Sensing Network Regulated by Small RNAs in Vibrio Harveyi

    NASA Astrophysics Data System (ADS)

    Shen, Jian-Wei

    2011-03-01

    Bacterial quorum sensing (QS) has attracted much interests and it is an important process of cell communication. Recently, Bassler et al. studied the phenomena of QS regulated by small RNAs and the experimental data showed that small RNAs played important role in the QS of Vibrio harveyi and it can permit the fine-tuning of gene regulation and maintenance of homeostasis. According to Michaelis—Menten kinetics and mass action law in this paper, we construct a mathematical model to investigate the mechanism induced QS by coexist of small RNA and signal molecular (AI) and show that there are periodic oscillation when the time delay and Hill coefficient exceed a critical value and the periodic oscillation produces the change of concentration and induces QS. These results are fit to the experimental results. In the meanwhile, we also get some theoretical value of Hopf Bifurcation on time deday. In addition, we also find this network is robust against noise.

  7. Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins?

    PubMed Central

    2016-01-01

    RNA silencing is a eukaryote‐specific phenomenon in which microRNAs and small interfering RNAs degrade messenger RNAs containing a complementary sequence. To this end, these small RNAs need to be loaded onto an Argonaute protein (AGO protein) to form the effector complex referred to as RNA‐induced silencing complex (RISC). RISC assembly undergoes multiple and sequential steps with the aid of Hsc70/Hsp90 chaperone machinery. The molecular mechanisms for this assembly process remain unclear, despite their significance for the development of gene silencing techniques and RNA interference‐based therapeutics. This review dissects the currently available structures of AGO proteins and proposes models and hypotheses for RISC assembly, covering the conformation of unloaded AGO proteins, the chaperone‐assisted duplex loading, and the slicer‐dependent and slicer‐independent duplex separation. The differences in the properties of RISC between prokaryotes and eukaryotes will also be clarified. WIREs RNA 2016, 7:637–660. doi: 10.1002/wrna.1356 For further resources related to this article, please visit the WIREs website. PMID:27184117

  8. Small RNAs in plants: recent development and application for crop improvement.

    PubMed

    Kamthan, Ayushi; Chaudhuri, Abira; Kamthan, Mohan; Datta, Asis

    2015-01-01

    The phenomenon of RNA interference (RNAi) which involves sequence-specific gene regulation by small non-coding RNAs, i.e., small interfering RNA (siRNA) and microRNA (miRNA) has emerged as one of most powerful approaches for crop improvement. RNAi based on siRNA is one of the widely used tools of reverse genetics which aid in revealing gene functions in many species. This technology has been extensively applied to alter the gene expression in plants with an aim to achieve desirable traits. RNAi has been used for enhancing the crop yield and productivity by manipulating the gene involved in biomass, grain yield and enhanced shelf life of fruits and vegetables. It has also been applied for developing resistance against various biotic (bacteria, fungi, viruses, nematodes, insects) and abiotic stresses (drought, salinity, cold, etc.). Nutritional improvements of crops have also been achieved by enriching the crops with essential amino acids, fatty acids, antioxidants and other nutrients beneficial for human health or by reducing allergens or anti-nutrients. microRNAs are key regulators of important plant processes like growth, development, and response to various stresses. In spite of similarity in size (20-24 nt), miRNA differ from siRNA in precursor structures, pathway of biogenesis, and modes of action. This review also highlights the miRNA based genetic modification technology where various miRNAs/artificial miRNAs and their targets can be utilized for improving several desirable plant traits. microRNA based strategies are much efficient than siRNA-based RNAi strategies due to its specificity and less undesirable off target effects. As per the FDA guidelines, small RNA (sRNA) based transgenics are much safer for consumption than those over-expressing proteins. This review thereby summarizes the emerging advances and achievement in the field of sRNAs and its application for crop improvement.

  9. Small RNAs in plants: recent development and application for crop improvement

    PubMed Central

    Kamthan, Ayushi; Chaudhuri, Abira; Kamthan, Mohan; Datta, Asis

    2015-01-01

    The phenomenon of RNA interference (RNAi) which involves sequence-specific gene regulation by small non-coding RNAs, i.e., small interfering RNA (siRNA) and microRNA (miRNA) has emerged as one of most powerful approaches for crop improvement. RNAi based on siRNA is one of the widely used tools of reverse genetics which aid in revealing gene functions in many species. This technology has been extensively applied to alter the gene expression in plants with an aim to achieve desirable traits. RNAi has been used for enhancing the crop yield and productivity by manipulating the gene involved in biomass, grain yield and enhanced shelf life of fruits and vegetables. It has also been applied for developing resistance against various biotic (bacteria, fungi, viruses, nematodes, insects) and abiotic stresses (drought, salinity, cold, etc.). Nutritional improvements of crops have also been achieved by enriching the crops with essential amino acids, fatty acids, antioxidants and other nutrients beneficial for human health or by reducing allergens or anti-nutrients. microRNAs are key regulators of important plant processes like growth, development, and response to various stresses. In spite of similarity in size (20–24 nt), miRNA differ from siRNA in precursor structures, pathway of biogenesis, and modes of action. This review also highlights the miRNA based genetic modification technology where various miRNAs/artificial miRNAs and their targets can be utilized for improving several desirable plant traits. microRNA based strategies are much efficient than siRNA-based RNAi strategies due to its specificity and less undesirable off target effects. As per the FDA guidelines, small RNA (sRNA) based transgenics are much safer for consumption than those over-expressing proteins. This review thereby summarizes the emerging advances and achievement in the field of sRNAs and its application for crop improvement. PMID:25883599

  10. Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia

    PubMed Central

    Marcial-Quino, Jaime; Gómez-Manzo, Saúl; Fierro, Francisco; Vanoye-Carlo, America; Rufino-González, Yadira; Sierra-Palacios, Edgar; Castillo-Villanueva, Adriana; Castillo-Rodríguez, Rosa Angélica; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto; Reyes-Vivas, Horacio

    2016-01-01

    Stem-loop quantitative reverse transcription PCR (RT-qPCR) is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL)-based design to detect—in a rapid, sensitive, specific, and reproducible way—the small nucleolar RNA (snoRNA) GlsR17 and its derived miRNA (miR2) of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G. lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G. lamblia. This methodology will be a powerful tool for studying gene regulation in G. lamblia, and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi) pathway in G. lamblia. PMID:27999395

  11. An improved method for identification of small non-coding RNAs in bacteria using support vector machine

    PubMed Central

    Barman, Ranjan Kumar; Mukhopadhyay, Anirban; Das, Santasabuj

    2017-01-01

    Bacterial small non-coding RNAs (sRNAs) are not translated into proteins, but act as functional RNAs. They are involved in diverse biological processes like virulence, stress response and quorum sensing. Several high-throughput techniques have enabled identification of sRNAs in bacteria, but experimental detection remains a challenge and grossly incomplete for most species. Thus, there is a need to develop computational tools to predict bacterial sRNAs. Here, we propose a computational method to identify sRNAs in bacteria using support vector machine (SVM) classifier. The primary sequence and secondary structure features of experimentally-validated sRNAs of Salmonella Typhimurium LT2 (SLT2) was used to build the optimal SVM model. We found that a tri-nucleotide composition feature of sRNAs achieved an accuracy of 88.35% for SLT2. We validated the SVM model also on the experimentally-detected sRNAs of E. coli and Salmonella Typhi. The proposed model had robustly attained an accuracy of 81.25% and 88.82% for E. coli K-12 and S. Typhi Ty2, respectively. We confirmed that this method significantly improved the identification of sRNAs in bacteria. Furthermore, we used a sliding window-based method and identified sRNAs from complete genomes of SLT2, S. Typhi Ty2 and E. coli K-12 with sensitivities of 89.09%, 83.33% and 67.39%, respectively. PMID:28383059

  12. 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...

  13. Roles of small RNAs in the effects of nutrition on apoptosis and spermatogenesis in the adult testis

    PubMed Central

    Guan, Yongjuan; Liang, Guanxiang; Hawken, Penelope A. R.; Malecki, Irek A.; Cozens, Greg; Vercoe, Philip E.; Martin, Graeme B.; Guan, Le Luo

    2015-01-01

    We tested whether reductions in spermatozoal quality induced by under-nutrition are associated with increased germ cell apoptosis and disrupted spermatogenesis, and whether these effects are mediated by small RNAs. Groups of 8 male sheep were fed for a 10% increase or 10% decrease in body mass over 65 days. Underfeeding increased the number of apoptotic germ cells (P < 0.05) and increased the expression of apoptosis-related genes (P < 0.05) in testicular tissue. We identified 44 miRNAs and 35 putative piRNAs that were differentially expressed in well-fed and underfed males (FDR < 0.05). Some were related to reproductive system development, apoptosis (miRNAs), and sperm production and quality (piRNAs). Novel-miR-144 (miR-98), was found to target three apoptotic genes (TP53, CASP3, FASL). The proportion of miRNAs as a total of small RNAs was greater in well-fed males than in underfed males (P < 0.05) and was correlated (r = 0.8, P < 0.05) with the proportion of piRNAs in well-fed and underfed males. In conclusion, the reductions in spermatozoal quality induced by under-nutrition are caused, at least partly, by disruptions to Sertoli cell function and increased germ cell apoptosis, mediated by changes in the expression of miRNAs and piRNAs. PMID:25996545

  14. Identification of 88 regulatory small RNAs in the TIGR4 strain of the human pathogen Streptococcus pneumoniae

    PubMed Central

    Acebo, Paloma; Martin-Galiano, Antonio J.; Navarro, Sara; Zaballos, Ángel; Amblar, Mónica

    2012-01-01

    Streptococcus pneumoniae is the main etiological agent of community-acquired pneumonia and a major cause of mortality and morbidity among children and the elderly. Genome sequencing of several pneumococcal strains revealed valuable information about the potential proteins and genetic diversity of this prevalent human pathogen. However, little is known about its transcriptional regulation and its small regulatory noncoding RNAs. In this study, we performed deep sequencing of the S. pneumoniae TIGR4 strain RNome to identify small regulatory RNA candidates expressed in this pathogen. We discovered 1047 potential small RNAs including intragenic, 5′- and/or 3′-overlapping RNAs and 88 small RNAs encoded in intergenic regions. With this approach, we recovered many of the previously identified intergenic small RNAs and identified 68 novel candidates, most of which are conserved in both sequence and genomic context in other S. pneumoniae strains. We confirmed the independent expression of 17 intergenic small RNAs and predicted putative mRNA targets for six of them using bioinformatics tools. Preliminary results suggest that one of these six is a key player in the regulation of competence development. This study is the biggest catalog of small noncoding RNAs reported to date in S. pneumoniae and provides a highly complete view of the small RNA network in this pathogen. PMID:22274957

  15. Small RNAs from Bemisia tabaci Are Transferred to Solanum lycopersicum Phloem during Feeding

    PubMed Central

    van Kleeff, Paula J. M.; Galland, Marc; Schuurink, Robert C.; Bleeker, Petra M.

    2016-01-01

    The phloem-feeding whitefly Bemisia tabaci is a serious pest to a broad range of host plants, including many economically important crops such as tomato. These insects serve as a vector for various devastating plant viruses. It is known that whiteflies are capable of manipulating host-defense responses, potentially mediated by effector molecules in the whitefly saliva. We hypothesized that, beside putative effector proteins, small RNAs (sRNA) are delivered by B. tabaci into the phloem, where they may play a role in manipulating host plant defenses. There is already evidence to suggest that sRNAs can mediate the host-pathogen dialogue. It has been shown that Botrytis cinerea, the causal agent of gray mold disease, takes advantage of the plant sRNA machinery to selectively silence host genes involved in defense signaling. Here we identified sRNAs originating from B. tabaci in the phloem of tomato plants on which they are feeding. sRNAs were isolated and sequenced from tomato phloem of whitefly-infested and control plants as well as from the nymphs themselves, control leaflets, and from the infested leaflets. Using stem-loop RT-PCR, three whitefly sRNAs have been verified to be present in whitefly-infested leaflets that were also present in the whitefly-infested phloem sample. Our results show that whitefly sRNAs are indeed present in tomato tissues upon feeding, and they appear to be mobile in the phloem. Their role in the host-insect interaction can now be investigated. PMID:27933079

  16. A sensitive non-radioactive northern blot method to detect small RNAs.

    PubMed

    Kim, Sang Woo; Li, Zhihua; Moore, Patrick S; Monaghan, A Paula; Chang, Yuan; Nichols, Mark; John, Bino

    2010-04-01

    The continuing discoveries of potentially active small RNAs at an unprecedented rate using high-throughput sequencing have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Currently, northern blot is the most widely used method for validating small RNAs that are identified by methods such as high-throughput sequencing. We describe a new northern blot-based protocol (LED) for small RNA (approximately 15-40 bases) detection using digoxigenin (DIG)-labeled oligonucleotide probes containing locked nucleic acids (LNA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for cross-linking the RNA to the membrane. LED generates clearly visible signals for RNA amounts as low as 0.05 fmol. This method requires as little as a few seconds of membrane exposure to outperform the signal intensity using overnight exposure of isotope-based methods, corresponding to approximately 1000-fold improvement in exposure-time. In contrast to commonly used radioisotope-based methods, which require freshly prepared and hazardous probes, LED probes can be stored for at least 6 months, facilitate faster and more cost-effective experiments, and are more environmentally friendly. A detailed protocol of LED is provided in the Supplementary Data.

  17. 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.

  18. Neisseria meningitidis Uses Sibling Small Regulatory RNAs To Switch from Cataplerotic to Anaplerotic Metabolism

    PubMed Central

    Huis in ‘t Veld, Robert A. G.; Schipper, Kim; Bovenkerk, Sandra; Kramer, Gertjan; Brouwer, Matthijs C.; van de Beek, Diederik; Speijer, Dave; van der Ende, Arie

    2017-01-01

    ABSTRACT Neisseria meningitidis (the meningococcus) is primarily a commensal of the human oropharynx that sporadically causes septicemia and meningitis. Meningococci adapt to diverse local host conditions differing in nutrient supply, like the nasopharynx, blood, and cerebrospinal fluid, by changing metabolism and protein repertoire. However, regulatory transcription factors and two-component systems in meningococci involved in adaptation to local nutrient variations are limited. We identified novel sibling small regulatory RNAs (Neisseria metabolic switch regulators [NmsRs]) regulating switches between cataplerotic and anaplerotic metabolism in this pathogen. Overexpression of NmsRs was tolerated in blood but not in cerebrospinal fluid. Expression of six tricarboxylic acid cycle enzymes was downregulated by direct action of NmsRs. Expression of the NmsRs themselves was under the control of the stringent response through the action of RelA. Small sibling regulatory RNAs of meningococci, controlling general metabolic switches, add an exciting twist to their versatile repertoire in bacterial pathogens. PMID:28325760

  19. Characterization of Small Interfering RNAs Derived from the Geminivirus/Betasatellite Complex Using Deep Sequencing

    PubMed Central

    Yang, Xiuling; Wang, Yu; Guo, Wei; Xie, Yan; Xie, Qi; Fan, Longjiang; Zhou, Xueping

    2011-01-01

    Background Small RNA (sRNA)-guided RNA silencing is a critical antiviral defense mechanism employed by a variety of eukaryotic organisms. Although the induction of RNA silencing by bipartite and monopartite begomoviruses has been described in plants, the nature of begomovirus/betasatellite complexes remains undefined. Methodology/Principal Findings Solanum lycopersicum plant leaves systemically infected with Tomato yellow leaf curl China virus (TYLCCNV) alone or together with its associated betasatellite (TYLCCNB), and Nicotiana benthamiana plant leaves systemically infected with TYLCCNV alone, or together with TYLCCNB or with mutant TYLCCNB were harvested for RNA extraction; sRNA cDNA libraries were then constructed and submitted to Solexa-based deep sequencing. Both sense and anti-sense TYLCCNV and TYLCCNB-derived sRNAs (V-sRNAs and S-sRNAs) accumulated preferentially as 22 nucleotide species in infected S. lycopersicum and N. benthamiana plants. High resolution mapping of V-sRNAs and S-sRNAs revealed heterogeneous distribution of V-sRNA and S-sRNA sequences across the TYLCCNV and TYLCCNB genomes. In TYLCCNV-infected S. lycopersicum or N. benthamiana and TYLCCNV and βC1-mutant TYLCCNB co-infected N. benthamiana plants, the primary TYLCCNV targets were AV2 and the 5′ terminus of AV1. In TYLCCNV and betasatellite-infected plants, the number of V-sRNAs targeting this region decreased and the production of V-sRNAs increased corresponding to the overlapping regions of AC2 and AC3, as well as the 3′ terminal of AC1. βC1 is the primary determinant mediating symptom induction and also the primary silencing target of the TYLCCNB genome even in its mutated form. Conclusions/Significance We report the first high-resolution sRNA map for a monopartite begomovirus and its associated betasatellite using Solexa-based deep sequencing. Our results suggest that viral transcript might act as RDR substrates resulting in dsRNA and secondary siRNA production. In addition, the

  20. Do EBV Encoded Small RNAs Interfere With Tumor Suppressor APC in EBV Associated Breast Cancers

    DTIC Science & Technology

    2005-08-01

    fibroadenomas of the breast in immuno compromised patients. In all latently infected cells EBV expresses two small non-polyadenylated RNAs (EBERs).Recent studies...numbers of rapidly growing fibroadenomas of the breast in immunocompromised patients.1 Because of close association of EBV with various epithelial and...estrogen receptor negative invasive breast cancers. EBV has also been detected in large numbers of rapidly growing fibroadenomas of the breast in immuno

  1. Rational Design of Small Molecules Targeting Oncogenic Noncoding RNAs from Sequence.

    PubMed

    Disney, Matthew D; Angelbello, Alicia J

    2016-12-20

    The discovery of RNA catalysis in the 1980s and the dissemination of the human genome sequence at the start of this century inspired investigations of the regulatory roles of noncoding RNAs in biology. In fact, the Encyclopedia of DNA Elements (ENCODE) project has shown that only 1-2% of the human genome encodes protein, yet 75% is transcribed into RNA. Functional studies both preceding and following the ENCODE project have shown that these noncoding RNAs have important roles in regulating gene expression, developmental timing, and other critical functions. RNA's diverse roles are often a consequence of the various folds that it adopts. The single-stranded nature of the biopolymer enables it to adopt intramolecular folds with noncanonical pairings to lower its free energy. These folds can be scaffolds to bind proteins or to form frameworks to interact with other RNAs. Not surprisingly, dysregulation of certain noncoding RNAs has been shown to be causative of disease. Given this as the background, it is easy to see why it would be useful to develop methods that target RNA and manipulate its biology in rational and predictable ways. The antisense approach has afforded strategies to target RNAs via Watson-Crick base pairing and has typically focused on targeting partially unstructured regions of RNA. Small molecule strategies to target RNA would be desirable not only because compounds could be lead optimized via medicinal chemistry but also because structured regions within an RNA of interest could be targeted to directly interfere with RNA folds that contribute to disease. Additionally, small molecules have historically been the most successful drug candidates. Until recently, the ability to design small molecules that target non-ribosomal RNAs has been elusive, creating the perception that they are "undruggable". In this Account, approaches to demystify targeting RNA with small molecules are described. Rather than bulk screening for compounds that bind to singular

  2. Comparative Profiling of Pseudomonas aeruginosa Strains Reveals Differential Expression of Novel Unique and Conserved Small RNAs

    PubMed Central

    Ferrara, Silvia; Brugnoli, Margherita; De Bonis, Angela; Righetti, Francesco; Delvillani, Francesco; Dehò, Gianni; Horner, David; Briani, Federica; Bertoni, Giovanni

    2012-01-01

    Pseudomonas aeruginosa is a highly adaptable bacterium that thrives in a broad range of ecological niches and can infect multiple hosts as diverse as plants, nematodes and mammals. In humans, it is an important opportunistic pathogen. This wide adaptability correlates with its broad genetic diversity. In this study, we used a deep-sequencing approach to explore the complement of small RNAs (sRNAs) in P. aeruginosa as the number of such regulatory molecules previously identified in this organism is relatively low, considering its genome size, phenotypic diversity and adaptability. We have performed a comparative analysis of PAO1 and PA14 strains which share the same host range but differ in virulence, PA14 being considerably more virulent in several model organisms. Altogether, we have identified more than 150 novel candidate sRNAs and validated a third of them by Northern blotting. Interestingly, a number of these novel sRNAs are strain-specific or showed strain-specific expression, strongly suggesting that they could be involved in determining specific phenotypic traits. PMID:22590564

  3. Small regulatory RNAs in lambdoid bacteriophages and phage-derived plasmids: Not only antisense.

    PubMed

    Nejman-Faleńczyk, Bożena; Bloch, Sylwia; Licznerska, Katarzyna; Felczykowska, Agnieszka; Dydecka, Aleksandra; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2015-03-01

    Until recently, only two small regulatory RNAs encoded by lambdoid bacteriophages were known. These transcripts are derived from paQ and pO promoters. The former one is supposed to act as an antisense RNA for expression of the Q gene, encoding a transcription antitermination protein. The latter transcript, called oop RNA, was initially proposed to have a double role, in establishing expression of the cI gene and in providing a primer for DNA replication. Although the initially proposed mechanisms by which oop RNA could influence the choice between two alternative developmental pathways of the phage and the initiation of phage DNA replication were found not true, the pO promoter has been demonstrated to be important for both regulation of phage development and control of DNA replication. Namely, the pO-derived transcript is an antisense RNA for expression of the cII gene, and pO is a part of a dual promoter system responsible for regulation of initiation of DNA synthesis from the oriλ region. Very recent studies identified a battery of small RNAs encoded by lambdoid bacteriophages existing as prophages in chromosomes of enterohemorrhagic Escherichia coli strains. Some of them have very interesting functions, like anti-small RNAs.

  4. Small Genetic Circuits and MicroRNAs: Big Players in Polymerase II Transcriptional Control in Plants

    PubMed Central

    Cumbie, Jason S.; Ivanchenko, Maria G.

    2016-01-01

    RNA Polymerase II (Pol II) regulatory cascades involving transcription factors (TFs) and their targets orchestrate the genetic circuitry of every eukaryotic organism. In order to understand how these cascades function, they can be dissected into small genetic networks, each containing just a few Pol II transcribed genes, that generate specific signal-processing outcomes. Small RNA regulatory circuits involve direct regulation of a small RNA by a TF and/or direct regulation of a TF by a small RNA and have been shown to play unique roles in many organisms. Here, we will focus on small RNA regulatory circuits containing Pol II transcribed microRNAs (miRNAs). While the role of miRNA-containing regulatory circuits as modular building blocks for the function of complex networks has long been on the forefront of studies in the animal kingdom, plant studies are poised to take a lead role in this area because of their advantages in probing transcriptional and posttranscriptional control of Pol II genes. The relative simplicity of tissue- and cell-type organization, miRNA targeting, and genomic structure make the Arabidopsis thaliana plant model uniquely amenable for small RNA regulatory circuit studies in a multicellular organism. In this Review, we cover analysis, tools, and validation methods for probing the component interactions in miRNA-containing regulatory circuits. We then review the important roles that plant miRNAs are playing in these circuits and summarize methods for the identification of small genetic circuits that strongly influence plant function. We conclude by noting areas of opportunity where new plant studies are imminently needed. PMID:26869700

  5. Small Genetic Circuits and MicroRNAs: Big Players in Polymerase II Transcriptional Control in Plants.

    PubMed

    Megraw, Molly; Cumbie, Jason S; Ivanchenko, Maria G; Filichkin, Sergei A

    2016-02-01

    RNA Polymerase II (Pol II) regulatory cascades involving transcription factors (TFs) and their targets orchestrate the genetic circuitry of every eukaryotic organism. In order to understand how these cascades function, they can be dissected into small genetic networks, each containing just a few Pol II transcribed genes, that generate specific signal-processing outcomes. Small RNA regulatory circuits involve direct regulation of a small RNA by a TF and/or direct regulation of a TF by a small RNA and have been shown to play unique roles in many organisms. Here, we will focus on small RNA regulatory circuits containing Pol II transcribed microRNAs (miRNAs). While the role of miRNA-containing regulatory circuits as modular building blocks for the function of complex networks has long been on the forefront of studies in the animal kingdom, plant studies are poised to take a lead role in this area because of their advantages in probing transcriptional and posttranscriptional control of Pol II genes. The relative simplicity of tissue- and cell-type organization, miRNA targeting, and genomic structure make the Arabidopsis thaliana plant model uniquely amenable for small RNA regulatory circuit studies in a multicellular organism. In this Review, we cover analysis, tools, and validation methods for probing the component interactions in miRNA-containing regulatory circuits. We then review the important roles that plant miRNAs are playing in these circuits and summarize methods for the identification of small genetic circuits that strongly influence plant function. We conclude by noting areas of opportunity where new plant studies are imminently needed.

  6. 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

  7. Small RNAs, big impact: small RNA pathways in transposon control and their effect on the host stress response.

    PubMed

    Wheeler, Bayly S

    2013-12-01

    Transposons are mobile genetic elements that are a major constituent of most genomes. Organisms regulate transposable element expression, transposition, and insertion site preference, mitigating the genome instability caused by uncontrolled transposition. A recent burst of research has demonstrated the critical role of small non-coding RNAs in regulating transposition in fungi, plants, and animals. While mechanistically distinct, these pathways work through a conserved paradigm. The presence of a transposon is communicated by the presence of its RNA or by its integration into specific genomic loci. These signals are then translated into small non-coding RNAs that guide epigenetic modifications and gene silencing back to the transposon. In addition to being regulated by the host, transposable elements are themselves capable of influencing host gene expression. Transposon expression is responsive to environmental signals, and many transposons are activated by various cellular stresses. TEs can confer local gene regulation by acting as enhancers and can also confer global gene regulation through their non-coding RNAs. Thus, transposable elements can act as stress-responsive regulators that control host gene expression in cis and trans.

  8. PMS1T, producing phased small-interfering RNAs, regulates photoperiod-sensitive male sterility in rice

    PubMed Central

    Fan, Yourong; Yang, Jiangyi; Mathioni, Sandra M.; Yu, Jinsheng; Shen, Jianqiang; Yang, Xuefei; Wang, Lei; Zhang, Qinghua; Cai, Zhaoxia; Xu, Caiguo; Li, Xianghua; Xiao, Jinghua; Zhang, Qifa

    2016-01-01

    Phased small-interfering RNAs (phasiRNAs) are a special class of small RNAs, which are generated in 21- or 24-nt intervals from transcripts of precursor RNAs. Although phasiRNAs have been found in a range of organisms, their biological functions in plants have yet to be uncovered. Here we show that phasiRNAs generated by the photopheriod-sensetive genic male sterility 1 (Pms1) locus were associated with photoperiod-sensitive male sterility (PSMS) in rice, a germplasm that started the two-line hybrid rice breeding. The Pms1 locus encodes a long-noncoding RNA PMS1T that was preferentially expressed in young panicles. PMS1T was targeted by miR2118 to produce 21-nt phasiRNAs that preferentially accumulated in the PSMS line under long-day conditions. A single nucleotide polymorphism in PMS1T nearby the miR2118 recognition site was critical for fertility change, likely leading to differential accumulation of the phasiRNAs. This result suggested possible roles of phasiRNAs in reproductive development of rice, demonstrating the potential importance of this RNA class as regulators in biological processes. PMID:27965387

  9. Plastic germline reprogramming of heritable small RNAs enables maintenance or erasure of epigenetic memories

    PubMed Central

    Houri-Ze'evi, Leah; Rechavi, Oded

    2016-01-01

    ABSTRACT In Caenorhabditis elegans small RNAs can regulate genes across generations. The mysterious tendency of heritable RNA interference (RNAi) responses to terminate after 3–5 generations has been referred to as “the bottleneck to RNAi inheritance.” We have recently shown that the re-setting of epigenetic inheritance after 3–5 generations is not due to passive dilution of the original RNA trigger, but instead results from an active, multigenerational, and small RNA-mediated regulatory pathway. In this “Point of View” manuscript we suggest that the process that leads to the erasure of the ancestral small RNA-encoded memory is a specialized type of germline reprogramming mechanism, analogous to the processes that robustly remove parental DNA methylation and histone modifications early in development in different organisms. Traditionally, germline reprogramming mechanisms that re-set chromatin are thought to stand in the way of inheritance of memories of parental experiences. We found that reprogramming of heritable small RNAs takes multiple generations to complete, enabling long-term inheritance of small RNA responses. Moreover, the duration of this reprogramming process can be prolonged significantly if new heritable RNAi responses are provoked. A dedicated signaling pathway that is responsive to environmental cues can tune the epigenetic state of the RNAi inheritance system, so that inheritance of particular small RNA species can be extended. PMID:27592591

  10. Small RNAs regulate the biocontrol property of fluorescent Pseudomonas strain Psd.

    PubMed

    Upadhyay, Anamika; Kochar, Mandira; Upadhyay, Ashutosh; Tripathy, Soumya; Rajam, Manchikatla Venkat; Srivastava, Sheela

    2017-03-01

    The production of biocontrol factors by Pseudomonads is reported to be controlled at the post-transcriptional level by the GacS/GacA signal transduction pathway. This involves RNA-binding translational repressor proteins, RsmA and RsmE, and the small regulatory RNAs (sRNAs) RsmX, RsmY, and RsmZ. While the former represses genes involved in secondary metabolite production, the latter relieves this repression at the end of exponential growth. We have studied the fluorescent Pseudomonas strain Psd, possessing good biocontrol potential, and confirmed the presence of rsmY and rsmZ by PCR amplification. Gene constructs for all the three small RNAs (RsmX, RsmY and RsmZ) carried on broad host-range plasmid, pME6032 were mobilized into strain Psd. Expression analysis of gacA in the recombinant strains over-expressing rsmX (Psd-pME7320), rsmY (Psd-pME6359) and rsmZ (Psd-pME6918) revealed a significant upregulation of the response regulator. Besides, a remarkable down-regulation of rsmA was also reported in all the strains. The variant strains were found to produce comparatively higher levels of phenazines. Indole acetic acid levels were higher to some extent, and strain Psd-pME6918 also showed elevated production of HCN. The tomato seedlings infected with Fusarium oxysporum and Verticillium dahliae in the presence of culture filtrate of the recombinant strains showed better plant protection response in comparison to the wild-type strain Psd. These results suggest that small RNAs are important determinants in regulation of the biocontrol property of strain Psd.

  11. Genomic dissection of small RNAs in wild rice (Oryza rufipogon): lessons for rice domestication.

    PubMed

    Wang, Yu; Bai, Xuefei; Yan, Chenghai; Gui, Yiejie; Wei, Xinghua; Zhu, Qian-Hao; Guo, Longbiao; Fan, Longjiang

    2012-11-01

    The lack of a MIRNA set and genome sequence of wild rice (Oryza rufipogon) has prevented us from determining the role of MIRNA genes in rice domestication. In this study, a genome, three small RNA populations and a degradome of O. rufipogon were sequenced by Illumina platform and the expression levels of microRNAs (miRNAs) were investigated by miRNA chips. A de novo O. rufipogon genome was assembled using c. 55× coverage of raw sequencing data and a total of 387 MIRNAs were identified in the O. rufipogon genome based on c. 5.2 million unique small RNA reads from three different tissues of O. rufipogon. Of these, O. rufipogon MIRNAs, 259 were not found in the cultivated rice, suggesting a loss of these MIRNAs in the cultivated rice. We also found that 48 MIRNAs were novel in the cultivated rice, suggesting that they were potential targets of domestication selection. Some miRNAs showed significant expression differences between wild and cultivated rice, suggesting that expression of miRNA could also be a target of domestication, as demonstrated for the miR164 family. Our results illustrated that MIRNA genes, like protein-coding genes, might have been significantly shaped during rice domestication and could be one of the driving forces that contributed to rice domestication.

  12. Obesity-Dependent Increases in Oocyte mRNAs Are Associated With Increases in Proinflammatory Signaling and Gut Microbial Abundance of Lachnospiraceae in Female Mice.

    PubMed

    Xie, Fang; Anderson, Christopher L; Timme, Kelsey R; Kurz, Scott G; Fernando, Samodha C; Wood, Jennifer R

    2016-04-01

    RNAs stored in the metaphase II-arrested oocyte play important roles in successful embryonic development. Their abundance is defined by transcriptional activity during oocyte growth and selective degradation of transcripts during LH-induced oocyte maturation. Our previous studies demonstrated that mRNA abundance is increased in mature ovulated oocytes collected from obese humans and mice and therefore may contribute to reduced oocyte developmental competence associated with metabolic dysfunction. In the current study mouse models of diet-induced obesity were used to determine whether obesity-dependent increases in proinflammatory signaling regulate ovarian abundance of oocyte-specific mRNAs. The abundance of oocyte-specific Bnc1, Dppa3, and Pou5f1 mRNAs as well as markers of proinflammatory signaling were significantly increased in ovaries of obese compared with lean mice which were depleted of fully grown preovulatory follicles. Chromatin-immunoprecipitation analyses also demonstrated increased association of phosphorylated signal transducer and activator of transcription 3 with the Pou5f1 promoter in ovaries of obese mice suggesting that proinflammatory signaling regulates transcription of this gene in the oocyte. The cecum microbial content of lean and obese female mice was subsequently examined to identify potential relationships between microbial composition and proinflammatory signaling in the ovary. Multivariate Association with Linear Models identified significant positive correlations between cecum abundance of the bacterial family Lachnospiraceae and ovarian abundance of Tnfa as well as Dppa3, Bnc1, and Pou5f1 mRNAs. Together, these data suggest that diet-induced changes in gut microbial composition may be contributing to ovarian inflammation which in turn alters ovarian gene expression and ultimately contributes to obesity-dependent reduction in oocyte quality and development of infertility in obese patients.

  13. Small Open Reading Frames, Non-Coding RNAs and Repetitive Elements in Bradyrhizobium japonicum USDA 110

    PubMed Central

    Hahn, Julia; Tsoy, Olga V.; Thalmann, Sebastian; Čuklina, Jelena; Gelfand, Mikhail S.

    2016-01-01

    Small open reading frames (sORFs) and genes for non-coding RNAs are poorly investigated components of most genomes. Our analysis of 1391 ORFs recently annotated in the soybean symbiont Bradyrhizobium japonicum USDA 110 revealed that 78% of them contain less than 80 codons. Twenty-one of these sORFs are conserved in or outside Alphaproteobacteria and most of them are similar to genes found in transposable elements, in line with their broad distribution. Stabilizing selection was demonstrated for sORFs with proteomic evidence and bll1319_ISGA which is conserved at the nucleotide level in 16 alphaproteobacterial species, 79 species from other taxa and 49 other Proteobacteria. Further we used Northern blot hybridization to validate ten small RNAs (BjsR1 to BjsR10) belonging to new RNA families. We found that BjsR1 and BjsR3 have homologs outside the genus Bradyrhizobium, and BjsR5, BjsR6, BjsR7, and BjsR10 have up to four imperfect copies in Bradyrhizobium genomes. BjsR8, BjsR9, and BjsR10 are present exclusively in nodules, while the other sRNAs are also expressed in liquid cultures. We also found that the level of BjsR4 decreases after exposure to tellurite and iron, and this down-regulation contributes to survival under high iron conditions. Analysis of additional small RNAs overlapping with 3’-UTRs revealed two new repetitive elements named Br-REP1 and Br-REP2. These REP elements may play roles in the genomic plasticity and gene regulation and could be useful for strain identification by PCR-fingerprinting. Furthermore, we studied two potential toxin genes in the symbiotic island and confirmed toxicity of the yhaV homolog bll1687 but not of the newly annotated higB homolog blr0229_ISGA in E. coli. Finally, we revealed transcription interference resulting in an antisense RNA complementary to blr1853, a gene induced in symbiosis. The presented results expand our knowledge on sORFs, non-coding RNAs and repetitive elements in B. japonicum and related bacteria. PMID

  14. Analysis of Small RNAs in Streptococcus mutans under Acid Stress—A New Insight for Caries Research

    PubMed Central

    Liu, Shanshan; Tao, Ye; Yu, Lixia; Zhuang, Peilin; Zhi, Qinghui; Zhou, Yan; Lin, Huancai

    2016-01-01

    Streptococcus mutans (S. mutans) is the major clinical pathogen responsible for dental caries. Its acid tolerance has been identified as a significant virulence factor for its survival and cariogenicity in acidic conditions. Small RNAs (sRNAs) are recognized as key regulators of virulence and stress adaptation. Here, we constructed three libraries of sRNAs with small size exposed to acidic conditions for the first time, followed by verification using qRT-PCR. The levels of two sRNAs and target genes predicted to be bioinformatically related to acid tolerance were further evaluated under different acid stress conditions (pH 7.5, 6.5, 5.5, and 4.5) at three time points (0.5, 1, and 2 h). Meanwhile, bacterial growth characteristics and vitality were assessed. We obtained 1879 sRNAs with read counts of at least 100. One hundred and ten sRNAs were perfectly mapped to reported msRNAs in S. mutans. Ten out of 18 sRNAs were validated by qRT-PCR. The survival of bacteria declined as the acid was increased from pH 7.5 to 4.5 at each time point. The bacteria can proliferate under each pH except pH 4.5 with time. The levels of sRNAs gradually decreased from pH 7.5 to 5.5, and slightly increased in pH 4.5; however, the expression levels of target mRNAs were up-regulated in acidic conditions than in pH 7.5. These results indicate that some sRNAs are specially induced at acid stress conditions, involving acid adaptation, and provide a new insight into exploring the complex acid tolerance for S. mutans. PMID:27649155

  15. The pivotal role of small non-coding RNAs in the regulation of seed development.

    PubMed

    Rodrigues, Andreia S; Miguel, Célia M

    2017-03-13

    Seeds represent a crucial stage of the seed plants life cycle. It is during seed development that the foundations of the future plant body, and the ability to give rise to a new plant capable of growing under sometimes adverse environmental conditions, are established. Small non-coding RNAs are major regulators of gene expression both at the post-transcriptional and transcriptional levels and, not surprisingly, these elements play major roles in seed development and germination. We review here the current knowledge about small RNA expression and functions in seed development, going from the morphogenesis phase comprehending embryo development and patterning, to the several steps of the maturation phase, ending in the transition to the germination. A special focus is given to the small RNAs for which functional studies have been conducted and their participation in regulatory networks operating in seeds. Many challenges remain ahead for dissecting the complex small RNA landscape in seeds, but this is a highly relevant issue in plant biology and advances in this area will most certainly impact plant breeding.

  16. Induction of Silencing in Plants by High-Pressure Spraying of In vitro-Synthesized Small RNAs

    PubMed Central

    Dalakouras, Athanasios; Wassenegger, Michèle; McMillan, John N.; Cardoza, Vinitha; Maegele, Ira; Dadami, Elena; Runne, Miriam; Krczal, Gabi; Wassenegger, Michael

    2016-01-01

    In this report, we describe a method for the delivery of small interfering RNAs (siRNAs) into plant cells. In vitro synthesized siRNAs that were designed to target the coding region of a GREEN FLUORESCENT PROTEIN (GFP) transgene were applied by various methods onto GFP-expressing transgenic Nicotiana benthamiana plants to trigger RNA silencing. In contrast to mere siRNA applications, including spraying, syringe injection, and infiltration of siRNAs that all failed to induce RNA silencing, high pressure spraying of siRNAs resulted in efficient local and systemic silencing of the GFP transgene, with comparable efficiency as was achieved with biolistic siRNA introduction. High-pressure spraying of siRNAs with sizes of 21, 22, and 24 nucleotides (nt) led to local GFP silencing. Small RNA deep sequencing revealed that no shearing of siRNAs was detectable by high-pressure spraying. Systemic silencing was basically detected upon spraying of 22 nt siRNAs. Local and systemic silencing developed faster and more extensively upon targeting the apical meristem than spraying of mature leaves. PMID:27625678

  17. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs

    SciTech Connect

    Travis, G.H.; Sutcliffe, J.G.

    1988-03-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA.

  18. Comprehensive Identification of Meningococcal Genes and Small Noncoding RNAs Required for Host Cell Colonization

    PubMed Central

    Capel, Elena; Zomer, Aldert L.; Nussbaumer, Thomas; Bole, Christine; Izac, Brigitte; Frapy, Eric; Meyer, Julie; Bouzinba-Ségard, Haniaa; Bille, Emmanuelle; Jamet, Anne; Cavau, Anne; Letourneur, Franck; Bourdoulous, Sandrine; Rattei, Thomas; Coureuil, Mathieu

    2016-01-01

    ABSTRACT Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia, affecting infants and adults worldwide. N. meningitidis is also a common inhabitant of the human nasopharynx and, as such, is highly adapted to its niche. During bacteremia, N. meningitidis gains access to the blood compartment, where it adheres to endothelial cells of blood vessels and causes dramatic vascular damage. Colonization of the nasopharyngeal niche and communication with the different human cell types is a major issue of the N. meningitidis life cycle that is poorly understood. Here, highly saturated random transposon insertion libraries of N. meningitidis were engineered, and the fitness of mutations during routine growth and that of colonization of endothelial and epithelial cells in a flow device were assessed in a transposon insertion site sequencing (Tn-seq) analysis. This allowed the identification of genes essential for bacterial growth and genes specifically required for host cell colonization. In addition, after having identified the small noncoding RNAs (sRNAs) located in intergenic regions, the phenotypes associated with mutations in those sRNAs were defined. A total of 383 genes and 8 intergenic regions containing sRNA candidates were identified to be essential for growth, while 288 genes and 33 intergenic regions containing sRNA candidates were found to be specifically required for host cell colonization. PMID:27486197

  19. Modification of small RNAs associated with suppression of RNA silencing by tobamovirus replicase protein.

    PubMed

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

    2007-10-01

    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.

  20. Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs.

    PubMed

    Coffman, Stephanie R; Lu, Jinfeng; Guo, Xunyang; Zhong, Jing; Jiang, Hongshan; Broitman-Maduro, Gina; Li, Wan-Xiang; Lu, Rui; Maduro, Morris; Ding, Shou-Wei

    2017-03-21

    Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors.IMPORTANCE The helicase and C-terminal domains of mammalian RLRs sense intracellular viral RNAs to initiate the interferon-regulated innate immunity against RNA virus infection. Both of the domains from

  1. Repertoire of virus-derived small RNAs produced by mosquito and mammalian cells in response to dengue virus infection.

    PubMed

    Schirtzinger, Erin E; Andrade, Christy C; Devitt, Nicholas; Ramaraj, Thiruvarangan; Jacobi, Jennifer L; Schilkey, Faye; Hanley, Kathryn A

    2015-02-01

    RNA interference (RNAi) is the major defense of many arthropods against arthropod-borne RNA viruses (arboviruses), but the role of RNAi in vertebrate immunity to arboviruses is not clear. RNA viruses can trigger RNAi in vertebrate cells, but the vertebrate interferon response may obscure this interaction. We quantified virus-derived small RNAs (vRNAs) generated by mosquito (U4.4) cells and interferon-deficient (Vero) and interferon-competent (HuH-7) mammalian cells infected with a single isolate of mosquito-borne dengue virus. Mosquito cells produced significantly more vRNAs than mammalian cells, and mosquito cell vRNAs were derived from both the positive- and negative-sense dengue genomes whereas mammalian cell vRNAs were derived primarily from positive-sense genome. Mosquito cell vRNAs were predominantly 21 nucleotides in length whereas mammalian cell vRNAs were between 12 and 36 nucleotides with a modest peak at 24 nucleotides. Hot-spots, regions of the virus genome that generated a disproportionate number of vRNAs, overlapped among the cell lines.

  2. Synergistic and independent actions of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs in Arabidopsis.

    PubMed

    Wang, Xiaoyan; Zhang, Shuxin; Dou, Yongchao; Zhang, Chi; Chen, Xuemei; Yu, Bin; Ren, Guodong

    2015-04-01

    All types of small RNAs in plants, piwi-interacting RNAs (piRNAs) in animals and a subset of siRNAs in Drosophila and C. elegans are subject to HEN1 mediated 3' terminal 2'-O-methylation. This modification plays a pivotal role in protecting small RNAs from 3' uridylation, trimming and degradation. In Arabidopsis, HESO1 is a major enzyme that uridylates small RNAs to trigger their degradation. However, U-tail is still present in null hen1 heso1 mutants, suggesting the existence of (an) enzymatic activities redundant with HESO1. Here, we report that UTP: RNA uridylyltransferase (URT1) is a functional paralog of HESO1. URT1 interacts with AGO1 and plays a predominant role in miRNA uridylation when HESO1 is absent. Uridylation of miRNA is globally abolished in a hen1 heso1 urt1 triple mutant, accompanied by an extensive increase of 3'-to-5' trimming. In contrast, disruption of URT1 appears not to affect the heterochromatic siRNA uridylation. This indicates the involvement of additional nucleotidyl transferases in the siRNA pathway. Analysis of miRNA tailings in the hen1 heso1 urt1 triple mutant also reveals the existence of previously unknown enzymatic activities that can add non-uridine nucleotides. Importantly, we show HESO1 may also act redundantly with URT1 in miRNA uridylation when HEN1 is fully competent. Taken together, our data not only reveal a synergistic action of HESO1 and URT1 in the 3' uridylation of miRNAs, but also independent activities of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs and an antagonistic relationship between uridylation and trimming. Our results may provide further insight into the mechanisms of small RNA 3' end modification and stability control.

  3. Daily Expression Pattern of Protein-Encoding Genes and Small Noncoding RNAs in Synechocystis sp. Strain PCC 6803

    PubMed Central

    Beck, Christian; Hertel, Stefanie; Rediger, Anne; Lehmann, Robert; Wiegard, Anika; Kölsch, Adrian; Heilmann, Beate; Georg, Jens; Hess, Wolfgang R.

    2014-01-01

    Many organisms harbor circadian clocks with periods close to 24 h. These cellular clocks allow organisms to anticipate the environmental cycles of day and night by synchronizing circadian rhythms with the rising and setting of the sun. These rhythms originate from the oscillator components of circadian clocks and control global gene expression and various cellular processes. The oscillator of photosynthetic cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, linked to a complex regulatory network. Synechocystis sp. strain PCC 6803 possesses the standard cyanobacterial kaiABC gene cluster plus multiple kaiB and kaiC gene copies and antisense RNAs for almost every kai transcript. However, there is no clear evidence of circadian rhythms in Synechocystis sp. PCC 6803 under various experimental conditions. It is also still unknown if and to what extent the multiple kai gene copies and kai antisense RNAs affect circadian timing. Moreover, a large number of small noncoding RNAs whose accumulation dynamics over time have not yet been monitored are known for Synechocystis sp. PCC 6803. Here we performed a 48-h time series transcriptome analysis of Synechocystis sp. PCC 6803, taking into account periodic light-dark phases, continuous light, and continuous darkness. We found that expression of functionally related genes occurred in different phases of day and night. Moreover, we found day-peaking and night-peaking transcripts among the small RNAs; in particular, the amounts of kai antisense RNAs correlated or anticorrelated with those of their respective kai target mRNAs, pointing toward the regulatory relevance of these antisense RNAs. Surprisingly, we observed that the amounts of 16S and 23S rRNAs in this cyanobacterium fluctuated in light-dark periods, showing maximum accumulation in the dark phase. Importantly, the amounts of all transcripts, including small noncoding RNAs, did not show any rhythm under continuous light or darkness, indicating the absence

  4. Quantification of Small Non-Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles

    PubMed Central

    Masotti, Andrea; Caputo, Viviana; Da Sacco, Letizia; Pizzuti, Antonio; Dallapiccola, Bruno; Bottazzo, Gian Franco

    2009-01-01

    MicroRNAs (miRNAs) are highly conserved ∼22-mer RNA molecules, encoded by plants and animals that regulate the expression of genes binding to the 3′-UTR of specific target mRNAs. The amount of miRNAs in a total RNA sample depends on the recovery efficiency that may be significantly affected by the different purification methods employed. Traditional approaches may be inefficient at recovering small RNAs, and common spectrophotometric determination is not adequate to quantify selectively these low molecular weight (LMW) species from total RNA samples. Here, we describe the use of qualitative and quantitative lab-on-a-chip tools for the analysis of these LMW RNA species. Our data emphasize the close correlation of LMW RNAs with the expression levels of some miRNAs. We therefore applied our result to the comparison of some miRNA expression profiles in different tissues. Finally, the methods we used in this paper allowed us to analyze the efficiency of extraction protocols, to study the small (but significant) differences among various preparations and to allow a proper comparison of some miRNA expression profiles in various tissues. PMID:19727414

  5. Delivery of small interfering RNAs in human cervical cancer cells by polyethylenimine-functionalized carbon nanotubes.

    PubMed

    Huang, Yuan-Pin; Lin, I-Jou; Chen, Chih-Chen; Hsu, Yi-Chiang; Chang, Chi-Chang; Lee, Mon-Juan

    2013-06-06

    Carbon nanotubes are capable of penetrating the cell membrane and are widely considered as potential carriers for gene or drug delivery. Because the C-C and C=C bonds in carbon nanotubes are nonpolar, functionalization is required for carbon nanotubes to interact with genes or drugs as well as to improve their biocompatibility. In this study, polyethylenimine (PEI)-functionalized single-wall (PEI-NH-SWNTs) and multiwall carbon nanotubes (PEI-NH-MWNTs) were produced by direct amination method. PEI functionalization increased the positive charge on the surface of SWNTs and MWNTs, allowing carbon nanotubes to interact electrostatically with the negatively charged small interfering RNAs (siRNAs) and to serve as nonviral gene delivery reagents. PEI-NH-MWNTs and PEI-NH-SWNTs had a better solubility in water than pristine carbon nanotubes, and further removal of large aggregates by centrifugation produced a stable suspension of reduced particle size and improved homogeneity and dispersity. The amount of grafted PEI estimated by thermogravimetric analysis was 5.08% (w/w) and 5.28% (w/w) for PEI-NH-SWNTs and PEI-NH-MWNTs, respectively. For the assessment of cytotoxicity, various concentrations of PEI-NH-SWNTs and PEI-NH-MWNTs were incubated with human cervical cancer cells, HeLa-S3, for 48 h. PEI-NH-SWNTs and PEI-NH-MWNTs induced cell deaths in a dose-dependent manner but were less cytotoxic compared to pure PEI. As determined by electrophoretic mobility shift assay, siRNAs directed against glyceraldehyde-3-phosphate dehydrogenase (siGAPDH) were completely associated with PEI-NH-SWNTs or PEI-NH-MWNTs at a PEI-NH-SWNT/siGAPDH or PEI-NH-MWNT/siGAPDH mass ratio of 80:1 or 160:1, respectively. Furthermore, PEI-NH-SWNTs and PEI-NH-MWNTs successfully delivered siGAPDH into HeLa-S3 cells at PEI-NH-SWNT/siGAPDH and PEI-NH-MWNT/siGAPDH mass ratios of 1:1 to 20:1, resulting in suppression of the mRNA level of GAPDH to an extent similar to that of DharmaFECT, a common transfection

  6. Delivery of small interfering RNAs in human cervical cancer cells by polyethylenimine-functionalized carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Huang, Yuan-Pin; Lin, I.-Jou; Chen, Chih-Chen; Hsu, Yi-Chiang; Chang, Chi-Chang; Lee, Mon-Juan

    2013-06-01

    Carbon nanotubes are capable of penetrating the cell membrane and are widely considered as potential carriers for gene or drug delivery. Because the C-C and C=C bonds in carbon nanotubes are nonpolar, functionalization is required for carbon nanotubes to interact with genes or drugs as well as to improve their biocompatibility. In this study, polyethylenimine (PEI)-functionalized single-wall (PEI-NH-SWNTs) and multiwall carbon nanotubes (PEI-NH-MWNTs) were produced by direct amination method. PEI functionalization increased the positive charge on the surface of SWNTs and MWNTs, allowing carbon nanotubes to interact electrostatically with the negatively charged small interfering RNAs (siRNAs) and to serve as nonviral gene delivery reagents. PEI-NH-MWNTs and PEI-NH-SWNTs had a better solubility in water than pristine carbon nanotubes, and further removal of large aggregates by centrifugation produced a stable suspension of reduced particle size and improved homogeneity and dispersity. The amount of grafted PEI estimated by thermogravimetric analysis was 5.08% ( w/ w) and 5.28% ( w/ w) for PEI-NH-SWNTs and PEI-NH-MWNTs, respectively. For the assessment of cytotoxicity, various concentrations of PEI-NH-SWNTs and PEI-NH-MWNTs were incubated with human cervical cancer cells, HeLa-S3, for 48 h. PEI-NH-SWNTs and PEI-NH-MWNTs induced cell deaths in a dose-dependent manner but were less cytotoxic compared to pure PEI. As determined by electrophoretic mobility shift assay, siRNAs directed against glyceraldehyde-3-phosphate dehydrogenase (siGAPDH) were completely associated with PEI-NH-SWNTs or PEI-NH-MWNTs at a PEI-NH-SWNT/siGAPDH or PEI-NH-MWNT/siGAPDH mass ratio of 80:1 or 160:1, respectively. Furthermore, PEI-NH-SWNTs and PEI-NH-MWNTs successfully delivered siGAPDH into HeLa-S3 cells at PEI-NH-SWNT/siGAPDH and PEI-NH-MWNT/siGAPDH mass ratios of 1:1 to 20:1, resulting in suppression of the mRNA level of GAPDH to an extent similar to that of DharmaFECT, a common transfection

  7. Frequency modulation of stochastic gene expression bursts by strongly interacting small RNAs.

    PubMed

    Kumar, Niraj; Jia, Tao; Zarringhalam, Kourosh; Kulkarni, Rahul V

    2016-10-01

    The sporadic nature of gene expression at the single-cell level-long periods of inactivity punctuated by bursts of mRNA or protein production-plays a critical role in diverse cellular processes. To elucidate the cellular role of bursting in gene expression, synthetic biology approaches have been used to design simple genetic circuits with bursty mRNA or protein production. Understanding how such genetic circuits can be designed with the ability to control burst-related parameters requires the development of quantitative stochastic models of gene expression. In this work, we analyze stochastic models for the regulation of gene expression bursts by strongly interacting small RNAs. For the parameter range considered, results based on mean-field approaches are significantly inaccurate and alternative analytical approaches are needed. Using simplifying approximations, we obtain analytical results for the corresponding steady-state distributions that are in agreement with results from stochastic simulations. These results indicate that regulation by small RNAs, in the strong interaction limit, can be used to effectively modulate the frequency of bursting. We explore the consequences of such regulation for simple genetic circuits involving feedback effects and switching between promoter states.

  8. Frequency modulation of stochastic gene expression bursts by strongly interacting small RNAs

    NASA Astrophysics Data System (ADS)

    Kumar, Niraj; Jia, Tao; Zarringhalam, Kourosh; Kulkarni, Rahul V.

    2016-10-01

    The sporadic nature of gene expression at the single-cell level—long periods of inactivity punctuated by bursts of mRNA or protein production—plays a critical role in diverse cellular processes. To elucidate the cellular role of bursting in gene expression, synthetic biology approaches have been used to design simple genetic circuits with bursty mRNA or protein production. Understanding how such genetic circuits can be designed with the ability to control burst-related parameters requires the development of quantitative stochastic models of gene expression. In this work, we analyze stochastic models for the regulation of gene expression bursts by strongly interacting small RNAs. For the parameter range considered, results based on mean-field approaches are significantly inaccurate and alternative analytical approaches are needed. Using simplifying approximations, we obtain analytical results for the corresponding steady-state distributions that are in agreement with results from stochastic simulations. These results indicate that regulation by small RNAs, in the strong interaction limit, can be used to effectively modulate the frequency of bursting. We explore the consequences of such regulation for simple genetic circuits involving feedback effects and switching between promoter states.

  9. Micromanagement of the immune system by microRNAs.

    PubMed

    Lodish, Harvey F; Zhou, Beiyan; Liu, Gwen; Chen, Chang-Zheng

    2008-02-01

    MicroRNAs (miRNAs) are an abundant class of evolutionarily conserved small non-coding RNAs that are thought to control gene expression by targeting mRNAs for degradation or translational repression. Emerging evidence suggests that miRNA-mediated gene regulation represents a fundamental layer of genetic programmes at the post-transcriptional level and has diverse functional roles in animals. Here, we provide an overview of the mechanisms by which miRNAs regulate gene expression, with specific focus on the role of miRNAs in regulating the development of immune cells and in modulating innate and adaptive immune responses.

  10. “Guest list” or “Black list”? Heritable Small RNAs as Immunogenic Memories

    PubMed Central

    Rechavi, Oded

    2016-01-01

    Small RNA-mediated gene silencing plays a pivotal role in genome immunity by recognizing and eliminating viruses and transposons which otherwise may colonize the genome. However, this can be challenging since individual genomic parasites are highly diverse, and employ multiple immune evasion techniques. In this review, I discuss a new theory proposing that the integrity of the germline is maintained by transgenerationally-transmitted RNA “memories” that record ancestral gene expression patterns, and delineate “Self” from “Foreign” sequences. To maintain such recollection two tactics are employed in parallel: “black listing” of invading nucleic acids, and “guest listing” of endogenous genes. Studies in a number of organisms have shown that this memorization is used by the next generation small RNAs to act as “Inherited Vaccines” that ambush invading elements, or as “Inherited Licenses” that grant the transcription of autogenous sequences. PMID:24231398

  11. Small RNA Sequencing for Profiling MicroRNAs in Long-Term Preserved Formalin-Fixed and Paraffin-Embedded Non-Small Cell Lung Cancer Tumor Specimens

    PubMed Central

    Kadota, Kyuichi; Kannisto, Eric; Jones, David R.; Adusumilli, Prasad S.

    2015-01-01

    Background The preservation of microRNAs in formalin-fixed and paraffin-embedded (FFPE) tissue makes them particularly useful for biomarker studies. The utility of small RNA sequencing for microRNA expression profiling of FFPE samples has yet to be determined. Methods Total RNA was extracted from de-paraffinized and proteinase K-treated FFPE specimens (15–20 years old) of 8 human lung adenocarcinoma tumors by affinity chromatography on silica columns. MicroRNAs in the RNA preparations were quantified by the Illumina HiSeq 2000 sequencing platform with sequencing libraries prepared with the TruSeq Small RNA Sample Preparation Kit (version 2.0) to obtain unpaired reads of 50 b for small RNA fragments. MicroRNAs were also quantified using Agilent Human miRNA (release 16.0) microarrays that can detect 1,205 mature microRNAs and by quantitative reverse transcription (RT)-PCR assays. Results Between 9.1–16.9 million reads were obtained by small RNA sequencing of extracted RNA samples. Of these, only 0.6–2.3% (mean = 1.5%) represented microRNAs. The sequencing method detected 454–625 microRNAs/sample (mean = 550) compared with 200–349 (mean = 286) microRNAs detected by microarray. In Spearman correlation analyses, the average correlation coefficient for the 126 microRNAs detected in all samples by both methods was 0.37, and >0.5 for 63 microRNAs. In correlation analyses of the sequencing- and RT-PCR-based measurements, the coefficients were 0.19–0.95 (mean = 0.73) and >0.7, respectively, for 7 of 9 examined microRNAs. The average inter-replicate Spearman correlation coefficient for the sequencing method was 0.81. Conclusions Small RNA sequencing can be used to obtain microRNA profiles of FFPE tissue specimens with performance characteristics similar to those of microarrays, in spite of the fragmentation of ribosomal and messenger RNAs that reduces the method's informative capacity. The accuracy of the method can conceivably be improved by increasing sequencing

  12. Identification of Mitochondrial Genome-Encoded Small RNAs Related to Egg Deterioration Caused by Postovulatory Aging in Rainbow Trout.

    PubMed

    Ma, Hao; Weber, Gregory M; Wei, Hairong; Yao, Jianbo

    2016-10-01

    Many factors have been reported to affect rainbow trout egg quality, among which, postovulatory aging is one of the most significant causes as reared rainbow trout do not usually volitionally oviposit the ovulated eggs. In order to uncover the genetic regulation underling egg deterioration caused by postovulatory aging in rainbow trout, mitochondrial genome-encoded small RNA (mitosRNAs) were analyzed from unfertilized eggs on Days 1, 7, and 14 postovulation with fertilization rates of 91.8, 73.4, and less than 50 %, respectively. A total of 248 mitosRNAs were identified from Illumina high-throughput sequencing of the small RNA libraries derived from the eggs of ten females. Ninety-eight of the small RNAs exhibited more than a threefold difference in expression between eggs from females exhibiting high fertilization rates at Day 1 and low fertilization rates at Day 14. The differentially expressed mitosRNAs were predominantly derived from mitochondrial D-loop, tRNA, rRNA, COII, and Cytb gene regions. Real-time quantitative PCR analysis was carried out for 14 differentially expressed mitosRNAs, of which, 12 were confirmed to be consistent with the sequencing reads. Further characterization of the differentially expressed mitosRNAs may lead to the development of new biomarkers for egg quality in rainbow trout.

  13. An in silico model for identification of small RNAs in whole bacterial genomes: characterization of antisense RNAs in pathogenic Escherichia coli and Streptococcus agalactiae strains

    PubMed Central

    Pichon, Christophe; du Merle, Laurence; Caliot, Marie Elise; Trieu-Cuot, Patrick; Le Bouguénec, Chantal

    2012-01-01

    Characterization of small non-coding ribonucleic acids (sRNA) among the large volume of data generated by high-throughput RNA-seq or tiling microarray analyses remains a challenge. Thus, there is still a need for accurate in silico prediction methods to identify sRNAs within a given bacterial species. After years of effort, dedicated software were developed based on comparative genomic analyses or mathematical/statistical models. Although these genomic analyses enabled sRNAs in intergenic regions to be efficiently identified, they all failed to predict antisense sRNA genes (asRNA), i.e. RNA genes located on the DNA strand complementary to that which encodes the protein. The statistical models enabled any genomic region to be analyzed theorically but not efficiently. We present a new model for in silico identification of sRNA and asRNA candidates within an entire bacterial genome. This model was successfully used to analyze the Gram-negative Escherichia coli and Gram-positive Streptococcus agalactiae. In both bacteria, numerous asRNAs are transcribed from the complementary strand of genes located in pathogenicity islands, strongly suggesting that these asRNAs are regulators of the virulence expression. In particular, we characterized an asRNA that acted as an enhancer-like regulator of the type 1 fimbriae production involved in the virulence of extra-intestinal pathogenic E. coli. PMID:22139924

  14. Synaptic vesicles isolated from the electric organ of Torpedo californica and from the central nervous system of Mus musculus contain small ribonucleic acids (sRNAs).

    PubMed

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S; Harlow, Mark L

    2017-06-01

    Synaptic vesicles (SVs) are presynaptic organelles that load and release small molecule neurotransmitters at chemical synapses. In addition to classic neurotransmitters, we have demonstrated that SVs isolated from the Peripheral Nervous Systems (PNS) of the electric organ of Torpedo californica, a model cholinergic synapse, and SVs isolated from the Central Nervous System (CNS) of Mus musculus (mouse) contain small ribonucleic acids (sRNAs; ≤ 50 nucleotides) (Scientific Reports, 5:1-14(14918) Li et al. (2015) [1]). Our previous publication provided the five most abundant sequences associated with the T. californica SVs, and the ten most abundant sequences associated with the mouse SVs, representing 59% and 39% of the total sRNA reads sequenced, respectively). We provide here a full repository of the SV sRNAs sequenced from T. californica and the mouse deposited in the NCBI as biosamples. Three data studies are included: SVs isolated from the electric organ of T. californica using standard techniques, SVs isolated from the electric organ of T. californica using standard techniques with an additional affinity purification step, and finally, SVs isolated from the CNS of mouse. The three biosamples are available at https://www.ncbi.nlm.nih.gov/biosample/ SRS1523467, SRS1523466, and SRS1523472 respectively.

  15. Genome-defence small RNAs exapted for epigenetic mating-type inheritance.

    PubMed

    Singh, Deepankar Pratap; Saudemont, Baptiste; Guglielmi, Gérard; Arnaiz, Olivier; Goût, Jean-François; Prajer, Malgorzata; Potekhin, Alexey; Przybòs, Ewa; Aubusson-Fleury, Anne; Bhullar, Simran; Bouhouche, Khaled; Lhuillier-Akakpo, Maoussi; Tanty, Véronique; Blugeon, Corinne; Alberti, Adriana; Labadie, Karine; Aury, Jean-Marc; Sperling, Linda; Duharcourt, Sandra; Meyer, Eric

    2014-05-22

    In the ciliate Paramecium, transposable elements and their single-copy remnants are deleted during the development of somatic macronuclei from germline micronuclei, at each sexual generation. Deletions are targeted by scnRNAs, small RNAs produced from the germ line during meiosis that first scan the maternal macronuclear genome to identify missing sequences, and then allow the zygotic macronucleus to reproduce the same deletions. Here we show that this process accounts for the maternal inheritance of mating types in Paramecium tetraurelia, a long-standing problem in epigenetics. Mating type E depends on expression of the transmembrane protein mtA, and the default type O is determined during development by scnRNA-dependent excision of the mtA promoter. In the sibling species Paramecium septaurelia, mating type O is determined by coding-sequence deletions in a different gene, mtB, which is specifically required for mtA expression. These independently evolved mechanisms suggest frequent exaptation of the scnRNA pathway to regulate cellular genes and mediate transgenerational epigenetic inheritance of essential phenotypic polymorphisms.

  16. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

    PubMed

    Meyer, Sarai; Chappell, James; Sankar, Sitara; Chew, Rebecca; Lucks, Julius B

    2016-01-01

    Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA engineering strategies can improve fold activation from 5.4-fold to 13.4-fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1-fold to 14.6-fold for an unrelated STAR regulator, opening the door to creating a range of additional STARs to use in a broad array of biotechnologies. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of RNA transcriptional repressors, enabling these optimized STARs to be used in sophisticated circuits.

  17. RNAi pathways in Mucor: A tale of proteins, small RNAs and functional diversity.

    PubMed

    Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M

    2016-05-01

    The existence of an RNA-mediated silencing mechanism in the opportunistic fungal pathogen Mucor circinelloides was first described in the early 2000. Since then, Mucor has reached an outstanding position within the fungal kingdom as a model system to achieve a deeper understanding of regulation of endogenous functions by the RNA interference (RNAi) machinery. M. circinelloides combines diverse components of its RNAi machinery to carry out functions not only limited to the defense against invasive nucleic acids, but also to regulate expression of its own genes by producing different classes of endogenous small RNA molecules (esRNAs). The recent discovery of a novel RNase that participates in a new RNA degradation pathway adds more elements to the gene silencing-mediated regulation. This review focuses on esRNAs in M. circinelloides, the different pathways involved in their biogenesis, and their roles in regulating specific physiological and developmental processes in response to environmental signals, highlighting the complexity of silencing-mediated regulation in fungi.

  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.

  19. A ruler protein in a complex for antiviral defense determines the length of small interfering CRISPR RNAs.

    PubMed

    Hatoum-Aslan, Asma; Samai, Poulami; Maniv, Inbal; Jiang, Wenyan; Marraffini, Luciano A

    2013-09-27

    Small RNAs undergo maturation events that precisely determine the length and structure required for their function. CRISPRs (clustered regularly interspaced short palindromic repeats) encode small RNAs (crRNAs) that together with CRISPR-associated (cas) genes constitute a sequence-specific prokaryotic immune system for anti-viral and anti-plasmid defense. crRNAs are subject to multiple processing events during their biogenesis, and little is known about the mechanism of the final maturation step. We show that in the Staphylococcus epidermidis type III CRISPR-Cas system, mature crRNAs are measured in a Cas10·Csm ribonucleoprotein complex to yield discrete lengths that differ by 6-nucleotide increments. We looked for mutants that impact this crRNA size pattern and found that an alanine substitution of a conserved aspartate residue of Csm3 eliminates the 6-nucleotide increments in the length of crRNAs. In vitro, recombinant Csm3 binds RNA molecules at multiple sites, producing gel-shift patterns that suggest that each protein binds 6 nucleotides of substrate. In vivo, changes in the levels of Csm3 modulate the crRNA size distribution without disrupting the 6-nucleotide periodicity. Our data support a model in which multiple Csm3 molecules within the Cas10·Csm complex bind the crRNA with a 6-nucleotide periodicity to function as a ruler that measures the extent of crRNA maturation.

  20. Identification of conserved and novel microRNAs in Aquilaria sinensis based on small RNA sequencing and transcriptome sequence data.

    PubMed

    Gao, Zhi-Hui; Wei, Jian-He; Yang, Yun; Zhang, Zheng; Xiong, Huan-Ying; Zhao, Wen-Ting

    2012-08-15

    Agarwood is in great demand for its high value in medicine, incense, and perfume across Asia, Middle East, and Europe. As agarwood is formed only when the Aquilaria trees are wounded or infected by some microbes, overharvesting and habitat loss are threatening some populations of agarwood-producing species. Aquilaria sinensis is such a significant economic tree species. To promote the production efficiency and protect the resource of A. sinensis, it would be critical to reveal the regulation mechanisms of stress-induced agarwood formation. MicroRNAs (miRNAs), a key gene expression regulator involved in various plant stress response and metabolic processes, might function in agarwood formation, but no report concerning miRNAs in Aquilaria is available. In this study, the small RNA high-throughput sequencing and 454 transcriptome data were adopted to identify both conserved and novel miRNAs in A. sinensis. Deep sequencing showed that the small RNA (sRNA) population of A. sinensis was complex and the length of sRNAs varied. By in silico analysis of the small RNA deep sequencing data and transcriptome data, we discovered 27 novel miRNAs in A. sinensis. Based on the mature miRNA sequence conservation, we identified 74 putative conserved miRNAs from A. sinensis and 10 of them were confirmed with hairpin forming precursor. Interestingly, a novel miRNA sequence was determined to be the miRNA of asi-miR408, but with accumulation much higher than asi-miR408. The expression levels of ten stress-responsive miRNAs were examined during the time-course after wound treatment. Eight were shown to be wound-responsive. This not only shows the existence of miRNAs in this Asian economically significant tree species but also indicated its critical role in stress-induced agarwood formation. The highly accumulated miRNA of asi-miR408 implied miRNAs would be functional as well as miRNAs in plants.

  1. Small Non-Coding RNAs: New Insights in Modulation of Host Immune Response by Intracellular Bacterial Pathogens

    PubMed Central

    Ahmed, Waqas; Zheng, Ke; Liu, Zheng-Fei

    2016-01-01

    Pathogenic bacteria possess intricate regulatory networks that temporally control the production of virulence factors and enable the bacteria to survive and proliferate within host cell. Small non-coding RNAs (sRNAs) have been identified as important regulators of gene expression in diverse biological contexts. Recent research has shown bacterial sRNAs involved in growth and development, cell proliferation, differentiation, metabolism, cell signaling, and immune response through regulating protein–protein interactions or via their ability to base pair with RNA and DNA. In this review, we provide a brief overview of mechanism of action employed by immune-related sRNAs, their known functions in immunity, and how they can be integrated into regulatory circuits that govern virulence, which will facilitate our understanding of pathogenesis and the development of novel, more effective therapeutic approaches to treat infections caused by intracellular bacterial pathogens. PMID:27803700

  2. Experimental RNomics and genomic comparative analysis reveal a large group of species-specific small non-message RNAs in the silkworm Bombyx mori

    PubMed Central

    Li, Dandan; Wang, Yanhong; Zhang, Kun; Jiao, Zhujin; Zhu, Xiaopeng; Skogerboe, Geir; Guo, Xiangqian; Chinnusamy, Viswanathan; Bi, Lijun; Huang, Yongping; Dong, Shuanglin; Chen, Runsheng; Kan, Yunchao

    2011-01-01

    Accumulating evidences show that small non-protein coding RNAs (ncRNAs) play important roles in development, stress response and other cellular processes. The silkworm is an important model for studies on insect genetics and control of lepidopterous pests. Here, we have performed the first systematic identification and analysis of intermediate size ncRNAs (50–500 nt) in the silkworm. We identified 189 novel ncRNAs, including 141 snoRNAs, six snRNAs, three tRNAs, one SRP and 38 unclassified ncRNAs. Forty ncRNAs showed significantly altered expression during silkworm development or across specific stage transitions. Genomic comparisons revealed that 123 of these ncRNAs are potentially silkworm-specific. Analysis of the genomic organization of the ncRNA loci showed that 32.62% of the novel snoRNA loci are intergenic, and that all the intronic snoRNAs follow the pattern of one-snoRNA-per-intron. Target site analysis predicted a total of 95 2′-O-methylation and pseudouridylation modification sites of rRNAs, snRNAs and tRNAs. Together, these findings provide new clues for future functional study of ncRNA during insect development and evolution. PMID:21227919

  3. Experimental RNomics and genomic comparative analysis reveal a large group of species-specific small non-message RNAs in the silkworm Bombyx mori.

    PubMed

    Li, Dandan; Wang, Yanhong; Zhang, Kun; Jiao, Zhujin; Zhu, Xiaopeng; Skogerboe, Geir; Guo, Xiangqian; Chinnusamy, Viswanathan; Bi, Lijun; Huang, Yongping; Dong, Shuanglin; Chen, Runsheng; Kan, Yunchao

    2011-05-01

    Accumulating evidences show that small non-protein coding RNAs (ncRNAs) play important roles in development, stress response and other cellular processes. The silkworm is an important model for studies on insect genetics and control of lepidopterous pests. Here, we have performed the first systematic identification and analysis of intermediate size ncRNAs (50-500 nt) in the silkworm. We identified 189 novel ncRNAs, including 141 snoRNAs, six snRNAs, three tRNAs, one SRP and 38 unclassified ncRNAs. Forty ncRNAs showed significantly altered expression during silkworm development or across specific stage transitions. Genomic comparisons revealed that 123 of these ncRNAs are potentially silkworm-specific. Analysis of the genomic organization of the ncRNA loci showed that 32.62% of the novel snoRNA loci are intergenic, and that all the intronic snoRNAs follow the pattern of one-snoRNA-per-intron. Target site analysis predicted a total of 95 2'-O-methylation and pseudouridylation modification sites of rRNAs, snRNAs and tRNAs. Together, these findings provide new clues for future functional study of ncRNA during insect development and evolution.

  4. RNY (YRNA)-derived small RNAs regulate cell death and inflammation in monocytes/macrophages.

    PubMed

    Hizir, Zoheir; Bottini, Silvia; Grandjean, Valerie; Trabucchi, Michele; Repetto, Emanuela

    2017-01-05

    The recent discovery of new classes of small RNAs has opened unknown territories to explore new regulations of physiopathological events. We have recently demonstrated that RNY (or Y RNA)-derived small RNAs (referred to as s-RNYs) are an independent class of clinical biomarkers to detect coronary artery lesions and are associated with atherosclerosis burden. Here, we have studied the role of s-RNYs in human and mouse monocytes/macrophages and have shown that in lipid-laden monocytes/macrophages s-RNY expression is timely correlated to the activation of both NF-κB and caspase 3-dependent cell death pathways. Loss- or gain-of-function experiments demonstrated that s-RNYs activate caspase 3 and NF-κB signaling pathways ultimately promoting cell death and inflammatory responses. As, in atherosclerosis, Ro60-associated s-RNYs generated by apoptotic macrophages are released in the blood of patients, we have investigated the extracellular function of the s-RNY/Ro60 complex. Our data demonstrated that s-RNY/Ro60 complex induces caspase 3-dependent cell death and NF-κB-dependent inflammation, when added to the medium of cultured monocytes/macrophages. Finally, we have shown that s-RNY function is mediated by Toll-like receptor 7 (TLR7). Indeed using chloroquine, which disrupts signaling of endosome-localized TLRs 3, 7, 8 and 9 or the more specific TLR7/9 antagonist, the phosphorothioated oligonucleotide IRS954, we blocked the effect of either intracellular or extracellular s-RNYs. These results position s-RNYs as relevant novel functional molecules that impacts on macrophage physiopathology, indicating their potential role as mediators of inflammatory diseases, such as atherosclerosis.

  5. Functional roles of non-coding Y RNAs.

    PubMed

    Kowalski, Madzia P; Krude, Torsten

    2015-09-01

    Non-coding RNAs are involved in a multitude of cellular processes but the biochemical function of many small non-coding RNAs remains unclear. The family of small non-coding Y RNAs is conserved in vertebrates and related RNAs are present in some prokaryotic species. Y RNAs are also homologous to the newly identified family of non-coding stem-bulge RNAs (sbRNAs) in nematodes, for which potential physiological functions are only now emerging. Y RNAs are essential for the initiation of chromosomal DNA replication in vertebrates and, when bound to the Ro60 protein, they are involved in RNA stability and cellular responses to stress in several eukaryotic and prokaryotic species. Additionally, short fragments of Y RNAs have recently been identified as abundant components in the blood and tissues of humans and other mammals, with potential diagnostic value. While the number of functional roles of Y RNAs is growing, it is becoming increasingly clear that the conserved structural domains of Y RNAs are essential for distinct cellular functions. Here, we review the biochemical functions associated with these structural RNA domains, as well as the functional conservation of Y RNAs in different species. The existing biochemical and structural evidence supports a domain model for these small non-coding RNAs that has direct implications for the modular evolution of functional non-coding RNAs.

  6. 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.

  7. Secretion of small/microRNAs including miR-638 into extracellular spaces by sphingomyelin phosphodiesterase 3.

    PubMed

    Kubota, Shiori; Chiba, Mitsuru; Watanabe, Miki; Sakamoto, Maki; Watanabe, Narumi

    2015-01-01

    A recent study demonstrated that intracellular small/microRNAs are released from cells, and some of these extracellular RNAs are embedded in vesicles, such as ceramide-rich exosomes, on lipid-bilayer membranes. In the present study, we examined the effects of sphingomyelin phosphodiesterase 3 (SMPD3), which generates ceramide from sphingomyelin, on the release of small/microRNAs from intracellular to extracellular spaces. In these experiments, SW480 human colorectal and HuH-7 human hepatocellular cancer cells were cultured for 48 h in serum-free media. Culture supernatants were then collected, and floating cells and debris were removed by centrifugation and filtration through a 0.22-µm filter. Extracellular small RNAs in purified culture supernatants were stable for 4 weeks at room temperature, after 20 freeze-thaw cycles and exposure to pH 2.0, and were resistant to ribonuclease A degradation. Amino acid sequence analyses of SMPD3 showed high homology between mammals, indicating evolutionary conservation. Therefore, to investigate the mechanisms of cellular small/microRNA export, SW480 and HuH-7 cells were treated with the SMPD3 inhibitor GW4869 in serum-free media. Culture supernatants were collected for microarray and/or reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments. The number of microRNAs in culture supernatants was decreased following treatment with GW4869. Among these, extracellular and intracellular miR-638 were dose-dependently decreased and increased, respectively. These data suggest that SMPD3 plays an important role in the release of microRNAs into extracellular spaces.

  8. Polymorphisms in microRNAs are associated with survival in non-small cell lung cancer

    PubMed Central

    Zhao, Yang; Wei, Qingyi; Hu, Lingming; Chen, Feng; Hu, Zhibin; Heist, Rebecca S.; Su, Li; Amos, Christopher I.; Shen, Hongbing; Christiani, David C.

    2014-01-01

    Background MicroRNAs (miRNAs) play important roles in the regulation of eukaryotic gene expression and are involved in human carcinogenesis. Single nucleotide polymorphisms (SNPs) in miRNA sequence may alter miRNA functions in gene regulation, which, in turn, may affect cancer risk and disease progression. Methods We conducted an analysis of associations of 142 miRNA SNPs with non-small cell lung cancer (NSCLC) survival using data from a genome-wide association study (GWAS) in a Caucasian population from the Massachusetts General Hospital (Boston, MA, US) including 452 early-stage and 526 late-stage NSCLC cases. Replication analyses were further performed in two external populations, one Caucasian cohort from The University of Texas MD Anderson Cancer Center (Houston, TX, US) and one Han Chinese cohort from Nanjing, China. Results We identified 7 significant SNPs in the discovery set. Results from the independent Caucasian cohort demonstrated that the C allele of rs2042253 (has-miRNA-5197) was significantly associated with decreased risk for death among the late-stage NSCLC patients (discovery set: HR=0.80, P=0.007; validation set: HR=0.86, P=0.035; combined analysis: HR=0.84, P=0.001). Conclusions These findings provide evidence that some miRNA SNPs are associated with NSCLC survival and can be used as predictive biomarkers. Impact This study provided an estimate of outcome probability for survival experience of NSCLC patients, which demonstrates that genetic factors, as well as classical non-genetic factors, may be used to predict individual outcome. PMID:25103824

  9. A diversity of U1 small nuclear RNAs in the silk moth Bombyx mori.

    PubMed

    Sierra-Montes, J M; Pereira-Simon, S; Freund, A V; Ruiz, L M; Szmulewicz, M N; Herrera, R J

    2003-01-01

    Variants of U1 small nuclear RNAs (snRNAs) have been previously detected in a permanent cell line (BmN) of the silk moth Bombyx mori. In this study, the existence of U1 snRNA isoforms in the silk gland (SG) of the organism is investigated. The polyploidy (approximately 200,000X the 2N somatic value) state of the B. mori silk gland cells represents a unique system to explore the potential presence and differential expression of multiple U1 variants in a normal tissue. B. mori U1-specific RT-PCR libraries from the silk gland were generated and five U1 isoforms were isolated and characterized. Nucleotide differences, structural alterations, as well as protein and RNA interaction sites were examined in these variants and compared to the previously reported isoforms from the transformed BmN cell line. In all these SG U1 variants, variant sites and inter-species differences are located in moderately conserved regions. Substitutional or compensatory changes were found in the double stranded areas and clustered in moderately conserved regions. Some of the changes generate stronger base pairing. Calculated free energy (DeltaG) values for the entire U1 snRNA secondary structures and for the individual stem/loops (I, II, III and IV) domains of the isoforms were generated and compared to determine their structural stability. Using phylogenetic analysis, an evolutionary parallelism is observed between the polymorphic sites in B. mori and variant locations found among animal and plant species.

  10. Deep sequencing of mycovirus-derived small RNAs from Botrytis species.

    PubMed

    Donaire, Livia; Ayllón, María A

    2016-08-31

    RNA silencing is an ancient regulatory mechanism operating in all eukaryotic cells. In fungi, it was first discovered in Neurospora crassa, although its potential as a defence mechanism against mycoviruses was first reported in Cryphonectria parasitica and, later, in several fungal species. There is little evidence of the antiviral potential of RNA silencing in the phytopathogenic species of the fungal genus Botrytis. Moreover, little is known about the RNA silencing components in these fungi, although the analysis of public genome databases identified two Dicer-like genes in B. cinerea, as in most of the ascomycetes sequenced to date. In this work, we used deep sequencing to study the virus-derived small RNA (vsiRNA) populations from different mycoviruses infecting field isolates of Botrytis spp. The mycoviruses under study belong to different genera and species, and have different types of genome [double-stranded RNA (dsRNA), (+)single-stranded RNA (ssRNA) and (-)ssRNA]. In general, vsiRNAs derived from mycoviruses are mostly of 21, 20 and 22 nucleotides in length, possess sense or antisense orientation, either in a similar ratio or with a predominance of sense polarity depending on the virus species, have predominantly U at their 5' end, and are unevenly distributed along the viral genome, showing conspicuous hotspots of vsiRNA accumulation. These characteristics reveal striking similarities with vsiRNAs produced by plant viruses, suggesting similar pathways of viral targeting in plants and fungi. We have shown that the fungal RNA silencing machinery acts against the mycoviruses used in this work in a similar manner independent of their viral or fungal origin.

  11. Mechanism of induction and suppression of antiviral immunity directed by virus-derived small RNAs in Drosophila.

    PubMed

    Aliyari, Roghiyh; Wu, Qingfa; Li, Hong-Wei; Wang, Xiao-Hong; Li, Feng; Green, Lance D; Han, Cliff S; Li, Wan-Xiang; Ding, Shou-Wei

    2008-10-16

    The small RNA-directed viral immunity pathway in plants and invertebrates begins with the production by Dicer nuclease of virus-derived siRNAs (viRNAs), which guide specific antiviral silencing by Argonaute protein in an RNA-induced silencing complex (RISC). Molecular identity of the viral RNA precursor of viRNAs remains a matter of debate. Using Flock house virus (FHV) infection of Drosophila as a model, we show that replication of FHV positive-strand RNA genome produces an approximately 400 bp dsRNA from its 5' terminus that serves as the major Dicer-2 substrate. ViRNAs thus generated are loaded in Argonaute-2 and methylated at their 3' ends. Notably, FHV-encoded RNAi suppressor B2 protein interacts with both viral dsRNA and RNA replicase and inhibits production of the 5'-terminal viRNAs. Our findings, therefore, provide a model in which small RNA-directed viral immunity is induced during the initiation of viral progeny (+)RNA synthesis and suppressed by B2 inside the viral RNA replication complex.

  12. Identification of differentially expressed small non-coding RNAs in the legume endosymbiont Sinorhizobium meliloti by comparative genomics.

    PubMed

    del Val, Coral; Rivas, Elena; Torres-Quesada, Omar; Toro, Nicolás; Jiménez-Zurdo, José I

    2007-12-01

    Bacterial small non-coding RNAs (sRNAs) are being recognized as novel widespread regulators of gene expression in response to environmental signals. Here, we present the first search for sRNA-encoding genes in the nitrogen-fixing endosymbiont Sinorhizobium meliloti, performed by a genome-wide computational analysis of its intergenic regions. Comparative sequence data from eight related alpha-proteobacteria were obtained, and the interspecies pairwise alignments were scored with the programs eQRNA and RNAz as complementary predictive tools to identify conserved and stable secondary structures corresponding to putative non-coding RNAs. Northern experiments confirmed that eight of the predicted loci, selected among the original 32 candidates as most probable sRNA genes, expressed small transcripts. This result supports the combined use of eQRNA and RNAz as a robust strategy to identify novel sRNAs in bacteria. Furthermore, seven of the transcripts accumulated differentially in free-living and symbiotic conditions. Experimental mapping of the 5'-ends of the detected transcripts revealed that their encoding genes are organized in autonomous transcription units with recognizable promoter and, in most cases, termination signatures. These findings suggest novel regulatory functions for sRNAs related to the interactions of alpha-proteobacteria with their eukaryotic hosts.

  13. Genome-wide characterization of rice black streaked dwarf virus-responsive microRNAs in rice leaves and roots by small RNA and degradome sequencing.

    PubMed

    Sun, Zongtao; He, Yuqing; Li, Junmin; Wang, Xu; Chen, Jianping

    2015-04-01

    MicroRNAs (miRNAs) are small, non-coding RNAs which typically function by guiding cleavage of target mRNAs. They play important roles in development, abiotic stress and responses to pathogens. Four small RNA libraries and four degradome libraries were constructed from the leaves and roots of healthy rice and plants infected with Rice black streaked dwarf virus (RBSDV). Analysis of the deep sequencing results showed that the expression patterns of 14 miRNAs in leaves and 16 miRNAs in roots changed significantly in response to RBSDV infection. Some responses were similar in roots and leaves, but many miRNAs responded differently in different tissues. The results were confirmed for selected miRNAs by quantitative real-time PCR. By using degradome sequencing, a total of 104 target transcripts for 17 conserved and 16 non-conserved miRNAs were shown to be responsive to RBSDV infection. Fifteen novel miRNAs were also identified by small RNA and degradome sequencing. The results provide new insights into the regulatory networks of miRNAs and their targets in different plant tissues in response to virus infection.

  14. Characterization of small RNAs and their targets of Fusarium oxysporum infected and non-infected cotton seedlings

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, we characterized small RNA (sRNA) or microRNA (miRNA) profiles during Fusarium oxysporum f.sp. vasinfectum (FOV) race 3 pathogenesis in cotton (Gossypium hirsutum L.) seedlings. sRNAs or miRNA are known to play important roles in gene expression, including stress responses, influencin...

  15. Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

    NASA Astrophysics Data System (ADS)

    Cooper, Edwin L.; Overstreet, Nicola

    2014-03-01

    Recent evidence supports that prokaryotes exhibit adaptive immunity in the form of CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) and Cas (CRISPR associated proteins). The CRISPR-Cas system confers resistance to exogenous genetic elements such as phages and plasmids by allowing for the recognition and silencing of these genetic elements. Moreover, CRISPR-Cas serves as a memory of past exposures. This suggests that the evolution of the immune system has counterparts among the prokaryotes, not exclusively among eukaryotes. Mathematical models have been proposed which simulate the evolutionary patterns of CRISPR, however large gaps in our understanding of CRISPR-Cas function and evolution still exist. The CRISPR-Cas system is analogous to small RNAs involved in resistance mechanisms throughout the tree of life, and a deeper understanding of the evolution of small RNA pathways is necessary before the relationship between these convergent systems is to be determined. Presented in this review are novel RNAi therapies based on CRISPR-Cas analogs and the potential for future therapies based on CRISPR-Cas system components.

  16. Allele Dependent Silencing of Collagen Type I Using Small Interfering RNAs Targeting 3'UTR Indels - a Novel Therapeutic Approach in Osteogenesis Imperfecta

    PubMed Central

    Lindahl, Katarina; Kindmark, Andreas; Laxman, Navya; Åström, Eva; Rubin, Carl-Johan; Ljunggren, Östen

    2013-01-01

    Osteogenesis imperfecta, also known as “brittle bone disease”, is a heterogeneous disorder of connective tissue generally caused by dominant mutations in the genes COL1A1 and COL1A2, encoding the α1 and α2 chains of type I (pro)collagen. Symptomatic patients are usually prescribed bisphosphonates, but this treatment is neither curative nor sufficient. A promising field is gene silencing through RNA interference. In this study small interfering RNAs (siRNAs) were designed to target each allele of 3'UTR insertion/deletion polymorphisms (indels) in COL1A1 (rs3840870) and COL1A2 (rs3917). For both indels, the frequency of heterozygous individuals was determined to be approximately 50% in Swedish cohorts of healthy controls as well as in patients with osteogenesis imperfecta. Cultures of primary human bone derived cells were transfected with siRNAs through magnet-assisted transfection. cDNA from transfected cells was sequenced in order to measure targeted allele/non-targeted allele ratios and the overall degree of silencing was assessed by quantitative PCR. Successful allele dependent silencing was observed, with promising results for siRNAs complementary to both the insertion and non-insertion harboring alleles. In COL1A1 cDNA the indel allele ratios were shifted from 1 to 0.09 and 0.19 for the insertion and non-insertion allele respectively while the equivalent resulting ratios for COL1A2 were 0.05 and 0.01. Reductions in mRNA abundance were also demonstrated; in cells treated with siRNAs targeting the COL1A1 alleles the average COL1A1 mRNA levels were reduced 65% and 78% compared to negative control levels and in cells treated with COL1A2 siRNAs the average COL1A2 mRNA levels were decreased 26% and 49% of those observed in the corresponding negative controls. In conclusion, allele dependent silencing of collagen type I utilizing 3'UTR indels common in the general population constitutes a promising mutation independent therapeutic approach for osteogenesis

  17. DETAILED ABUNDANCES OF STARS WITH SMALL PLANETS DISCOVERED BY KEPLER. I. THE FIRST SAMPLE

    SciTech Connect

    Schuler, Simon C.; Vaz, Zachary A.; Santrich, Orlando J. Katime; Cunha, Katia; Smith, Verne V.; King, Jeremy R.; Teske, Johanna K.; Ghezzi, Luan; Howell, Steve B.; Isaacson, Howard E-mail: zachary.vaz@spartans.ut.edu E-mail: kcunha@noao.edu E-mail: jking2@clemson.edu E-mail: lghezzi@cfa.harvard.edu E-mail: hisaacson@berkeley.edu

    2015-12-10

    We present newly derived stellar parameters and the detailed abundances of 19 elements of seven stars with small planets discovered by NASA's Kepler Mission. Each star, save one, has at least one planet with a radius ≤1.6 R{sub ⊕}, suggesting a primarily rocky composition. The stellar parameters and abundances are derived from high signal-to-noise ratio, high-resolution echelle spectroscopy obtained with the 10 m Keck I telescope and High Resolution Echelle Spectrometer using standard spectroscopic techniques. The metallicities of the seven stars range from −0.32 to +0.13 dex, with an average metallicity that is subsolar, supporting previous suggestions that, unlike Jupiter-type giant planets, small planets do not form preferentially around metal-rich stars. The abundances of elements other than iron are in line with a population of Galactic disk stars, and despite our modest sample size, we find hints that the compositions of stars with small planets are similar to stars without known planets and with Neptune-size planets, but not to those of stars with giant planets. This suggests that the formation of small planets does not require exceptional host-star compositions and that small planets may be ubiquitous in the Galaxy. We compare our derived abundances (which have typical uncertainties of ≲0.04 dex) to the condensation temperature of the elements; a correlation between the two has been suggested as a possible signature of rocky planet formation. None of the stars demonstrate the putative rocky planet signature, despite at least three of the stars having rocky planets estimated to contain enough refractory material to produce the signature, if real. More detailed abundance analyses of stars known to host small planets are needed to verify our results and place ever more stringent constraints on planet formation models.

  18. The Abundance and Physical State of Polycyclic Aromatic Hydrocarbons in the Small Magellanic Cloud

    NASA Astrophysics Data System (ADS)

    Sandstrom, Karin M.; Bolatto, A. D.; Draine, B.; Stanimirovic, S.; Bot, C.

    2010-01-01

    We present the results of two studies investigating the abundance and physical state of polycyclic aromatic hydrocarbons (PAHs) in the Small Magellanic Cloud (SMC). PAHs play important roles in the thermal balance and chemistry of the interstellar medium and their mid-IR emission bands are bright and detectable out to high redshift. Observations with ISO and Spitzer have shown that PAHs are deficient in low-metallicity galaxies. In particular, galaxies with 12 + log(O/H) < 8 show mid-IR colors and spectra consistent with low PAH abundance. The SMC provides a unique opportunity to map the PAH emission in a low-metallicity (12 + log(O/H) 8) galaxy at high spatial resolution to learn what processes set the abundance and physical state of PAHs. To study the PAH abundance, we model dust SEDs across the SMC using Spitzer Survey of the SMC (S3MC) photometry from 3.6 to 160 microns. We also use overlapping spectral mapping observations from 5 to 38 microns from the Spitzer Spectroscopic Survey of the Small Magellanic Cloud (S4MC) in selected regions to test the reliability of the PAH abundance as determined from SED fits using the Draine et al (2007) models. Our results show that the average PAH abundance in the SMC is low compared to the Milky Way (q_pah 0.6% vs. 4%) and variable--with high abundance (q_pah 1-2%) in molecular clouds and low abundance in the diffuse ISM. Based on these results, we suggest that PAHs are destroyed in the diffuse ISM of the SMC and/or PAHs are forming in molecular clouds. In addition, we use S4MC observations to diagnose the physical state of the PAHs from the ratios of their mid-IR bands. We find that PAHs in the SMC tend to be smaller and less ionized than in higher metallicity galaxies.

  19. Locked Nucleic Acid and Flow Cytometry-Fluorescence In Situ Hybridization for the Detection of Bacterial Small Noncoding RNAs

    PubMed Central

    Robertson, Kelly L.

    2012-01-01

    We describe the development and testing of a high-throughput method that enables the detection of small noncoding RNAs (ncRNAs) from single bacterial cells using locked nucleic acid probes (LNA) and flow cytometry-fluorescence in situ hybridization (flow-FISH). The LNA flow-FISH method and quantitative reverse transcription-PCR (qRT-PCR) were used to monitor the expression of three ncRNAs (6S, CsrB, and TPP-2) in Vibrio campbellii ATCC BAA-1116 cultures during lag phase, mid-log phase, and stationary phase. Both LNA flow-FISH and qRT-PCR revealed that CsrB and TPP-2 were highly expressed during lag phase but markedly reduced in mid-log phase and stationary phase, whereas 6S demonstrated no to little expression during lag phase but increased thereafter. Importantly, while LNA flow-FISH and qRT-PCR demonstrated similar overall expression trends, only LNA flow-FISH, which enabled the detection of ncRNAs in individual cells as opposed to the lysate-based ensemble measurements generated by qRT-PCR, was able to capture the cell-to-cell heterogeneity in ncRNA expression. As such, this study demonstrates a new method that simultaneously enables the in situ detection of ncRNAs and the determination of gene expression heterogeneity within an isogenic bacterial population. PMID:22057868

  20. Comparative genome‐wide analysis of small RNAs of major Gram‐positive pathogens: from identification to application

    PubMed Central

    Mraheil, Mobarak A.; Billion, André; Kuenne, Carsten; Pischimarov, Jordan; Kreikemeyer, Bernd; Engelmann, Susanne; Hartke, Axel; Giard, Jean‐Christophe; Rupnik, Maja; Vorwerk, Sonja; Beier, Markus; Retey, Julia; Hartsch, Thomas; Jacob, Anette; Cemič, Franz; Hemberger, Jürgen; Chakraborty, Trinad; Hain, Torsten

    2010-01-01

    Summary In the recent years, the number of drug‐ and multi‐drug‐resistant microbial strains has increased rapidly. Therefore, the need to identify innovative approaches for development of novel anti‐infectives and new therapeutic targets is of high priority in global health care. The detection of small RNAs (sRNAs) in bacteria has attracted considerable attention as an emerging class of new gene expression regulators. Several experimental technologies to predict sRNA have been established for the Gram‐negative model organism Escherichia coli. In many respects, sRNA screens in this model system have set a blueprint for the global and functional identification of sRNAs for Gram‐positive microbes, but the functional role of sRNAs in colonization and pathogenicity for Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis and Clostridium difficile is almost completely unknown. Here, we report the current knowledge about the sRNAs of these socioeconomically relevant Gram‐positive pathogens, overview the state‐of‐the‐art high‐throughput sRNA screening methods and summarize bioinformatics approaches for genome‐wide sRNA identification and target prediction. Finally, we discuss the use of modified peptide nucleic acids (PNAs) as a novel tool to inactivate potential sRNA and their applications in rapid and specific detection of pathogenic bacteria. PMID:21255362

  1. Transcriptome sequencing uncovers novel long noncoding and small nucleolar RNAs dysregulated in head and neck squamous cell carcinoma

    PubMed Central

    Zou, Angela E.; Ku, Jonjei; Honda, Thomas K.; Yu, Vicky; Kuo, Selena Z.; Zheng, Hao; Xuan, Yinan; Saad, Maarouf A.; Hinton, Andrew; Brumund, Kevin T.; Lin, Jonathan H.; Wang-Rodriguez, Jessica; Ongkeko, Weg M.

    2015-01-01

    Head and neck squamous cell carcinoma persists as one of the most common and deadly malignancies, with early detection and effective treatment still posing formidable challenges. To expand our currently sparse knowledge of the noncoding alterations involved in the disease and identify potential biomarkers and therapeutic targets, we globally profiled the dysregulation of small nucleolar and long noncoding RNAs in head and neck tumors. Using next-generation RNA-sequencing data from 40 pairs of tumor and matched normal tissues, we found 2808 long noncoding RNA (lncRNA) transcripts significantly differentially expressed by a fold change magnitude ≥2. Meanwhile, RNA-sequencing analysis of 31 tumor-normal pairs yielded 33 significantly dysregulated small nucleolar RNAs (snoRNA). In particular, we identified two dramatically down-regulated lncRNAs and one down-regulated snoRNA whose expression levels correlated significantly with overall patient survival, suggesting their functional significance and clinical relevance in head and neck cancer pathogenesis. We confirmed the dysregulation of these noncoding RNAs in head and neck cancer cell lines derived from different anatomic sites, and determined that ectopic expression of the two lncRNAs inhibited key EMT and stem cell genes and reduced cellular proliferation and migration. As a whole, noncoding RNAs are pervasively dysregulated in head and squamous cell carcinoma. The precise molecular roles of the three transcripts identified warrants further characterization, but our data suggest that they are likely to play substantial roles in head and neck cancer pathogenesis and are significantly associated with patient survival. PMID:25904139

  2. Long noncoding RNAs: new insights into non-small cell lung cancer biology, diagnosis and therapy.

    PubMed

    Ricciuti, Biagio; Mencaroni, Clelia; Paglialunga, Luca; Paciullo, Francesco; Crinò, Lucio; Chiari, Rita; Metro, Giulio

    2016-02-01

    Recent advances in tiling array and high throughput analyses revealed that at least 87.3 % of the human genome is actively transcribed, though <3 % of the human genome encodes proteins. This unexpected truth suggests that most of the transcriptome is constituted by noncoding RNA. Among them, high-resolution microarray and massively parallel sequencing analyses identified long noncoding RNAs (lncRNAs) as nonprotein-coding transcripts. lncRNAs are largely polyadenylated and >200 nucleotides in length transcripts, involved in gene expression through epigenetic and transcriptional regulation, splicing, imprinting and subcellular transport. Although lncRNAs functions are largely uncharacterized, accumulating data indicate that they are involved in fundamental biological functions. Conversely, their dysregulation has increasingly been recognized to contribute to the development and progression of several human malignancies, especially lung cancer, which represents the leading cause of cancer-related deaths worldwide. We conducted a comprehensive review of the published literature focusing on lncRNAs function and disruption in nonsmall cell lung cancer biology, also highlighting their value as biomarkers and potential therapeutic targets. lncRNAs are involved in NSCLC pathogenesis, modulating fundamental cellular processes such as proliferation, cell growth, apoptosis, migration, stem cell maintenance and epithelial to mesenchymal transition, also serving as signaling transducers, molecular decoys and scaffolds. Also, lncRNAs represent very promising biomarkers in early-stage NSCLC patients and may become particularly useful in noninvasive screening protocols. lncRNAs may be used as predictive biomarkers for chemotherapy and targeted therapies sensitivity. Furthermore, selectively targeting oncogenic lncRNAs could provide a new therapeutic tool in treating NSCLC patients. lncRNAs disruption plays a pivotal role in NSCLC development and progression. These molecules also

  3. Novel Insights into Insect-Microbe Interactions—Role of Epigenomics and Small RNAs

    PubMed Central

    Kim, Dohyup; Thairu, Margaret W.; Hansen, Allison K.

    2016-01-01

    It has become increasingly clear that microbes form close associations with the vast majority of animal species, especially insects. In fact, an array of diverse microbes is known to form shared metabolic pathways with their insect hosts. A growing area of research in insect-microbe interactions, notably for hemipteran insects and their mutualistic symbionts, is to elucidate the regulation of this inter-domain metabolism. This review examines two new emerging mechanisms of gene regulation and their importance in host-microbe interactions. Specifically, we highlight how the incipient areas of research on regulatory “dark matter” such as epigenomics and small RNAs, can play a pivotal role in the evolution of both insect and microbe gene regulation. We then propose specific models of how these dynamic forms of gene regulation can influence insect-symbiont-plant interactions. Future studies in this area of research will give us a systematic understanding of how these symbiotic microbes and animals reciprocally respond to and regulate their shared metabolic processes. PMID:27540386

  4. Nanoparticles for targeted delivery of therapeutics and small interfering RNAs in hepatocellular carcinoma.

    PubMed

    Varshosaz, Jaleh; Farzan, Maryam

    2015-11-14

    Hepatocellular carcinoma (HCC) is the 5(th) most common malignancy which is responsible for more than half million annual mortalities; also, it is the third leading cause of cancer related death. Unfavorable systemic side-effects of chemotherapeutic agents and susceptibility to the degradation of small interfering RNAs (siRNAs), which can knock down a specific gene involved in the disease, have hampered their clinical application. So, it could be beneficial to develop an efficient carrier for the stabilization and specific delivery of drugs and siRNA to cells. Targeted nanoparticles have gained considerable attention as an efficient drug and gene delivery system, which is due to their capability in achieving the highest accumulation of cytotoxic agents in tumor tissue, modifiable drug pharmacokinetic- and bio-distribution, improved effectiveness of treatment, and limited side-effects. Recent studies have shed more light on the advantages of novel drug loaded carrier systems vs free drugs. Most of the animal studies have reported improvement in treatment efficacy and survival rate using novel carrier systems. Targeted delivery may be achieved passively or actively. In passive targeting, no ligand as homing device is used, while targeting is achieved by incorporating the therapeutic agent into a macromolecule or nanoparticle that passively reaches the target organ. However, in active targeting, the therapeutic agent or carrier system is conjugated to a tissue or cell-specific receptor which is over-expressed in a special malignancy using a ligand called a homing device. This review covers a broad spectrum of targeted nanoparticles as therapeutic and non-viral siRNA delivery systems, which are developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and their characteristics and opportunities for the clinical applications of drugs and therapeutic siRNA are discussed in this article. Asialoglycoprotein receptors, low-density lipoprotein

  5. Nanoparticles for targeted delivery of therapeutics and small interfering RNAs in hepatocellular carcinoma

    PubMed Central

    Varshosaz, Jaleh; Farzan, Maryam

    2015-01-01

    Hepatocellular carcinoma (HCC) is the 5th most common malignancy which is responsible for more than half million annual mortalities; also, it is the third leading cause of cancer related death. Unfavorable systemic side-effects of chemotherapeutic agents and susceptibility to the degradation of small interfering RNAs (siRNAs), which can knock down a specific gene involved in the disease, have hampered their clinical application. So, it could be beneficial to develop an efficient carrier for the stabilization and specific delivery of drugs and siRNA to cells. Targeted nanoparticles have gained considerable attention as an efficient drug and gene delivery system, which is due to their capability in achieving the highest accumulation of cytotoxic agents in tumor tissue, modifiable drug pharmacokinetic- and bio-distribution, improved effectiveness of treatment, and limited side-effects. Recent studies have shed more light on the advantages of novel drug loaded carrier systems vs free drugs. Most of the animal studies have reported improvement in treatment efficacy and survival rate using novel carrier systems. Targeted delivery may be achieved passively or actively. In passive targeting, no ligand as homing device is used, while targeting is achieved by incorporating the therapeutic agent into a macromolecule or nanoparticle that passively reaches the target organ. However, in active targeting, the therapeutic agent or carrier system is conjugated to a tissue or cell-specific receptor which is over-expressed in a special malignancy using a ligand called a homing device. This review covers a broad spectrum of targeted nanoparticles as therapeutic and non-viral siRNA delivery systems, which are developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and their characteristics and opportunities for the clinical applications of drugs and therapeutic siRNA are discussed in this article. Asialoglycoprotein receptors, low-density lipoprotein

  6. Sweating the small stuff: microRNAs and genetic changes define pancreatic cancer.

    PubMed

    Tang, Siuwah; Bonaroti, Jillian; Unlu, Sebnem; Liang, Xiaoyan; Tang, Daolin; Zeh, Herbert J; Lotze, Michael T

    2013-07-01

    MicroRNAs (miRNAs) are 18- to 22-nucleotide-long, single-stranded, noncoding RNAs that regulate important biological processes including differentiation, proliferation, and response to cellular stressors such as hypoxia, nutrient depletion, and traversion of the cell cycle by controlling protein expression within the cell. Many investigators have profiled cancer tissue and serum miRNAs to identify potential therapeutic targets, understand the pathways involved in tumorigenesis, and identify diagnostic tumor signatures. In the setting of pancreatic cancer, obtaining pancreatic tissue is invasive and impractical for early diagnosis. Several groups have profiled miRNAs that are present in the blood as a means to diagnose tumor progression and predict prognosis/survival or drug resistance. Several miRNA signatures found in pancreatic tissue and the peripheral blood, as well as the pathways that are associated with pancreatic cancer, are reviewed here in detail. Three miRNA biomarkers (miR-21, miR-155, and miR-200) have been repetitively identified in both pancreatic cancer tissue and patients' blood. Those miRNAs regulate and are regulated by the central genetic and epigenetic changes observed in pancreatic cancer including p53, transforming growth factor β, p16(INK4A), BRCA1/2, and Kras. These miRNAs are involved in DNA repair, cell cycle, and cell invasion and also play important roles in promoting metastases.

  7. Creating conditional dual fluorescence labeled transgenic animals for studying function of small noncoding RNAs.

    PubMed

    Yang, Kun; Gao, Yun; Yang, Mingfu; Xu, Zuoshang; Chen, Qian

    2017-01-01

    Because the function of most noncoding (nc) RNAs is unknown, Cre-lox transgenic mice are useful tools to determine their functions in a tissue or developmental stage-specific manner. However, the technology faces challenges because expression of ncRNA-transgene lacks protein product. No antibody or peptide-tag can be used to trace ncRNA expression in mouse tissues in real time. Furthermore, transgene integration at different locus or orientations in the genome may result in recombination of genomic fragments in the Cre-lox system. Establishing a reliable method that can be used to determine the precise copy number and orientation of the transgene is critical to the field. We developed a fast and straightforward method to determine ncRNA-transgene copy number, orientation, and insertion site in the genome. Furthermore, upon tissue-specific expression of ncRNA, a Cre-loxP-mediated dual-fluorescence expression system facilitates fluorescence signal switching from green to red, which enables real-time monitoring of ncRNA expression by fluorescence signals. As proof of concept, we demonstrate that after microRNA (miRNA)-Flox mice crossed with Col2a1-Cre mice, miRNA transgene expression could be detected successfully by red fluorescence signals in various cartilaginous tissues. This method of creating small ncRNA transgenic mice facilitates both tissue-specific ncRNA expression and real-time visualization of its expression. It is particularly suitable for in vivo studies of the functional roles and lineage tracing of small ncRNA.

  8. The prrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

    PubMed Central

    Reinhart, Alexandria A.; Powell, Daniel A.; Nguyen, Angela T.; O'Neill, Maura; Djapgne, Louise; Wilks, Angela; Ernst, Robert K.

    2014-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part of P. aeruginosa's iron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem in P. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2 mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identified phuS, encoding a heme binding protein involved in heme acquisition, and vreR, encoding a previously identified regulator of P. aeruginosa virulence genes, as novel targets of prrF-mediated heme regulation. Finally, we showed that the prrF locus encoding the PrrF and PrrH sRNAs is required for P. aeruginosa virulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2 deletion mutant protects against future challenge with wild-type P. aeruginosa. Combined, these data demonstrate that the prrF-encoded sRNAs are critical regulators of P. aeruginosa virulence. PMID:25510881

  9. 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.

  10. The Exosome and Trans-Acting Small Interfering RNAs Regulate Cuticular Wax Biosynthesis during Arabidopsis Inflorescence Stem Development1[OPEN

    PubMed Central

    Lam, Patricia; Zhao, Lifang; Eveleigh, Nathan; Yu, Yu; Chen, Xuemei

    2015-01-01

    The primary aerial surfaces of land plants are covered with a cuticle, a protective layer composed of the cutin polyester matrix and cuticular waxes. Previously, we discovered a unique mechanism of regulating cuticular wax biosynthesis during Arabidopsis (Arabidopsis thaliana) stem elongation that involves ECERIFERUM7 (CER7), a core subunit of the exosome. Because loss-of-function mutations in CER7 result in reduced expression of the wax biosynthetic gene CER3, we proposed that CER7 is involved in degrading a messenger RNA encoding a CER3 repressor. To identify this putative repressor, we performed a cer7 suppressor screen that resulted in the isolation of the posttranscriptional gene-silencing components RNA-DEPENDENT RNA POLYMERASE1 and SUPPRESSOR OF GENE SILENCING3, indicating that small RNAs regulate CER3 expression. To establish the identity of the effector RNA species and determine whether these RNAs control CER3 transcript levels directly, we cloned additional genes identified in our suppressor screen and performed next-generation sequencing of small RNA populations that differentially accumulate in the cer7 mutant in comparison with the wild type. Our results demonstrate that the trans-acting small interfering RNA class of small RNAs are the effector molecules involved in direct silencing of CER3 and that the expression of five additional genes (EARLY RESPONSE TO DEHYDRATION14, AUXIN RESISTANT1, a translation initiation factor SUI1 family protein, and two genes of unknown function) is controlled by both CER7 and trans-acting small interfering RNAs. PMID:25502190

  11. Inforna 2.0: A Platform for the Sequence-Based Design of Small Molecules Targeting Structured RNAs.

    PubMed

    Disney, Matthew D; Winkelsas, Audrey M; Velagapudi, Sai Pradeep; Southern, Mark; Fallahi, Mohammad; Childs-Disney, Jessica L

    2016-06-17

    The development of small molecules that target RNA is challenging yet, if successful, could advance the development of chemical probes to study RNA function or precision therapeutics to treat RNA-mediated disease. Previously, we described Inforna, an approach that can mine motifs (secondary structures) within target RNAs, which is deduced from the RNA sequence, and compare them to a database of known RNA motif-small molecule binding partners. Output generated by Inforna includes the motif found in both the database and the desired RNA target, lead small molecules for that target, and other related meta-data. Lead small molecules can then be tested for binding and affecting cellular (dys)function. Herein, we describe Inforna 2.0, which incorporates all known RNA motif-small molecule binding partners reported in the scientific literature, a chemical similarity searching feature, and an improved user interface and is freely available via an online web server. By incorporation of interactions identified by other laboratories, the database has been doubled, containing 1936 RNA motif-small molecule interactions, including 244 unique small molecules and 1331 motifs. Interestingly, chemotype analysis of the compounds that bind RNA in the database reveals features in small molecule chemotypes that are privileged for binding. Further, this updated database expanded the number of cellular RNAs to which lead compounds can be identified.

  12. Identification and characterization of a viroid resembling apple dimple fruit viroid in fig (Ficus carica L.) by next generation sequencing of small RNAs.

    PubMed

    Chiumenti, M; Torchetti, E M; Di Serio, F; Minafra, A

    2014-08-08

    Viroids are small (246-401 nt) circular and non coding RNAs infecting higher plants. They are targeted by host Dicer-like enzymes (DCLs) that generate small RNAs of 21-24 nt (sRNAs), which are involved in the host RNA silencing pathways. The accumulation in plant tissues of such viroid-derived small RNAs (vd-sRNAs) is a clear sign of an ongoing viroid infection. In this study, next generation sequencing of a sRNAs library and assembling of the sequenced vd-sRNAs were instrumental for the identification of a viroid resembling apple dimple fruit viroid (ADFVd) in a fig accession. After confirming by molecular methods the presence of this viroid in the fig tree, its population was characterized, showing that the ADFVd master sequence from fig diverges from that of the ADFVd reference variant from apple. Moreover, since this viroid accumulates at a low level in fig, a semi-nested RT-PCR assay was developed for detecting it in other fig accessions. ADFVd seems to have a wider host range than thought before and this poses questions about its epidemiology. A further characterization of ADFVd-sRNAs showed similar accumulation of (+) or (-) vd-sRNAs that mapped on the viroid genome generating hotspot profiles. Moreover, similarly to other nuclear-replicating viroids, vd-sRNAs of 21, 22 and 24 nt in size prevailed in the distribution profiles. Altogether, these data support the involvement of double-stranded RNAs and different DCLs, targeting the same restricted viroid regions, in the genesis of ADFVd-sRNAs.

  13. Therapeutic Potential of a Combination of Two Gene-Specific Small Interfering RNAs against Clinical Strains of Acanthamoeba▿

    PubMed Central

    Lorenzo-Morales, Jacob; Martín-Navarro, Carmen M.; López-Arencibia, Atteneri; Santana-Morales, María A.; Afonso-Lehmann, Raquel N.; Maciver, Sutherland K.; Valladares, Basilio; Martínez-Carretero, Enrique

    2010-01-01

    Pathogenic strains of the genus Acanthamoeba are causative agents of severe infections, such as fatal encephalitis and a sight-threatening amoebic keratitis. Antimicrobial therapy for these infections is generally empirical, and patient recovery is often problematic, due to the existence of a highly resistant cyst stage in these amoebae. In previous studies, small interfering RNAs (siRNAs) against the catalytic domains of extracellular serine proteases and glycogen phosphorylase from Acanthamoeba were designed and evaluated for future therapeutic use. The silencing of proteases resulted in Acanthamoeba failing to degrade human corneal cells, and silencing of glycogen phosphorylase caused amoebae to be unable to form mature cysts. After the siRNA design and concentration were optimized in order to avoid toxicity problems, cultures of Acanthamoeba were treated with a combination of both siRNAs, and cells were evaluated under an inverted microscope. This siRNA-based treatment dramatically affected the growth rate and cellular survival of the amoebae. These results were observed less than 48 h after the initiation of the treatment. In order to check possible toxic effects of the siRNA combination, three eukaryotic cell lines (HeLa, murine macrophages, and osteosarcoma cells) were treated with the same molecules, and cytotoxicity was examined by measuring lactate dehydrogenase release. The future use of the combination of these siRNAs is proposed as a potential therapeutic approach against pathogenic strains of Acanthamoeba. PMID:20855732

  14. Implications of MicroRNAs in the Treatment of Gefitinib-Resistant Non-Small Cell Lung Cancer

    PubMed Central

    Sin, Thomas K.; Wang, Fengfeng; Meng, Fei; Wong, S. C. Cesar; Cho, William C. S.; Siu, Parco M.; Chan, Lawrence W. C.; Yung, Benjamin Y. M.

    2016-01-01

    Non-small cell lung cancer (NSCLC) represents about 85% of the reported cases of lung cancer. Acquired resistance to targeted therapy with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, is not uncommon. It is thus vital to explore novel strategies to restore sensitivity to gefitinib. Provided that microRNAs (miRNAs) negatively regulate their gene targets at the transcriptional level, it is speculated that miRNA mimetics may reduce the expression, activity and signal transduction of EGFR so that sensitization of tumour sites to gefitinib-induced cytotoxicity can be achieved. Indeed, a growing body of evidence has shown that the manipulation of endogenous levels of miRNA not only attenuates the EGFR/PI3K/Akt phosphorylation cascade, but also restores apoptotic cell death in in vitro models of experimentally-induced gefitinib resistance and provoked tumour regression/shrinkage in xenograft models. These data are in concordant with the clinical data showing that the differential expression profiles of miRNA in tumour tissues and blood associate strongly with drug response and overall survival. Furthermore, another line of studies indicate that the chemopreventive effects of a variety of natural compounds may involve miRNAs. The present review aims to discuss the therapeutic capacity of miRNAs in relation to recent discoveries on EGFR-TKI resistance, including chronic drug exposure and mutations. PMID:26891293

  15. Implications of MicroRNAs in the Treatment of Gefitinib-Resistant Non-Small Cell Lung Cancer.

    PubMed

    Sin, Thomas K; Wang, Fengfeng; Meng, Fei; Wong, S C Cesar; Cho, William C S; Siu, Parco M; Chan, Lawrence W C; Yung, Benjamin Y M

    2016-02-15

    Non-small cell lung cancer (NSCLC) represents about 85% of the reported cases of lung cancer. Acquired resistance to targeted therapy with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, is not uncommon. It is thus vital to explore novel strategies to restore sensitivity to gefitinib. Provided that microRNAs (miRNAs) negatively regulate their gene targets at the transcriptional level, it is speculated that miRNA mimetics may reduce the expression, activity and signal transduction of EGFR so that sensitization of tumour sites to gefitinib-induced cytotoxicity can be achieved. Indeed, a growing body of evidence has shown that the manipulation of endogenous levels of miRNA not only attenuates the EGFR/PI3K/Akt phosphorylation cascade, but also restores apoptotic cell death in in vitro models of experimentally-induced gefitinib resistance and provoked tumour regression/shrinkage in xenograft models. These data are in concordant with the clinical data showing that the differential expression profiles of miRNA in tumour tissues and blood associate strongly with drug response and overall survival. Furthermore, another line of studies indicate that the chemopreventive effects of a variety of natural compounds may involve miRNAs. The present review aims to discuss the therapeutic capacity of miRNAs in relation to recent discoveries on EGFR-TKI resistance, including chronic drug exposure and mutations.

  16. Small non-coding RNAs (sncRNA) regulate gene silencing and modify homeostatic status in animals faced with porcine reproductive and respiratory syndrome virus (PRRSV)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It has been established that reduced susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) has a genetic component. This genetic component may take the form of small non-coding RNAs (sncRNA), which are molecules that function as regulators of gene expression. Various sncRNAs ...

  17. Assessing small mammal abundance with track-tube indices and mark-recapture population estimates

    USGS Publications Warehouse

    Wiewel, A.S.; Clark, W.R.; Sovada, M.A.

    2007-01-01

    We compared track-tube sampling with mark-recapture livetrapping and evaluated a track-tube index, defined as the number of track tubes with identifiable small mammal tracks during a 4-night period, as a predictor of small mammal abundance estimates in North Dakota grasslands. Meadow voles (Microtus pennsylvanicus) were the most commonly recorded species by both methods, but were underrepresented in track-tube sampling, whereas 13-lined ground squirrels (Spermophilus tridecemlineatus) and Franklin's ground squirrels (S. franklinii) were overrepresented in track-tube sampling. Estimates of average species richness were lower from track tubes than from livetrapping. Regression models revealed that the track-tube index was at best a moderately good predictor of small mammal population estimates because both the form (linear versus curvilinear) and slope of the relationship varied between years. In addition, 95% prediction intervals indicated low precision when predicting population estimates from new track-tube index observations. Track tubes required less time and expense than mark-recapture and eliminated handling of small mammals. Using track tubes along with mark-recapture in a double sampling for regression framework would have potential value when attempting to estimate abundance of small mammals over large areas. ?? 2007 American Society of Mammalogists.

  18. Rootstock-to-scion transfer of transgene-derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry.

    PubMed

    Zhao, Dongyan; Song, Guo-qing

    2014-12-01

    Small interfering RNAs (siRNAs) are silencing signals in plants. Virus-resistant transgenic rootstocks developed through siRNA-mediated gene silencing may enhance virus resistance of nontransgenic scions via siRNAs transported from the transgenic rootstocks. However, convincing evidence of rootstock-to-scion movement of siRNAs of exogenous genes in woody plants is still lacking. To determine whether exogenous siRNAs can be transferred, nontransgenic sweet cherry (scions) was grafted on transgenic cherry rootstocks (TRs), which was transformed with an RNA interference (RNAi) vector expressing short hairpin RNAs of the genomic RNA3 of Prunus necrotic ringspot virus (PNRSV-hpRNA). Small RNA sequencing was conducted using bud tissues of TRs and those of grafted (rootstock/scion) trees, locating at about 1.2 m above the graft unions. Comparison of the siRNA profiles revealed that the PNRSV-hpRNA was efficient in producing siRNAs and eliminating PNRSV in the TRs. Furthermore, our study confirmed, for the first time, the long-distance (1.2 m) transfer of PNRSV-hpRNA-derived siRNAs from the transgenic rootstock to the nontransgenic scion in woody plants. Inoculation of nontransgenic scions with PNRSV revealed that the transferred siRNAs enhanced PNRSV resistance of the scions grafted on the TRs. Collectively, these findings provide the foundation for 'using transgenic rootstocks to produce products of nontransgenic scions in fruit trees'.

  19. Novel low abundance and transient RNAs in yeast revealed by tiling microarrays and ultra high-throughput sequencing are not conserved across closely related yeast species.

    PubMed

    Lee, Albert; Hansen, Kasper Daniel; Bullard, James; Dudoit, Sandrine; Sherlock, Gavin

    2008-12-01

    A complete description of the transcriptome of an organism is crucial for a comprehensive understanding of how it functions and how its transcriptional networks are controlled, and may provide insights into the organism's evolution. Despite the status of Saccharomyces cerevisiae as arguably the most well-studied model eukaryote, we still do not have a full catalog or understanding of all its genes. In order to interrogate the transcriptome of S. cerevisiae for low abundance or rapidly turned over transcripts, we deleted elements of the RNA degradation machinery with the goal of preferentially increasing the relative abundance of such transcripts. We then used high-resolution tiling microarrays and ultra high-throughput sequencing (UHTS) to identify, map, and validate unannotated transcripts that are more abundant in the RNA degradation mutants relative to wild-type cells. We identified 365 currently unannotated transcripts, the majority presumably representing low abundance or short-lived RNAs, of which 185 are previously unknown and unique to this study. It is likely that many of these are cryptic unstable transcripts (CUTs), which are rapidly degraded and whose function(s) within the cell are still unclear, while others may be novel functional transcripts. Of the 185 transcripts we identified as novel to our study, greater than 80 percent come from regions of the genome that have lower conservation scores amongst closely related yeast species than 85 percent of the verified ORFs in S. cerevisiae. Such regions of the genome have typically been less well-studied, and by definition transcripts from these regions will distinguish S. cerevisiae from these closely related species.

  20. Diverse human extracellular RNAs are widely detected in human plasma

    PubMed Central

    Freedman, Jane E.; Gerstein, Mark; Mick, Eric; Rozowsky, Joel; Levy, Daniel; Kitchen, Robert; Das, Saumya; Shah, Ravi; Danielson, Kirsty; Beaulieu, Lea; Navarro, Fabio C. P.; Wang, Yaoyu; Galeev, Timur R.; Holman, Alex; Kwong, Raymond Y.; Murthy, Venkatesh; Tanriverdi, Selim E.; Koupenova, Milka; Mikhalev, Ekaterina; Tanriverdi, Kahraman

    2016-01-01

    There is growing appreciation for the importance of non-protein-coding genes in development and disease. Although much is known about microRNAs, limitations in bioinformatic analyses of RNA sequencing have precluded broad assessment of other forms of small-RNAs in humans. By analysing sequencing data from plasma-derived RNA from 40 individuals, here we identified over a thousand human extracellular RNAs including microRNAs, piwi-interacting RNA (piRNA), and small nucleolar RNAs. Using a targeted quantitative PCR with reverse transcription approach in an additional 2,763 individuals, we characterized almost 500 of the most abundant extracellular transcripts including microRNAs, piRNAs and small nucleolar RNAs. The presence in plasma of many non-microRNA small-RNAs was confirmed in an independent cohort. We present comprehensive data to demonstrate the broad and consistent detection of diverse classes of circulating non-cellular small-RNAs from a large population. PMID:27112789

  1. Small transcriptome analysis indicates that the enzyme RppH influences both the quality and quantity of sRNAs in Neisseria gonorrhoeae

    PubMed Central

    Wachter, Jenny; Hill, Stuart A.

    2015-01-01

    Prokaryotic mRNA turnover can be initiated by the removal of pyrophosphate from the 5′ end of a transcript using the RNA pyrophosphohydrolase enzyme RppH. Following the initial dephosphorylation step, RNaseE then degrades the message into small oligonucleotide segments. This study assessed the small RNA transcriptome of Neisseria gonorrhoeae strain MS11 in two genetic backgrounds; using wild type cells as well as cells carrying a rppH insertional mutation. It was found that the presence of the RppH enzyme affected both the quantity and length of small RNAs (sRNAs) in various chromosomal locations and involved sense transcripts (seRNAs), transcripts originating from the opposite strand (asRNAs) as well as inter-genic-derived RNAs (IGRs). In comparing the two transcriptomes, we found that not all small RNAs were expressed in both genetic backgrounds, suggesting that RppH apparently targets only a subset of transcripts. Overall, this study shows that small RNAs can be detected from the majority of genes within the chromosome, as well as from inter-genic regions, and that more sRNA transcripts are detected in the absence of the RppH enzyme. PMID:25688066

  2. Identifying (non-)coding RNAs and small peptides: challenges and opportunities.

    PubMed

    Pauli, Andrea; Valen, Eivind; Schier, Alexander F

    2015-01-01

    Over the past decade, high-throughput studies have identified many novel transcripts. While their existence is undisputed, their coding potential and functionality have remained controversial. Recent computational approaches guided by ribosome profiling have indicated that translation is far more pervasive than anticipated and takes place on many transcripts previously assumed to be non-coding. Some of these newly discovered translated transcripts encode short, functional proteins that had been missed in prior screens. Other transcripts are translated, but it might be the process of translation rather than the resulting peptides that serves a function. Here, we review annotation studies in zebrafish to discuss the challenges of placing RNAs onto the continuum that ranges from functional protein-encoding mRNAs to potentially non-functional peptide-producing RNAs to non-coding RNAs. As highlighted by the discovery of the novel signaling peptide Apela/ELABELA/Toddler, accurate annotations can give rise to exciting opportunities to identify the functions of previously uncharacterized transcripts.

  3. VirusDetect: An automated pipeline for efficient virus discovery using deep sequencing of small RNAs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Accurate detection of viruses in plants and animals is critical for agriculture production and human health. Deep sequencing and assembly of virus-derived siRNAs has proven to be a highly efficient approach for virus discovery. However, to date no computational tools specifically designed for both k...

  4. Light-regulated mRNA condensation by a photosensitive surfactant works as a series photoswitch of translation activity in the presence of small RNAs.

    PubMed

    Rudiuk, Sergii; Saito, Hirohide; Hara, Tomoaki; Inoue, Tan; Yoshikawa, Kenichi; Baigl, Damien

    2011-11-14

    AzoTAB, a photosensitive azobenzene cationic surfactant, which phototriggers translation activity through light-regulated condensation of mRNA, is added to a translation solution containing several mRNAs, which can be selectively silenced by specific small RNAs. We find that gene silencing by small RNAs remains functional regardless of AzoTAB concentration and UV illumination. In the absence of UV, the translation of all genes present in the medium is partially to fully inhibited depending on AzoTAB concentration. In contrast, the application of a short UV stimulus (365 nm for 1.5 min) results in the selective photoactivation of genes that are not silenced by small RNA. These results show that light-regulated condensation by AzoTAB works as a sequence-independent series photoswitch added to parallel sequence-specific regulation by small RNAs.

  5. Non-coding RNAs in Mammary Gland Development and Disease.

    PubMed

    Sandhu, Gurveen K; Milevskiy, Michael J G; Wilson, Wesley; Shewan, Annette M; Brown, Melissa A

    2016-01-01

    Non-coding RNAs (ncRNAs) are untranslated RNA molecules that function to regulate the expression of numerous genes and associated biochemical pathways and cellular functions. NcRNAs include small interfering RNAs (siRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs). They participate in the regulation of all developmental processes and are frequently aberrantly expressed or functionally defective in disease. This Chapter will focus on the role of ncRNAs, in particular miRNAs and lncRNAs, in mammary gland development and disease.

  6. A genetic approach for finding small RNAs regulators of genes of interest identifies RybC as regulating the DpiA/DpiB two-component system.

    PubMed

    Mandin, Pierre; Gottesman, Susan

    2009-05-01

    In Escherichia coli, the largest class of small regulatory RNAs binds to the RNA chaperone Hfq and regulates the stability and/or translation of specific mRNAs. While recent studies have shown that some mRNAs could be subject to post-transcriptional regulation by sRNAs (e.g. mRNAs found by co-immunoprecipitation with Hfq), no method has yet been described to identify small RNAs that regulate them. We developed a method to easily make translational fusions of genes of interest to the lacZ reporter gene, under the control of a P(BAD)-inducible promoter. A multicopy plasmid library of the E. coli genome can then be used to screen for small RNAs that affect the activity of the fusion. This screening method was first applied to the dpiB gene from the dpiBA operon, which encodes a two-component signal transduction system involved in the SOS response to beta-lactams. One small RNA, RybC, was found to negatively regulate the expression of dpiB. Using mutants in the dpiB-lacZ fusion and compensatory mutations in the RybC sRNA, we demonstrate that RybC directly base pairs with the dpiBA mRNA.

  7. Genome-wide differential expression of genes and small RNAs in testis of two different porcine breeds and at two different ages

    PubMed Central

    Li, Yao; Li, Jialian; Fang, Chengchi; Shi, Liang; Tan, Jiajian; Xiong, Yuanzhu; Bin Fan; Li, Changchun

    2016-01-01

    Some documented evidences proved small RNAs (sRNA) and targeted genes are involved in mammalian testicular development and spermatogenesis. However, the detailed molecular regulation mechanisms of them remain largely unknown so far. In this study, we obtained a total of 10,716 mRNAs, 67 miRNAs and 16,953 piRNAs which were differentially expressed between LC and LW pig breeds or between the two sexual maturity stages. Of which, we identified 16 miRNAs and 28 targeted genes possibly related to spermatogenesis; 14 miRNA and 18 targeted genes probably associated with cell adhesion related testis development. We also annotated 579 piRNAs which could potentially regulate cell death, nucleosome organization and other basic biology process, which implied that those piRNAs might be involved in sexual maturation difference. The integrated network analysis results suggested that some differentially expressed genes were involved in spermatogenesis through the ECM–receptor interaction, focal adhesion, Wnt and PI3K–Akt signaling pathways, some particular miRNAs have the negative regulation roles and some special piRNAs have the positive and negative regulation roles in testicular development. Our data provide novel insights into the molecular expression and regulation similarities and diversities of spermatogenesis and testicular development in different pig breeds at different stages of sexual maturity. PMID:27229484

  8. Fencing and mowing as effective methods for reducing tick abundance on very small, infested plots.

    PubMed

    Del Fabbro, Simone

    2015-03-01

    The tick Ixodes ricinus (L.) transmits a large variety of pathogens to humans and is therefore a matter of concern for public health. Different strategies for reducing the risk of tick bite, and thus of infection, have been developed and vary according to the kind of exposure (occupational, recreational, peridomestic). The present study (carried out in an endemic region for both Lyme borreliosis and tick-borne encephalitis) aimed to assess the efficacy of two simple and cheap interventions for reducing I. ricinus abundance around residential properties surrounded by wooded areas. The immediate impact of exclosures (host-targeted control methods) and mowing (vegetation management) on very small surfaces (<1ha) were evaluated both alone and in combination. Results suggest that fencing (even if applied on very small surfaces), by preventing the entrance of tick reproductive hosts, can decrease the abundance of parasites in a short time, and that mowing can contribute to reach the goal. This control method could be of great value in small portions of heavily infested areas that have to be kept tick-free to reduce the risk of peridomestic exposure or to permit their recreational use (e.g. picnic areas within natural parks). Benefits appear even greater when considering that these interventions are environmental safe, cheap, technically simple and effective even in close proximity to heavy infested woodlands.

  9. 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

  10. Tidal amplitude and fish abundance in the mouth region of a small estuary.

    PubMed

    Becker, A; Whitfield, A K; Cowley, P D; Cole, V J; Taylor, M D

    2016-09-01

    Using an acoustic underwater camera (Dual Frequency IDentification SONar, DIDSON), the abundance and direction of movement of fishes > 80 mm total length (LT ) in the mouth of a small South African estuary during spring and neap tidal cycles were observed. While the sizes of fishes recorded were consistent across both tide cycles, the number of fishes passing the camera was significantly greater during the smaller neap tides. Schooling behaviour was more pronounced for fishes that were travelling into the estuary compared to fishes swimming towards the ocean.

  11. Community-Weighted Mean Plant Traits Predict Small Scale Distribution of Insect Root Herbivore Abundance.

    PubMed

    Sonnemann, Ilja; Pfestorf, Hans; Jeltsch, Florian; Wurst, Susanne

    2015-01-01

    Small scale distribution of insect root herbivores may promote plant species diversity by creating patches of different herbivore pressure. However, determinants of small scale distribution of insect root herbivores, and impact of land use intensity on their small scale distribution are largely unknown. We sampled insect root herbivores and measured vegetation parameters and soil water content along transects in grasslands of different management intensity in three regions in Germany. We calculated community-weighted mean plant traits to test whether the functional plant community composition determines the small scale distribution of insect root herbivores. To analyze spatial patterns in plant species and trait composition and insect root herbivore abundance we computed Mantel correlograms. Insect root herbivores mainly comprised click beetle (Coleoptera, Elateridae) larvae (43%) in the investigated grasslands. Total insect root herbivore numbers were positively related to community-weighted mean traits indicating high plant growth rates and biomass (specific leaf area, reproductive- and vegetative plant height), and negatively related to plant traits indicating poor tissue quality (leaf C/N ratio). Generalist Elaterid larvae, when analyzed independently, were also positively related to high plant growth rates and furthermore to root dry mass, but were not related to tissue quality. Insect root herbivore numbers were not related to plant cover, plant species richness and soil water content. Plant species composition and to a lesser extent plant trait composition displayed spatial autocorrelation, which was not influenced by land use intensity. Insect root herbivore abundance was not spatially autocorrelated. We conclude that in semi-natural grasslands with a high share of generalist insect root herbivores, insect root herbivores affiliate with large, fast growing plants, presumably because of availability of high quantities of food. Affiliation of insect root

  12. Community- Weighted Mean Plant Traits Predict Small Scale Distribution of Insect Root Herbivore Abundance

    PubMed Central

    Jeltsch, Florian; Wurst, Susanne

    2015-01-01

    Small scale distribution of insect root herbivores may promote plant species diversity by creating patches of different herbivore pressure. However, determinants of small scale distribution of insect root herbivores, and impact of land use intensity on their small scale distribution are largely unknown. We sampled insect root herbivores and measured vegetation parameters and soil water content along transects in grasslands of different management intensity in three regions in Germany. We calculated community-weighted mean plant traits to test whether the functional plant community composition determines the small scale distribution of insect root herbivores. To analyze spatial patterns in plant species and trait composition and insect root herbivore abundance we computed Mantel correlograms. Insect root herbivores mainly comprised click beetle (Coleoptera, Elateridae) larvae (43%) in the investigated grasslands. Total insect root herbivore numbers were positively related to community-weighted mean traits indicating high plant growth rates and biomass (specific leaf area, reproductive- and vegetative plant height), and negatively related to plant traits indicating poor tissue quality (leaf C/N ratio). Generalist Elaterid larvae, when analyzed independently, were also positively related to high plant growth rates and furthermore to root dry mass, but were not related to tissue quality. Insect root herbivore numbers were not related to plant cover, plant species richness and soil water content. Plant species composition and to a lesser extent plant trait composition displayed spatial autocorrelation, which was not influenced by land use intensity. Insect root herbivore abundance was not spatially autocorrelated. We conclude that in semi-natural grasslands with a high share of generalist insect root herbivores, insect root herbivores affiliate with large, fast growing plants, presumably because of availability of high quantities of food. Affiliation of insect root

  13. Microbial Abundances in Salt Marsh Soils: A Molecular Approach for Small Spatial Scales

    NASA Astrophysics Data System (ADS)

    Granse, Dirk; Mueller, Peter; Weingartner, Magdalena; Hoth, Stefan; Jensen, Kai

    2016-04-01

    The rate of biological decomposition greatly determines the carbon sequestration capacity of salt marshes. Microorganisms are involved in the decomposition of biomass and the rate of decomposition is supposed to be related to microbial abundance. Recent studies quantified microbial abundance by means of quantitative polymerase chain reaction (QPCR), a method that also allows determining the microbial community structure by applying specific primers. The main microbial community structure can be determined by using primers specific for 16S rRNA (Bacteria) and 18S rRNA (Fungi) of the microbial DNA. However, the investigation of microbial abundance pattern at small spatial scales, such as locally varying abiotic conditions within a salt-marsh system, requires high accuracy in DNA extraction and QPCR methods. Furthermore, there is evidence that a single extraction may not be sufficient to reliably quantify rRNA gene copies. The aim of this study was to establish a suitable DNA extraction method and stable QPCR conditions for the measurement of microbial abundances in semi-terrestrial environments. DNA was extracted from two soil samples (top WE{5}{cm}) by using the PowerSoil DNA Extraction Kit (Mo Bio Laboratories, Inc., Carlsbad, CA) and applying a modified extraction protocol. The DNA extraction was conducted in four consecutive DNA extraction loops from three biological replicates per soil sample by reusing the PowerSoil bead tube. The number of Fungi and Bacteria rRNA gene copies of each DNA extraction loop and a pooled DNA solution (extraction loop 1 - 4) was measured by using the QPCR method with taxa specific primer pairs (Bacteria: B341F, B805R; Fungi: FR1, FF390). The DNA yield of the replicates varied at DNA extraction loop 1 between WE{25 and 85}{ng

  14. In silico genome wide mining of conserved and novel miRNAs in the brain and pineal gland of Danio rerio using small RNA sequencing data.

    PubMed

    Agarwal, Suyash; Nagpure, Naresh Sahebrao; Srivastava, Prachi; Kushwaha, Basdeo; Kumar, Ravindra; Pandey, Manmohan; Srivastava, Shreya

    2016-03-01

    MicroRNAs (miRNAs) are small, non-coding RNA molecules that bind to the mRNA of the target genes and regulate the expression of the gene at the post-transcriptional level. Zebrafish is an economically important freshwater fish species globally considered as a good predictive model for studying human diseases and development. The present study focused on uncovering known as well as novel miRNAs, target prediction of the novel miRNAs and the differential expression of the known miRNA using the small RNA sequencing data of the brain and pineal gland (dark and light treatments) obtained from NCBI SRA. A total of 165, 151 and 145 known zebrafish miRNAs were found in the brain, pineal gland (dark treatment) and pineal gland (light treatment), respectively. Chromosomes 4 and 5 of zebrafish reference assembly GRCz10 were found to contain maximum number of miR genes. The miR-181a and miR-182 were found to be highly expressed in terms of number of reads in the brain and pineal gland, respectively. Other ncRNAs, such as tRNA, rRNA and snoRNA, were curated against Rfam. Using GRCz10 as reference, the subsequent bioinformatic analyses identified 25, 19 and 9 novel miRNAs from the brain, pineal gland (dark treatment) and pineal gland (light treatment), respectively. Targets of the novel miRNAs were identified, based on sequence complementarity between miRNAs and mRNA, by searching for antisense hits in the 3'-UTR of reference RNA sequences of the zebrafish. The discovery of novel miRNAs and their targets in the zebrafish genome can be a valuable scientific resource for further functional studies not only in zebrafish but also in other economically important fishes.

  15. Identification of RNase L-Dependent, 3′-End-Modified, Viral Small RNAs in Sindbis Virus-Infected Mammalian Cells

    PubMed Central

    Girardi, Erika; Chane-Woon-Ming, Béatrice; Messmer, Mélanie; Kaukinen, Pasi; Pfeffer, Sébastien

    2013-01-01

    ABSTRACT Small RNAs play a critical role in host-pathogen interaction. Indeed, small RNA-mediated silencing or RNA interference (RNAi) is one of the earliest forms of antiviral immunity. Although it represents the main defense system against viruses in many organisms, the antiviral role of RNAi has not been clearly proven in higher vertebrates. However, it is well established that their response to viral infection relies on the recognition of viral RNAs by host pattern recognition receptors (PRRs) to trigger activation of the interferon pathway. In the present work, we report the existence of a novel small noncoding RNA population produced in mammalian cells upon RNA virus infection. Using Sindbis virus (SINV) as a prototypic arbovirus model, we profiled the small RNA population of infected cells in both human and African green monkey cell lines. Here, we provide evidence for the presence of discrete small RNAs of viral origin that are not associated with the RNA-induced silencing complex (RISC), that are highly expressed and detected by Northern blot analysis, and that accumulate as 21- to 28-nucleotide (nt) species during infection. We report that the cellular antiviral endoribonuclease RNase L cleaves the viral genome, producing in turn the small RNAs. Surprisingly, we uncovered the presence of a modification on the 3′-end nucleotide of SINV-derived viral small RNAs (SvsRNAs) that might be at the origin of their stability. Altogether, our findings show that stable modified small viral RNAs could represent a novel way to modulate host-virus interaction upon SINV infection. PMID:24255120

  16. Experimental approaches to identify small RNAs and their diverse roles in bacteria--what we have learnt in one decade of MicA research.

    PubMed

    Van Puyvelde, Sandra; Vanderleyden, Jozef; De Keersmaecker, Sigrid C J

    2015-10-01

    Nowadays the identification of small RNAs (sRNAs) and characterization of their role within regulatory networks takes a prominent place in deciphering complex bacterial phenotypes. Compared to the study of other components of bacterial cells, this is a relatively new but fast-growing research field. Although reports on new sRNAs appear regularly, some sRNAs are already subject of research for a longer time. One of such sRNAs is MicA, a sRNA best described for its role in outer membrane remodeling, but probably having a much broader function than anticipated. An overview of what we have learnt from MicA led to the conclusion that even for this well-described sRNA, we still do not have the overall picture. More general, the story of MicA might become an experimental lead for unraveling the many sRNAs with unknown functions. In this review, three important topics in the sRNA field are covered, exemplified from the perspective of MicA: (i) identification of new sRNAs, (ii) target identification and unraveling the biological function, (iii) structural analysis. The complex mechanisms of action of MicA deliver some original insights in the sRNA field which includes the existence of dimer formation or simultaneous cis and trans regulation, and might further inspire the understanding of the function of other sRNAs.

  17. Role of the Salmonella enterica serovar Typhi Fur regulator and small RNAs RfrA and RfrB in iron homeostasis and interaction with host cells.

    PubMed

    Leclerc, Jean-Mathieu; Dozois, Charles M; Daigle, France

    2013-03-01

    Iron is an essential element but can be toxic at high concentrations. Therefore, its acquisition and storage require tight control. Salmonella encodes the global regulator Fur (ferric uptake regulator) and the small regulatory non-coding RNAs (sRNAs) RfrA and RfrB, homologues of RyhB. The role of these iron homeostasis regulators was investigated in Salmonella enterica serovar Typhi (S. Typhi). Strains containing either single or combined deletions of these regulators were obtained. The mutants were tested for growth in low and high iron conditions, resistance to oxidative stress, expression and production of siderophores, and during interaction with host cells. The fur mutant showed a growth defect and was sensitive to hydrogen peroxide. The expression of the sRNAs was responsible for these defects. Siderophore expression by S. Typhi and both sRNAs were regulated by iron and by Fur. Fur contributed to invasion of epithelial cells, and was shown for the first time to play a role in phagocytosis and intracellular survival of S. Typhi in human macrophages. The sRNAs RfrA and RfrB were not required for interaction with epithelial cells, but both sRNAs were important for optimal intracellular replication in macrophages. In S. Typhi, Fur is a repressor of both sRNAs, and loss of either RfrA or RfrB resulted in distinct phenotypes, suggesting a non-redundant role for these regulatory RNAs.

  18. MISIS-2: A bioinformatics tool for in-depth analysis of small RNAs and representation of consensus master genome in viral quasispecies.

    PubMed

    Seguin, Jonathan; Otten, Patricia; Baerlocher, Loïc; Farinelli, Laurent; Pooggin, Mikhail M

    2016-07-01

    In most eukaryotes, small RNA (sRNA) molecules such as miRNAs, siRNAs and piRNAs regulate gene expression and repress transposons and viruses. AGO/PIWI family proteins sort functional sRNAs based on size, 5'-nucleotide and other sequence features. In plants and some animals, viral sRNAs are extremely diverse and cover the entire viral genome sequences, which allows for de novo reconstruction of a complete viral genome by deep sequencing and bioinformatics analysis of viral sRNAs. Previously, we have developed a tool MISIS to view and analyze sRNA maps of viruses and cellular genome regions which spawn multiple sRNAs. Here we describe a new release of MISIS, MISIS-2, which enables to determine and visualize a consensus sequence and count sRNAs of any chosen sizes and 5'-terminal nucleotide identities. Furthermore we demonstrate the utility of MISIS-2 for identification of single nucleotide polymorphisms (SNPs) at each position of a reference sequence and reconstruction of a consensus master genome in evolving viral quasispecies. MISIS-2 is a Java standalone program. It is freely available along with the source code at the website http://www.fasteris.com/apps.

  19. A Cluster of Four Homologous Small RNAs Modulates C1 Metabolism and the Pyruvate Dehydrogenase Complex in Rhodobacter sphaeroides under Various Stress Conditions

    PubMed Central

    Billenkamp, Fabian; Peng, Tao; Berghoff, Bork A.

    2015-01-01

    ABSTRACT In bacteria, regulatory RNAs play an important role in the regulation and balancing of many cellular processes and stress responses. Among these regulatory RNAs, trans-encoded small RNAs (sRNAs) are of particular interest since one sRNA can lead to the regulation of multiple target mRNAs. In the purple bacterium Rhodobacter sphaeroides, several sRNAs are induced by oxidative stress. In this study, we focused on the functional characterization of four homologous sRNAs that are cotranscribed with the gene for the conserved hypothetical protein RSP_6037, a genetic arrangement described for only a few sRNAs until now. Each of the four sRNAs is characterized by two stem-loops that carry CCUCCUCCC motifs in their loops. They are induced under oxidative stress, as well as by various other stress conditions, and were therefore renamed here sRNAs CcsR1 to CcsR4 (CcsR1–4) for conserved CCUCCUCCC motif stress-induced RNAs 1 to 4. Increased CcsR1–4 expression decreases the expression of genes involved in C1 metabolism or encoding components of the pyruvate dehydrogenase complex either directly by binding to their target mRNAs or indirectly. One of the CcsR1–4 target mRNAs encodes the transcriptional regulator FlhR, an activator of glutathione-dependent methanol/formaldehyde metabolism. Downregulation of this glutathione-dependent pathway increases the pool of glutathione, which helps to counteract oxidative stress. The FlhR-dependent downregulation of the pyruvate dehydrogenase complex reduces a primary target of reactive oxygen species and reduces aerobic electron transport, a main source of reactive oxygen species. Our findings reveal a previously unknown strategy used by bacteria to counteract oxidative stress. IMPORTANCE Phototrophic organisms have to cope with photo-oxidative stress due to the function of chlorophylls as photosensitizers for the formation of singlet oxygen. Our study assigns an important role in photo-oxidative stress resistance to a

  20. Going mobile: non-cell-autonomous small RNAs shape the genetic landscape of plants.

    PubMed

    Pyott, Douglas E; Molnar, Attila

    2015-04-01

    RNA silencing is a form of genetic regulation, which is conserved across eukaryotes and has wide ranging biological functions. Recently, there has been a growing appreciation for the importance of mobility in RNA silencing pathways, particularly in plants. Moreover, in addition to the importance for mobile RNA silencing in an evolutionary context, the potential for utilizing mobile short silencing RNAs in biotechnological applications is becoming apparent. This review aims to set current knowledge of this topic in a historical context and provides examples to illustrate the importance of mobile RNA silencing in both natural and artificially engineered systems in plants.

  1. Next-Generation Sequencing of Protein-Coding and Long Non-protein-Coding RNAs in Two Types of Exosomes Derived from Human Whole Saliva.

    PubMed

    Ogawa, Yuko; Tsujimoto, Masafumi; Yanoshita, Ryohei

    2016-01-01

    Exosomes are small extracellular vesicles containing microRNAs and mRNAs that are produced by various types of cells. We previously used ultrafiltration and size-exclusion chromatography to isolate two types of human salivary exosomes (exosomes I, II) that are different in size and proteomes. We showed that salivary exosomes contain large repertoires of small RNAs. However, precise information regarding long RNAs in salivary exosomes has not been fully determined. In this study, we investigated the compositions of protein-coding RNAs (pcRNAs) and long non-protein-coding RNAs (lncRNAs) of exosome I, exosome II and whole saliva (WS) by next-generation sequencing technology. Although 11% of all RNAs were commonly detected among the three samples, the compositions of reads mapping to known RNAs were similar. The most abundant pcRNA is ribosomal RNA protein, and pcRNAs of some salivary proteins such as S100 calcium-binding protein A8 (protein S100-A8) were present in salivary exosomes. Interestingly, lncRNAs of pseudogenes (presumably, processed pseudogenes) were abundant in exosome I, exosome II and WS. Translationally controlled tumor protein gene, which plays an important role in cell proliferation, cell death and immune responses, was highly expressed as pcRNA and pseudogenes in salivary exosomes. Our results show that salivary exosomes contain various types of RNAs such as pseudogenes and small RNAs, and may mediate intercellular communication by transferring these RNAs to target cells as gene expression regulators.

  2. Hankin and Reeves' approach to estimating fish abundance in small streams: Limitations and alternatives

    USGS Publications Warehouse

    Thompson, W.L.

    2003-01-01

    Hankin and Reeves' (1988) approach to estimating fish abundance in small streams has been applied in stream fish studies across North America. However, their population estimator relies on two key assumptions: (1) removal estimates are equal to the true numbers of fish, and (2) removal estimates are highly correlated with snorkel counts within a subset of sampled stream units. Violations of these assumptions may produce suspect results. To determine possible sources of the assumption violations, I used data on the abundance of steelhead Oncorhynchus mykiss from Hankin and Reeves' (1988) in a simulation composed of 50,000 repeated, stratified systematic random samples from a spatially clustered distribution. The simulation was used to investigate effects of a range of removal estimates, from 75% to 100% of true fish abundance, on overall stream fish population estimates. The effects of various categories of removal-estimates-to-snorkel-count correlation levels (r = 0.75-1.0) on fish population estimates were also explored. Simulation results indicated that Hankin and Reeves' approach may produce poor results unless removal estimates exceed at least 85% of the true number of fish within sampled units and unless correlations between removal estimates and snorkel counts are at least 0.90. A potential modification to Hankin and Reeves' approach is the inclusion of environmental covariates that affect detection rates of fish into the removal model or other mark-recapture model. A potential alternative approach is to use snorkeling combined with line transect sampling to estimate fish densities within stream units. As with any method of population estimation, a pilot study should be conducted to evaluate its usefulness, which requires a known (or nearly so) population of fish to serve as a benchmark for evaluating bias and precision of estimators.

  3. Evaluating abundance estimate precision and the assumptions of a count-based index for small mammals

    USGS Publications Warehouse

    Wiewel, A.S.; Adams, A.A.Y.; Rodda, G.H.

    2009-01-01

    Conservation and management of small mammals requires reliable knowledge of population size. We investigated precision of markrecapture and removal abundance estimates generated from live-trapping and snap-trapping data collected at sites on Guam (n 7), Rota (n 4), Saipan (n 5), and Tinian (n 3), in the Mariana Islands. We also evaluated a common index, captures per unit effort (CPUE), as a predictor of abundance. In addition, we evaluated cost and time associated with implementing live-trapping and snap-trapping and compared species-specific capture rates of selected live- and snap-traps. For all species, markrecapture estimates were consistently more precise than removal estimates based on coefficients of variation and 95 confidence intervals. The predictive utility of CPUE was poor but improved with increasing sampling duration. Nonetheless, modeling of sampling data revealed that underlying assumptions critical to application of an index of abundance, such as constant capture probability across space, time, and individuals, were not met. Although snap-trapping was cheaper and faster than live-trapping, the time difference was negligible when site preparation time was considered. Rattus diardii spp. captures were greatest in Haguruma live-traps (Standard Trading Co., Honolulu, HI) and Victor snap-traps (Woodstream Corporation, Lititz, PA), whereas Suncus murinus and Mus musculus captures were greatest in Sherman live-traps (H. B. Sherman Traps, Inc., Tallahassee, FL) and Museum Special snap-traps (Woodstream Corporation). Although snap-trapping and CPUE may have utility after validation against more rigorous methods, validation should occur across the full range of study conditions. Resources required for this level of validation would likely be better allocated towards implementing rigorous and robust methods.

  4. Exo-miRExplorer: A Comprehensive Resource for Exploring and Comparatively Analyzing Exogenous MicroRNAs

    PubMed Central

    Zheng, Ling-Ling; Deng, Kai-Wen; Deng, An-Cheng; Wu, Jie; Yang, Jian-Hua; Lun, Zhao-Rong; Qu, Liang-Hu

    2017-01-01

    MicroRNAs (miRNAs) are small regulatory RNAs that play important roles in animals, plants, and viruses. Deep-sequencing technology has been widely adopted in miRNA investigations. However, it is still a big mysterious why nearly all sequencing data contain miRNA sequences from exogenous species, called exo-miRNAs. In this study, we developed a novel platform, exo-miRExplorer, for mining and identifying exo-miRNAs from high-throughput small RNA sequencing experiments which originated from tissues and cell lines of multiple organisms. Thousands of exo-miRNAs are characterized with their expression abundance, the RNA families, original organisms and the sequencing platforms presented in exo-miRExplorer. Subsequently, we used exo-miRExplorer to perform further analysis. Comparative analysis of the exo-miRNAs between different sequencing datasets revealed significant correlation of exo-miRNAs between experiments in the same study. The plant-derived exo-miRNAs analysis provided robust evidence for non-diet source of exo-miRNAs. Virus-derived exo-miRNA analysis showed that pathogen RNAs could transfer to host cells and exist in deep-sequencing result at abundance level. In conclusion, exo-miRExplorer provides users with an integrative resource to facilitate detection and analysis of exo-miRNAs. exo-miRExplorer is available at the following URL: http://rna.sysu.edu.cn/exomiRDB/. PMID:28203233

  5. Small-scale fuel variation alters fire intensity and shrub abundance in a pine savanna.

    PubMed

    Thaxton, Jarrod M; Platt, William J

    2006-05-01

    Small-scale variation in fire intensity and effects may be an important source of environmental heterogeneity in frequently burned plant communities. We hypothesized that variation in fire intensity resulting from local differences in fuel loads produces heterogeneity in pine savanna ground cover by altering shrub abundance. To test this hypothesis, we experimentally manipulated prefire fuel loads to mimic naturally occurring fuel-load heterogeneity associated with branch falls, needle fall near large pines, and animal disturbances in a frequently burned longleaf pine (Pinus palustris) savanna in Louisiana, USA. We applied one of four fuel treatments (unaltered control, fine-fuel removal, fine-fuel addition, wood addition) to each of 540 (1-m2) quadrats prior to growing-season prescribed fires in each of two years (1999 and 2001). In both years fuel addition increased (and fuel removal decreased) fuel consumption and maximum fire temperatures relative to unaltered controls. Fuel addition, particularly wood, increased damage to shrubs, increased shrub mortality, and decreased resprout density relative to controls. We propose that local variation in fire intensity may contribute to maintenance of high species diversity in pine savannas by reducing shrub abundance and creating openings in an otherwise continuous ground cover.

  6. Sensing Small Changes in Protein Abundance: Stimulation of Caco-2 Cells by Human Whey Proteins.

    PubMed

    Cundiff, Judy K; McConnell, Elizabeth J; Lohe, Kimberly J; Maria, Sarah D; McMahon, Robert J; Zhang, Qiang

    2016-01-04

    Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FCs) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein, we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5-40% changes in protein abundance, with the variance controls of ± 5% FC for around 95% of TMT ratios when sampling 9-12 biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development.

  7. Landscape of Fluid Sets of Hairpin-Derived 21-/24-nt-Long Small RNAs at Seed Set Uncovers Special Epigenetic Features in Picea glauca

    PubMed Central

    2017-01-01

    Conifers’ exceptionally large genome (20–30 Gb) is scattered with 60% retrotransposon (RT) components and we have little knowledge on their origin and evolutionary implications. RTs may impede the expression of flanking genes and provide sources of the formation of novel small RNA (sRNAs) populations to constrain events of transposon (TE) proliferation/transposition. Here we show a declining expression of 24-nt-long sRNAs and low expression levels of their key processing gene, pgRTL2 (RNASE THREE LIKE 2) at seed set in Picea glauca. The sRNAs in 24-nt size class are significantly less enriched in type and read number than 21-nt sRNAs and have not been documented in other species. The architecture of MIR loci generating highly expressed 24-/21-nt sRNAs is featured by long terminal repeat—retrotransposons (LTR-RTs) in families of Ty3/Gypsy and Ty1/Copia elements. This implies that the production of sRNAs may be predominantly originated from TE fragments on chromosomes. Furthermore, a large proportion of highly expressed 24-nt sRNAs does not have predictable targets against unique genes in Picea, suggestive of their potential pathway in DNA methylation modifications on, for instance, TEs. Additionally, the classification of computationally predicted sRNAs suggests that 24-nt sRNA targets may bear particular functions in metabolic processes while 21-nt sRNAs target genes involved in many different biological processes. This study, therefore, directs our attention to a possible extrapolation that lacking of 24-nt sRNAs at the late conifer seed developmental phase may result in less constraints in TE activities, thus contributing to the massive expansion of genome size. PMID:28082604

  8. Small role with big impact: miRNAs as communicators in the cross-talk between cancer-associated fibroblasts and cancer cells

    PubMed Central

    Wang, Zhanhuai; Tan, Yinuo; Yu, Wei; Zheng, Shu; Zhang, Suzhan; Sun, Lifeng; Ding, Kefeng

    2017-01-01

    Cancer microenvironment is composed of numerous components that can support cancer cell proliferation, promote cancer progression and contribute to cancer treatment resistance. The major components of caner microenvironment are fibroblasts, endothelial cells, immune cells as well as cytokines, chemokines, and extracellular matrix (ECM) all of which surround tumor cells as the core and cross talk with each other. Among them, cancer-associated fibroblasts (CAFs) play an important role in promoting cancer progression by secreting various pro-inflammatory factors. MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate protein expression both in cancer cell and normal stromal cells. Changes of miRNAs expression in cancer-associated fibroblasts can be induced both by cancer cells and other stromal cells. This change can arise through direct interaction or by secreted paracrine factors or even by secreted miRNAs. The desregulated miRNAs in cancer-associated fibroblasts then enhance the CAFs phenotype and assist their cancer promotion ability. Explore the regulatory function of miRNAs in the complex communication between cancer cells and cancer microenvironment is important to understand the process of tumor progression and may help to develop new therapeutic strategies. This review provides an updated content of latest research advances about the relevance of miRNAs in the interaction between cancer cells and the CAFs. We will describe miRNAs which are differential expressed by NFs and CAFs, their function in regulating fibroblasts activation as well as miRNAs expressed in CAFs as prognostic factors in cancer stroma in recent studies. We will also discuss miRNA as an important player in CAFs mediated regulation of cancer progression and metastasis, cancer metabolism, cancer stem cell property and chemoresistance. PMID:28367098

  9. Host substitution by Ixodes persulcatus (Acari: Ixodidae) larvae in the years of deep depression in the abundance of small mammals.

    PubMed

    Uspensky, I; Rubina, M

    1992-01-01

    A phenomenon of host substitution by the taiga tick (Ixodes persulcatus) larvae was observed by the authors in the western part of the Soviet Far East. In the mountain forests where the highest abundance of I. persulcatus was found, larvae usually fed on small mammals (primarily on Clethrionomys rufocanus, C. rutilus and Apodemus speciosus). Numerous larvae were found, however, on the mountain hare (Lepus timidus) in the years of deep depression in the abundance of small mammals. The host substitution is considered as one of the mechanisms stabilizing the abundance of I. persulcatus adults.

  10. The expression level of small non-coding RNAs derived from the first exon of protein-coding genes is predictive of cancer status.

    PubMed

    Zovoilis, Athanasios; Mungall, Andrew J; Moore, Richard; Varhol, Richard; Chu, Andy; Wong, Tina; Marra, Marco; Jones, Steven J M

    2014-04-01

    Small non-coding RNAs (smRNAs) are known to be significantly enriched near the transcriptional start sites of genes. However, the functional relevance of these smRNAs remains unclear, and they have not been associated with human disease. Within the cancer genome atlas project (TCGA), we have generated small RNA datasets for many tumor types. In prior cancer studies, these RNAs have been regarded as transcriptional "noise," due to their apparent chaotic distribution. In contrast, we demonstrate their striking potential to distinguish efficiently between cancer and normal tissues and classify patients with cancer to subgroups of distinct survival outcomes. This potential to predict cancer status is restricted to a subset of these smRNAs, which is encoded within the first exon of genes, highly enriched within CpG islands and negatively correlated with DNA methylation levels. Thus, our data show that genome-wide changes in the expression levels of small non-coding RNAs within first exons are associated with cancer.

  11. Land use determinants of small mammal abundance and distribution in a plague endemic area of Lushoto District, Tanzania.

    PubMed

    Hieronimo, Proches; Kimaro, Didas N; Kihupi, Nganga I; Gulinck, Hubert; Mulungu, Loth S; Msanya, Balthazar M; Leirs, Herwig; Deckers, Jozef A

    2014-07-01

    Small mammals are considered to be involved in the transmission cycle of bubonic plague, still occurring in different parts of the world, including the Lushoto District in Tanzania. The objective of this study was to determine the relationship between land use types and practices and small mammal abundance and distribution. A field survey was used to collect data in three landscapes differing in plague incidences. Data collection was done both in the wet season (April-June 2012) and dry season (August-October 2012). Analysis of variance and Boosted Regression Trees (BRT) modelling technique were used to establish the relationship between land use and small mammal abundance and distribution. Significant variations (p ≤ 0.05) of small mammal abundance among land use types were identified. Plantation forest with farming, natural forest and fallow had higher populations of small mammals than the other aggregated land use types. The influence of individual land use types on small mammal abundance level showed that, in both dry and wet seasons, miraba and fallow tended to favour small mammals' habitation whereas land tillage practices had the opposite effect. In addition, during the wet season crop types such as potato and maize appeared to positively influence the distribution and abundance of small mammals which was attributed to both shelter and food availability. Based on the findings from this study it is recommended that future efforts to predict and map spatial and temporal human plague infection risk at fine scale should consider the role played by land use and associated human activities on small mammal abundance and distribution.

  12. Small mammal abundance and habitat relationships on deciduous forested sites with different susceptibility to gypsy moth defoliation

    NASA Astrophysics Data System (ADS)

    Yahner, Richard H.; Smith, Harvey R.

    1991-01-01

    Small mammals are important predators of gypsy moths ( Lymantria dispar L.), which are major defoliators of deciduous forests in the northeastern United States. Abundance and habitat relationships of small mammals were studied during summers 1984 and 1985 on forested sites at Moshannon and Rothrock state forests in two physiographic regions of Pennsylvania (Allegheny High Plateaus Province and Valley and Ridge Province, respectively) that varied in potential susceptibility to defoliation. The white-footed mouse ( Peromyscus leucopus), which is a major vertebrate predator of gypsy moths, was the most common small mammal on all sites. Of the four common species, northern short-tailed shrews ( Blarina brevicauda), southern red-backed voles ( Clethrionomys gapperi), and white-footed mice were more abundant at Moshannon compared to Rothrock State Forest, but masked shrews ( Sorex cinereus) were more abundant at Rothrock. Elevation was a major factor affecting abundance and distribution of small mammals. Because of the greater abundance of small mammals and more suitable physiographic features at Moshannon compared to Rothrock State Forest, small mammals may be more effective as predators on gypsy moths in the Allegheny High Plateaus than the Valley and Ridge Province of Pennsylvania.

  13. CELF4 Regulates Translation and Local Abundance of a Vast Set of mRNAs, Including Genes Associated with Regulation of Synaptic Function

    PubMed Central

    Sun, Wenzhi; Mahaffey, Connie L.; Curk, Tomaž; Rot, Gregor; Ule, Jernej; Frankel, Wayne N.

    2012-01-01

    RNA–binding proteins have emerged as causal agents of complex neurological diseases. Mice deficient for neuronal RNA–binding protein CELF4 have a complex neurological disorder with epilepsy as a prominent feature. Human CELF4 has recently been associated with clinical features similar to those seen in mutant mice. CELF4 is expressed primarily in excitatory neurons, including large pyramidal cells of the cerebral cortex and hippocampus, and it regulates excitatory but not inhibitory neurotransmission. We examined mechanisms underlying neuronal hyperexcitability in Celf4 mutants by identifying CELF4 target mRNAs and assessing their fate in the absence of CELF4 in view of their known functions. CELF4 binds to at least 15%–20% of the transcriptome, with striking specificity for the mRNA 3′ untranslated region. CELF4 mRNA targets encode a variety of proteins, many of which are well established in neuron development and function. While the overall abundance of these mRNA targets is often dysregulated in Celf4 deficient mice, the actual expression changes are modest at the steady-state level. In contrast, by examining the transcriptome of polysome fractions and the mRNA distribution along the neuronal cell body-neuropil axis, we found that CELF4 is critical for maintaining mRNA stability and availability for translation. Among biological processes associated with CELF4 targets that accumulate in neuropil of mutants, regulation of synaptic plasticity and transmission are the most prominent. Together with a related study of the impact of CELF4 loss on sodium channel Nav1.6 function, we suggest that CELF4 deficiency leads to abnormal neuronal function by combining a specific effect on neuronal excitation with a general impairment of synaptic transmission. These results also expand our understanding of the vital roles RNA–binding proteins play in regulating and shaping the activity of neural circuits. PMID:23209433

  14. Integrating Small RNA Sequencing with QTL Mapping for Identification of miRNAs and Their Target Genes Associated with Heat Tolerance at the Flowering Stage in Rice

    PubMed Central

    Liu, Qing; Yang, Tifeng; Yu, Ting; Zhang, Shaohong; Mao, Xingxue; Zhao, Junliang; Wang, Xiaofei; Dong, Jingfang; Liu, Bin

    2017-01-01

    Although, microRNAs (miRNAs) have been reported to be associated with heat tolerance at the seedling stage in rice, their involvement in heat tolerance at the flowering stage is still unknown. In this study, small RNA profiling was conducted in a heat-tolerant variety Gan-Xiang-Nuo (GXN) and a heat-sensitive variety Hua-Jing-Xian-74 (HJX), respectively. Totally, 102 miRNAs were differentially expressed (DE) under heat stress. Compared to HJX, GXN had more DE miRNAs and its DE miRNAs changed earlier under heat stress. Plant Ontology (PO) analysis of the target genes revealed that many DE miRNAs were involved in flower development. As a parallel experiment, QTL mapping was also conducted and four QTLs for heat tolerance at the flowering stage were identified using chromosome single-segment substitution lines derived from GXN and HJX. Further, through integrating analysis of DE miRNAs with QTLs, we identified 8 target genes corresponding to 26 miRNAs within the four QTL regions. Some meaningful target genes such as LOC_Os12g42400, SGT1, and pectinesterase were within the QTL regions. The negative correlation between miR169r-5p and its target gene LOC_Os12g42400 was confirmed under heat stress, and overexpression of miR169r-5p enhanced heat tolerance at flowering stage in rice. Our results demonstrate that the integrated analysis of genome-wide miRNA profiling with QTL mapping can facilitate identification of miRNAs and their target genes associated with the target traits and the limited candidates identified in this study offer an important source for further functional analysis and molecular breeding for heat tolerance in rice. PMID:28174587

  15. Novel Meiotic miRNAs and Indications for a Role of PhasiRNAs in Meiosis

    PubMed Central

    Dukowic-Schulze, Stefanie; Sundararajan, Anitha; Ramaraj, Thiruvarangan; Kianian, Shahryar; Pawlowski, Wojciech P.; Mudge, Joann; Chen, Changbin

    2016-01-01

    Small RNAs (sRNA) add additional layers to the regulation of gene expression, with siRNAs directing gene silencing at the DNA level by RdDM (RNA-directed DNA methylation), and micro RNAs (miRNAs) directing post-transcriptional regulation of specific target genes, mostly by mRNA cleavage. We used manually isolated male meiocytes from maize (Zea mays) to investigate sRNA and DNA methylation landscapes during zygotene, an early stage of meiosis during which steps of meiotic recombination and synapsis of paired homologous chromosomes take place. We discovered two novel miRNAs from meiocytes, zma-MIR11969 and zma-MIR11970, and identified putative target genes. Furthermore, we detected abundant phasiRNAs of 21 and 24 nt length. PhasiRNAs are phased small RNAs which occur in 21 or 24 nt intervals, at a few hundred loci, specifically in male reproductive tissues in grasses. So far, the function of phasiRNAs remained elusive. Data from isolated meiocytes now revealed elevated DNA methylation at phasiRNA loci, especially in the CHH context, suggesting a role for phasiRNAs in cis DNA methylation. In addition, we consider a role of these phasiRNAs in chromatin remodeling/dynamics during meiosis. However, this is not well supported yet and will need more additional data. Here, we only lay out the idea due to other relevant literature and our additional observation of a peculiar GC content pattern at phasiRNA loci. Chromatin remodeling is also indicated by the discovery that histone genes were enriched for sRNA of 22 nt length. Taken together, we gained clues that lead us to hypothesize sRNA-driven DNA methylation and possibly chromatin remodeling during male meiosis in the monocot maize which is in line with and extends previous knowledge. PMID:27313591

  16. Hankin and Reeves' Approach to Estimating Fish Abundance in Small Streams : Limitations and Potential Options.

    SciTech Connect

    Thompson, William L.

    2000-11-01

    Hankin and Reeves' (1988) approach to estimating fish abundance in small streams has been applied in stream-fish studies across North America. However, as with any method of population estimation, there are important assumptions that must be met for estimates to be minimally biased and reasonably precise. Consequently, I investigated effects of various levels of departure from these assumptions via simulation based on results from an example application in Hankin and Reeves (1988) and a spatially clustered population. Coverage of 95% confidence intervals averaged about 5% less than nominal when removal estimates equaled true numbers within sampling units, but averaged 62% - 86% less than nominal when they did not, with the exception where detection probabilities of individuals were >0.85 and constant across sampling units (95% confidence interval coverage = 90%). True total abundances averaged far (20% - 41%) below the lower confidence limit when not included within intervals, which implies large negative bias. Further, average coefficient of variation was about 1.5 times higher when removal estimates did not equal true numbers within sampling units (C{bar V} = 0.27 [SE = 0.0004]) than when they did (C{bar V} = 0.19 [SE = 0.0002]). A potential modification to Hankin and Reeves' approach is to include environmental covariates that affect detection rates of fish into the removal model or other mark-recapture model. A potential alternative is to use snorkeling in combination with line transect sampling to estimate fish densities. Regardless of the method of population estimation, a pilot study should be conducted to validate the enumeration method, which requires a known (or nearly so) population of fish to serve as a benchmark to evaluate bias and precision of population estimates.

  17. A RADIAL VELOCITY AND CALCIUM TRIPLET ABUNDANCE SURVEY OF FIELD SMALL MAGELLANIC CLOUD GIANTS

    SciTech Connect

    De Propris, Roberto; Rich, R. Michael; Mallery, Ryan C.; Howard, Christian D.

    2010-05-10

    We present the results of a pilot wide-field radial velocity and metal abundance survey of red giants in 10 fields in the Small Magellanic Cloud (SMC). The targets lie at projected distances of 0.9 and 1.9 kpc from the SMC center (m - M = 18.79) to the north, east, south, and west. Two more fields are to the east at distances of 3.9 and 5.1 kpc. In this last field, we find only a few to no SMC giants, suggesting that the edge of the SMC in this direction lies approximately at 6 kpc from its center. In all eastern fields, we observe a double peak in the radial velocities of stars, with a component at the classical SMC recession velocity of {approx}160 km s{sup -1} and a high-velocity component at about 200 km s{sup -1}, similar to observations in H I. In the most distant field (3.9 kpc), the low-velocity component is at 106 km s{sup -1}. The metal abundance distribution in all fields is broad and centered at about [Fe/H] {approx}-1.25, reaching to solar and possibly slightly supersolar values and down to [Fe/H] of about -2.5. In the two innermost (0.9 kpc) northern and southern fields, we observe a secondary peak at metallicities of about {approx}-0.6. This may be evidence of a second episode of star formation in the center, possibly triggered by the interactions that created the Stream and Bridge.

  18. miRNAs trigger widespread epigenetically activated siRNAs from transposons in Arabidopsis.

    PubMed

    Creasey, Kate M; Zhai, Jixian; Borges, Filipe; Van Ex, Frederic; Regulski, Michael; Meyers, Blake C; Martienssen, Robert A

    2014-04-17

    In plants, post-transcriptional gene silencing (PTGS) is mediated by DICER-LIKE 1 (DCL1)-dependent microRNAs (miRNAs), which also trigger 21-nucleotide secondary short interfering RNAs (siRNAs) via RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), DCL4 and ARGONAUTE 1 (AGO1), whereas transcriptional gene silencing (TGS) of transposons is mediated by 24-nucleotide heterochromatic (het)siRNAs, RDR2, DCL3 and AGO4 (ref. 4). Transposons can also give rise to abundant 21-nucleotide 'epigenetically activated' small interfering RNAs (easiRNAs) in DECREASED DNA METHYLATION 1 (ddm1) and DNA METHYLTRANSFERASE 1 (met1) mutants, as well as in the vegetative nucleus of pollen grains and in dedifferentiated plant cell cultures. Here we show that easiRNAs in Arabidopsis thaliana resemble secondary siRNAs, in that thousands of transposon transcripts are specifically targeted by more than 50 miRNAs for cleavage and processing by RDR6. Loss of RDR6, DCL4 or DCL1 in a ddm1 background results in loss of 21-nucleotide easiRNAs and severe infertility, but 24-nucleotide hetsiRNAs are partially restored, supporting an antagonistic relationship between PTGS and TGS. Thus miRNA-directed easiRNA biogenesis is a latent mechanism that specifically targets transposon transcripts, but only when they are epigenetically reactivated during reprogramming of the germ line. This ancient recognition mechanism may have been retained both by transposons to evade long-term heterochromatic silencing and by their hosts for genome defence.

  19. Transposon Defense by Endo-siRNAs, piRNAs and Somatic pilRNAs in Drosophila: Contributions of Loqs-PD and R2D2

    PubMed Central

    Mirkovic-Hösle, Milijana; Förstemann, Klaus

    2014-01-01

    Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing activities that target transposons: endogenous siRNAs (esiRNAs or endo-siRNAs) and Piwi-interacting small RNAs (piRNAs). The biogenesis of endo-siRNAs involves the Dicer-2 co-factors Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2, which primarily helps to direct siRNAs into the RNA interference effector Ago2. Nonetheless, loss of either protein is not sufficient to produce a phenotype comparable with a dcr-2 mutation. We provide further deep sequencing evidence supporting the notion that R2D2 and Loqs-PD have partially overlapping function. Certain transposons display a preference for either dsRBD-protein during production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs) that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway. PMID:24454776

  20. Predicting small mammal and flea abundance using landform and soil properties in a plague endemic area in Lushoto District, Tanzania.

    PubMed

    Meliyo, Joel L; Kimaro, Didas N; Msanya, Balthazar M; Mulungu, Loth S; Hieronimo, Proches; Kihupi, Nganga I; Gulinck, Hubert; Deckers, Jozef A

    2014-07-01

    Small mammals particularly rodents, are considered the primary natural hosts of plague. Literature suggests that plague persistence in natural foci has a root cause in soils. The objective of this study was to investigate the relationship between on the one hand landforms and associated soil properties, and on the other hand small mammals and fleas in West Usambara Mountains in Tanzania, a plague endemic area. Standard field survey methods coupled with Geographical Information System (GIS) technique were used to examine landform and soils characteristics. Soil samples were analysed in the laboratory for physico-chemical properties. Small mammals were trapped on pre-established landform positions and identified to genus/species level. Fleas were removed from the trapped small mammals and counted. Exploration of landform and soil data was done using ArcGIS Toolbox functions and descriptive statistical analysis. The relationships between landforms, soils, small mammals and fleas were established by generalised linear regression model (GLM) operated in R statistics software. Results show that landforms and soils influence the abundance of small mammals and fleas and their spatial distribution. The abundance of small mammals and fleas increased with increase in elevation. Small mammal species richness also increases with elevation. A landform-soil model shows that available phosphorus, slope aspect and elevation were statistically significant predictors explaining richness and abundance of small mammals. Fleas' abundance and spatial distribution were influenced by hill-shade, available phosphorus and base saturation. The study suggests that landforms and soils have a strong influence on the richness and evenness of small mammals and their fleas' abundance hence could be used to explain plague dynamics in the area.

  1. The reduction in small ribosomal subunit abundance in ethanol-stressed cells of Bacillus subtilis is mediated by a SigB-dependent antisense RNA.

    PubMed

    Mars, Ruben A T; Mendonça, Karoline; Denham, Emma L; van Dijl, Jan Maarten

    2015-10-01

    One of the best-characterized general stress responses in bacteria is the σB-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The σB regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these σB-dependent RNAs, named S1136-S1134, was recently mapped as being transcribed from the S1136 promoter on the opposite strand of the essential rpsD gene, which encodes the ribosomal primary-binding protein S4. Accordingly, S1136-S1134 transcription results in an rpsD-overlapping antisense RNA (asRNA). Upon exposure of B. subtilis to ethanol, the S1136 promoter was found to be induced, while rpsD transcription was downregulated. By quantitative PCR, we show that the activation of transcription from the S1136 promoter is directly responsible for the downregulation of rpsD upon ethanol exposure. We also show that this downregulation of rpsD leads to a reduced level of the small (30S) ribosomal subunit upon ethanol stress. The activation of the S1136 promoter thus represents the first example of antisense transcription-mediated regulation in the general stress response of B. subtilis and implicates the reduction of ribosomal protein abundance as a new aspect in the σB-dependent stress response. We propose that the observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides under conditions of ethanol stress.

  2. Dicer-Independent Primal RNAs Trigger RNAi and Heterochromatin Formation

    PubMed Central

    Halic, Mario; Moazed, Danesh

    2010-01-01

    SUMMARY Assembly of fission yeast pericentromeric heterochromatin and generation of small interfering RNAs (siRNAs) from noncoding centromeric transcripts are mutually dependent processes. How this interdependent positive feedback loop is first triggered is a fundamental unanswered question. Here, we show that two distinct Argonaute (Ago1)-dependent pathways mediate small RNA generation. RNA-dependent RNA polymerase complex (RDRC) and Dicer act on specific noncoding RNAs to generate siRNAs by a mechanism that requires the slicer activity of Ago1 but is independent of pre-existing heterochromatin. In the absence of RDRC or Dicer, a distinct class of small RNAs, called primal small RNAs (priRNAs), associates with Ago1. priRNAs are degradation products of abundant transcripts, which bind to Ago1 and target antisense transcripts that result from bidirectional transcription of DNA repeats. Our results suggest that a transcriptome surveillance mechanism based on random association of RNA degradation products with Argonaute triggers siRNA amplification and heterochromatin assembly within DNA repeats. PMID:20178743

  3. Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets

    PubMed Central

    Wang, Xiu-Jie; Reyes, José L; Chua, Nam-Hai; Gaasterland, Terry

    2004-01-01

    Background A class of eukaryotic non-coding RNAs termed microRNAs (miRNAs) interact with target mRNAs by sequence complementarity to regulate their expression. The low abundance of some miRNAs and their time- and tissue-specific expression patterns make experimental miRNA identification difficult. We present here a computational method for genome-wide prediction of Arabidopsis thaliana microRNAs and their target mRNAs. This method uses characteristic features of known plant miRNAs as criteria to search for miRNAs conserved between Arabidopsis and Oryza sativa. Extensive sequence complementarity between miRNAs and their target mRNAs is used to predict miRNA-regulated Arabidopsis transcripts. Results Our prediction covered 63% of known Arabidopsis miRNAs and identified 83 new miRNAs. Evidence for the expression of 25 predicted miRNAs came from northern blots, their presence in the Arabidopsis Small RNA Project database, and massively parallel signature sequencing (MPSS) data. Putative targets functionally conserved between Arabidopsis and O. sativa were identified for most newly identified miRNAs. Independent microarray data showed that the expression levels of some mRNA targets anti-correlated with the accumulation pattern of their corresponding regulatory miRNAs. The cleavage of three target mRNAs by miRNA binding was validated in 5' RACE experiments. Conclusions We identified new plant miRNAs conserved between Arabidopsis and O. sativa and report a wide range of transcripts as potential miRNA targets. Because MPSS data are generated from polyadenylated RNA molecules, our results suggest that at least some miRNA precursors are polyadenylated at certain stages. The broad range of putative miRNA targets indicates that miRNAs participate in the regulation of a variety of biological processes. PMID:15345049

  4. Severe Inbreeding and Small Effective Number of Breeders in a Formerly Abundant Marine Fish

    PubMed Central

    O'Leary, Shannon J.; Hice, Lyndie A.; Feldheim, Kevin A.; Frisk, Michael G.; McElroy, Anne E.; Fast, Mark D.; Chapman, Demian D.

    2013-01-01

    In contrast to freshwater fish it is presumed that marine fish are unlikely to spawn with close relatives due to the dilution effect of large breeding populations and their propensity for movement and reproductive mixing. Inbreeding is therefore not typically a focal concern of marine fish management. We measured the effective number of breeders in 6 New York estuaries for winter flounder (Pseudopleuronectes americanus), a formerly abundant fish, using 11 microsatellite markers (6–56 alleles per locus). The effective number of breeders for 1–2 years was remarkably small, with point estimates ranging from 65–289 individuals. Excess homozygosity was detected at 10 loci in all bays (FIS = 0.169–0.283) and individuals exhibited high average internal relatedness (IR; mean = 0.226). These both indicate that inbreeding is very common in all bays, after testing for and ruling out alternative explanations such as technical and sampling artifacts. This study demonstrates that even historically common marine fish can be prone to inbreeding, a factor that should be considered in fisheries management and conservation plans. PMID:23762473

  5. An abundance of small exoplanets around stars with a wide range of metallicities.

    PubMed

    Buchhave, Lars A; Latham, David W; Johansen, Anders; Bizzarro, Martin; Torres, Guillermo; Rowe, Jason F; Batalha, Natalie M; Borucki, William J; Brugamyer, Erik; Caldwell, Caroline; Bryson, Stephen T; Ciardi, David R; Cochran, William D; Endl, Michael; Esquerdo, Gilbert A; Ford, Eric B; Geary, John C; Gilliland, Ronald L; Hansen, Terese; Isaacson, Howard; Laird, John B; Lucas, Philip W; Marcy, Geoffrey W; Morse, Jon A; Robertson, Paul; Shporer, Avi; Stefanik, Robert P; Still, Martin; Quinn, Samuel N

    2012-06-13

    The abundance of heavy elements (metallicity) in the photospheres of stars similar to the Sun provides a 'fossil' record of the chemical composition of the initial protoplanetary disk. Metal-rich stars are much more likely to harbour gas giant planets, supporting the model that planets form by accumulation of dust and ice particles. Recent ground-based surveys suggest that this correlation is weakened for Neptunian-sized planets. However, how the relationship between size and metallicity extends into the regime of terrestrial-sized exoplanets is unknown. Here we report spectroscopic metallicities of the host stars of 226 small exoplanet candidates discovered by NASA's Kepler mission, including objects that are comparable in size to the terrestrial planets in the Solar System. We find that planets with radii less than four Earth radii form around host stars with a wide range of metallicities (but on average a metallicity close to that of the Sun), whereas large planets preferentially form around stars with higher metallicities. This observation suggests that terrestrial planets may be widespread in the disk of the Galaxy, with no special requirement of enhanced metallicity for their formation.

  6. Natural-abundance stable carbon isotopes of small-subunit ribosomal RNA (SSU rRNA) from Guaymas Basin (Mexico)

    NASA Astrophysics Data System (ADS)

    MacGregor, B. J.; Mendlovitz, H.; Albert, D.; Teske, A. P.

    2012-12-01

    Small-subunit ribosomal RNA (SSU rRNA) is a phylogenetically informative molecule found in all species. Because it is poorly preserved in most environments, it is a useful marker for active microbial populations. We are using the natural-abundance stable carbon isotopic composition of specific microbial groups to help identify the carbon substrates contributing to microbial biomass in a variety of marine environments. At Guaymas Basin, hydrothermal fluids interact with abundant sedimentary organic carbon to produce natural gas and petroleum. Where this reaches the sediment surface, it can support dense patches of seafloor life, including Beggiatoa mats. We report here on the stable carbon isotopic composition of SSU rRNA from a Beggiatoa mat transect, a cold background site, a warm site with high oil concentration, and a second Beggiatoa mat. The central part of the transect mat overlay the steepest temperature gradient, and was visually dominated by orange Beggiatoa. This was fringed by white Beggiatoa mat and bare, but still warm, sediment. Methane concentrations were saturating beneath the orange and white mats and at the oily site, lower beneath bare sediment, and below detection at the background site. Our initial hypotheses were that rRNA isotopic composition would be strongly influenced by methane supply, and that archaeal rRNA might be lighter than bacterial due to contributions from methanogens and anaerobic methane oxidizers. We used biotin-labeled oligonucleotides to capture Bacterial and Archaeal SSU rRNA for isotopic determination. Background-site rRNA was isotopically heaviest, and bacterial RNA from below 2 cm at the oily site was lightest, consistent with control by methane. Within the transect mat, however, the pattern was more complicated; at some sediment depths, rRNA from the mat periphery was isotopically lightest. Part of this may be due to the spatially and temporally variable paths followed by hydrothermal fluid, which can include horizontal

  7. A 28-day oral toxicity evaluation of small interfering RNAs and a long double-stranded RNA targeting vacuolar ATPase in mice.

    PubMed

    Petrick, Jay S; Moore, William M; Heydens, William F; Koch, Michael S; Sherman, James H; Lemke, Shawna L

    2015-02-01

    New biotechnology-derived crop traits have been developed utilizing the natural process of RNA interference (RNAi). However, plant-produced double stranded RNAs (dsRNAs) are not known to present a hazard to mammals because numerous biological barriers limit uptake and potential for activity. To evaluate this experimentally, dsRNA sequences matching the mouse vATPase gene (an established target for control of corn rootworms) were evaluated in a 28-day toxicity study with mice. Test groups were orally gavaged with escalating doses of either a pool of four 21-mer vATPase small interfering RNAs (siRNAs) or a 218-base pair vATPase dsRNA. There were no treatment-related effects on body weight, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints. The highest dose levels tested were considered to be the no observed adverse effect levels (NOAELs) for the 21-mer siRNAs (48 mg/kg/day) and the 218 bp dsRNA (64 mg/kg/day). As an additional exploratory endpoint, vATPase gene expression, was evaluated in selected gastrointestinal tract and systemic tissues. The results of this assay did not indicate treatment-related suppression of vATPase. The results of this study indicate that orally ingested dsRNAs, even those targeting a gene in the test species, do not produce adverse health effects in mammals.

  8. Small interfering RNAs expressed from a Pol III promoter suppress the EWS/Fli-1 transcript in an Ewing sarcoma cell line.

    PubMed

    Dohjima, Taikoh; Lee, Nan Sook; Li, Haitang; Ohno, Takatoshi; Rossi, John J

    2003-06-01

    The EWS/Fli-1 fusion gene encodes an oncogenic fusion protein. The fusion is a product of the translocation t(11;22) (q24;q12), which is detected in 85% of Ewing sarcoma and primitive neuroectodermal tumor cells. Utilizing intracellularly expressed 21- to 23-nucleotide small interfering RNAs (siRNAs) targeting the EWS/Fli-1 fusion transcript in an Ewing sarcoma cell line, we achieved a greater than 80% reduction in the EWS/Fli-1 transcript. The reduction in transcript levels was accompanied by growth inhibition of an Ewing cell line. In addition to quantitating the reduction of the fusion transcript, we carefully monitored reduction of the endogenous EWS and Fli-1 mRNAs as well. One of the two siRNAs targeted to the fusion transcript also partially downregulated the Fli-1 mRNA, further potentiating the growth inhibition. These results highlight both the power of siRNAs and the potential side reactions that need to be carefully monitored. In addition, these results provide the first demonstration of expressed siRNAs downregulating an oncogenic fusion transcript. The results and observations from these studies should prove useful in targeting other fusion transcripts characteristic of sarcomas and erythroleukemias.

  9. Hybrid Selection of Small RNAs by Using Simian Virus 40 DNA: Evidence that the Simian Virus 40-Associated Small RNA Is Synthesized by Specific Cleavage from Large Viral Transcripts

    PubMed Central

    Alwine, James C.

    1982-01-01

    The simian virus 40 (SV40)-associated small RNA (SAS-RNA), approximately 64 nucleotides, is virally encoded within a region of the viral late (+) DNA strand which encodes no known protein. The SAS-RNA arises in abundance late in SV40 lytic infection. Previous data indicate that the synthesis of the SAS-RNA may be under the control of the normal late viral promoter; i.e., inhibition of transcription from the late promoter results in cessation of SAS-RNA synthesis. The synthesis of SAS-RNA was examined to determine whether the SAS-RNA is the product of cleavage from noncoding regions of nuclear late transcripts or an independent transcription product like 5S RNA, or the adenovirus VA-RNAs. The data described below suggest that SAS-RNA is cleaved from large late transcripts. In vitro transcription of DNA fragments containing the SAS-RNA coding region yielded no SAS-RNA synthesis; this result was supported by DNA sequence analysis, which indicated no promoter-like regions either within or flanking the SAS-RNA coding region. In support of a cleavage mechanism, the SAS-RNA has a 3′-phosphate end, an occurrence which is indicative of nuclease cleavage. In addition, 5′-end labeling of the SAS-RNA was possible only after calf alkaline phosphatase treatment; this indicates that the SAS-RNA is not capped. Hybrid selection analysis was used to demonstrate that separation of the SAS-RNA coding region from the normal late promoter resulted in elimination of SAS-RNA synthesis. This was demonstrated in SV40-transformed cells in which integration of a single copy of SV40 breaks the continuity of the late coding region, so that the SAS-RNA coding region is physically separated from the normal late promoter. The lack of SAS-RNA synthesis indicates that the SAS-RNA coding region cannot function as a primary transcription unit. The same result and conclusion were obtained by using a permissive cell line transformed by SV40 (COS-1 cells); here it was found that the integrated SAS

  10. A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

    PubMed

    Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

    1996-04-30

    Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation.

  11. Sequence-based design of bioactive small molecules that target precursor microRNAs.

    PubMed

    Velagapudi, Sai Pradeep; Gallo, Steven M; Disney, Matthew D

    2014-04-01

    Oligonucleotides are designed to target RNA using base pairing rules, but they can be hampered by poor cellular delivery and nonspecific stimulation of the immune system. Small molecules are preferred as lead drugs or probes but cannot be designed from sequence. Herein, we describe an approach termed Inforna that designs lead small molecules for RNA from solely sequence. Inforna was applied to all human microRNA hairpin precursors, and it identified bioactive small molecules that inhibit biogenesis by binding nuclease-processing sites (44% hit rate). Among 27 lead interactions, the most avid interaction is between a benzimidazole (1) and precursor microRNA-96. Compound 1 selectively inhibits biogenesis of microRNA-96, upregulating a protein target (FOXO1) and inducing apoptosis in cancer cells. Apoptosis is ablated when FOXO1 mRNA expression is knocked down by an siRNA, validating compound selectivity. Markedly, microRNA profiling shows that 1 only affects microRNA-96 biogenesis and is at least as selective as an oligonucleotide.

  12. Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation

    PubMed Central

    Stutika, Catrin; Mietzsch, Mario; Gogol-Döring, Andreas; Weger, Stefan; Sohn, Madlen; Chen, Wei; Heilbronn, Regine

    2016-01-01

    Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic. PMID:27611072

  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.

  14. Competition between virus-derived and endogenous small RNAs regulates gene expression in Caenorhabditis elegans.

    PubMed

    Sarkies, Peter; Ashe, Alyson; Le Pen, Jérémie; McKie, Mikel A; Miska, Eric A

    2013-08-01

    Positive-strand RNA viruses encompass more than one-third of known virus genera and include many medically and agriculturally relevant human, animal, and plant pathogens. The nematode Caenorhabditis elegans and its natural pathogen, the positive-strand RNA virus Orsay, have recently emerged as a new animal model to understand the mechanisms and evolution of innate immune responses. In particular, the RNA interference (RNAi) pathway is required for C. elegans resistance to viral infection. Here we report the first genome-wide analyses of gene expression upon viral infection in C. elegans. Using the laboratory strain N2, we identify a novel C. elegans innate immune response specific to viral infection. A subset of these changes is driven by the RNAi response to the virus, which redirects the Argonaute protein RDE-1 from its endogenous small RNA cofactors, leading to loss of repression of endogenous RDE-1 targets. Additionally, we show that a C. elegans wild isolate, JU1580, has a distinct gene expression signature in response to viral infection. This is associated with a reduction in microRNA (miRNA) levels and an up-regulation of their target genes. Intriguingly, alterations in miRNA levels upon JU1580 infection are associated with a transformation of the antiviral transcriptional response into an antibacterial-like response. Together our data support a model whereby antiviral RNAi competes with endogenous small RNA pathways, causing widespread transcriptional changes. This provides an elegant mechanism for C. elegans to orchestrate its antiviral response, which may have significance for the relationship between small RNA pathways and immune regulation in other organisms.

  15. Gene regulation by noncoding RNAs

    PubMed Central

    Patil, Veena S.; Zhou, Rui; Rana, Tariq M.

    2015-01-01

    The past two decades have seen an explosion in research on noncoding RNAs and their physiological and pathological functions. Several classes of small (20–30 nucleotides) and long (>200 nucleotides) noncoding RNAs have been firmly established as key regulators of gene expression in myriad processes ranging from embryonic development to innate immunity. In this review, we focus on our current understanding of the molecular mechanisms underlying the biogenesis and function of small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). In addition, we briefly review the relevance of small and long noncoding RNAs to human physiology and pathology and their potential to be exploited as therapeutic agents. PMID:24164576

  16. The diversity and abundance of small arthropods in onion, Allium cepa, seed crops, and their potential role in pollination.

    PubMed

    Walker, M K; Howlett, B G; Wallace, A R; McCallum, J A; Teulon, D A J

    2011-01-01

    Onion, Allium cepa L. (Asparagales: Amaryllidaceae), crop fields grown for seed production require arthropod pollination for adequate seed yield. Although many arthropod species visit A. cepa flowers, for most there is little information on their role as pollinators. Small flower visiting arthropods (body width < 3 mm) in particular are rarely assessed. A survey of eight flowering commercial A. cepa seed fields in the North and South Islands of New Zealand using window traps revealed that small arthropods were highly abundant among all except one field. Insects belonging to the orders Diptera and Thysanoptera were the most abundant and Hymenoptera, Collembola, Psocoptera, Hemiptera, and Coleoptera were also present. To test whether small arthropods might contribute to pollination, seed sets from umbels caged within 3 mm diameter mesh cages were compared with similarly caged, hand-pollinated umbels and uncaged umbels. Caged umbels that were not hand-pollinated set significantly fewer seeds (average eight seeds/umbel, n = 10) than caged hand-pollinated umbels (average 146 seeds/umbel) and uncaged umbels (average 481 seeds/umbel). Moreover, sticky traps placed on umbels within cages captured similar numbers of small arthropods as sticky traps placed on uncaged umbels, suggesting cages did not inhibit the movement of small arthropods to umbels. Therefore, despite the high abundance of small arthropods within fields, evidence to support their role as significant pollinators of commercial A. cepa seed crops was not found.

  17. 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

  18. Accurate detection for a wide range of mutation and editing sites of microRNAs from small RNA high-throughput sequencing profiles

    PubMed Central

    Zheng, Yun; Ji, Bo; Song, Renhua; Wang, Shengpeng; Li, Ting; Zhang, Xiaotuo; Chen, Kun; Li, Tianqing; Li, Jinyan

    2016-01-01

    Various types of mutation and editing (M/E) events in microRNAs (miRNAs) can change the stabilities of pre-miRNAs and/or complementarities between miRNAs and their targets. Small RNA (sRNA) high-throughput sequencing (HTS) profiles can contain many mutated and edited miRNAs. Systematic detection of miRNA mutation and editing sites from the huge volume of sRNA HTS profiles is computationally difficult, as high sensitivity and low false positive rate (FPR) are both required. We propose a novel method (named MiRME) for an accurate and fast detection of miRNA M/E sites using a progressive sequence alignment approach which refines sensitivity and improves FPR step-by-step. From 70 sRNA HTS profiles with over 1.3 billion reads, MiRME has detected thousands of statistically significant M/E sites, including 3′-editing sites, 57 A-to-I editing sites (of which 32 are novel), as well as some putative non-canonical editing sites. We demonstrated that a few non-canonical editing sites were not resulted from mutations in genome by integrating the analysis of genome HTS profiles of two human cell lines, suggesting the existence of new editing types to further diversify the functions of miRNAs. Compared with six existing studies or methods, MiRME has shown much superior performance for the identification and visualization of the M/E sites of miRNAs from the ever-increasing sRNA HTS profiles. PMID:27229138

  19. Zooplankton Composition and Abundance as Indicators of Eutrophication in Two Small Man-made Lakes

    PubMed Central

    Ismail, Azma Hanim; Adnan, Anis Amalina Mohd

    2016-01-01

    The distribution and abundance of zooplankton species of Harapan and Aman Lakes were investigated in relation to physical parameters and chlorophyll-a content. Both lakes were characterised by the occurrence of algal bloom problem. The composition of zooplankton was collected at monthly intervals from November 2013 to February 2014. The total number of taxa in Harapan and Aman Lakes were 23 and 27, respectively. Rotifera was the highest abundance group represent 64% of the total species recorded followed by Copepoda (29%) and Cladocera (7%). Three dominant zooplankton that been recorded in both the lakes are Brachionus forficula, Brachionus nilsoni, and Trichocerca sp. High abundance of these species indicates that the lakes are eutrophic water bodies. Overall, zooplankton species distribution and abundance in the study sites are influenced by various environmental factors such as water transparency and chlorophyll-a content. PMID:27965738

  20. Characterization of siRNAs derived from cucumber mosaic virus in infected tobacco plants.

    PubMed

    Qiu, Yanhong; Zhang, Yongjiang; Hu, Fan; Zhu, Shuifang

    2017-03-27

    This study characterized the viral small interfering RNAs (vsiRNAs) from Nicotiana tabacum cv. Samsun infected with a cucumber mosaic virus (CMV) 2b-deficient mutant. Most vsiRNAs were 21 -22 nucleotides in length and the 5'-terminal ends were dominated by A and U, respectively. The observed vsiRNAs were heterogeneously distributed throughout the CMV genome; however, most of the vsiRNAs were derived from sense strands, as opposed to antisense strands. These results demonstrate the conserved and specific function of Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins in tobacco. Finally, it was revealed that vsiRNAs target abundant host genes, indicating complex roles for CMV vsiRNAs during the development of symptoms.

  1. Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells

    PubMed Central

    Liu, Teresa T.; Arango-Argoty, Gustavo; Li, Zhihua; Lin, Yuefeng; Kim, Sang Woo; Dueck, Anne; Ozsolak, Fatih; Monaghan, A. Paula; Meister, Gunter; DeFranco, Donald B.; John, Bino

    2015-01-01

    The highly conserved, multifunctional YB-1 is a powerful breast cancer prognostic indicator. We report on a pervasive role for YB-1 in which it associates with thousands of nonpolyadenylated short RNAs (shyRNAs) that are further processed into small RNAs (smyRNAs). Many of these RNAs have previously been identified as functional noncoding RNAs (http://www.johnlab.org/YB1). We identified a novel, abundant, 3′-modified short RNA antisense to Dicer1 (Shad1) that colocalizes with YB-1 to P-bodies and stress granules. The expression of Shad1 was shown to correlate with that of YB-1 and whose inhibition leads to an increase in cell proliferation. Additionally, Shad1 influences the expression of additional prognostic markers of cancer progression such as DLX2 and IGFBP2. We propose that the examination of these noncoding RNAs could lead to better understanding of prostate cancer progression. PMID:25904138

  2. Noncoding RNAs that associate with YB-1 alter proliferation in prostate cancer cells.

    PubMed

    Liu, Teresa T; Arango-Argoty, Gustavo; Li, Zhihua; Lin, Yuefeng; Kim, Sang Woo; Dueck, Anne; Ozsolak, Fatih; Monaghan, A Paula; Meister, Gunter; DeFranco, Donald B; John, Bino

    2015-06-01

    The highly conserved, multifunctional YB-1 is a powerful breast cancer prognostic indicator. We report on a pervasive role for YB-1 in which it associates with thousands of nonpolyadenylated short RNAs (shyRNAs) that are further processed into small RNAs (smyRNAs). Many of these RNAs have previously been identified as functional noncoding RNAs (http://www.johnlab.org/YB1). We identified a novel, abundant, 3'-modified short RNA antisense to Dicer1 (Shad1) that colocalizes with YB-1 to P-bodies and stress granules. The expression of Shad1 was shown to correlate with that of YB-1 and whose inhibition leads to an increase in cell proliferation. Additionally, Shad1 influences the expression of additional prognostic markers of cancer progression such as DLX2 and IGFBP2. We propose that the examination of these noncoding RNAs could lead to better understanding of prostate cancer progression.

  3. Finding a fox: an evaluation of survey methods to estimate abundance of a small desert carnivore.

    PubMed

    Dempsey, Steven J; Gese, Eric M; Kluever, Bryan M

    2014-01-01

    The status of many carnivore species is a growing concern for wildlife agencies, conservation organizations, and the general public. Historically, kit foxes (Vulpes macrotis) were classified as abundant and distributed in the desert and semi-arid regions of southwestern North America, but is now considered rare throughout its range. Survey methods have been evaluated for kit foxes, but often in populations where abundance is high and there is little consensus on which technique is best to monitor abundance. We conducted a 2-year study to evaluate four survey methods (scat deposition surveys, scent station surveys, spotlight survey, and trapping) for detecting kit foxes and measuring fox abundance. We determined the probability of detection for each method, and examined the correlation between the relative abundance as estimated by each survey method and the known minimum kit fox abundance as determined by radio-collared animals. All surveys were conducted on 15 5-km transects during the 3 biological seasons of the kit fox. Scat deposition surveys had both the highest detection probabilities (p = 0.88) and were most closely related to minimum known fox abundance (r2 = 0.50, P = 0.001). The next best method for kit fox detection was the scent station survey (p = 0.73), which had the second highest correlation to fox abundance (r2 = 0.46, P<0.001). For detecting kit foxes in a low density population we suggest using scat deposition transects during the breeding season. Scat deposition surveys have low costs, resilience to weather, low labor requirements, and pose no risk to the study animals. The breeding season was ideal for monitoring kit fox population size, as detections consisted of the resident population and had the highest detection probabilities. Using appropriate monitoring techniques will be critical for future conservation actions for this rare desert carnivore.

  4. Finding a Fox: An Evaluation of Survey Methods to Estimate Abundance of a Small Desert Carnivore

    PubMed Central

    Dempsey, Steven J.; Gese, Eric M.; Kluever, Bryan M.

    2014-01-01

    The status of many carnivore species is a growing concern for wildlife agencies, conservation organizations, and the general public. Historically, kit foxes (Vulpes macrotis) were classified as abundant and distributed in the desert and semi-arid regions of southwestern North America, but is now considered rare throughout its range. Survey methods have been evaluated for kit foxes, but often in populations where abundance is high and there is little consensus on which technique is best to monitor abundance. We conducted a 2-year study to evaluate four survey methods (scat deposition surveys, scent station surveys, spotlight survey, and trapping) for detecting kit foxes and measuring fox abundance. We determined the probability of detection for each method, and examined the correlation between the relative abundance as estimated by each survey method and the known minimum kit fox abundance as determined by radio-collared animals. All surveys were conducted on 15 5-km transects during the 3 biological seasons of the kit fox. Scat deposition surveys had both the highest detection probabilities (p = 0.88) and were most closely related to minimum known fox abundance (r2 = 0.50, P = 0.001). The next best method for kit fox detection was the scent station survey (p = 0.73), which had the second highest correlation to fox abundance (r2 = 0.46, P<0.001). For detecting kit foxes in a low density population we suggest using scat deposition transects during the breeding season. Scat deposition surveys have low costs, resilience to weather, low labor requirements, and pose no risk to the study animals. The breeding season was ideal for monitoring kit fox population size, as detections consisted of the resident population and had the highest detection probabilities. Using appropriate monitoring techniques will be critical for future conservation actions for this rare desert carnivore. PMID:25148102

  5. Climate warming increases biodiversity of small rodents by favoring rare or less abundant species in a grassland ecosystem.

    PubMed

    Jiang, Guangshun; Liu, Jun; Xu, Lei; Yu, Guirui; He, Honglin; Zhang, Zhibin

    2013-06-01

    Our Earth is facing the challenge of accelerating climate change, which imposes a great threat to biodiversity. Many published studies suggest that climate warming may cause a dramatic decline in biodiversity, especially in colder and drier regions. In this study, we investigated the effects of temperature, precipitation and a normalized difference vegetation index on biodiversity indices of rodent communities in the current or previous year for both detrended and nondetrended data in semi-arid grassland of Inner Mongolia during 1982-2006. Our results demonstrate that temperature showed predominantly positive effects on the biodiversity of small rodents; precipitation showed both positive and negative effects; a normalized difference vegetation index showed positive effects; and cross-correlation function values between rodent abundance and temperature were negatively correlated with rodent abundance. Our results suggest that recent climate warming increased the biodiversity of small rodents by providing more benefits to population growth of rare or less abundant species than that of more abundant species in Inner Mongolia grassland, which does not support the popular view that global warming would decrease biodiversity in colder and drier regions. We hypothesized that higher temperatures might benefit rare or less abundant species (with smaller populations and more folivorous diets) by reducing the probability of local extinction and/or by increasing herbaceous food resources.

  6. Differential mRNA Accumulation upon Early Arabidopsis thaliana Infection with ORMV and TMV-Cg Is Associated with Distinct Endogenous Small RNAs Level

    PubMed Central

    Zavallo, Diego; Manacorda, Carlos Augusto; Rodriguez, Maria Cecilia; Asurmendi, Sebastian

    2015-01-01

    Small RNAs (sRNAs) play important roles in plant development and host-pathogen interactions. Several studies have highlighted the relationship between viral infections, endogenous sRNA accumulation and transcriptional changes associated with symptoms. However, few studies have described a global analysis of endogenous sRNAs by comparing related viruses at early stages of infection, especially before viral accumulation reaches systemic tissues. An sRNA high-throughput sequencing of Arabidopsis thaliana leaf samples infected either with Oilseed rape mosaic virus (ORMV) or crucifer-infecting Tobacco mosaic virus (TMV-Cg) with slightly different symptomatology at two early stages of infection (2 and 4dpi) was performed. At early stages, both viral infections strongly alter the patterns of several types of endogenous sRNA species in distal tissues with no virus accumulation suggesting a systemic signaling process foregoing to virus spread. A correlation between sRNAs derived from protein coding genes and the associated mRNA transcripts was also detected, indicating that an unknown recursive mechanism is involved in a regulatory circuit encompassing this sRNA/mRNA equilibrium. This work represents the initial step in uncovering how differential accumulation of endogenous sRNAs contributes to explain the massive alteration of the transcriptome associated with plant-virus interactions. PMID:26237414

  7. Efficient inhibition of the formation of joint adhesions by ERK2 small interfering RNAs

    SciTech Connect

    Li, Fengfeng; Ruan, Hongjiang; Fan, Cunyi; Zeng, Bingfang; Wang, Chunyang; Wang, Xiang

    2010-01-01

    Transforming growth factor-{beta}1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which extracellular signal-regulated kinase (ERK)2 is considered to be crucial. Based on these theories, we examined the effects of a lentivirus-mediated small interfering RNA (siRNA) targeting ERK2 on the suppression of joint adhesion formation in vivo. The effects were assessed in vivo from different aspects including the adhesion score, histology and joint contracture angle. We found that the adhesions in the ERK2 siRNA group became soft and weak, and were easily stretched. Accordingly, the flexion contracture angles in the ERK2 siRNA group were also reduced (P < 0.05 compared with the control group). The animals appeared healthy, with no signs of impaired wound healing. In conclusion, local delivery of a lentivirus-mediated siRNA targeting ERK2 can ameliorate joint adhesion formation effectively and safely.

  8. How climate warming impacts the distribution and abundance of two small flatfish species in the North Sea

    NASA Astrophysics Data System (ADS)

    van Hal, Ralf; Smits, Kalle; Rijnsdorp, Adriaan D.

    2010-07-01

    Climate change, specifically temperature, affects the distribution and densities of species in marine and terrestrial ecosystems. Here, we looked at the effect of temperature during winter and spawning period on latitudinal range shifts and changes in abundance of two non-commercial North Sea fish species, solenette ( Buglossidium luteum) and scaldfish ( Arnoglossus laterna). Both species have increased in abundance and moved to the north since the late 1980s, coinciding with a series of mild winters. In 1996, following a very cold winter, the abundance of both species temporarily decreased as they retracted to the south. The shift in temperature affected adult habitat conditions, allowing them to immigrate into new areas where they subsequently reproduced successfully. We can conclude this because recruitment improved following the increase in abundance. The recruitment relates significantly to the higher adult stock and higher temperatures. The predictions of higher average temperatures and milder winters in the North Sea make it likely that these species will increase further in abundance and move northward. The observed increase in abundance of these small flatfish species will affect the North Sea food web and therefore commercial species, e.g. plaice, by predation on juveniles and competition for benthic food resources.

  9. Selective recruitment of mRNAs and miRNAs to polyribosomes in response to rhizobia infection in Medicago truncatula.

    PubMed

    Reynoso, Mauricio Alberto; Blanco, Flavio Antonio; Bailey-Serres, Julia; Crespi, Martín; Zanetti, María Eugenia

    2013-01-01

    Translation of mRNAs is a key regulatory step that contributes to the coordination and modulation of eukaryotic gene expression during development or adaptation to the environment. mRNA stability or translatability can be regulated by the action of small regulatory RNAs (sRNAs), which control diverse biological processes. Under low nitrogen conditions, leguminous plants associate with soil bacteria and develop a new organ specialized in nitrogen fixation: the nodule. To gain insight into the translational regulation of mRNAs during nodule formation, the association of mRNAs and sRNAs to polysomes was characterized in roots of the model legume Medicago truncatula during the symbiotic interaction with Sinorhizobium meliloti. Quantitative comparison of steady-state and polysomal mRNAs for 15 genes involved in nodulation identified a group of transcripts with slight or no change in total cellular abundance that were significantly upregulated at the level of association with polysomes in response to rhizobia. This group included mRNAs encoding receptors like kinases required either for nodule organogenesis, bacterial infection or both, and transcripts encoding GRAS and NF-Y transcription factors (TFs). Quantitative analysis of sRNAs in total and polysomal RNA samples revealed that mature microRNAs (miRNAs) were associated with the translational machinery, notably, miR169 and miR172, which target the NF-YA/HAP2 and AP2 TFs, respectively. Upon inoculation, levels of miR169 pronouncedly decreased in polysomal complexes, concomitant with the increased accumulation of the NF-YA/HAP2 protein. These results indicate that both mRNAs and miRNAs are subject to differential recruitment to polysomes, and expose the importance of selective mRNA translation during root nodule symbiosis.

  10. The use of small interfering RNAs to inhibit adipocyte differentiation in human preadipocytes and fetal-femur-derived mesenchymal cells

    SciTech Connect

    Xu, Y.; Mirmalek-Sani, S.-H.; Yang, X.; Zhang, J.; Oreffo, R.O.C. . E-mail: roco@soton.ac.uk

    2006-06-10

    RNA interference (RNAi) has been used in functional genomics and offers innovative approaches in the development of novel therapeutics. Human mesenchymal stem cells offer a unique cell source for tissue engineering/regeneration strategies. The current study examined the potential of small interfering RNAs (siRNA) against human peroxisome proliferator activated receptor gamma (PPAR{gamma}) to suppress adipocyte differentiation (adipogenesis) in human preadipocytes and fetal-femur-derived mesenchymal cells. Adipogenesis was investigated using cellular and biochemical analysis. Transient transfection with PPAR{gamma}-siRNA using a liposomal-based strategy resulted in a significant inhibition of adipogenesis in human preadipocytes and fetal-femur-derived mesenchymal cells, compared to controls (cell, liposomal and negative siRNA). The inhibitory effect of PPAR{gamma}-siRNA was supported by testing human PPAR{gamma} mRNA and adipogenic associated genes using reverse transcription polymerase chain reaction (RT-PCR) to adiponectin receptor 1 and 2 as well as examination of fatty acid binding protein 3 (FABP{sub 3}) expression, an adipocyte-specific marker. The current studies indicate that PPAR{gamma}-siRNA is a useful tool to study adipogenesis in human cells, with potential applications both therapeutic and in the elucidation of mesenchymal cell differentiation in the modulation of cell differentiation in human mesenchymal cells.

  11. Identification of novel and candidate miRNAs in rice by high throughput sequencing

    PubMed Central

    Sunkar, Ramanjulu; Zhou, Xuefeng; Zheng, Yun; Zhang, Weixiong; Zhu, Jian-Kang

    2008-01-01

    Background Small RNA-guided gene silencing at the transcriptional and post-transcriptional levels has emerged as an important mode of gene regulation in plants and animals. Thus far, conventional sequencing of small RNA libraries from rice led to the identification of most of the conserved miRNAs. Deep sequencing of small RNA libraries is an effective approach to uncover rare and lineage- and/or species-specific microRNAs (miRNAs) in any organism. Results In order to identify new miRNAs and possibly abiotic-stress regulated small RNAs in rice, three small RNA libraries were constructed from control rice seedlings and seedlings exposed to drought or salt stress, and then subjected to pyrosequencing. A total of 58,781, 43,003 and 80,990 unique genome-matching small RNAs were obtained from the control, drought and salt stress libraries, respectively. Sequence analysis confirmed the expression of most of the conserved miRNAs in rice. Importantly, 23 new miRNAs mostly each derived from a unique locus in rice genome were identified. Six of the new miRNAs are conserved in other monocots. Additionally, we identified 40 candidate miRNAs. Allowing not more than 3 mis-matches between a miRNA and its target mRNA, we predicted 20 targets for 9 of the new miRNAs. Conclusion Deep sequencing proved to be an effective strategy that allowed the discovery of 23 low-abundance new miRNAs and 40 candidate miRNAs in rice. PMID:18312648

  12. Effects of flooding on abundance of native and nonnative fishes downstream from a small impoundment

    USGS Publications Warehouse

    Schultz, A.A.; Maughan, O.E.; Bonar, Scott A.; Matter, W.J.

    2003-01-01

    Flooding can benefit native fishes in southwestern streams by disproportionately displacing nonnative fishes. We examined how the presence of an upstream impoundment affected this relationship in lower Sonoita Creek, Arizona. Nonnative species not found in the reservoir decreased in abundance in lower Sonoita Creek after flooding. The catch and relative abundance of some nonnative species found in both the reservoir and the creek increased in lower Sonoita Creek after flooding. Movement of nonnative fishes out of the reservoir via the spillway during periods of high water probably contributes to the persistence and abundance of these species downstream. Both preventing nonnative fishes from escaping reservoirs and the release of flushing flows would aid conservation of native southwestern fishes downstream.

  13. Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs.

    PubMed

    Cho, Changhee; Lee, Sang Yup

    2017-02-01

    Clostridium is considered a promising microbial host for the production of valuable industrial chemicals. However, Clostridium is notorious for the difficulty of genetic manipulations, and consequently metabolic engineering. Thus, much effort has been exerted to develop novel tools for genetic and metabolic engineering of Clostridium strains. Here, we report the development of a synthetic small regulatory RNA (sRNA)-based system for controlled gene expression in Clostridium acetobutylicum, consisting of a target recognition site, MicC sRNA scaffold, and an RNA chaperone Hfq. To examine the functional operation of sRNA system in C. acetobutylicum, expression control was first examined with the Evoglow fluorescent protein as a model protein. Initially, a C. acetobutylicum protein annotated as Hfq was combined with the synthetic sRNA based on the Escherichia coli MicC scaffold to knockdown Evoglow expression. However, C. acetobutylicum Hfq did not bind to E. coli MicC, while MicC scaffold-based synthetic sRNA itself was able to knockdown the expression of Evoglow. When E. coli hfq gene was introduced, the knockdown efficiency assessed by measuring fluorescence intensity, could be much enhanced. Then, this E. coli MicC scaffold-Hfq system was used to knock down adhE1 gene expression in C. acetobutylicum. Knocking down the adhE1 gene expression using the synthetic sRNA led to a 40% decrease in butanol production (2.5 g/L), compared to that (4.5 g/L) produced by the wild-type strain harboring an empty vector. The sRNA system was further extended to knock down the pta gene expression in the buk mutant C. acetobutylicum strain PJC4BK for enhanced butanol production. The PJC4BK (pPta-Hfq(Eco) ) strain, which has the pta gene expression knocked down, was able to produce 16.9 g/L of butanol, which is higher than that (14.9 g/L) produced by the PJC4BK strain, mainly due to reduced acetic acid production. Fed-batch culture of PJC4BK (pPta-Hfq(Eco) ) strain coupled with

  14. Analysis of Microarray Data on Gene Expression and Methylation to Identify Long Non-coding RNAs in Non-small Cell Lung Cancer

    PubMed Central

    Feng, Nannan; Ching, Travers; Wang, Yu; Liu, Ben; Lin, Hongyan; Shi, Oumin; Zhang, Xiaohong; Zheng, Min; Zheng, Xin; Gao, Ming; Zheng, Zhi-jie; Yu, Herbert; Garmire, Lana; Qian, Biyun

    2016-01-01

    To identify what long non-coding RNAs (lncRNAs) are involved in non-small cell lung cancer (NSCLC), we analyzed microarray data on gene expression and methylation. Gene expression chip and HumanMethylation450BeadChip were used to interrogate genome-wide expression and methylation in tumor samples. Differential expression and methylation were analyzed through comparing tumors with adjacent non-tumor tissues. LncRNAs expressed differentially and correlated with coding genes and DNA methylation were validated in additional tumor samples using RT-qPCR and pyrosequencing. In vitro experiments were performed to evaluate lncRNA’s effects on tumor cells. We identified 8,500 lncRNAs expressed differentially between tumor and non-tumor tissues, of which 1,504 were correlated with mRNA expression. Two of the lncRNAs, LOC146880 and ENST00000439577, were positively correlated with expression of two cancer-related genes, KPNA2 and RCC2, respectively. High expression of LOC146880 and ENST00000439577 were also associated with poor survival. Analysis of lncRNA expression in relation to DNA methylation showed that LOC146880 expression was down-regulated by DNA methylation in its promoter. Lowering the expression of LOC146880 or ENST00000439577 in tumor cells could inhibit cell proliferation, invasion and migration. Analysis of microarray data on gene expression and methylation allows us to identify two lncRNAs, LOC146880 and ENST00000439577, which may promote the progression of NSCLC. PMID:27849024

  15. Cloning and expression profiling of testis-expressed piRNA-like RNAs

    PubMed Central

    Ro, Seungil; Park, Chanjae; Song, Rui; Nguyen, Dan; Jin, Jingling; Sanders, Kenton M.; McCarrey, John R.; Yan, Wei

    2007-01-01

    Using a novel small RNA cloning method, we identified 630 piRNA-like RNAs (pilRNAs) from the mouse testis, and 498 of them are novel. These pilRNA genes were mapped to all chromosomes as 71 clusters, and the majority of them (∼84%) are derived from intergenic, intronic, and exonic sequences. One of the structural characteristics for pilRNAs is that a single locus can encode numerous homologous pilRNAs with overlapping sequences. Hundreds or even thousands of pilRNAs from a single pilRNA gene cluster are all produced from a single long transcript. Expression profiling for 64 pilRNAs revealed that ∼14% of all the pilRNAs analyzed displayed a ubiquitous expression pattern, although the majority of (∼86%) pilRNAs were preferentially or exclusively expressed in meiotic and haploid male germ cells of the testis. Our semiquantitative analyses also suggest that the testis is the organ with the highest expression of pilRNAs both in number and in abundance. The large number, high abundance, unique genomic locations, and biogenesis all suggest that pilRNAs have important regulatory roles not only in spermatogenesis but also in other biological processes. PMID:17698640

  16. Folding of small RNAs displaying the GNC base-pattern: implications for the self-organization of the genetic system.

    PubMed

    Lehmann, Jean; Riedo, Bruno; Dietler, Giovanni

    2004-04-07

    Theoretical arguments and statistical analyses of present-day coding sequences have long suggested the existence of primitive patterns in RNA sequences, which were thought to have been predominant at the time of the origin of the genetic code. The main propositions were centered around the base-patterns GNC and RNY, where R = A or G , Y = C or U and N = A, G, C or U. A theoretical model of the primitive process of translation explaining the origin of this type of pattern was recently published in the Journal of Theoretical Biology. On the basis of this previous analysis, and on physico-chemical evidence supporting the idea of the GNC base-pattern as the most primitive one, the present paper shows the results of folding simulations of small RNA strands displaying this pattern, which enabled us to specify the characteristics of the suggested primitive form of tRNA. This analysis is notably based on a conjecture of Eigen and Schuster of an early structural (or pattern) similarity between mRNA and tRNA, and, more specifically, of a "joint function of messenger and adaptor". Working with this conjecture, we show that the convergence of the primitive pool of RNAs toward a system containing a high proportion of sequences displaying the GNC base-pattern (according to the evolutionary model) is accompanied by a significant gain in stability of the translation process. In particular, it is demonstrated how the reading frame would be automatically discriminated without the presence of a start codon.

  17. Noncoding RNAs in endocrine malignancy.

    PubMed

    Kentwell, Jessica; Gundara, Justin S; Sidhu, Stan B

    2014-05-01

    Only recently has it been uncovered that the mammalian transcriptome includes a large number of noncoding RNAs (ncRNAs) that play a variety of important regulatory roles in gene expression and other biological processes. Among numerous kinds of ncRNAs, short noncoding RNAs, such as microRNAs, have been extensively investigated with regard to their biogenesis, function, and importance in carcinogenesis. Long noncoding RNAs (lncRNAs) have only recently been implicated in playing a key regulatory role in cancer biology. The deregulation of ncRNAs has been demonstrated to have important roles in the regulation and progression of cancer development. In this review, we describe the roles of both short noncoding RNAs (including microRNAs, small nuclear RNAs, and piwi-interacting RNAs) and lncRNAs in carcinogenesis and outline the possible underlying genetic mechanisms, with particular emphasis on clinical applications. The focus of our review includes studies from the literature on ncRNAs in traditional endocrine-related cancers, including thyroid, parathyroid, adrenal gland, and gastrointestinal neuroendocrine malignancies. The current and potential future applications of ncRNAs in clinical cancer research is also discussed, with emphasis on diagnosis and future treatment.

  18. 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

  19. The rnc Gene Promotes Exopolysaccharide Synthesis and Represses the vicRKX Gene Expressions via MicroRNA-Size Small RNAs in Streptococcus mutans

    PubMed Central

    Mao, Meng-Ying; Yang, Ying-Ming; Li, Ke-Zeng; Lei, Lei; Li, Meng; Yang, Yan; Tao, Xiang; Yin, Jia-Xin; Zhang, Ru; Ma, Xin-Rong; Hu, Tao

    2016-01-01

    Dental caries is a biofilm-dependent disease that largely relies on the ability of Streptococcus mutans to synthesize exopolysaccharides. Although the rnc gene is suggested to be involved in virulence mechanisms in many other bacteria, the information regarding it in S. mutans is very limited. Here, using deletion or overexpression mutant assay, we demonstrated that rnc in S. mutans significantly positively regulated exopolysaccharide synthesis and further altered biofilm formation. Meanwhile, the cariogenecity of S. mutans was decreased by deletion of rnc in a specific pathogen-free (SPF) rat model. Interestingly, analyzing the expression at mRNA level, we found the downstream vic locus was repressed by rnc in S. mutans. Using deep sequencing and bioinformatics analysis, for the first time, three putative microRNA-size small RNAs (msRNAs) targeting vicRKX were predicted in S. mutans. The expression levels of these msRNAs were negatively correlated with vicRKX but positively correlated with rnc, indicating rnc probably repressed vicRKX expression through msRNAs at the post-transcriptional level. In all, the results present that rnc has a potential role in the regulation of exopolysaccharide synthesis and can affect vicRKX expressions via post-transcriptional repression in S. mutans. This study provides an alternative avenue for further research aimed at preventing caries. PMID:27242713

  20. Broth versus Surface-Grown Cells: Differential Regulation of RsmY/Z Small RNAs in Pseudomonas aeruginosa by the Gac/HptB System

    PubMed Central

    Jean-Pierre, Fabrice; Tremblay, Julien; Déziel, Eric

    2017-01-01

    Two-component systems are capable of profoundly affecting genetic regulation in bacteria by detecting environmental stimuli, allowing them to quickly adapt. In Pseudomonas aeruginosa, the small RNAs (sRNAs) RsmY and RsmZ are under the control of the GacS/A system. They have been described as ones of the major key players in the control of planktonic and surface-associated behaviors. Genetic regulation by these sRNAs is achieved by the titration of the negative post-transcriptional regulator RsmA which affects the expression of over 500 genes. There is increasing evidence pinpointing the importance of RsmY and RsmZ in the planktonic-sessile P. aeruginosa lifestyles switch control. Using swarming motility as a model, we show here that these sRNA are differentially regulated depending on the selected growth conditions (i.e., planktonic versus surface grown-cells). Also, we report that opposite to planktonically grown cells, rsmZ regulation does not implicate the response regulator GacA in swarming cells. Furthermore, we present data indicating that RsmY/Z expression influence swarming motility via the protein HptB which acts as a negative regulator of these sRNAs and that they do not strictly converge to RsmA as previously reported. PMID:28119684

  1. Physical Conditions of the Small Magellanic Cloud HII Region NGC 456 and the Underestimated Heavy Element Abundances of the Universe

    NASA Astrophysics Data System (ADS)

    Pena-Guerrero, Maria Angeles; Peimbert, A.

    2011-01-01

    We study the O/H abundance determinations in HII regions in the presence of temperature inhomogeneities (as proposed by Peimbert in 1967), t2. The first object of our study is NGC 456, which the second brightest HII region of the Small Magellanic Clouds (SMC); we find it has an abundance 1.75 times higher than previously determined. Traditionally it is assumed that these objects have the same temperature throughout the whole volume; this temperature is then used to calculate abundances. Our determination was done using Collisionally Excited Lines (CEL) and the t2 obtained from HeI lines; it is consistent with abundances determined with Recombination Lines (RL). The most well known strong-line ratio or metallicity indicator used to determine the O/H ratio is O23= ([OII]3727Å + [OIII]4959,5007Å) / Hβ introduced by Pagel et al. (1979). The log(O23) vs. 12+log(O/H) diagram constitutes an important tool in the study of objects with low intrinsecal brightness or high redshift. To account for the presence of thermal inhomogeneities, the upper branch of the O23-O/H diagram can be recalibrated using RLs whereas in the lower branch of the diagram RLs cannot be used due to their faintness. For low metallicity objects the formalism of t2 is applied to CELs to determine abundances and recalibrate the O23-O/H diagram. We find that the point corresponding to NGC 456 in the diagram shifts up by 0.24 dex in the 12+log(O/H) axis. This is consistent with preliminary results for the next 3 objects of our sample and with the behavior of other HII regions from the literature were t2 has been measured. The systematic shift in the curve of the O23-O/H diagram implies that abundances in the Universe need to be corrected by a factor of approximately 2.

  2. Decreased small mammal and on-host tick abundance in association with invasive red imported fire ants (Solenopsis invicta).

    PubMed

    Castellanos, Adrian A; Medeiros, Matthew C I; Hamer, Gabriel L; Morrow, Michael E; Eubanks, Micky D; Teel, Pete D; Hamer, Sarah A; Light, Jessica E

    2016-09-01

    Invasive species may impact pathogen transmission by altering the distributions and interactions among native vertebrate reservoir hosts and arthropod vectors. Here, we examined the direct and indirect effects of the red imported fire ant (Solenopsis invicta) on the native tick, small mammal and pathogen community in southeast Texas. Using a replicated large-scale field manipulation study, we show that small mammals were more abundant on treatment plots where S. invicta populations were experimentally reduced. Our analysis of ticks on small mammal hosts demonstrated a threefold increase in the ticks caught per unit effort on treatment relative to control plots, and elevated tick loads (a 27-fold increase) on one common rodent species. We detected only one known human pathogen (Rickettsia parkeri), present in 1.4% of larvae and 6.7% of nymph on-host Amblyomma maculatum samples but with no significant difference between treatment and control plots. Given that host and vector population dynamics are key drivers of pathogen transmission, the reduced small mammal and tick abundance associated with S. invicta may alter pathogen transmission dynamics over broader spatial scales.

  3. Decreased small mammal and on-host tick abundance in association with invasive red imported fire ants (Solenopsis invicta)

    PubMed Central

    Medeiros, Matthew C. I.; Hamer, Gabriel L.; Morrow, Michael E.; Eubanks, Micky D.; Teel, Pete D.

    2016-01-01

    Invasive species may impact pathogen transmission by altering the distributions and interactions among native vertebrate reservoir hosts and arthropod vectors. Here, we examined the direct and indirect effects of the red imported fire ant (Solenopsis invicta) on the native tick, small mammal and pathogen community in southeast Texas. Using a replicated large-scale field manipulation study, we show that small mammals were more abundant on treatment plots where S. invicta populations were experimentally reduced. Our analysis of ticks on small mammal hosts demonstrated a threefold increase in the ticks caught per unit effort on treatment relative to control plots, and elevated tick loads (a 27-fold increase) on one common rodent species. We detected only one known human pathogen (Rickettsia parkeri), present in 1.4% of larvae and 6.7% of nymph on-host Amblyomma maculatum samples but with no significant difference between treatment and control plots. Given that host and vector population dynamics are key drivers of pathogen transmission, the reduced small mammal and tick abundance associated with S. invicta may alter pathogen transmission dynamics over broader spatial scales. PMID:27651533

  4. ANALYSIS OF TWO SMALL MAGELLANIC CLOUD H II REGIONS CONSIDERING THERMAL INHOMOGENEITIES: IMPLICATIONS FOR THE DETERMINATIONS OF EXTRAGALACTIC CHEMICAL ABUNDANCES

    SciTech Connect

    Pena-Guerrero, Maria A.; Peimbert, Antonio; Peimbert, Manuel; Ruiz, Maria Teresa E-mail: antonio@astroscu.unam.mx E-mail: mtruiz@das.uchile.cl

    2012-02-20

    We present long-slit spectrophotometry considering the presence of thermal inhomogeneities (t{sup 2}) of two H II regions in the Small Magellanic Cloud (SMC): NGC 456 and NGC 460. Physical conditions and chemical abundances were determined for three positions in NGC 456 and one position in NGC 460, first under the assumption of uniform temperature and then allowing for the possibility of thermal inhomogeneities. We determined t{sup 2} values based on three different methods: (1) by comparing the temperature derived using oxygen forbidden lines with the temperature derived using helium recombination lines (RLs), (2) by comparing the abundances derived from oxygen forbidden lines with those derived from oxygen RLs, and (3) by comparing the abundances derived from ultraviolet carbon forbidden lines with those derived from optical carbon RLs. The first two methods averaged t{sup 2} = 0.067 {+-} 0.013 for NGC 456 and t{sup 2} = 0.036 {+-} 0.027 for NGC 460. These values of t{sup 2} imply that when gaseous abundances are determined with collisionally excited lines they are underestimated by a factor of nearly two. From these objects and others in the literature, we find that in order to account for thermal inhomogeneities and dust depletion, the O/H ratio in low-metallicity H II regions should be corrected by 0.25-0.45 dex depending on the thermal structure of the nebula or by 0.35 dex if such information is not available.

  5. Two novel aspects of the kinetics of gene expression including miRNAs

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir

    2013-04-01

    In eukaryotic cells, many genes are transcribed into non-coding RNAs. Small RNAs or, more specifically, microRNAs (miRNAs) form an abundant sub-class of such RNAs. miRNAs are transcribed as long noncoding RNA and then generated via a processing pathway down to the 20-24-nucleotide length. The key ability of miRNAs is to associate with target mRNAs and to suppress their translation and/or facilitate degradation. Using the mean-field kinetic equations and Monte Carlo simulations, we analyze two aspects of this interplay. First, we describe the situation when the formation of mRNA or miRNA is periodically modulated by a transcription factor which itself is not perturbed by these species. Depending on the ratio between the mRNA and miRNA formation rates, the corresponding induced periodic kinetics are shown to be either nearly harmonic or shaped as anti-phase pulses. The second part of the work is related to recent experimental studies indicating that differentiation of stem cells often involves changes in gene transcription into miRNAs and/or the interference between miRNAs, mRNAs and proteins. In particular, the regulatory protein obtained via mRNA translation may suppress the miRNA formation, and the latter may suppress in turn the miRNA-mRNA association and degradation. The corresponding bistable kinetics are described in detail.

  6. MicroRNAs as Potential Biomarkers in Cancer: Opportunities and Challenges

    PubMed Central

    Lu, Haiqi; Wang, Xian

    2015-01-01

    MicroRNAs (miRNAs) are a group of small noncoding RNAs (ncRNAs) that posttranscriptionally regulate gene expression by targeting their corresponding messenger RNAs (mRNAs). Dysregulated miRNAs have been considered as a new type of ‘‘oncomiRs” or ‘‘tumor suppressors,” playing essential roles in cancer initiation and progression. Using genome-wide detection methods, ubiquitously aberrant expression profiles of miRNAs have been identified in a broad array of human cancers, showing great potential as novel diagnostic and prognostic biomarkers of cancer with high specificity and sensitivity. The detectable miRNAs in tissue, blood, and other body fluids with high stability provide an abundant source for miRNA-based biomarkers in human cancers. Despite the fact that an increasing number of potential miRNA biomarkers have been reported, the transition of miRNAs-based biomarkers from bench to bedside still necessitates addressing several challenges. In this review, we will summarize our current understanding of miRNAs as potential biomarkers in human cancers. PMID:25874201

  7. Potato virus Y HCPro suppression of antiviral silencing in Nicotiana benthamiana plants correlates with its ability to bind in vivo to 21- and 22-nucleotide small RNAs of viral sequence.

    PubMed

    Del Toro, Francisco J; Donaire, Livia; Aguilar, Emmanuel; Chung, Bong-Nam; Tenllado, Francisco; Canto, Tomás

    2017-04-05

    We have investigated short and small RNAs (sRNAs) that were bound to a biologically active hexahistidine-tagged Potato virus Y (PVY) HCPro suppressor of silencing, expressed from a heterologous virus vector in Nicotiana benthamiana plants, and purified under non-denaturing conditions. We found that RNAs in purified preparations were differentially enriched in sRNAs of 21 and to a much lesser extent of 22 nucleotides (nt) in length and of viral sequence (vsRNAs) when compared to those found in a control plant protein background bound to the nickel resin in the absence of HCPro, or in a purified alanine substitution HCPro mutant (HCPro mutB) control that lacked suppressor of silencing activity. In both controls, sRNAs were composed almost entirely by molecules of plant sequence, indicating that the resin-bound protein background had no affinity for vsRNAs, and also that HCPro mutB failed to bind to vsRNAs. Therefore, PVY HCPro suppressor activity correlated with its ability to bind to 21 and 22 nt vsRNAs. HCPro constituted at least 54 % of the total protein content in purified preparations and we were able to calculate its contribution to the 21 and the 22 nt pool of sRNAs present in the purified samples and its binding strength relative to the background. We also found that in the 21 nt vsRNAs of the HCPro preparation 5' terminal adenines were overrepresented relative to the controls, but this was not observed in vsRNAs of other sizes, or of plant sequence.IMPORTANCE It was previously shown that HCPro can bind to long and small RNAs (sRNAs) in vitro, and in the case of Turnip mosaic virus HCPro also in vivo in arabidopsis AGO2-deficient plants. Our data show that PVY HCPro does bind in vivo to sRNAs during infection in wild-type Nicotiana benthamiana plants when expressed from a heterologous virus vector. Using a suppression of silencing-deficient mutant HCPro that can accumulate in this host when expressed from a virus vector we also show that sRNA binding

  8. Identification of mRNA-like non-coding RNAs and validation of a mighty one named MAR in Panax ginseng.

    PubMed

    Wang, Meizhen; Wu, Bin; Chen, Chao; Lu, Shanfa

    2015-03-01

    Increasing evidence suggests that long non-coding RNAs (lncRNAs) play significant roles in plants. However, little is known about lncRNAs in Panax ginseng C. A. Meyer, an economically significant medicinal plant species. A total of 3,688 mRNA-like non-coding RNAs (mlncRNAs), a class of lncRNAs, were identified in P. ginseng. Approximately 40% of the identified mlncRNAs were processed into small RNAs, implying their regulatory roles via small RNA-mediated mechanisms. Eleven miRNA-generating mlncRNAs also produced siRNAs, suggesting the coordinated production of miRNAs and siRNAs in P. ginseng. The mlncRNA-derived small RNAs might be 21-, 22-, or 24-nt phased and could be generated from both or only one strand of mlncRNAs, or from super long hairpin structures. A full-length mlncRNA, termed MAR (multiple-function-associated mlncRNA), was cloned. It generated the most abundant siRNAs. The MAR siRNAs were predominantly 24-nt and some of them were distributed in a phased pattern. A total of 228 targets were predicted for 71 MAR siRNAs. Degradome sequencing validated 68 predicted targets involved in diverse metabolic pathways, suggesting the significance of MAR in P. ginseng. Consistently, MAR was detected in all tissues analyzed and responded to methyl jasmonate (MeJA) treatment. It sheds light on the function of mlncRNAs in plants.

  9. MicroRNAs as putative mediators of treatment response in prostate cancer.

    PubMed

    O'Kelly, Fardod; Marignol, Laure; Meunier, Armelle; Lynch, Thomas H; Perry, Antoinette S; Hollywood, Donal

    2012-05-22

    MicroRNAs (miRNAs) are an abundant class of noncoding RNAs that function to regulate post-transcriptional gene expression, predominantly by translational repression. In addition to their role in prostate cancer initiation and progression, recent evidence suggests that miRNAs might also participate in treatment response across a range of therapies including radiation treatment, chemotherapy and androgen suppression. The mechanism of this regulation is thought to be multifactorial and is currently poorly understood. To date, only a small number of studies have examined the functional role of miRNAs in response to prostate cancer treatment. Elucidating the role of miRNAs in treatment response following radiotherapy, chemotherapy and androgen suppression will provide new avenues of investigation for the development of novel therapies for the treatment of prostate cancer.

  10. Inhibition of basal JNK activity by small interfering RNAs enhances cisplatin sensitivity and decreases DNA repair in T98G glioblastoma cells.

    PubMed

    Parra, Eduardo; Gutiérrez, Luis; Ferreira, Jorge

    2015-01-01

    Inhibition of basal Jun kinase (JNK) activity by small interfering RNAs (siRNAs) enhances cisplatin sensitivity and decreases DNA repair in T98G glioblastoma cells. Although the JNK pathway has been extensively studied in recent years, little is known concerning the signaling pathways that control their expression in glioma cells. The aim of the present study was to assess the role of c-Jun-NH2-terminal kinases (JNKs) in the regulation of T98G glioblastoma cells treated with cisplatin in the presence or absence of siRNAs against JNK1 and JNK2. Addition of either small interfering JNK1-siRNA or JNK2-siRNA induced decreased DNA repair and sensitized the T98G glioblastoma cells to the DNA damaging drug cisplatin (cis-diamminedichloroplatinum). This effect was associated with reduced cell survival and loss of anchorage‑independent colony formation. The results indicate that effective inhibition of the JNK pathway significantly sensitizes glioblastoma cells to cisplatin, a compound of proven clinical value whose spectrum of application is limited by resistance phenomena.

  11. Characterisation of microRNAs from apple (Malus domestica 'Royal Gala') vascular tissue and phloem sap

    PubMed Central

    2010-01-01

    Background Plant microRNAs (miRNAs) are a class of small, non-coding RNAs that play an important role in development and environmental responses. Hundreds of plant miRNAs have been identified to date, mainly from the model species for which there are available genome sequences. The current challenge is to characterise miRNAs from plant species with agricultural and horticultural importance, to aid our understanding of important regulatory mechanisms in crop species and enable improvement of crops and rootstocks. Results Based on the knowledge that many miRNAs occur in large gene families and are highly conserved among distantly related species, we analysed expression of twenty-one miRNA sequences in different tissues of apple (Malus x domestica 'Royal Gala'). We identified eighteen sequences that are expressed in at least one of the tissues tested. Some, but not all, miRNAs expressed in apple tissues including the phloem tissue were also detected in the phloem sap sample derived from the stylets of woolly apple aphids. Most of the miRNAs detected in apple phloem sap were also abundant in the phloem sap of herbaceous species. Potential targets for apple miRNAs were identified that encode putative proteins shown to be targets of corresponding miRNAs in a number of plant species. Expression patterns of potential targets were analysed and correlated with expression of corresponding miRNAs. Conclusions This study validated tissue-specific expression of apple miRNAs that target genes responsible for plant growth, development, and stress response. A subset of characterised miRNAs was also present in the apple phloem translocation stream. A comparative analysis of phloem miRNAs in herbaceous species and woody perennials will aid our understanding of non-cell autonomous roles of miRNAs in plants. PMID:20682080

  12. Characterization and Functional Analysis of Extracellular Vesicles and Muscle-Abundant miRNAs (miR-1, miR-133a, and miR-206) in C2C12 Myocytes and mdx Mice

    PubMed Central

    Matsuzaka, Yasunari; Tanihata, Jun; Komaki, Hirofumi; Ishiyama, Akihiko; Oya, Yasushi; Rüegg, Urs; Takeda, Shin-ichi; Hashido, Kazuo

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder. Here, we show that the CD63 antigen, which is located on the surface of extracellular vesicles (EVs), is associated with increased levels of muscle-abundant miRNAs, namely myomiRs miR-1, miR-133a, and miR-206, in the sera of DMD patients and mdx mice. Furthermore, the release of EVs from the murine myoblast C2C12 cell line was found to be modulated by intracellular ceramide levels in a Ca2+-dependent manner. Next, to investigate the effects of EVs on cell survival, C2C12 myoblasts and myotubes were cultured with EVs from the sera of mdx mice or C2C12 cells overexpressing myomiRs in presence of cellular stresses. Both the exposure of C2C12 myoblasts and myotubes to EVs from the serum of mdx mice, and the overexpression of miR-133a in C2C12 cells in presence of cellular stress resulted in a significant decrease in cell death. Finally, to assess whether miRNAs regulate skeletal muscle regeneration in vivo, we intraperitoneally injected GW4869 (an inhibitor of exosome secretion) into mdx mice for 5 and 10 days. Levels of miRNAs and creatine kinase in the serum of GW4869-treated mdx mice were significantly downregulated compared with those of controls. The tibialis anterior muscles of the GW4869-treated mdx mice showed a robust decrease in Evans blue dye uptake. Collectively, these results indicate that EVs and myomiRs might protect the skeletal muscle of mdx mice from degeneration. PMID:27977725

  13. How does landscape use influence small mammal diversity, abundance and biomass in hedgerow networks of farming landscapes?

    NASA Astrophysics Data System (ADS)

    Michel, Nadia; Burel, Françoise; Butet, Alain

    2006-07-01

    Over the last decades, profound changes in agricultural practices in the world have led to modifications of land-use as well as landscape structure and composition. Major changes resulted in enlargement of parcel size, increase of cultivated areas and drastic reduction of permanent elements such as woods, hedges or natural meadows. In this context we chose to investigate the composition and structure of small mammal communities in the hedgerow networks of three landscape units of Western France (Brittany) differing by their level of agricultural land-use intensity and hedgerow network density: a slightly intensified dense hedgerow network landscape unit (BOC1), a moderately intensified and fragmented hedgerow network landscape unit (BOC2) and a highly intensified landscape unit on an area reclaimed from the sea (POL). Characterization of small mammal communities was performed using live trapping on permanent habitats (eight hedges per landscape unit). In each of the 24 trapping units, a standardized method was used consisting of a baited 100-m trap-line. Diversity indices were used to compare the three communities. Species richness didn't vary across landscapes whereas Shannon's index of diversity underlined a clear difference between, on the one hand, the most intensified landscape unit (POL) which displayed the lowest diversity and, on the other hand, the two other less intensified units. The abundance of small mammals differed between the three sites: they were significantly more numerous in the hedges of the most intensified site than in hedges of the two other sites. Differences between species also appeared: for example, the Bank vole ( Clethrionomys glareolus) was very characteristic of POL, whereas the Pygmy shrew ( Sorex minutus) was much more associated with BOC sites. Within hedges, like for abundance, small mammal biomass was the highest in the most intensified site (POL > BOC2 = BOC1). On the contrary, at the landscape scale, biomass was the lowest in

  14. Deep Sequencing of Virus-Derived Small Interfering RNAs and RNA from Viral Particles Shows Highly Similar Mutational Landscapes of a Plant Virus Population

    PubMed Central

    Rupar, Matevž; Gutierrez-Aguirre, Ion; Curk, Tomaž; Kreuze, Jan F.

    2015-01-01

    ABSTRACT RNA viruses exist within a host as a population of mutant sequences, often referred to as quasispecies. Within a host, sequences of RNA viruses constitute several distinct but interconnected pools, such as RNA packed in viral particles, double-stranded RNA, and virus-derived small interfering RNAs. We aimed to test if the same representation of within-host viral population structure could be obtained by sequencing different viral sequence pools. Using ultradeep Illumina sequencing, the diversity of two coexisting Potato virus Y sequence pools present within a plant was investigated: RNA isolated from viral particles and virus-derived small interfering RNAs (the derivatives of a plant RNA silencing mechanism). The mutational landscape of the within-host virus population was highly similar between both pools, with no notable hotspots across the viral genome. Notably, all of the single-nucleotide polymorphisms with a frequency of higher than 1.6% were found in both pools. Some unique single-nucleotide polymorphisms (SNPs) with very low frequencies were found in each of the pools, with more of them occurring in the small RNA (sRNA) pool, possibly arising through genetic drift in localized virus populations within a plant and the errors introduced during the amplification of silencing signal. Sequencing of the viral particle pool enhanced the efficiency of consensus viral genome sequence reconstruction. Nonhomologous recombinations were commonly detected in the viral particle pool, with a hot spot in the 3′ untranslated and coat protein regions of the genome. We stress that they present an important but often overlooked aspect of virus population diversity. IMPORTANCE This study is the most comprehensive whole-genome characterization of a within-plant virus population to date and the first study comparing diversity of different pools of viral sequences within a host. We show that both virus-derived small RNAs and RNA from viral particles could be used for

  15. Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA

    PubMed Central

    Miyakoshi, Masatoshi; Chao, Yanjie; Vogel, Jörg

    2015-01-01

    There is an expanding list of examples by which one mRNA can posttranscriptionally influence the expression of others. This can involve RNA sponges that sequester regulatory RNAs of mRNAs in the same regulon, but the underlying molecular mechanism of such mRNA cross talk remains little understood. Here, we report sponge-mediated mRNA cross talk in the posttranscriptional network of GcvB, a conserved Hfq-dependent small RNA with one of the largest regulons known in bacteria. We show that mRNA decay from the gltIJKL locus encoding an amino acid ABC transporter generates a stable fragment (SroC) that base-pairs with GcvB. This interaction triggers the degradation of GcvB by RNase E, alleviating the GcvB-mediated mRNA repression of other amino acid-related transport and metabolic genes. Intriguingly, since the gltIJKL mRNA itself is a target of GcvB, the SroC sponge seems to enable both an internal feed-forward loop to activate its parental mRNA in cis and activation of many trans-encoded mRNAs in the same pathway. Disabling this mRNA cross talk affects bacterial growth when peptides are the sole carbon and nitrogen sources. PMID:25630703

  16. Dietary RNAs: New stories regarding oral delivery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    MicroRNAs (miRNAs), a class of small RNAs, are important regulators of various developmental processes in both plants and animals. Several years ago, a report showed the detection of diet-derived plant miRNAs in mammalian tissues and their regulation of mammalian genes, challenging the traditional f...

  17. Eubacterial components similar to small nuclear ribonucleoproteins: identification of immunoprecipitable proteins and capped RNAs in a cyanobacterium and a gram-positive eubacterium.

    PubMed Central

    Kovacs, S A; O'Neil, J; Watcharapijarn, J; Moe-Kirvan, C; Vijay, S; Silva, V

    1993-01-01

    Small nuclear ribonucleoprotein (snRNP) particles play an important role in the processing of pre-mRNA. snRNPs have been identified immunologically in a variety of cells, but none have ever been observed in prokaryotic systems. This report provides the first evidence for the presence of snRNP-like components in two types of prokaryotic cells: those of the cyanobacterium Synechococcus leopoliensis and those of the gram-positive eubacterium Bacillus subtilis. These components consist of snRNP-immunoreactive proteins and RNAs, including some with the snRNP-unique 5' m2,2,7G (m3G) cap. Immunoreactivity was determined by immunoprecipitation procedures, with either antinuclear-antibody-positive (RNP- and Sm-monospecific) patient sera or a m3G monoclonal antibody, with radiolabelled cell extracts that were preadsorbed with antinuclear-antibody-negative sera. S. leopoliensis immunoprecipitates showed the presence of high-molecular-mass proteins (14 to 70 kDa) and RNAs (138 to 243 nucleotides) that are analogous in size to proteins and RNAs found in human (HEp-2) cell immunoprecipitates but absent in Escherichia coli immunoprecipitates. Thin-layer chromatography of S. leopoliensis immunoprecipitates confirmed the presence of a capped nucleotide similar to a capped nucleotide in HEp-2 immunoprecipitates; no such nucleotide was observed in E. coli immunoprecipitates. Immunoreactive RNAs (117-170 nucleotides) were identified in a second eubacterium, B. subtilis, as well. This work suggests that snRNPs or their evolutionary predecessors predate the emergence of eukaryotic cells. Images PMID:8458830

  18. A Single dicer Gene Is Required for Efficient Gene Silencing Associated with Two Classes of Small Antisense RNAs in Mucor circinelloides▿ †

    PubMed Central

    de Haro, Juan P.; Calo, Silvia; Cervantes, María; Nicolás, Francisco E.; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M.

    2009-01-01

    RNA silencing in the zygomycete Mucor circinelloides exhibits uncommon features, such as induction by self-replicative sense transgenes and the accumulation of two size classes of antisense small interfering RNAs (siRNAs). To investigate whether this silencing phenomenon follows the rules of a canonical RNA-silencing mechanism, we used hairpin RNA (hpRNA)-producing constructs as silencing triggers and analyzed the efficiency and stability of silencing in different genetic backgrounds. We show here that the dsRNA-induced silencing mechanism is also associated with the accumulation of two sizes of antisense siRNAs and that this mechanism is not mediated by the previously known dcl-1 (dicer-like) gene, which implies the existence of an additional dicer gene. An M. circinelloides dcl-2 gene was cloned and characterized, and the corresponding null mutant was generated by gene replacement. This mutant is severely impaired in the silencing mechanism induced by self-replicative sense or inverted-repeat transgenes, providing the first genetic evidence of a canonical silencing mechanism in this class of fungus and pointing to a role for dcl-2 in the mechanism. Moreover, a functional dcl-2 gene is required for the normal accumulation of the two sizes of antisense RNAs, as deduced from the analysis of dcl-2− transformants containing hpRNA-expressing plasmids. In addition to its critical role in transgene-induced silencing, the dcl-2 gene seems to play a role in the control of vegetative development, since the dcl-2 null mutants showed a significant decrease in their production of asexual spores. PMID:19666782

  19. Small Interfering RNAs That Trigger Posttranscriptional Gene Silencing Are Not Required for the Histone H3 Lys9 Methylation Necessary for Transgenic Tandem Repeat Stabilization in Neurospora crassa†

    PubMed Central

    Chicas, Agustin; Forrest, Emma C.; Sepich, Silvia; Cogoni, Carlo; Macino, Giuseppe

    2005-01-01

    In Neurospora crassa, the introduction of a transgene can lead to small interfering RNA (siRNA)-mediated posttranscriptional gene silencing (PTGS) of homologous genes. siRNAs can also guide locus-specific methylation of Lys9 of histone H3 (Lys9H3) in Schizosaccharomyces pombe. Here we tested the hypothesis that transgenically derived siRNAs may contemporaneously both activate the PTGS mechanism and induce chromatin modifications at the transgene and the homologous endogenous gene. We carried out chromatin immunoprecipitation using a previously characterized albino-1 (al-1) silenced strain but detected no alterations in the pattern of histone modifications at the endogenous al-1 locus, suggesting that siRNAs produced from the transgenic locus do not trigger modifications in trans of those histones tested. Instead, we found that the transgenic locus was hypermethylated at Lys9H3 in our silenced strain and remained hypermethylated in the quelling defective mutants (qde), further demonstrating that the PTGS machinery is dispensable for Lys9H3 methylation. However, we found that a mutant in the histone Lys9H3 methyltransferase dim-5 was unable to maintain PTGS, with transgenic copies being rapidly lost, resulting in reversion of the silenced phenotype. These results indicate that the defect in PTGS of the Δdim-5 strain is due to the inability to maintain the transgene in tandem, suggesting a role for DIM-5 in stabilizing such repeated sequences. We conclude that in Neurospora, siRNAs produced from the transgenic locus are used in the RNA-induced silencing complex-mediated PTGS pathway and do not communicate with an RNAi-induced initiation of transcriptional gene silencing complex to effect chromatin-based silencing. PMID:15831483

  20. Small interfering RNAs that trigger posttranscriptional gene silencing are not required for the histone H3 Lys9 methylation necessary for transgenic tandem repeat stabilization in Neurospora crassa.

    PubMed

    Chicas, Agustin; Forrest, Emma C; Sepich, Silvia; Cogoni, Carlo; Macino, Giuseppe

    2005-05-01

    In Neurospora crassa, the introduction of a transgene can lead to small interfering RNA (siRNA)-mediated posttranscriptional gene silencing (PTGS) of homologous genes. siRNAs can also guide locus-specific methylation of Lys9 of histone H3 (Lys9H3) in Schizosaccharomyces pombe. Here we tested the hypothesis that transgenically derived siRNAs may contemporaneously both activate the PTGS mechanism and induce chromatin modifications at the transgene and the homologous endogenous gene. We carried out chromatin immunoprecipitation using a previously characterized albino-1 (al-1) silenced strain but detected no alterations in the pattern of histone modifications at the endogenous al-1 locus, suggesting that siRNAs produced from the transgenic locus do not trigger modifications in trans of those histones tested. Instead, we found that the transgenic locus was hypermethylated at Lys9H3 in our silenced strain and remained hypermethylated in the quelling defective mutants (qde), further demonstrating that the PTGS machinery is dispensable for Lys9H3 methylation. However, we found that a mutant in the histone Lys9H3 methyltransferase dim-5 was unable to maintain PTGS, with transgenic copies being rapidly lost, resulting in reversion of the silenced phenotype. These results indicate that the defect in PTGS of the Deltadim-5 strain is due to the inability to maintain the transgene in tandem, suggesting a role for DIM-5 in stabilizing such repeated sequences. We conclude that in Neurospora, siRNAs produced from the transgenic locus are used in the RNA-induced silencing complex-mediated PTGS pathway and do not communicate with an RNAi-induced initiation of transcriptional gene silencing complex to effect chromatin-based silencing.

  1. Loss of RNA–Dependent RNA Polymerase 2 (RDR2) Function Causes Widespread and Unexpected Changes in the Expression of Transposons, Genes, and 24-nt Small RNAs

    PubMed Central

    Jia, Yi; Lisch, Damon R.; Ohtsu, Kazuhiro; Scanlon, Michael J.; Nettleton, Dan; Schnable, Patrick S.

    2009-01-01

    Transposable elements (TEs) comprise a substantial portion of many eukaryotic genomes and are typically transcriptionally silenced. RNA–dependent RNA polymerase 2 (RDR2) is a component of the RNA–directed DNA methylation (RdDM) silencing pathway. In maize, loss of mediator of paramutation1 (mop1) encoded RDR2 function results in reactivation of transcriptionally silenced Mu transposons and a substantial reduction in the accumulation of 24 nt short-interfering RNAs (siRNAs) that recruit RNA silencing components. An RNA–seq experiment conducted on shoot apical meristems (SAMs) revealed that, as expected based on a model in which RDR2 generates 24 nt siRNAs that suppress expression, most differentially expressed DNA TEs (78%) were up-regulated in the mop1 mutant. In contrast, most differentially expressed retrotransposons (68%) were down-regulated. This striking difference suggests that distinct silencing mechanisms are applied to different silencing templates. In addition, >6,000 genes (24% of analyzed genes), including nearly 80% (286/361) of genes in chromatin modification pathways, were differentially expressed. Overall, two-thirds of differentially regulated genes were down-regulated in the mop1 mutant. This finding suggests that RDR2 plays a significant role in regulating the expression of not only transposons, but also of genes. A re-analysis of existing small RNA data identified both RDR2–sensitive and RDR2–resistant species of 24 nt siRNAs that we hypothesize may at least partially explain the complex changes in the expression of genes and transposons observed in the mop1 mutant. PMID:19936292

  2. Loss of RNA-dependent RNA polymerase 2 (RDR2) function causes widespread and unexpected changes in the expression of transposons, genes, and 24-nt small RNAs.

    PubMed

    Jia, Yi; Lisch, Damon R; Ohtsu, Kazuhiro; Scanlon, Michael J; Nettleton, Daniel; Schnable, Patrick S

    2009-11-01

    Transposable elements (TEs) comprise a substantial portion of many eukaryotic genomes and are typically transcriptionally silenced. RNA-dependent RNA polymerase 2 (RDR2) is a component of the RNA-directed DNA methylation (RdDM) silencing pathway. In maize, loss of mediator of paramutation1 (mop1) encoded RDR2 function results in reactivation of transcriptionally silenced Mu transposons and a substantial reduction in the accumulation of 24 nt short-interfering RNAs (siRNAs) that recruit RNA silencing components. An RNA-seq experiment conducted on shoot apical meristems (SAMs) revealed that, as expected based on a model in which RDR2 generates 24 nt siRNAs that suppress expression, most differentially expressed DNA TEs (78%) were up-regulated in the mop1 mutant. In contrast, most differentially expressed retrotransposons (68%) were down-regulated. This striking difference suggests that distinct silencing mechanisms are applied to different silencing templates. In addition, >6,000 genes (24% of analyzed genes), including nearly 80% (286/361) of genes in chromatin modification pathways, were differentially expressed. Overall, two-thirds of differentially regulated genes were down-regulated in the mop1 mutant. This finding suggests that RDR2 plays a significant role in regulating the expression of not only transposons, but also of genes. A re-analysis of existing small RNA data identified both RDR2-sensitive and RDR2-resistant species of 24 nt siRNAs that we hypothesize may at least partially explain the complex changes in the expression of genes and transposons observed in the mop1 mutant.

  3. Endogenous siRNAs Derived from Transposons and mRNAs in Drosophila Somatic Cells

    PubMed Central

    Ghildiyal, Megha; Seitz, Hervé; Horwich, Michael D.; Li, Chengjian; Du, Tingting; Lee, Soohyun; Xu, Jia; Kittler, Ellen L.W.; Zapp, Maria L.; Weng, Zhiping; Zamore, Phillip D.

    2009-01-01

    Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line. PMID:18403677

  4. Stratigraphy of small shield volcanoes on Venus: Criteria for determining stratigraphic relationships and assessment of relative age and temporal abundance

    NASA Astrophysics Data System (ADS)

    Ivanov, Mikhail A.; Head, James W.

    2004-10-01

    Small volcanic edifices, shields with a diameter less than about 20 km, are common and sometimes very abundant features on the plains of Venus. Typically, they form tight or loose clusters of features known as shield fields. Small shields are interpreted to be formed due to small-scale eruptions through numerous and distinct sources, a mode of formation apparently significantly different from the mechanism thought to be responsible for the emplacement of the vast regional plains of Venus. Did the eruption style of small shields occur repeatedly throughout the visible part of the geologic record of Venus? Or was this style more concentrated in a specific epoch or epochs of geologic history? Do the clusters of shields represent localized development of sources over a thermal anomaly such as a plume, or do they represent exposures or kipukas of a more regional unit or units? A major step toward answering these questions is an understanding of small shield stratigraphy. Multiple criteria have been developed to assess the stratigraphic relationships of individual small shields and that of shield fields with the adjacent units. In our analysis, we expanded and developed the previous criteria and added detailed criteria to describe specific patterns of deformation within shield fields, cross-cutting, and embayment relationships between shield fields and surrounding units. We also used secondary characteristics of shield fields such as radar albedo difference, changes in shield density and size, etc. In our study, we applied these criteria and analyzed in detail stratigraphic relationships of shield fields in a random sample of features (64 fields) and in the global geotraverse along 30°N (77 fields). The total number of analyzed shield fields (141) represents about 22% of the general population of these features catalogued by Crumpler and Aubele [2000]. The majority of the fields (98, or ~69%) predate emplacement of material of vast regional plains with wrinkle ridges

  5. MicroRNAs As Potential Targets for Abiotic Stress Tolerance in Plants

    PubMed Central

    Shriram, Varsha; Kumar, Vinay; Devarumath, Rachayya M.; Khare, Tushar S.; Wani, Shabir H.

    2016-01-01

    The microRNAs (miRNAs) are small (20–24 nt) sized, non-coding, single stranded riboregulator RNAs abundant in higher organisms. Recent findings have established that plants assign miRNAs as critical post-transcriptional regulators of gene expression in sequence-specific manner to respond to numerous abiotic stresses they face during their growth cycle. These small RNAs regulate gene expression via translational inhibition. Usually, stress induced miRNAs downregulate their target mRNAs, whereas, their downregulation leads to accumulation and function of positive regulators. In the past decade, investigations were mainly aimed to identify plant miRNAs, responsive to individual or multiple environmental factors, profiling their expression patterns and recognizing their roles in stress responses and tolerance. Altered expressions of miRNAs implicated in plant growth and development have been reported in several plant species subjected to abiotic stress conditions such as drought, salinity, extreme temperatures, nutrient deprivation, and heavy metals. These findings indicate that miRNAs may hold the key as potential targets for genetic manipulations to engineer abiotic stress tolerance in crop plants. This review is aimed to provide recent updates on plant miRNAs, their biogenesis and functions, target prediction and identification, computational tools and databases available for plant miRNAs, and their roles in abiotic stress-responses and adaptive mechanisms in major crop plants. Besides, the recent case studies for overexpressing the selected miRNAs for miRNA-mediated enhanced abiotic stress tolerance of transgenic plants have been discussed. PMID:27379117

  6. Synthesis of small interfering RNAs containing acetal-type nucleoside analogs at their 3'-ends and analysis of their silencing activity and their ability to bind to the Argonaute2 PAZ domain.

    PubMed

    Inada, Natsumi; Nakamoto, Kosuke; Yokogawa, Takashi; Ueno, Yoshihito

    2015-10-20

    In this study, we aimed to create small interfering RNAs (siRNAs) with increased silencing activities and nuclease resistance properties. Therefore, we designed and synthesized five types of siRNA containing acetal-type nucleoside analogs at their 3'-dangling ends. We found that the siRNA containing 1-O-(2,2,2-trifluoroethyl)-β-D-ribofuranose at the 3'-dangling end was the most potent among the synthesized siRNAs and showed more resistance to nucleolytic degradation by a 3' exonuclease than a natural RNA did. Thus, modification of siRNAs by addition of 1-O-(2,2,2-trifluoroethyl)-β-D-ribofuranose may hold promise as a means of improving the silencing activity and nuclease resistance of siRNAs.

  7. MicroRNAs in the etiology of colorectal cancer: pathways and clinical implications

    PubMed Central

    Strubberg, Ashlee M.

    2017-01-01

    ABSTRACT MicroRNAs (miRNAs) are small single-stranded RNAs that repress mRNA translation and trigger mRNA degradation. Of the ∼1900 miRNA-encoding genes present in the human genome, ∼250 miRNAs are reported to have changes in abundance or altered functions in colorectal cancer. Thousands of studies have documented aberrant miRNA levels in colorectal cancer, with some miRNAs reported to actively regulate tumorigenesis. A recurrent phenomenon with miRNAs is their frequent participation in feedback loops, which probably serve to reinforce or magnify biological outcomes to manifest a particular cellular phenotype. Here, we review the roles of oncogenic miRNAs (oncomiRs), tumor suppressive miRNAs (anti-oncomiRs) and miRNA regulators in colorectal cancer. Given their stability in patient-derived samples and ease of detection with standard and novel techniques, we also discuss the potential use of miRNAs as biomarkers in the diagnosis of colorectal cancer and as prognostic indicators of this disease. MiRNAs also represent attractive candidates for targeted therapies because their function can be manipulated through the use of synthetic antagonists and miRNA mimics. PMID:28250048

  8. Genome-wide analysis of leafbladeless1-regulated and phased small RNAs underscores the importance of the TAS3 ta-siRNA pathway to maize development.

    PubMed

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

    2014-12-01

    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

  9. 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.