Sample records for abundant small rnas

  1. Identification and Differential Abundance of Mitochondrial Genome Encoding Small RNAs (mitosRNA) in Breast Muscles of Modern Broilers and Unselected Chicken Breed

    PubMed Central

    Bottje, Walter G.; Khatri, Bhuwan; Shouse, Stephanie A.; Seo, Dongwon; Mallmann, Barbara; Orlowski, Sara K.; Pan, Jeonghoon; Kong, Seongbae; Owens, Casey M.; Anthony, Nicholas B.; Kim, Jae K.; Kong, Byungwhi C.

    2017-01-01

    Background: Although small non-coding RNAs are mostly encoded by the nuclear genome, thousands of small non-coding RNAs encoded by the mitochondrial genome, termed as mitosRNAs were recently reported in human, mouse and trout. In this study, we first identified chicken mitosRNAs in breast muscle using small RNA sequencing method and the differential abundance was analyzed between modern pedigree male (PeM) broilers (characterized by rapid growth and large muscle mass) and the foundational Barred Plymouth Rock (BPR) chickens (characterized by slow growth and small muscle mass). Methods: Small RNA sequencing was performed with total RNAs extracted from breast muscles of PeM and BPR (n = 6 per group) using the 1 × 50 bp single end read method of Illumina sequencing. Raw reads were processed by quality assessment, adapter trimming, and alignment to the chicken mitochondrial genome (GenBank Accession: X52392.1) using the NGen program. Further statistical analyses were performed using the JMP Genomics 8. Differentially expressed (DE) mitosRNAs between PeM and BPR were confirmed by quantitative PCR. Results: Totals of 183,416 unique small RNA sequences were identified as potential chicken mitosRNAs. After stringent filtering processes, 117 mitosRNAs showing >100 raw read counts were abundantly produced from all 37 mitochondrial genes (except D-loop region) and the length of mitosRNAs ranged from 22 to 46 nucleotides. Of those, abundance of 44 mitosRNAs were significantly altered in breast muscles of PeM compared to those of BPR: all mitosRNAs were higher in PeM breast except those produced from 16S-rRNA gene. Possibly, the higher mitosRNAs abundance in PeM breast may be due to a higher mitochondrial content compared to BPR. Our data demonstrate that in addition to 37 known mitochondrial genes, the mitochondrial genome also encodes abundant mitosRNAs, that may play an important regulatory role in muscle growth via mitochondrial gene expression control. PMID:29104541

  2. A Novel Class of Somatic Small RNAs Similar to Germ Cell Pachytene PIWI-interacting Small RNAs*

    PubMed Central

    Ortogero, Nicole; Schuster, Andrew S.; Oliver, Daniel K.; Riordan, Connor R.; Hong, Annie S.; Hennig, Grant W.; Luong, Dickson; Bao, Jianqiang; Bhetwal, Bhupal P.; Ro, Seungil; McCarrey, John R.; Yan, Wei

    2014-01-01

    PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3′-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs. PMID:25320077

  3. Identification of small RNAs abundant in Burkholderia cenocepacia biofilms reveal putative regulators with a potential role in carbon and iron metabolism.

    PubMed

    Sass, Andrea; Kiekens, Sanne; Coenye, Tom

    2017-11-15

    Small RNAs play a regulatory role in many central metabolic processes of bacteria, as well as in developmental processes such as biofilm formation. Small RNAs of Burkholderia cenocepacia, an opportunistic pathogenic beta-proteobacterium, are to date not well characterised. To address that, we performed genome-wide transcriptome structure analysis of biofilm grown B. cenocepacia J2315. 41 unannotated short transcripts were identified in intergenic regions of the B. cenocepacia genome. 15 of these short transcripts, highly abundant in biofilms, widely conserved in Burkholderia sp. and without known function, were selected for in-depth analysis. Expression profiling showed that most of these sRNAs are more abundant in biofilms than in planktonic cultures. Many are also highly abundant in cells grown in minimal media, suggesting they are involved in adaptation to nutrient limitation and growth arrest. Their computationally predicted targets include a high proportion of genes involved in carbon metabolism. Expression and target genes of one sRNA suggest a potential role in regulating iron homoeostasis. The strategy used for this study to detect sRNAs expressed in B. cenocepacia biofilms has successfully identified sRNAs with a regulatory function.

  4. Circular RNAs are abundant, conserved, and associated with ALU repeats

    PubMed Central

    Jeck, William R.; Sorrentino, Jessica A.; Wang, Kai; Slevin, Michael K.; Burd, Christin E.; Liu, Jinze; Marzluff, William F.; Sharpless, Norman E.

    2013-01-01

    Circular RNAs composed of exonic sequence have been described in a small number of genes. Thought to result from splicing errors, circular RNA species possess no known function. To delineate the universe of endogenous circular RNAs, we performed high-throughput sequencing (RNA-seq) of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease, RNase R. We identified >25,000 distinct RNA species in human fibroblasts that contained non-colinear exons (a “backsplice”) and were reproducibly enriched by exonuclease degradation of linear RNA. These RNAs were validated as circular RNA (ecircRNA), rather than linear RNA, and were more stable than associated linear mRNAs in vivo. In some cases, the abundance of circular molecules exceeded that of associated linear mRNA by >10-fold. By conservative estimate, we identified ecircRNAs from 14.4% of actively transcribed genes in human fibroblasts. Application of this method to murine testis RNA identified 69 ecircRNAs in precisely orthologous locations to human circular RNAs. Of note, paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and mice. Though HIPK3 circular RNAs contain an AUG translation start, it and other ecircRNAs were not bound to ribosomes. Circular RNAs could be degraded by siRNAs and, therefore, may act as competing endogenous RNAs. Bioinformatic analysis revealed shared features of circularized exons, including long bordering introns that contained complementary ALU repeats. These data show that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression. PMID:23249747

  5. RIP-seq of BmAgo2-associated small RNAs reveal various types of small non-coding RNAs in the silkworm, Bombyx mori

    PubMed Central

    2013-01-01

    Background Small non-coding RNAs (ncRNAs) are important regulators of gene expression in eukaryotes. Previously, only microRNAs (miRNAs) and piRNAs have been identified in the silkworm, Bombyx mori. Furthermore, only ncRNAs (50-500nt) of intermediate size have been systematically identified in the silkworm. Results Here, we performed a systematic identification and analysis of small RNAs (18-50nt) associated with the Bombyx mori argonaute2 (BmAgo2) protein. Using RIP-seq, we identified various types of small ncRNAs associated with BmAGO2. These ncRNAs showed a multimodal length distribution, with three peaks at ~20nt, ~27nt and ~33nt, which included tRNA-, transposable element (TE)-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. The tRNA-derived fragments (tRFs) were found at an extremely high abundance and accounted for 69.90% of the BmAgo2-associated small RNAs. Northern blotting confirmed that many tRFs were expressed or up-regulated only in the BmNPV-infected cells, implying that the tRFs play a prominent role by binding to BmAgo2 during BmNPV infection. Additional evidence suggested that there are potential cleavage sites on the D, anti-codon and TψC loops of the tRNAs. TE-derived small RNAs and piRNAs also accounted for a significant proportion of the BmAgo2-associated small RNAs, suggesting that BmAgo2 could be involved in the maintenance of genome stability by suppressing the activities of transposons guided by these small RNAs. Finally, Northern blotting was also used to confirm the Bombyx 5.8 s rRNA-derived small RNAs, demonstrating that various novel small RNAs exist in the silkworm. Conclusions Using an RIP-seq method in combination with Northern blotting, we identified various types of small RNAs associated with the BmAgo2 protein, including tRNA-, TE-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. Our findings provide new clues for future functional studies of the role of small RNAs in insect

  6. A survey of small RNAs in human sperm

    PubMed Central

    Krawetz, Stephen A.; Kruger, Adele; Lalancette, Claudia; Tagett, Rebecca; Anton, Ester; Draghici, Sorin; Diamond, Michael P.

    2011-01-01

    BACKGROUND There has been substantial interest in assessing whether RNAs (mRNAs and sncRNAs, i.e. small non-coding) delivered from mammalian spermatozoa play a functional role in early embryo development. While the cadre of spermatozoal mRNAs has been characterized, comparatively little is known about the distribution or function of the estimated 24 000 sncRNAs within each normal human spermatozoon. METHODS RNAs of <200 bases in length were isolated from the ejaculates from three donors of proved fertility. RNAs of 18–30 nucleotides in length were then used to construct small RNA Digital Gene Expression libraries for Next Generation Sequencing. Known sncRNAs that uniquely mapped to a single location in the human genome were identified. RESULTS Bioinformatic analysis revealed the presence of multiple classes of small RNAs in human spermatozoa. The primary classes resolved included microRNA (miRNAs) (≈7%), Piwi-interacting piRNAs (≈17%), repeat-associated small RNAs (≈65%). A minor subset of short RNAs within the transcription start site/promoter fraction (≈11%) frames the histone promoter-associated regions enriched in genes of early embryonic development. These have been termed quiescent RNAs. CONCLUSIONS A complex population of male derived sncRNAs that are available for delivery upon fertilization was revealed. Sperm miRNA-targeted enrichment in the human oocyte is consistent with their role as modifiers of early post-fertilization. The relative abundance of piRNAs and repeat-associated RNAs suggests that they may assume a role in confrontation and consolidation. This may ensure the compatibility of the genomes at fertilization. PMID:21989093

  7. Novel Regulatory Small RNAs in Streptococcus pyogenes

    PubMed Central

    Tesorero, Rafael A.; Yu, Ning; Wright, Jordan O.; Svencionis, Juan P.; Cheng, Qiang; Kim, Jeong-Ho; Cho, Kyu Hong

    2013-01-01

    Streptococcus pyogenes (Group A Streptococcus or GAS) is a Gram-positive bacterial pathogen that has shown complex modes of regulation of its virulence factors to cause diverse diseases. Bacterial small RNAs are regarded as novel widespread regulators of gene expression in response to environmental signals. Recent studies have revealed that several small RNAs (sRNAs) have an important role in S. pyogenes physiology and pathogenesis by regulating gene expression at the translational level. To search for new sRNAs in S. pyogenes, we performed a genomewide analysis through computational prediction followed by experimental verification. To overcome the limitation of low accuracy in computational prediction, we employed a combination of three different computational algorithms (sRNAPredict, eQRNA and RNAz). A total of 45 candidates were chosen based on the computational analysis, and their transcription was analyzed by reverse-transcriptase PCR and Northern blot. Through this process, we discovered 7 putative novel trans-acting sRNAs. Their abundance varied between different growth phases, suggesting that their expression is influenced by environmental or internal signals. Further, to screen target mRNAs of an sRNA, we employed differential RNA sequencing analysis. This study provides a significant resource for future study of small RNAs and their roles in physiology and pathogenesis of S. pyogenes. PMID:23762235

  8. An Atlas of Soybean Small RNAs Identifies Phased siRNAs from Hundreds of Coding Genes[W

    PubMed Central

    Kakrana, Atul; Huang, Kun; Zhai, Jixian; Yan, Zhe; Valdés-López, Oswaldo; Prince, Silvas; Musket, Theresa A.; Stacey, Gary

    2014-01-01

    Small RNAs are ubiquitous, versatile repressors and include (1) microRNAs (miRNAs), processed from mRNA forming stem-loops; and (2) small interfering RNAs (siRNAs), the latter derived in plants by a process typically requiring an RNA-dependent RNA polymerase. We constructed and analyzed an expression atlas of soybean (Glycine max) small RNAs, identifying over 500 loci generating 21-nucleotide phased siRNAs (phasiRNAs; from PHAS loci), of which 483 overlapped annotated protein-coding genes. Via the integration of miRNAs with parallel analysis of RNA end (PARE) data, 20 miRNA triggers of 127 PHAS loci were detected. The primary class of PHAS loci (208 or 41% of the total) corresponded to NB-LRR genes; some of these small RNAs preferentially accumulate in nodules. Among the PHAS loci, novel representatives of TAS3 and noncanonical phasing patterns were also observed. A noncoding PHAS locus, triggered by miR4392, accumulated preferentially in anthers; the phasiRNAs are predicted to target transposable elements, with their peak abundance during soybean reproductive development. Thus, phasiRNAs show tremendous diversity in dicots. We identified novel miRNAs and assessed the veracity of soybean miRNAs registered in miRBase, substantially improving the soybean miRNA annotation, facilitating an improvement of miRBase annotations and identifying at high stringency novel miRNAs and their targets. PMID:25465409

  9. A Transgenic Transcription Factor (TaDREB3) in Barley Affects the Expression of MicroRNAs and Other Small Non-Coding RNAs

    PubMed Central

    Hackenberg, Michael; Shi, Bu-Jun; Gustafson, Perry; Langridge, Peter

    2012-01-01

    Transcription factors (TFs), microRNAs (miRNAs), small interfering RNAs (siRNAs) and other functional non-coding small RNAs (sRNAs) are important gene regulators. Comparison of sRNA expression profiles between transgenic barley over-expressing a drought tolerant TF (TaDREB3) and non-transgenic control barley revealed many group-specific sRNAs. In addition, 42% of the shared sRNAs were differentially expressed between the two groups (|log2| >1). Furthermore, TaDREB3-derived sRNAs were only detected in transgenic barley despite the existence of homologous genes in non-transgenic barley. These results demonstrate that the TF strongly affects the expression of sRNAs and siRNAs could in turn affect the TF stability. The TF also affects size distribution and abundance of sRNAs including miRNAs. About half of the sRNAs in each group were derived from chloroplast. A sRNA derived from tRNA-His(GUG) encoded by the chloroplast genome is the most abundant sRNA, accounting for 42.2% of the total sRNAs in transgenic barley and 28.9% in non-transgenic barley. This sRNA, which targets a gene (TC245676) involved in biological processes, was only present in barley leaves but not roots. 124 and 136 miRNAs were detected in transgenic and non-transgenic barley, respectively. miR156 was the most abundant miRNA and up-regulated in transgenic barley, while miR168 was the most abundant miRNA and up-regulated in non-transgenic barley. Eight out of 20 predicted novel miRNAs were differentially expressed between the two groups. All the predicted novel miRNA targets were validated using a degradome library. Our data provide an insight into the effect of TF on the expression of sRNAs in barley. PMID:22870277

  10. Wheat hybridization and polyploidization results in deregulation of small RNAs.

    PubMed

    Kenan-Eichler, Michal; Leshkowitz, Dena; Tal, Lior; Noor, Elad; Melamed-Bessudo, Cathy; Feldman, Moshe; Levy, Avraham A

    2011-06-01

    Speciation via interspecific or intergeneric hybridization and polyploidization triggers genomic responses involving genetic and epigenetic alterations. Such modifications may be induced by small RNAs, which affect key cellular processes, including gene expression, chromatin structure, cytosine methylation and transposable element (TE) activity. To date, the role of small RNAs in the context of wide hybridization and polyploidization has received little attention. In this work, we performed high-throughput sequencing of small RNAs of parental, intergeneric hybrid, and allopolyploid plants that mimic the genomic changes occurring during bread wheat speciation. We found that the percentage of small RNAs corresponding to miRNAs increased with ploidy level, while the percentage of siRNAs corresponding to TEs decreased. The abundance of most miRNA species was similar to midparent values in the hybrid, with some deviations, as seen in overrepresentation of miR168, in the allopolyploid. In contrast, the number of siRNAs corresponding to TEs strongly decreased upon allopolyploidization, but not upon hybridization. The reduction in corresponding siRNAs, together with decreased CpG methylation, as shown here for the Veju element, represent hallmarks of TE activation. TE-siRNA downregulation in the allopolyploid may contribute to genome destabilization at the initial stages of speciation. This phenomenon is reminiscent of hybrid dysgenesis in Drosophila.

  11. 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. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

  12. 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.

  13. Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training.

    PubMed

    Smalheiser, Neil R; Lugli, Giovanni; Thimmapuram, Jyothi; Cook, Edwin H; Larson, John

    2011-01-01

    We previously proposed that endogenous siRNAs may regulate synaptic plasticity and long-term gene expression in the mammalian brain. Here, a hippocampal-dependent task was employed in which adult mice were trained to execute a nose-poke in a port containing one of two simultaneously present odors in order to obtain a reward. Mice demonstrating olfactory discrimination training were compared to pseudo-training and nose-poke control groups; size-selected hippocampal RNA was subjected to Illumina deep sequencing. Sequences that aligned uniquely and exactly to the genome without uncertain nucleotide assignments, within exons or introns of MGI annotated genes, were examined further. The data confirm that small RNAs having features of endogenous siRNAs are expressed in brain; that many of them derive from genes that regulate synaptic plasticity (and have been implicated in neuropsychiatric diseases); and that hairpin-derived endo-siRNAs and the 20- to 23-nt size class of small RNAs show a significant increase during an early stage of training. The most abundant putative siRNAs arose from an intronic inverted repeat within the SynGAP1 locus; this inverted repeat was a substrate for dicer in vitro, and SynGAP1 siRNA was specifically associated with Argonaute proteins in vivo. Unexpectedly, a dramatic increase with training (more than 100-fold) was observed for a class of 25- to 30-nt small RNAs derived from specific sites within snoRNAs and abundant noncoding RNAs (Y1 RNA, RNA component of mitochondrial RNAse P, 28S rRNA, and 18S rRNA). Further studies are warranted to characterize the role(s) played by endogenous siRNAs and noncoding RNA-derived small RNAs in learning and memory.

  14. Diversity and functional convergence of small noncoding RNAs in male germ cell differentiation and fertilization

    PubMed Central

    García-López, Jesús; Alonso, Lola; Cárdenas, David B.; Artaza-Alvarez, Haydeé; Hourcade, Juan de Dios; Martínez, Sergio; Brieño-Enríquez, Miguel A.; del Mazo, Jesús

    2015-01-01

    The small noncoding RNAs (sncRNAs) are considered as post-transcriptional key regulators of male germ cell development. In addition to microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), other sncRNAs generated from small nucleolar RNAs (snoRNAs), tRNAs, or rRNAs processing may also play important regulatory roles in spermatogenesis. By next-generation sequencing (NGS), we characterized the sncRNA populations detected at three milestone stages in male germ differentiation: primordial germ cells (PGCs), pubertal spermatogonia cells, and mature spermatozoa. To assess their potential transmission through the spermatozoa during fertilization, the sncRNAs of mouse oocytes and zygotes were also analyzed. Both, microRNAs and snoRNA-derived small RNAs are abundantly expressed in PGCs but transiently replaced by piRNAs in spermatozoa and endo-siRNAs in oocytes and zygotes. Exhaustive analysis of miRNA sequence variants also shows an increment of noncanonical microRNA forms along male germ cell differentiation. RNAs-derived from tRNAs and rRNAs interacting with PIWI proteins are not generated by the ping-pong pathway and could be a source of primary piRNAs. Moreover, our results strongly suggest that the small RNAs-derived from tRNAs and rRNAs are interacting with PIWI proteins, and specifically with MILI. Finally, computational analysis revealed their potential involvement in post-transcriptional regulation of mRNA transcripts suggesting functional convergence among different small RNA classes in germ cells and zygotes. PMID:25805854

  15. Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production- and development-correlated expression and new small RNA classes.

    PubMed

    Zhao, Ying-Tao; Wang, Meng; Fu, San-Xiong; Yang, Wei-Cai; Qi, Cun-Kou; Wang, Xiu-Jie

    2012-02-01

    MicroRNAs (miRNAs) and small interfering RNAs are important regulators of plant development and seed formation, yet their population and abundance in the oil crop Brassica napus are still not well understood, especially at different developmental stages and among cultivars with varied seed oil contents. Here, we systematically analyzed the small RNA expression profiles of Brassica napus seeds at early embryonic developmental stages in high-oil-content and low-oil-content B. napus cultivars, both cultured in two environments. A total of 50 conserved miRNAs and 9 new miRNAs were identified, together with some new miRNA targets. Expression analysis revealed some miRNAs with varied expression levels in different seed oil content cultivars or at different embryonic developmental stages. A large number of 23-nucleotide small RNAs with specific nucleotide composition preferences were also identified, which may present new classes of functional small RNAs.

  16. Heavy Chronic Intermittent Ethanol Exposure Alters Small Noncoding RNAs in Mouse Sperm and Epididymosomes.

    PubMed

    Rompala, Gregory R; Mounier, Anais; Wolfe, Cody M; Lin, Qishan; Lefterov, Iliya; Homanics, Gregg E

    2018-01-01

    While the risks of maternal alcohol abuse during pregnancy are well-established, several preclinical studies suggest that chronic preconception alcohol consumption by either parent may also have significance consequences for offspring health and development. Notably, since isogenic male mice used in these studies are not involved in gestation or rearing of offspring, the cross-generational effects of paternal alcohol exposure suggest a germline-based epigenetic mechanism. Many recent studies have demonstrated that the effects of paternal environmental exposures such as stress or malnutrition can be transmitted to the next generation via alterations to small noncoding RNAs in sperm. Therefore, we used high throughput sequencing to examine the effect of preconception ethanol on small noncoding RNAs in sperm. We found that chronic intermittent ethanol exposure altered several small noncoding RNAs from three of the major small RNA classes in sperm, tRNA-derived small RNA (tDR), mitochondrial small RNA, and microRNA. Six of the ethanol-responsive small noncoding RNAs were evaluated with RT-qPCR on a separate cohort of mice and five of the six were confirmed to be altered by chronic ethanol exposure, supporting the validity of the sequencing results. In addition to altered sperm RNA abundance, chronic ethanol exposure affected post-transcriptional modifications to sperm small noncoding RNAs, increasing two nucleoside modifications previously identified in mitochondrial tRNA. Furthermore, we found that chronic ethanol reduced epididymal expression of a tRNA methyltransferase, Nsun2 , known to directly regulate tDR biogenesis. Finally, ethanol-responsive sperm tDR are similarly altered in extracellular vesicles of the epididymis (i.e., epididymosomes), supporting the hypothesis that alterations to sperm tDR emerge in the epididymis and that epididymosomes are the primary source of small noncoding RNAs in sperm. These results add chronic ethanol to the growing list of

  17. Heavy Chronic Intermittent Ethanol Exposure Alters Small Noncoding RNAs in Mouse Sperm and Epididymosomes

    PubMed Central

    Rompala, Gregory R.; Mounier, Anais; Wolfe, Cody M.; Lin, Qishan; Lefterov, Iliya; Homanics, Gregg E.

    2018-01-01

    While the risks of maternal alcohol abuse during pregnancy are well-established, several preclinical studies suggest that chronic preconception alcohol consumption by either parent may also have significance consequences for offspring health and development. Notably, since isogenic male mice used in these studies are not involved in gestation or rearing of offspring, the cross-generational effects of paternal alcohol exposure suggest a germline-based epigenetic mechanism. Many recent studies have demonstrated that the effects of paternal environmental exposures such as stress or malnutrition can be transmitted to the next generation via alterations to small noncoding RNAs in sperm. Therefore, we used high throughput sequencing to examine the effect of preconception ethanol on small noncoding RNAs in sperm. We found that chronic intermittent ethanol exposure altered several small noncoding RNAs from three of the major small RNA classes in sperm, tRNA-derived small RNA (tDR), mitochondrial small RNA, and microRNA. Six of the ethanol-responsive small noncoding RNAs were evaluated with RT-qPCR on a separate cohort of mice and five of the six were confirmed to be altered by chronic ethanol exposure, supporting the validity of the sequencing results. In addition to altered sperm RNA abundance, chronic ethanol exposure affected post-transcriptional modifications to sperm small noncoding RNAs, increasing two nucleoside modifications previously identified in mitochondrial tRNA. Furthermore, we found that chronic ethanol reduced epididymal expression of a tRNA methyltransferase, Nsun2, known to directly regulate tDR biogenesis. Finally, ethanol-responsive sperm tDR are similarly altered in extracellular vesicles of the epididymis (i.e., epididymosomes), supporting the hypothesis that alterations to sperm tDR emerge in the epididymis and that epididymosomes are the primary source of small noncoding RNAs in sperm. These results add chronic ethanol to the growing list of

  18. 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

  19. Insight into small RNA abundance and expression in high- and low-temperature stress response using deep sequencing in Arabidopsis.

    PubMed

    Baev, Vesselin; Milev, Ivan; Naydenov, Mladen; Vachev, Tihomir; Apostolova, Elena; Mehterov, Nikolay; Gozmanva, Mariyana; Minkov, Georgi; Sablok, Gaurav; Yahubyan, Galina

    2014-11-01

    Small RNA profiling and assessing its dependence on changing environmental factors have expanded our understanding of the transcriptional and post-transcriptional regulation of plant stress responses. Insufficient data have been documented earlier to depict the profiling of small RNA classes in temperature-associated stress which has a wide implication for climate change biology. In the present study, we report a comparative assessment of the genome-wide profiling of small RNAs in Arabidopsis thaliana using two conditional responses, induced by high- and low-temperature. Genome-wide profiling of small RNAs revealed an abundance of 21 nt small RNAs at low temperature, while high temperature showed an abundance of 21 nt and 24 nt small RNAs. The two temperature treatments altered the expression of a specific subset of mature miRNAs and displayed differential expression of a number of miRNA isoforms (isomiRs). Comparative analysis demonstrated that a large number of protein-coding genes can give rise to differentially expressed small RNAs following temperature shifts. Low temperature caused accumulation of small RNAs, corresponding to the sense strand of a number of cold-responsive genes. In contrast, high temperature stimulated the production of small RNAs of both polarities from genes encoding functionally diverse proteins. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  20. Human tRNA-derived small RNAs in the global regulation of RNA silencing

    PubMed Central

    Haussecker, Dirk; Huang, Yong; Lau, Ashley; Parameswaran, Poornima; Fire, Andrew Z.; Kay, Mark A.

    2010-01-01

    Competition between mammalian RNAi-related gene silencing pathways is well documented. It is therefore important to identify all classes of small RNAs to determine their relationship with RNAi and how they affect each other functionally. Here, we identify two types of 5′-phosphate, 3′-hydroxylated human tRNA-derived small RNAs (tsRNAs). tsRNAs differ from microRNAs in being essentially restricted to the cytoplasm and in associating with Argonaute proteins, but not MOV10. The first type belongs to a previously predicted Dicer-dependent class of small RNAs that we find can modestly down-regulate target genes in trans. The 5′ end of type II tsRNA was generated by RNaseZ cleavage downstream from a tRNA gene, while the 3′ end resulted from transcription termination by RNA polymerase III. Consistent with their preferential association with the nonslicing Argonautes 3 and 4, canonical gene silencing activity was not observed for type II tsRNAs. The addition, however, of an oligonucleotide that was sense to the reporter gene, but antisense to an overexpressed version of the type II tsRNA, triggered robust, >80% gene silencing. This correlated with the redirection of the thus reconstituted fully duplexed double-stranded RNA into Argonaute 2, whereas Argonautes 3 and 4 were skewed toward less structured small RNAs, particularly single-strand RNAs. We observed that the modulation of tsRNA levels had minor effects on the abundance of microRNAs, but more pronounced changes in the silencing activities of both microRNAs and siRNAs. These findings support that tsRNAs are involved in the global control of small RNA silencing through differential Argonaute association, suggesting that small RNA-mediated gene regulation may be even more finely regulated than previously realized. PMID:20181738

  1. Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs.

    PubMed

    Khan, Aly A; Betel, Doron; Miller, Martin L; Sander, Chris; Leslie, Christina S; Marks, Debora S

    2009-06-01

    Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect--that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.

  2. Sources and functions of extracellular small RNAs in human circulation

    PubMed Central

    Fritz, Joëlle V.; Heintz-Buschart, Anna; Ghosal, Anubrata; Wampach, Linda; Etheridge, Alton; Galas, David; Wilmes, Paul

    2017-01-01

    Various biotypes of endogenous small RNAs (sRNAs) have been detected in human circulation including microRNAs, tRNA, rRNA and yRNA fragments. These extracellular sRNAs (ex-sRNAs) are packaged and secreted by many different cell types. Ex-sRNAs exhibit differences in abundance in several disease contexts and have therefore been proposed as well-suited biomarkers. Furthermore, exosome-borne ex-sRNAs have been reported to elicit physiological responses in receiving cells. Albeit controversial, exogenous ex-sRNAs derived from plants and microorganisms have also been described in human blood. Essential questions which remain to be conclusively addressed in the field concern the (i) presence and mechanistic sources of exogenous ex-sRNA in human bodily fluids, (ii) detection and measurement of ex-sRNA in human circulation, (iii) selectivity of ex-sRNA export and import, (iv) sensitivity and specificity of ex-sRNA delivery to cellular targets, and (v) cell-, tissue-, organ- and organism-wide impacts of ex-sRNAs. We will survey the present state of knowledge of most of these questions in this review. PMID:27215587

  3. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  5. The Big Role of Small RNAs in Anxiety and Stress-Related Disorders.

    PubMed

    Malan-Müller, S; Hemmings, S M J

    2017-01-01

    In the study of complex, heterogeneous disorders, such as anxiety and stress-related disorders, epigenetic factors provide an additional level of heritable complexity. MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function as epigenetic modulators of gene expression by binding to target messenger RNAs (mRNAs) and subsequently blocking translation or accelerating their degradation. In light of their abundance in the central nervous system (CNS) and their involvement in synaptic plasticity and neuronal differentiation, miRNAs represent an exciting frontier to be explored in the etiology and treatment of anxiety and stress-related disorders. This chapter will present a thorough review of miRNAs, their functions, and mRNA targets in the CNS, focusing on their role in anxiety and stress-related disorders as described by studies performed in animals and human subjects. © 2017 Elsevier Inc. All rights reserved.

  6. 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

  7. The Mechanisms of Virulence Regulation by Small Noncoding RNAs in Low GC Gram-Positive Pathogens

    PubMed Central

    Pitman, Stephanie; Cho, Kyu Hong

    2015-01-01

    The discovery of small noncoding regulatory RNAs (sRNAs) in bacteria has grown tremendously recently, giving new insights into gene regulation. The implementation of computational analysis and RNA sequencing has provided new tools to discover and analyze potential sRNAs. Small regulatory RNAs that act by base-pairing to target mRNAs have been found to be ubiquitous and are the most abundant class of post-transcriptional regulators in bacteria. The majority of sRNA studies has been limited to E. coli and other gram-negative bacteria. However, examples of sRNAs in gram-positive bacteria are still plentiful although the detailed gene regulation mechanisms behind them are not as well understood. Strict virulence control is critical for a pathogen’s survival and many sRNAs have been found to be involved in that process. This review outlines the targets and currently known mechanisms of trans-acting sRNAs involved in virulence regulation in various gram-positive pathogens. In addition, their shared characteristics such as CU interaction motifs, the role of Hfq, and involvement in two-component regulators, riboswitches, quorum sensing, or toxin/antitoxin systems are described. PMID:26694351

  8. Highly Complementary Target RNAs Promote Release of Guide RNAs from Human Argonaute2

    PubMed Central

    De, Nabanita; Young, Lisa; Lau, Pick-Wei; Meisner, Nicole-Claudia; Morrissey, David V.; MacRae, Ian J.

    2013-01-01

    SUMMARY Argonaute proteins use small RNAs to guide the silencing of complementary target RNAs in many eukaryotes. Although small RNA biogenesis pathways are well studied, mechanisms for removal of guide RNAs from Argonaute are poorly understood. Here we show that the Argonaute2 (Ago2) guide RNA complex is extremely stable, with a half-life on the order of days. However, highly complementary target RNAs destabilize the complex and significantly accelerate release of the guide RNA from Ago2. This “unloading” activity can be enhanced by mismatches between the target and the guide 5′ end and attenuated by mismatches to the guide 3′ end. The introduction of 3′ mismatches leads to more potent silencing of abundant mRNAs in mammalian cells. These findings help to explain why the 3′ ends of mammalian microRNAs (miRNAs) rarely match their targets, suggest a mechanism for sequence-specific small RNA turnover, and offer insights for controlling small RNAs in mammalian cells. PMID:23664376

  9. 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 (5.8S L rRNA) 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.

  10. 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

  11. Host-Pathogen interactions modulated by small RNAs.

    PubMed

    Islam, Waqar; Islam, Saif Ul; Qasim, Muhammad; Wang, Liande

    2017-07-03

    Biological processes such as defense mechanisms and microbial offense strategies are regulated through RNA induced interference in eukaryotes. Genetic mutations are modulated through biogenesis of small RNAs which directly impacts upon host development. Plant defense mechanisms are regulated and supported by a diversified group of small RNAs which are involved in streamlining several RNA interference pathways leading toward the initiation of pathogen gene silencing mechanisms. In the similar context, pathogens also utilize the support of small RNAs to launch their offensive attacks. Also there are strong evidences about the active involvement of these RNAs in symbiotic associations. Interestingly, small RNAs are not limited to the individuals in whom they are produced; they also show cross kingdom influences through variable interactions with other species thus leading toward the inter-organismic gene silencing. The phenomenon is understandable in the microbes which utilize these mechanisms to overcome host defense line. Understanding the mechanism of triggering host defense strategies can be a valuable step toward the generation of disease resistant host plants. We think that the cross kingdom trafficking of small RNA is an interesting insight that is needed to be explored for its vitality.

  12. Host-Pathogen interactions modulated by small RNAs

    PubMed Central

    Islam, Waqar; Islam, Saif ul; Qasim, Muhammad; Wang, Liande

    2017-01-01

    ABSTRACT Biological processes such as defense mechanisms and microbial offense strategies are regulated through RNA induced interference in eukaryotes. Genetic mutations are modulated through biogenesis of small RNAs which directly impacts upon host development. Plant defense mechanisms are regulated and supported by a diversified group of small RNAs which are involved in streamlining several RNA interference pathways leading toward the initiation of pathogen gene silencing mechanisms. In the similar context, pathogens also utilize the support of small RNAs to launch their offensive attacks. Also there are strong evidences about the active involvement of these RNAs in symbiotic associations. Interestingly, small RNAs are not limited to the individuals in whom they are produced; they also show cross kingdom influences through variable interactions with other species thus leading toward the inter-organismic gene silencing. The phenomenon is understandable in the microbes which utilize these mechanisms to overcome host defense line. Understanding the mechanism of triggering host defense strategies can be a valuable step toward the generation of disease resistant host plants. We think that the cross kingdom trafficking of small RNA is an interesting insight that is needed to be explored for its vitality. PMID:28430077

  13. Small RNAs Reflect Grandparental Environments in Apomictic Dandelion

    PubMed Central

    Morgado, Lionel; Preite, Veronica; Oplaat, Carla; Anava, Sarit; Ferreira de Carvalho, Julie; Rechavi, Oded; Johannes, Frank; Verhoeven, Koen J.F.

    2017-01-01

    Abstract Plants can show long-term effects of environmental stresses and in some cases a stress “memory” has been reported to persist across generations, potentially mediated by epigenetic mechanisms. However, few documented cases exist of transgenerational effects that persist for multiple generations and it remains unclear if or how epigenetic mechanisms are involved. Here, we show that the composition of small regulatory RNAs in apomictic dandelion lineages reveals a footprint of drought stress and salicylic acid treatment experienced two generations ago. Overall proportions of 21 and 24 nt RNA pools were shifted due to grandparental treatments. While individual genes did not show strong up- or downregulation of associated sRNAs, the subset of genes that showed the strongest shifts in sRNA abundance was significantly enriched for several GO terms including stress-specific functions. This suggests that a stress-induced signal was transmitted across multiple unexposed generations leading to persistent changes in epigenetic gene regulation. PMID:28472380

  14. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Isolation and characterization of the genes for two small RNAs of herpesvirus papio and their comparison with Epstein-Barr virus-encoded EBER RNAs.

    PubMed Central

    Howe, J G; Shu, M D

    1988-01-01

    Genes for the Epstein-Barr virus-encoded RNAs (EBERs), two low-molecular-weight RNAs encoded by the human gammaherpesvirus Epstein-Barr virus (EBV), hybridize to two small RNAs in a baboon cell line that contains a similar virus, herpesvirus papio (HVP). The genes for the HVP RNAs (HVP-1 and HVP-2) are located together in the small unique region at the left end of the viral genome and are transcribed by RNA polymerase III in a rightward direction, similar to the EBERs. There is significant similarity between EBER1 and HVP-1 RNA, except for an insert of 22 nucleotides which increases the length of HVP-1 RNA to 190 nucleotides. There is less similarity between the sequences of EBER2 and HVP-2 RNA, but both have a length of about 170 nucleotides. The predicted secondary structure of each HVP RNA is remarkably similar to that of the respective EBER, implying that the secondary structures are important for function. Upstream from the initiation sites of all four RNA genes are several highly conserved sequences which may function in the regulation of transcription. The HVP RNAs, together with the EBERs, are highly abundant in transformed cells and are efficiently bound by the cellular La protein. Images PMID:2839701

  16. Isolation and characterization of the genes for two small RNAs of herpesvirus papio and their comparison with Epstein-Barr virus-encoded EBER RNAs.

    PubMed

    Howe, J G; Shu, M D

    1988-08-01

    Genes for the Epstein-Barr virus-encoded RNAs (EBERs), two low-molecular-weight RNAs encoded by the human gammaherpesvirus Epstein-Barr virus (EBV), hybridize to two small RNAs in a baboon cell line that contains a similar virus, herpesvirus papio (HVP). The genes for the HVP RNAs (HVP-1 and HVP-2) are located together in the small unique region at the left end of the viral genome and are transcribed by RNA polymerase III in a rightward direction, similar to the EBERs. There is significant similarity between EBER1 and HVP-1 RNA, except for an insert of 22 nucleotides which increases the length of HVP-1 RNA to 190 nucleotides. There is less similarity between the sequences of EBER2 and HVP-2 RNA, but both have a length of about 170 nucleotides. The predicted secondary structure of each HVP RNA is remarkably similar to that of the respective EBER, implying that the secondary structures are important for function. Upstream from the initiation sites of all four RNA genes are several highly conserved sequences which may function in the regulation of transcription. The HVP RNAs, together with the EBERs, are highly abundant in transformed cells and are efficiently bound by the cellular La protein.

  17. 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

  18. MicroRNAs in the Pineal Gland

    PubMed Central

    Clokie, Samuel J. H.; Lau, Pierre; Kim, Hyun Hee; Coon, Steven L.; Klein, David C.

    2012-01-01

    MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ∼75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3′-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis. PMID:22908386

  19. Human Virus-Derived Small RNAs Can Confer Antiviral Immunity in Mammals.

    PubMed

    Qiu, Yang; Xu, Yanpeng; Zhang, Yao; Zhou, Hui; Deng, Yong-Qiang; Li, Xiao-Feng; Miao, Meng; Zhang, Qiang; Zhong, Bo; Hu, Yuanyang; Zhang, Fu-Chun; Wu, Ligang; Qin, Cheng-Feng; Zhou, Xi

    2017-06-20

    RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates; however, whether RNAi plays antiviral roles in mammals remains unclear. Here, using human enterovirus 71 (HEV71) as a model, we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired, the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner and loaded into AGO, and they were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice, whereas Dicer deficiency rescued HEV71 infection independently of type I interferon response. Thus, RNAi can function as an antiviral immunity, which is induced and suppressed by a human virus, in mammals. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Extracellular small RNAs: what, where, why?

    PubMed Central

    Hoy, Anna M.; Buck, Amy H.

    2012-01-01

    miRNAs (microRNAs) are a class of small RNA that regulate gene expression by binding to mRNAs and modulating the precise amount of proteins that get expressed in a cell at a given time. This form of gene regulation plays an important role in developmental systems and is critical for the proper function of numerous biological pathways. Although miRNAs exert their functions inside the cell, these and other classes of RNA are found in body fluids in a cell-free form that is resistant to degradation by RNases. A broad range of cell types have also been shown to secrete miRNAs in association with components of the RISC (RNA-induced silencing complex) and/or encapsulation within vesicles, which can be taken up by other cells. In the present paper, we provide an overview of the properties of extracellular miRNAs in relation to their capacity as biomarkers, stability against degradation and mediators of cell–cell communication. PMID:22817753

  1. Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs.

    PubMed

    Miesen, Pascal; Ivens, Alasdair; Buck, Amy H; van Rij, Ronald P

    2016-02-01

    In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species.

  2. ADAR2 induces reproducible changes in sequence and abundance of mature microRNAs in the mouse brain

    PubMed Central

    Vesely, Cornelia; Tauber, Stefanie; Sedlazeck, Fritz J.; Tajaddod, Mansoureh; von Haeseler, Arndt; Jantsch, Michael F.

    2014-01-01

    Adenosine deaminases that act on RNA (ADARs) deaminate adenosines to inosines in double-stranded RNAs including miRNA precursors. A to I editing is widespread and required for normal life. By comparing deep sequencing data of brain miRNAs from wild-type and ADAR2 deficient mouse strains, we detect editing sites and altered miRNA processing at high sensitivity. We detect 48 novel editing events in miRNAs. Some editing events reach frequencies of up to 80%. About half of all editing events depend on ADAR2 while some miRNAs are preferentially edited by ADAR1. Sixty-four percent of all editing events are located within the seed region of mature miRNAs. For the highly edited miR-3099, we experimentally prove retargeting of the edited miRNA to novel 3′ UTRs. We show further that an abundant editing event in miR-497 promotes processing by Drosha of the corresponding pri-miRNA. We also detect reproducible changes in the abundance of specific miRNAs in ADAR2-deficient mice that occur independent of adjacent A to I editing events. This indicates that ADAR2 binding but not editing of miRNA precursors may influence their processing. Correlating with changes in miRNA abundance we find misregulation of putative targets of these miRNAs in the presence or absence of ADAR2. PMID:25260591

  3. Emerging Roles of Small Epstein-Barr Virus Derived Non-Coding RNAs in Epithelial Malignancy

    PubMed Central

    Lung, Raymond Wai-Ming; Tong, Joanna Hung-Man; To, Ka-Fai

    2013-01-01

    Latent Epstein-Barr virus (EBV) infection is an etiological factor in the progression of several human epithelial malignancies such as nasopharyngeal carcinoma (NPC) and a subset of gastric carcinoma. Reports have shown that EBV produces several viral oncoproteins, yet their pathological roles in carcinogenesis are not fully elucidated. Studies on the recently discovered of EBV-encoded microRNAs (ebv-miRNAs) showed that these small molecules function as post-transcriptional gene regulators and may play a role in the carcinogenesis process. In NPC and EBV positive gastric carcinoma (EBVaGC), 22 viral miRNAs which are located in the long alternative splicing EBV transcripts, named BamH1 A rightward transcripts (BARTs), are abundantly expressed. The importance of several miR-BARTs in carcinogenesis has recently been demonstrated. These novel findings enhance our understanding of the oncogenic properties of EBV and may lead to a more effective design of therapeutic regimens to combat EBV-associated malignancies. This article will review the pathological roles of miR-BARTs in modulating the expression of cancer-related genes in both host and viral genomes. The expression of other small non-coding RNAs in NPC and the expression pattern of miR-BARTs in rare EBV-associated epithelial cancers will also be discussed. PMID:23979421

  4. RISC assembly: Coordination between small RNAs and Argonaute proteins.

    PubMed

    Kobayashi, Hotaka; Tomari, Yukihide

    2016-01-01

    Non-coding RNAs generally form ribonucleoprotein (RNP) complexes with their partner proteins to exert their functions. Small RNAs, including microRNAs, small interfering RNAs, and PIWI-interacting RNAs, assemble with Argonaute (Ago) family proteins into the effector complex called RNA-induced silencing complex (RISC), which mediates sequence-specific target gene silencing. RISC assembly is not a simple binding between a small RNA and Ago; rather, it follows an ordered multi-step pathway that requires specific accessory factors. Some steps of RISC assembly and RISC-mediated gene silencing are dependent on or facilitated by particular intracellular platforms, suggesting their spatial regulation. In this review, we summarize the currently known mechanisms for RISC assembly of each small RNA class and propose a revised model for the role of the chaperone machinery in the duplex-initiated RISC assembly pathway. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs

    PubMed Central

    Miesen, Pascal; Ivens, Alasdair; Buck, Amy H.; van Rij, Ronald P.

    2016-01-01

    In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species. PMID:26914027

  6. Dynamic evolution and biogenesis of small RNAs during sex reversal.

    PubMed

    Liu, Jie; Luo, Majing; Sheng, Yue; Hong, Qiang; Cheng, Hanhua; Zhou, Rongjia

    2015-05-06

    Understanding origin, evolution and functions of small RNA (sRNA) genes has been a great challenge in the past decade. Molecular mechanisms underlying sexual reversal in vertebrates, particularly sRNAs involved in this process, are largely unknown. By deep-sequencing of small RNA transcriptomes in combination with genomic analysis, we identified a large amount of piRNAs and miRNAs including over 1,000 novel miRNAs, which were differentially expressed during gonad reversal from ovary to testis via ovotesis. Biogenesis and expressions of miRNAs were dynamically changed during the reversal. Notably, phylogenetic analysis revealed dynamic expansions of miRNAs in vertebrates and an evolutionary trajectory of conserved miR-17-92 cluster in the Eukarya. We showed that the miR-17-92 cluster in vertebrates was generated through multiple duplications from ancestor miR-92 in invertebrates Tetranychus urticae and Daphnia pulex from the Chelicerata around 580 Mya. Moreover, we identified the sexual regulator Dmrt1 as a direct target of the members miR-19a and -19b in the cluster. These data suggested dynamic biogenesis and expressions of small RNAs during sex reversal and revealed multiple expansions and evolutionary trajectory of miRNAs from invertebrates to vertebrates, which implicate small RNAs in sexual reversal and provide new insight into evolutionary and molecular mechanisms underlying sexual reversal.

  7. Combination of small RNAs for skeletal muscle regeneration.

    PubMed

    Kim, NaJung; Yoo, James J; Atala, Anthony; Lee, Sang Jin

    2016-03-01

    Selectively controlling the expression of the target genes through RNA interference (RNAi) has significant therapeutic potential for injuries or diseases of tissues. We used this strategy to accelerate and enhance skeletal muscle regeneration for the treatment of muscular atrophy. In this study, we used myostatin small interfering (si)RNA (siGDF-8), a major inhibitory factor in the development and postnatal regeneration of skeletal muscle and muscle-specific microRNAs (miR-1 and -206) to further accelerate muscle regeneration. This combination of 3 small RNAs significantly improved the gene expression of myogenic regulatory factors in vitro, suggesting myogenic activation. Moreover, cell proliferation and myotube formation improved without compromising each other, which indicates the myogenic potential of this combination of small RNAs. The recovery of chemically injured tibialis anterior muscles in rats was significantly accelerated, both functionally and structurally. This novel combination of siRNA and miRNAs has promising therapeutic potential to improve in situ skeletal muscle regeneration. © FASEB.

  8. A novel class of small RNAs bind to MILI protein in mouse testes.

    PubMed

    Aravin, Alexei; Gaidatzis, Dimos; Pfeffer, Sébastien; Lagos-Quintana, Mariana; Landgraf, Pablo; Iovino, Nicola; Morris, Patricia; Brownstein, Michael J; Kuramochi-Miyagawa, Satomi; Nakano, Toru; Chien, Minchen; Russo, James J; Ju, Jingyue; Sheridan, Robert; Sander, Chris; Zavolan, Mihaela; Tuschl, Thomas

    2006-07-13

    Small RNAs bound to Argonaute proteins recognize partially or fully complementary nucleic acid targets in diverse gene-silencing processes. A subgroup of the Argonaute proteins--known as the 'Piwi family'--is required for germ- and stem-cell development in invertebrates, and two Piwi members--MILI and MIWI--are essential for spermatogenesis in mouse. Here we describe a new class of small RNAs that bind to MILI in mouse male germ cells, where they accumulate at the onset of meiosis. The sequences of the over 1,000 identified unique molecules share a strong preference for a 5' uridine, but otherwise cannot be readily classified into sequence families. Genomic mapping of these small RNAs reveals a limited number of clusters, suggesting that these RNAs are processed from long primary transcripts. The small RNAs are 26-31 nucleotides (nt) in length--clearly distinct from the 21-23 nt of microRNAs (miRNAs) or short interfering RNAs (siRNAs)--and we refer to them as 'Piwi-interacting RNAs' or piRNAs. Orthologous human chromosomal regions also give rise to small RNAs with the characteristics of piRNAs, but the cloned sequences are distinct. The identification of this new class of small RNAs provides an important starting point to determine the molecular function of Piwi proteins in mammalian spermatogenesis.

  9. Small silencing RNAs: state-of-the-art.

    PubMed

    Grimm, Dirk

    2009-07-25

    Over just a single decade, we have witnessed the rapid maturation of the field of RNA interference - the sequence-specific gene silencing mediated by small double-stranded RNAs - directly from its infancy into adulthood. With exciting data currently emerging from first clinical trials, it is now more likely than ever that RNAi drugs will soon provide another potent class of agents in our battle against infectious and genetic diseases. Accelerating this process and adding to RNAi's promise is our steadily expanding arsenal of innovative RNAi-based experimental tools and clinically applicable technologies. This article will critically review a selection of relevant recent advances in RNAi therapeutics, from novel asymmetric or bi-functional siRNA designs, deliberate use of small RNAs to regulate nuclear transcription, engineering of potent adeno-associated viral vectors for shRNA expression, exploitation of endogenous miRNAs to control transgene expression or vector tropism, to elegant attempts to inhibit cellular miRNAs involved in human disease. This review will also present cautionary notes on the potential risks inherent to in vivo RNAi applications, before discussing the latest surprising findings on circulating miRNAs in human body fluids, and concluding with an outlook into the possible future of RNAi as an increasingly powerful biomedical tool.

  10. Small non coding RNAs in adipocyte biology and obesity.

    PubMed

    Amri, Ez-Zoubir; Scheideler, Marcel

    2017-11-15

    Obesity has reached epidemic proportions world-wide and constitutes a substantial risk factor for hypertension, type 2 diabetes, cardiovascular diseases and certain cancers. So far, regulation of energy intake by dietary and pharmacological treatments has met limited success. The main interest of current research is focused on understanding the role of different pathways involved in adipose tissue function and modulation of its mass. Whole-genome sequencing studies revealed that the majority of the human genome is transcribed, with thousands of non-protein-coding RNAs (ncRNA), which comprise small and long ncRNAs. ncRNAs regulate gene expression at the transcriptional and post-transcriptional level. Numerous studies described the involvement of ncRNAs in the pathogenesis of many diseases including obesity and associated metabolic disorders. ncRNAs represent potential diagnostic biomarkers and promising therapeutic targets. In this review, we focused on small ncRNAs involved in the formation and function of adipocytes and obesity. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. 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. © 2014 Scandinavian Plant Physiology Society.

  12. 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

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

    PubMed

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

    2016-01-01

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

  14. Identification of two small RNAs within the first 1.5-kb of the herpes simplex virus type 1-encoded latency-associated transcript.

    PubMed

    Peng, Weiping; Vitvitskaia, Olga; Carpenter, Dale; Wechsler, Steven L; Jones, Clinton

    2008-01-01

    The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected neurons. In the rabbit or mouse ocular models of infection, expression of the first 1.5 kb of LAT coding sequences is sufficient for and necessary for wild-type levels of spontaneous reactivation from latency. The antiapoptosis functions of LAT, which maps to the same 1.5 kb of LAT, are important for the latency-reactivation cycle because replacement of LAT with other antiapoptosis genes (the baculovirus IAP gene or the bovine herpesvirus type 1 latency-related gene) restores wild-type levels of reactivation to a LAT null mutant. A recent study identified a micro-RNA within LAT that can inhibit apoptosis (Gupta et al, Nature 442: 82-85). In this study, the authors analyzed the first 1.5 kb of LAT for additional small RNAs that may have regulatory functions. Two LAT-specific small RNAs were detected in productively infected human neuroblastoma cells within the first 1.5 kb of LAT, in a region that is important for inhibiting apoptosis. Although these small RNAs possess extensive secondary structure and a stem-loop structure, bands migrating near 23 bases were not detected suggesting these small RNAs are not true micro-RNAs. Both of the small LAT-specific RNAs have the potential to base pair with the ICP4 mRNA. These two small LAT RNAs may play a role in the latency-reactivation cycle by reducing apoptosis and/or by reducing ICP4 RNA expression.

  15. High-throughput sequencing of small RNAs and analysis of differentially expressed microRNAs associated with pistil development in Japanese apricot

    PubMed Central

    2012-01-01

    Background MicroRNAs (miRNAs) are a class of endogenous, small, non-coding RNAs that regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in high plants. However, the diversity of miRNAs and their roles in floral development in Japanese apricot (Prunus mume Sieb. et Zucc) remains largely unexplored. Imperfect flowers with pistil abortion seriously decrease production yields. To understand the role of miRNAs in pistil development, pistil development-related miRNAs were identified by Solexa sequencing in Japanese apricot. Results Solexa sequencing was used to identify and quantitatively profile small RNAs from perfect and imperfect flower buds of Japanese apricot. A total of 22,561,972 and 24,952,690 reads were sequenced from two small RNA libraries constructed from perfect and imperfect flower buds, respectively. Sixty-one known miRNAs, belonging to 24 families, were identified. Comparative profiling revealed that seven known miRNAs exhibited significant differential expression between perfect and imperfect flower buds. A total of 61 potentially novel miRNAs/new members of known miRNA families were also identified by the presence of mature miRNAs and corresponding miRNA*s in the sRNA libraries. Comparative analysis showed that six potentially novel miRNAs were differentially expressed between perfect and imperfect flower buds. Target predictions of the 13 differentially expressed miRNAs resulted in 212 target genes. Gene ontology (GO) annotation revealed that high-ranking miRNA target genes are those implicated in the developmental process, the regulation of transcription and response to stress. Conclusions This study represents the first comparative identification of miRNAomes between perfect and imperfect Japanese apricot flowers. Seven known miRNAs and six potentially novel miRNAs associated with pistil development were identified, using high-throughput sequencing of small RNAs. The findings, both computationally

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

    PubMed Central

    Travis, G H; Sutcliffe, J G

    1988-01-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we 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. Images PMID:2894033

  17. Mobile small RNAs regulate genome-wide DNA methylation.

    PubMed

    Lewsey, Mathew G; Hardcastle, Thomas J; Melnyk, Charles W; Molnar, Attila; Valli, Adrián; Urich, Mark A; Nery, Joseph R; Baulcombe, David C; Ecker, Joseph R

    2016-02-09

    RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21-24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession.

  18. 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

  19. Expression profile of small RNAs in Acacia mangium secondary xylem tissue with contrasting lignin content - potential regulatory sequences in monolignol biosynthetic pathway

    PubMed Central

    2011-01-01

    Background Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component during wood formation, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes in controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the entire monolignol biosynthesis pathway. As small RNAs have critical functions in various biological process during wood formation, small RNA profiling is an important tool for the identification of complete set of differentially expressed small RNAs between low lignin and high lignin secondary xylem. Results In line with this, we have generated two small RNAs libraries from samples with contrasting lignin content using Illumina GAII sequencer. About 10 million sequence reads were obtained in secondary xylem of Am48 with high lignin content (41%) and a corresponding 14 million sequence reads were obtained in secondary xylem of Am54 with low lignin content (21%). Our results suggested that A. mangium small RNAs are composed of a set of 12 highly conserved miRNAs families found in plant miRNAs database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. The predicted target genes of those differentially expressed conserved and non-conserved miRNAs include transcription factors associated with regulation of the lignin biosynthetic pathway genes. Some of these small RNAs play an important role in

  20. Expression profile of small RNAs in Acacia mangium secondary xylem tissue with contrasting lignin content - potential regulatory sequences in monolignol biosynthetic pathway.

    PubMed

    Ong, Seong Siang; Wickneswari, Ratnam

    2011-11-30

    Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component during wood formation, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes in controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the entire monolignol biosynthesis pathway. As small RNAs have critical functions in various biological process during wood formation, small RNA profiling is an important tool for the identification of complete set of differentially expressed small RNAs between low lignin and high lignin secondary xylem. In line with this, we have generated two small RNAs libraries from samples with contrasting lignin content using Illumina GAII sequencer. About 10 million sequence reads were obtained in secondary xylem of Am48 with high lignin content (41%) and a corresponding 14 million sequence reads were obtained in secondary xylem of Am54 with low lignin content (21%). Our results suggested that A. mangium small RNAs are composed of a set of 12 highly conserved miRNAs families found in plant miRNAs database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. The predicted target genes of those differentially expressed conserved and non-conserved miRNAs include transcription factors associated with regulation of the lignin biosynthetic pathway genes. Some of these small RNAs play an important role in epigenetic silencing

  1. High-resolution identification and abundance profiling of cassava (Manihot esculenta Crantz) microRNAs.

    PubMed

    Khatabi, Behnam; Arikit, Siwaret; Xia, Rui; Winter, Stephan; Oumar, Doungous; Mongomake, Kone; Meyers, Blake C; Fondong, Vincent N

    2016-01-28

    Small RNAs (sRNAs) are endogenous sRNAs that play regulatory roles in plant growth, development, and biotic and abiotic stress responses. In plants, one subset of sRNAs, microRNAs (miRNAs) exhibit tissue-differential expression and regulate gene expression mainly through direct cleavage of mRNA or indirectly via production of secondary phased siRNAs (phasiRNAs) that silence cognate target transcripts in trans. Here, we have identified cassava (Manihot esculenta Crantz) miRNAs using high resolution sequencing of sRNA libraries from leaf, stem, callus, male and female flower tissues. To analyze the data, we built a cassava genome database and, via sequence analysis and secondary structure prediction, 38 miRNAs not previously reported in cassava were identified. These new cassava miRNAs included two miRNAs not previously been reported in any plant species. The miRNAs exhibited tissue-differential accumulation as confirmed by quantitative RT-PCR and Northern blot analysis, largely reflecting levels observed in sequencing data. Some of the miRNAs identified were predicted to trigger production of secondary phased siRNAs (phasiRNAs) from 80 PHAS loci. Cassava is a woody perennial shrub, grown principally for its starch-rich storage roots, which are rich in calories. In this study, new miRNAs were identified and their expression was validated using qRT-PCR of RNA from five different tissues. The data obtained expand the list of annotated miRNAs and provide additional new resources for cassava improvement research.

  2. Functional Interplay between Small Non-Coding RNAs and RNA Modification in the Brain.

    PubMed

    Leighton, Laura J; Bredy, Timothy W

    2018-06-07

    Small non-coding RNAs are essential for transcription, translation and gene regulation in all cell types, but are particularly important in neurons, with known roles in neurodevelopment, neuroplasticity and neurological disease. Many small non-coding RNAs are directly involved in the post-transcriptional modification of other RNA species, while others are themselves substrates for modification, or are functionally modulated by modification of their target RNAs. In this review, we explore the known and potential functions of several distinct classes of small non-coding RNAs in the mammalian brain, focusing on the newly recognised interplay between the epitranscriptome and the activity of small RNAs. We discuss the potential for this relationship to influence the spatial and temporal dynamics of gene activation in the brain, and predict that further research in the field of epitranscriptomics will identify interactions between small RNAs and RNA modifications which are essential for higher order brain functions such as learning and memory.

  3. A comparative study of sequence- and structure-based features of small RNAs and other RNAs of bacteria.

    PubMed

    Barik, Amita; Das, Santasabuj

    2018-01-02

    Small RNAs (sRNAs) in bacteria have emerged as key players in transcriptional and post-transcriptional regulation of gene expression. Here, we present a statistical analysis of different sequence- and structure-related features of bacterial sRNAs to identify the descriptors that could discriminate sRNAs from other bacterial RNAs. We investigated a comprehensive and heterogeneous collection of 816 sRNAs, identified by northern blotting across 33 bacterial species and compared their various features with other classes of bacterial RNAs, such as tRNAs, rRNAs and mRNAs. We observed that sRNAs differed significantly from the rest with respect to G+C composition, normalized minimum free energy of folding, motif frequency and several RNA-folding parameters like base-pairing propensity, Shannon entropy and base-pair distance. Based on the selected features, we developed a predictive model using Random Forests (RF) method to classify the above four classes of RNAs. Our model displayed an overall predictive accuracy of 89.5%. These findings would help to differentiate bacterial sRNAs from other RNAs and further promote prediction of novel sRNAs in different bacterial species.

  4. Validation of Small RNAs In Xylella fastidiosa by qRT-PCR

    USDA-ARS?s Scientific Manuscript database

    Xylella fastidiosa causes many economically important crop diseases including almond leaf scorch disease and Pierce’ disease of grapevine. Although non-coding small RNAs (sRNAs) are regarded as ubiquitous regulatory elements in bacteria, research attention to sRNAs in X. fastidiosa has been limited...

  5. Massive Analysis of Rice Small RNAs: Mechanistic Implications of Regulated MicroRNAs and Variants for Differential Target RNA Cleavage[W][OA

    PubMed Central

    Jeong, Dong-Hoon; Park, Sunhee; Zhai, Jixian; Gurazada, Sai Guna Ranjan; De Paoli, Emanuele; Meyers, Blake C.; Green, Pamela J.

    2011-01-01

    Small RNAs have a variety of important roles in plant development, stress responses, and other processes. They exert their influence by guiding mRNA cleavage, translational repression, and chromatin modification. To identify previously unknown rice (Oryza sativa) microRNAs (miRNAs) and those regulated by environmental stress, 62 small RNA libraries were constructed from rice plants and used for deep sequencing with Illumina technology. The libraries represent several tissues from control plants and plants subjected to different environmental stress treatments. More than 94 million genome-matched reads were obtained, resulting in more than 16 million distinct small RNA sequences. This allowed an evaluation of ~400 annotated miRNAs with current criteria and the finding that among these, ~150 had small interfering RNA–like characteristics. Seventy-six new miRNAs were found, and miRNAs regulated in response to water stress, nutrient stress, or temperature stress were identified. Among the new examples of miRNA regulation were members of the same miRNA family that were differentially regulated in different organs and had distinct sequences Some of these distinct family members result in differential target cleavage and provide new insight about how an agriculturally important rice phenotype could be regulated in the panicle. This high-resolution analysis of rice miRNAs should be relevant to plant miRNAs in general, particularly in the Poaceae. PMID:22158467

  6. Small and Smaller-sRNAs and MicroRNAs in the Regulation of Toxin Gene Expression in Prokaryotic Cells: A Mini-Review.

    PubMed

    Bloch, Sylwia; Węgrzyn, Alicja; Węgrzyn, Grzegorz; Nejman-Faleńczyk, Bożena

    2017-05-30

    Non-coding small RNAs (sRNAs) have been identified in the wide range of bacteria (also pathogenic species) and found to play an important role in the regulation of many processes, including toxin gene expression. The best characterized prokaryotic sRNAs regulate gene expression by base pairing with mRNA targets and fall into two broad classes: cis -encoded sRNAs (also called antisense RNA) and trans -acting sRNAs. Molecules from the second class are frequently considered as the most related to eukaryotic microRNAs. Interestingly, typical microRNA-size RNA molecules have also been reported in prokaryotic cells, although they have received little attention up to now. In this work we have collected information about all three types of small prokaryotic RNAs in the context of the regulation of toxin gene expression.

  7. New Insight into Inter-kingdom Communication: Horizontal Transfer of Mobile Small RNAs.

    PubMed

    Zhou, Geyu; Zhou, Yu; Chen, Xi

    2017-01-01

    Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), are conventionally regarded as critical molecular regulators of various intracellular processes. However, recent accumulating evidence indicates that sRNAs can be transferred within cells and tissues and even across species. In plants, nematodes and microbes, these mobile sRNAs can mediate inter-kingdom communication, environmental sensing, gene expression regulation, host-parasite defense and many other biological functions. Strikingly, a recent study by our group suggested that ingested plant miRNAs are transferred to blood, accumulate in tissues and regulate transcripts in consuming animals. While our and other independent groups' subsequent studies further explored the emerging field of sRNA-mediated crosstalk between species, some groups reported negative results and questioned its general applicability. Thus, further studies carefully evaluating the horizontal transfer of exogenous sRNAs and its potential biological functions are urgently required. Here, we review the current state of knowledge in the field of the horizontal transfer of mobile sRNAs, suggest its future directions and key points for examination and discuss its potential mechanisms and application prospects in nutrition, agriculture and medicine.

  8. Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.

    PubMed

    Gangras, Pooja; Dayeh, Daniel M; Mabin, Justin W; Nakanishi, Kotaro; Singh, Guramrit

    2018-01-01

    Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.

  9. Small and Smaller—sRNAs and MicroRNAs in the Regulation of Toxin Gene Expression in Prokaryotic Cells: A Mini-Review

    PubMed Central

    Bloch, Sylwia; Węgrzyn, Alicja; Węgrzyn, Grzegorz; Nejman-Faleńczyk, Bożena

    2017-01-01

    Non-coding small RNAs (sRNAs) have been identified in the wide range of bacteria (also pathogenic species) and found to play an important role in the regulation of many processes, including toxin gene expression. The best characterized prokaryotic sRNAs regulate gene expression by base pairing with mRNA targets and fall into two broad classes: cis-encoded sRNAs (also called antisense RNA) and trans-acting sRNAs. Molecules from the second class are frequently considered as the most related to eukaryotic microRNAs. Interestingly, typical microRNA-size RNA molecules have also been reported in prokaryotic cells, although they have received little attention up to now. In this work we have collected information about all three types of small prokaryotic RNAs in the context of the regulation of toxin gene expression. PMID:28556797

  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. USDA Potato Small RNA Database

    USDA-ARS?s Scientific Manuscript database

    Small RNAs (sRNAs) are now understood to be involved in gene regulation, function and development. High throughput sequencing (HTS) of sRNAs generates large data sets for analyzing the abundance, source and roles for specific sRNAs. These sRNAs result from transcript degradation as well as specific ...

  12. 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

  13. Plant Responses to Pathogen Attack: Small RNAs in Focus.

    PubMed

    Islam, Waqar; Noman, Ali; Qasim, Muhammad; Wang, Liande

    2018-02-08

    Small RNAs (sRNA) are a significant group of gene expression regulators for multiple biological processes in eukaryotes. In plants, many sRNA silencing pathways produce extensive array of sRNAs with specialized roles. The evidence on record advocates for the functions of sRNAs during plant microbe interactions. Host sRNAs are reckoned as mandatory elements of plant defense. sRNAs involved in plant defense processes via different pathways include both short interfering RNA (siRNA) and microRNA (miRNA) that actively regulate immunity in response to pathogenic attack via tackling pathogen-associated molecular patterns (PAMPs) and other effectors. In response to pathogen attack, plants protect themselves with the help of sRNA-dependent immune systems. That sRNA-mediated plant defense responses play a role during infections is an established fact. However, the regulations of several sRNAs still need extensive research. In this review, we discussed the topical advancements and findings relevant to pathogen attack and plant defense mediated by sRNAs. We attempted to point out diverse sRNAs as key defenders in plant systems. It is hoped that sRNAs would be exploited as a mainstream player to achieve food security by tackling different plant diseases.

  14. Plant Responses to Pathogen Attack: Small RNAs in Focus

    PubMed Central

    2018-01-01

    Small RNAs (sRNA) are a significant group of gene expression regulators for multiple biological processes in eukaryotes. In plants, many sRNA silencing pathways produce extensive array of sRNAs with specialized roles. The evidence on record advocates for the functions of sRNAs during plant microbe interactions. Host sRNAs are reckoned as mandatory elements of plant defense. sRNAs involved in plant defense processes via different pathways include both short interfering RNA (siRNA) and microRNA (miRNA) that actively regulate immunity in response to pathogenic attack via tackling pathogen-associated molecular patterns (PAMPs) and other effectors. In response to pathogen attack, plants protect themselves with the help of sRNA-dependent immune systems. That sRNA-mediated plant defense responses play a role during infections is an established fact. However, the regulations of several sRNAs still need extensive research. In this review, we discussed the topical advancements and findings relevant to pathogen attack and plant defense mediated by sRNAs. We attempted to point out diverse sRNAs as key defenders in plant systems. It is hoped that sRNAs would be exploited as a mainstream player to achieve food security by tackling different plant diseases. PMID:29419801

  15. 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

  16. 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.

  17. Bioinformatics Analysis of Small RNAs in Pima (Gossypium barbadense L.)

    PubMed Central

    Hu, Hongtao; Yu, Dazhao; Liu, Hong

    2015-01-01

    Small RNAs (sRNAs) are ~20 to 24 nucleotide single-stranded RNAs that play crucial roles in regulation of gene expression. In plants, sRNAs are classified into microRNAs (miRNAs), repeat-associated siRNAs (ra-siRNAs), phased siRNAs (pha-siRNAs), cis and trans natural antisense transcript siRNAs (cis- and trans-nat siRNAs). Pima (Gossypium barbadense L.) is one of the most economically important fiber crops, producing the best and longest spinnable fiber. Although some miRNAs are profiled in Pima, little is known about siRNAs, the largest subclass of plant sRNAs. In order to profile these gene regulators in Pima, a comprehensive analysis of sRNAs was conducted by mining publicly available sRNA data, leading to identification of 678 miRNAs, 3,559,126 ra-siRNAs, 627 pha-siRNAs, 136,600 cis-nat siRNAs and 79,994 trans-nat siRNAs. The 678 miRNAs, belonging to 98 conserved and 402 lineage-specific families, were produced from 2,138 precursors, of which 297 arose from introns, exons, or intron/UTR-exon junctions of protein-coding genes. Ra-siRNAs were produced from various repeat loci, while most (97%) were yielded from retrotransposons, especially LTRs (long terminal repeats). The genes encoding auxin-signaling-related proteins, NBS-LRRs and transcription factors were major sources of pha-siRNAs, while two conserved TAS3 homologs were found as well. Most cis-NATs in Pima overlapped in enclosed and convergent orientations, while a few hybridized in divergent and coincided orientations. Most cis- and trans-nat siRNAs were produced from overlapping regions. Additionally, characteristics of length and the 5’-first nucleotide of each sRNA class were analyzed as well. Results in this study created a valuable molecular resource that would facilitate studies on mechanism of controlling gene expression. PMID:25679373

  18. 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

  19. Global alteration of microRNAs and transposon-derived small RNAs in cotton (Gossypium hirsutum) during Cotton leafroll dwarf polerovirus (CLRDV) infection.

    PubMed

    Romanel, Elisson; Silva, Tatiane F; Corrêa, Régis L; Farinelli, Laurent; Hawkins, Jennifer S; Schrago, Carlos E G; Vaslin, Maite F S

    2012-11-01

    Small RNAs (sRNAs) are a class of non-coding RNAs ranging from 20- to 40-nucleotides (nts) that are present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, the maintenance of genome stability and the antiviral response. Viruses, however, can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the impact of viral infection on the plant sRNA pathway, we deep sequenced the sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV), which is a member of the economically important virus family Luteoviridae. A total of 60 putative conserved cotton miRNAs were identified, including 19 new miRNA families that had not been previously described in cotton. Some of these miRNAs were clearly misregulated during viral infection, and their possible role in symptom development and disease progression is discussed. Furthermore, we found that the 24-nt heterochromatin-associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation of at least one cotton transposable element. This is the first study to explore the global alterations of sRNAs in virus-infected cotton plants. Our results indicate that some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks.

  20. Genome-wide identification of Hfq-regulated small RNAs in the fire blight pathogen Erwinia amylovora discovered small RNAs with virulence regulatory function.

    PubMed

    Zeng, Quan; Sundin, George W

    2014-05-31

    Erwinia amylovora is a phytopathogenic bacterium and causal agent of fire blight disease in apples and pears. Although many virulence factors have been characterized, the coordination of expression of these virulence factors in E. amylovora is still not clear. Regulatory small RNAs (sRNAs) are important post-transcriptional regulatory components in bacteria. A large number of sRNAs require the RNA chaperone Hfq for both stability and functional activation. In E. amylovora, Hfq was identified as a major regulator of virulence and various virulence traits. However, information is still lacking about Hfq-dependent sRNAs on a genome scale, including the virulence regulatory functions of these sRNAs in E. amylovora. Using both an RNA-seq analysis and a Rho-independent terminator search, 40 candidate Hfq-dependent sRNAs were identified in E. amylovora. The expression and sizes of 12 sRNAs and the sequence boundaries of seven sRNAs were confirmed by Northern blot and 5' RACE assay respectively. Sequence conservation analysis identified sRNAs conserved only in the Erwinia genus as well as E. amylovora species-specific sRNAs. In addition, a dynamic re-patterning of expression of Hfq-dependent sRNAs was observed at 6 and 12 hours after induction in Hrp-inducing minimal medium. Furthermore, sRNAs that control virulence traits were characterized, among which ArcZ positively controls the type III secretion system (T3SS), amylovoran exopolysaccahride production, biofilm formation, and motility, and negatively modulates attachment while RmaA (Hrs6) and OmrAB both negatively regulate amylovoran production and positively regulate motility. This study has significantly enhanced our understanding of the Hfq-dependent sRNAs in E. amylovora at the genome level. The identification of multiple virulence-regulating sRNAs also suggests that post-transcriptional regulation by sRNAs may play a role in the deployment of virulence factors needed during varying stages of pathogenesis during

  1. Primary Characterization of Small RNAs in Symbiotic Nitrogen-Fixing Bacteria.

    PubMed

    Robledo, Marta; García-Tomsig, Natalia I; Jiménez-Zurdo, José I

    2018-01-01

    High-throughput transcriptome profiling (RNAseq) has uncovered large and heterogeneous populations of small noncoding RNA species (sRNAs) with potential regulatory roles in bacteria. A large fraction of sRNAs are differentially regulated and rely on protein-assisted antisense interactions to trans-encoded target mRNAs to fine-tune posttranscriptional reprogramming of gene expression in response to external cues. However, annotation and function of sRNAs are still largely overlooked in nonmodel bacteria with complex lifestyles. Here, we describe experimental protocols successfully applied for the accurate annotation, expression profiling and target mRNA identification of trans-acting sRNAs in the nitrogen-fixing α-rhizobium Sinorhizobium meliloti. The protocols presented here can be similarly applied for the characterization of trans-sRNAs in genetically tractable α-proteobacteria of agronomical or clinical relevance interacting with eukaryotic hosts.

  2. Riding in silence: a little snowboarding, a lot of small RNAs

    PubMed Central

    2010-01-01

    The recent symposium, RNA silencing: Mechanism, Biology and Applications, organized by Phillip D. Zamore (University of Massachusetts Medical School) and Beverly Davidson (University of Iowa), and held in Keystone, Colorado, brought together scientists working on diverse aspects of RNA silencing, a field that comprises a multitude of gene regulatory pathways guided by microRNAs, small interfering RNAs and PIWI-interacting RNAs. PMID:20230614

  3. 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. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Cloning and analysis of fetal ovary microRNAs in cattle.

    PubMed

    Tripurani, Swamy K; Xiao, Caide; Salem, Mohamed; Yao, Jianbo

    2010-07-01

    Ovarian folliculogenesis and early embryogenesis are complex processes, which require tightly regulated expression and interaction of a multitude of genes. Small endogenous RNA molecules, termed microRNAs (miRNAs), are involved in the regulation of gene expression during folliculogenesis and early embryonic development. To identify miRNAs in bovine oocytes/ovaries, a bovine fetal ovary miRNA library was constructed. Sequence analysis of random clones from the library identified 679 miRNA sequences, which represent 58 distinct bovine miRNAs. Of these distinct miRNAs, 42 are known bovine miRNAs present in the miRBase database and the remaining 16 miRNAs include 15 new bovine miRNAs that are homologous to miRNAs identified in other species, and one novel miRNA, which does not match any miRNAs in the database. The precursor sequences for 14 of the new 15 miRNAs as well as the novel miRNA were identified from the bovine genome database and their hairpin structures were predicted. Expression analysis of the 58 miRNAs in fetal ovaries in comparison to somatic tissue pools identified 8 miRNAs predominantly expressed in fetal ovaries. Further analysis of the eight miRNAs in germinal vesicle (GV) stage oocytes identified two miRNAs (bta-mir424 and bta-mir-10b), that are highly abundant in GV oocytes. Both miRNAs show similar expression patterns during oocyte maturation and preimplantation development of bovine embryos, being abundant in GV and MII stage oocytes, as well as in early stage embryos (until 16-cell stage). The amount of the novel miRNA is relatively small in oocytes and early cleavage embryos but greater in blastocysts, suggesting a role of this miRNA in blastocyst cell differentiation. Copyright 2010 Elsevier B.V. All rights reserved.

  5. Inventory of high-abundance mRNAs in skeletal muscle of normal men.

    PubMed

    Welle, S; Bhatt, K; Thornton, C A

    1999-05-01

    G42875rial analysis of gene expression (SAGE) method was used to generate a catalog of 53,875 short (14 base) expressed sequence tags from polyadenylated RNA obtained from vastus lateralis muscle of healthy young men. Over 12,000 unique tags were detected. The frequency of occurrence of each tag reflects the relative abundance of the corresponding mRNA. The mRNA species that were detected 10 or more times, each comprising >/=0.02% of the mRNA population, accounted for 64% of the mRNA mass but <10% of the total number of mRNA species detected. Almost all of the abundant tags matched mRNA or EST sequences cataloged in GenBank. Mitochondrial transcripts accounted for approximately 20% of the polyadenylated RNA. Transcripts encoding proteins of the myofibrils were the most abundant nuclear-encoded mRNAs. Transcripts encoding ribosomal proteins, and those encoding proteins involved in energy metabolism, also were very abundant. The database can be used as a reference for investigations of alterations in gene expression associated with conditions that influence muscle function, such as muscular dystrophies, aging, and exercise.

  6. Rapid and Efficient Isolation of High-Quality Small RNAs from Recalcitrant Plant Species Rich in Polyphenols and Polysaccharides

    PubMed Central

    Pu, Jinji; Guo, Jianrong; Fan, Zaifeng

    2014-01-01

    Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are important regulators of plant development and gene expression. The acquisition of high-quality small RNAs is the first step in the study of its expression and function analysis, yet the extraction method of small RNAs in recalcitrant plant tissues with various secondary metabolites is not well established, especially for tropical and subtropical plant species rich in polysaccharides and polyphenols. Here, we developed a simple and efficient method for high quality small RNAs extraction from recalcitrant plant species. Prior to RNA isolation, a precursory step with a CTAB-PVPP buffer system could efficiently remove compounds and secondary metabolites interfering with RNAs from homogenized lysates. Then, total RNAs were extracted by Trizol reagents followed by a differential precipitation of high-molecular-weight (HMW) RNAs using polyethylene glycol (PEG) 8000. Finally, small RNAs could be easily recovered from supernatant by ethanol precipitation without extra elimination steps. The isolated small RNAs from papaya showed high quality through a clear background on gel and a distinct northern blotting signal with miR159a probe, compared with other published protocols. Additionally, the small RNAs extracted from papaya were successfully used for validation of both predicted miRNAs and the putative conserved tasiARFs. Furthermore, the extraction method described here was also tested with several other subtropical and tropical plant tissues. The purity of the isolated small RNAs was sufficient for such applications as end-point stem-loop RT-PCR and northern blotting analysis, respectively. The simple and feasible extraction method reported here is expected to have excellent potential for isolation of small RNAs from recalcitrant plant tissues rich in polyphenols and polysaccharides. PMID:24787387

  7. 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.

  8. 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

  9. 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

  10. The function of small RNAs in plant biotic stress response.

    PubMed

    Huang, Juan; Yang, Meiling; Zhang, Xiaoming

    2016-04-01

    Small RNAs (sRNAs) play essential roles in plants upon biotic stress. Plants utilize RNA silencing machinery to facilitate pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity to defend against pathogen attack or to facilitate defense against insect herbivores. Pathogens, on the other hand, are also able to generate effectors and sRNAs to counter the host immune response. The arms race between plants and pathogens/insect herbivores has triggered the evolution of sRNAs, RNA silencing machinery and pathogen effectors. A great number of studies have been performed to investigate the roles of sRNAs in plant defense, bringing in the opportunity to utilize sRNAs in plant protection. Transgenic plants with pathogen-derived resistance ability or transgenerational defense have been generated, which show promising potential as solutions for pathogen/insect herbivore problems in the field. Here we summarize the recent progress on the function of sRNAs in response to biotic stress, mainly in plant-pathogen/insect herbivore interaction, and the application of sRNAs in disease and insect herbivore control. © 2016 Institute of Botany, Chinese Academy of Sciences.

  11. Virus-encoded microRNAs

    PubMed Central

    Grundhoff, Adam; Sullivan, Christopher S.

    2011-01-01

    microRNAs (miRNAs) are the subject of enormous interest. They are small non-coding RNAs that play a regulatory role in numerous and diverse cellular processes such as immune function, apoptosis and tumorigenesis. Several virus families have been shown to encode miRNAs, and an appreciation for their roles in the viral infectious cycle continues to grow. Despite the identification of numerous (>225) viral miRNAs, an in depth functional understanding of most virus-encoded miRNAs is lacking. Here we focus on a few viral miRNAs with well-defined functions. We use these examples to extrapolate general themes of viral miRNA activities including autoregulation of gene expression, avoidance of host defenses, and a likely important role in maintaining latent and persistent infections. We hypothesize that although the molecular mechanisms and machinery are similar, the majority of viral miRNAs may utilize a target strategy that differs from host miRNAs. That is, many viral miRNAs may have evolved to regulate viral-encoded transcripts or networks of host genes that are unique to viral miRNAs. Included in this latter category are a likely abundant class of viral miRNAs that may regulate only one or a few principal host genes. Key steps forward for the field are discussed, including the need for additional functional studies that utilize surgical viral miRNA mutants combined with relevant models of infection. PMID:21277611

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

    PubMed

    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

    2016-04-06

    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.

  13. Modifications in small nuclear RNAs and their roles in spliceosome assembly and function.

    PubMed

    Bohnsack, Markus T; Sloan, Katherine E

    2018-06-01

    Modifications in cellular RNAs have emerged as key regulators of all aspects of gene expression, including pre-mRNA splicing. During spliceosome assembly and function, the small nuclear RNAs (snRNAs) form numerous dynamic RNA-RNA and RNA-protein interactions, which are required for spliceosome assembly, correct positioning of the spliceosome on substrate pre-mRNAs and catalysis. The human snRNAs contain several base methylations as well as a myriad of pseudouridines and 2'-O-methylated nucleotides, which are largely introduced by small Cajal body-specific-RNPs. Modified nucleotides typically cluster in functionally important regions of the snRNAs, suggesting that their presence could optimise the interactions of snRNAs with each other or with pre-mRNAs, or may affect the binding of spliceosomal proteins. snRNA modifications appear to play important roles in snRNP biogenesis and spliceosome assembly, and have also been proposed to influence the efficiency and fidelity of pre-mRNAs splicing. Interestingly, alterations in the modification status of snRNAs have recently been observed in different cellular conditions, implying that some snRNA modifications are dynamic and raising the possibility that these modifications may fine-tune the spliceosome for particular functions. Here, we review the current knowledge on the snRNA modification machinery and discuss the timing, functions and dynamics of modifications in snRNAs.

  14. 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

  15. 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/.

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

    PubMed

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

    2016-01-01

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

  17. Sex specific expression and distribution of small RNAs in papaya.

    PubMed

    Aryal, Rishi; Jagadeeswaran, Guru; Zheng, Yun; Yu, Qingyi; Sunkar, Ramanjulu; Ming, Ray

    2014-01-13

    Regulatory function of small non-coding RNAs (sRNA) in response to environmental and developmental cues has been established. Additionally, sRNA, also plays an important role in maintaining the heterochromatin and centromere structures of the chromosome. Papaya, a trioecious species with recently evolved sex chromosomes, has emerged as an excellent model system to study sex determination and sex chromosome evolution in plants. However, role of small RNA in papaya sex determination is yet to be explored. We analyzed the high throughput sRNAs reads in the Illumina libraries prepared from male, female, and hermaphrodite flowers of papaya. Using the sRNA reads, we identified 29 miRNAs that were not previously reported from papaya. Including this and two previous studies, a total of 90 miRNAs has been identified in papaya. We analyzed the expression of these miRNAs in each sex types. A total of 65 miRNAs, including 31 conserved and 34 novel mirNA, were detected in at least one library. Fourteen of the 65 miRNAs were differentially expressed among different sex types. Most of the miRNA expressed higher in male flowers were related to the auxin signaling pathways, whereas the miRNAs expressed higher in female flowers were the potential regulators of the apical meristem identity genes. Aligning the sRNA reads identified the sRNA hotspots adjacent to the gaps of the X and Y chromosomes. The X and Y chromosomes sRNA hotspots has a 7.8 and 4.4 folds higher expression of sRNA, respectively, relative to the chromosome wide average. Approximately 75% of the reads aligned to the X chromosome hotspot was identical to that of the Y chromosome hotspot. By analyzing the large-scale sRNA sequences from three sex types, we identified the sRNA hotspots flanking the gaps of papaya X, Y, and Yh chromosome. The sRNAs expression patterns in these regions were reminiscent of the pericentromeric region indicating that the only remaining gap in each of these chromosomes is likely the

  18. Repression of small toxic protein synthesis by the Sib and OhsC small RNAs.

    PubMed

    Fozo, Elizabeth M; Kawano, Mitsuoki; Fontaine, Fanette; Kaya, Yusuf; Mendieta, Kathy S; Jones, Kristi L; Ocampo, Alejandro; Rudd, Kenneth E; Storz, Gisela

    2008-12-01

    The sequences encoding the QUAD1 RNAs were initially identified as four repeats in Escherichia coli. These repeats, herein renamed SIB, are conserved in closely related bacteria, although the number of repeats varies. All five Sib RNAs in E. coli MG1655 are expressed, and no phenotype was observed for a five-sib deletion strain. However, a phenotype reminiscent of plasmid addiction was observed for overexpression of the Sib RNAs, and further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18- to 19-amino-acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed and this toxicity could be prevented by coexpression of the corresponding Sib RNA. Two other RNAs encoded divergently in the yfhL-acpS intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression of both IbsC and ShoB led to immediate changes in membrane potential suggesting both proteins affect the cell envelope. Whole genome expression analysis showed that overexpression of IbsC and ShoB, as well as the small hydrophobic LdrD and TisB proteins, has both overlapping and unique consequences for the cell.

  19. Repression of small toxic protein synthesis by the Sib and OhsC small RNAs

    PubMed Central

    Fozo, Elizabeth M.; Kawano, Mitsuoki; Fontaine, Fanette; Kaya, Yusuf; Mendieta, Kathy S.; Jones, Kristi L.; Ocampo, Alejandro; Rudd, Kenneth E.; Storz, Gisela

    2008-01-01

    Summary The sequences encoding the QUAD1 RNAs were initially identified as four repeats in Escherichia coli. These repeats, herein renamed SIB, are conserved in closely related bacteria, though the number of repeats varies. All five Sib RNAs in E. coli MG1655 are expressed, and no phenotype was observed for a five sib deletion strain. However, a phenotype reminiscent of plasmid addiction was observed for overexpression of the Sib RNAs, and further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18–19 amino acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed and this toxicity could be prevented by co-expression of the corresponding Sib RNA. Two other RNAs encoded divergently in the yfhL-acpS intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression of both IbsC and ShoB led to immediate changes in membrane potential suggesting both proteins affect the cell envelope. Whole genome expression analysis showed that overexpression of IbsC and ShoB, as well as the small hydrophobic LdrD and TisB proteins, has both overlapping and unique consequences for the cell. PMID:18710431

  20. The presence, role and clinical use of spermatozoal RNAs

    PubMed Central

    Jodar, Meritxell; Selvaraju, Sellappan; Sendler, Edward; Diamond, Michael P.; Krawetz, Stephen A.

    2013-01-01

    BACKGROUND Spermatozoa are highly differentiated, transcriptionally inert cells characterized by a compact nucleus with minimal cytoplasm. Nevertheless they contain a suite of unique RNAs that are delivered to oocyte upon fertilization. They are likely integrated as part of many different processes including genome recognition, consolidation-confrontation, early embryonic development and epigenetic transgenerational inherence. Spermatozoal RNAs also provide a window into the developmental history of each sperm thereby providing biomarkers of fertility and pregnancy outcome which are being intensely studied. METHODS Literature searches were performed to review the majority of spermatozoal RNA studies that described potential functions and clinical applications with emphasis on Next-Generation Sequencing. Human, mouse, bovine and stallion were compared as their distribution and composition of spermatozoal RNAs, using these techniques, have been described. RESULTS Comparisons highlighted the complexity of the population of spermatozoal RNAs that comprises rRNA, mRNA and both large and small non-coding RNAs. RNA-seq analysis has revealed that only a fraction of the larger RNAs retain their structure. While rRNAs are the most abundant and are highly fragmented, ensuring a translationally quiescent state, other RNAs including some mRNAs retain their functional potential, thereby increasing the opportunity for regulatory interactions. Abundant small non-coding RNAs retained in spermatozoa include miRNAs and piRNAs. Some, like miR-34c are essential to the early embryo development required for the first cellular division. Others like the piRNAs are likely part of the genomic dance of confrontation and consolidation. Other non-coding spermatozoal RNAs include transposable elements, annotated lnc-RNAs, intronic retained elements, exonic elements, chromatin-associated RNAs, small-nuclear ILF3/NF30 associated RNAs, quiescent RNAs, mse-tRNAs and YRNAs. Some non-coding RNAs are

  1. Genome-wide identification of miRNAs and lncRNAs in Cajanus cajan.

    PubMed

    Nithin, Chandran; Thomas, Amal; Basak, Jolly; Bahadur, Ranjit Prasad

    2017-11-15

    Non-coding RNAs (ncRNAs) are important players in the post transcriptional regulation of gene expression (PTGR). On one hand, microRNAs (miRNAs) are an abundant class of small ncRNAs (~22nt long) that negatively regulate gene expression at the levels of messenger RNAs stability and translation inhibition, on the other hand, long ncRNAs (lncRNAs) are a large and diverse class of transcribed non-protein coding RNA molecules (> 200nt) that play both up-regulatory as well as down-regulatory roles at the transcriptional level. Cajanus cajan, a leguminosae pulse crop grown in tropical and subtropical areas of the world, is a source of high value protein to vegetarians or very poor populations globally. Hence, genome-wide identification of miRNAs and lncRNAs in C. cajan is extremely important to understand their role in PTGR with a possible implication to generate improve variety of crops. We have identified 616 mature miRNAs in C. cajan belonging to 118 families, of which 578 are novel and not reported in MirBase21. A total of 1373 target sequences were identified for 180 miRNAs. Of these, 298 targets were characterized at the protein level. Besides, we have also predicted 3919 lncRNAs. Additionally, we have identified 87 of the predicted lncRNAs to be targeted by 66 miRNAs. miRNA and lncRNAs in plants are known to control a variety of traits including yield, quality and stress tolerance. Owing to its agricultural importance and medicinal value, the identified miRNA, lncRNA and their targets in C. cajan may be useful for genome editing to improve better quality crop. A thorough understanding of ncRNA-based cellular regulatory networks will aid in the improvement of C. cajan agricultural traits.

  2. Dnmt2 mediates intergenerational transmission of paternally acquired metabolic disorders through sperm small non-coding RNAs.

    PubMed

    Zhang, Yunfang; Zhang, Xudong; Shi, Junchao; Tuorto, Francesca; Li, Xin; Liu, Yusheng; Liebers, Reinhard; Zhang, Liwen; Qu, Yongcun; Qian, Jingjing; Pahima, Maya; Liu, Ying; Yan, Menghong; Cao, Zhonghong; Lei, Xiaohua; Cao, Yujing; Peng, Hongying; Liu, Shichao; Wang, Yue; Zheng, Huili; Woolsey, Rebekah; Quilici, David; Zhai, Qiwei; Li, Lei; Zhou, Tong; Yan, Wei; Lyko, Frank; Zhang, Ying; Zhou, Qi; Duan, Enkui; Chen, Qi

    2018-05-01

    The discovery of RNAs (for example, messenger RNAs, non-coding RNAs) in sperm has opened the possibility that sperm may function by delivering additional paternal information aside from solely providing the DNA 1 . Increasing evidence now suggests that sperm small non-coding RNAs (sncRNAs) can mediate intergenerational transmission of paternally acquired phenotypes, including mental stress 2,3 and metabolic disorders 4-6 . How sperm sncRNAs encode paternal information remains unclear, but the mechanism may involve RNA modifications. Here we show that deletion of a mouse tRNA methyltransferase, DNMT2, abolished sperm sncRNA-mediated transmission of high-fat-diet-induced metabolic disorders to offspring. Dnmt2 deletion prevented the elevation of RNA modifications (m 5 C, m 2 G) in sperm 30-40 nt RNA fractions that are induced by a high-fat diet. Also, Dnmt2 deletion altered the sperm small RNA expression profile, including levels of tRNA-derived small RNAs and rRNA-derived small RNAs, which might be essential in composing a sperm RNA 'coding signature' that is needed for paternal epigenetic memory. Finally, we show that Dnmt2-mediated m 5 C contributes to the secondary structure and biological properties of sncRNAs, implicating sperm RNA modifications as an additional layer of paternal hereditary information.

  3. Undesired Small RNAs Originate from an Artificial microRNA Precursor in Transgenic Petunia (Petunia hybrida)

    PubMed Central

    Guo, Yulong; Han, Yao; Ma, Jing; Wang, Huiping; Sang, Xianchun; Li, Mingyang

    2014-01-01

    Although artificial microRNA (amiRNA) technology has been used frequently in gene silencing in plants, little research has been devoted to investigating the accuracy of amiRNA precursor processing. In this work, amiRNAchs1 (amiRchs1), based on the Arabidopsis miR319a precursor, was expressed in order to suppress the expression of CHS genes in petunia. The transgenic plants showed the CHS gene-silencing phenotype. A modified 5′ RACE technique was used to map small-RNA-directed cleavage sites and to detect processing intermediates of the amiRchs1 precursor. The results showed that the target CHS mRNAs were cut at the expected sites and that the amiRchs1 precursor was processed from loop to base. The accumulation of small RNAs in amiRchs1 transgenic petunia petals was analyzed using the deep-sequencing technique. The results showed that, alongside the accumulation of the desired artificial microRNAs, additional small RNAs that originated from other regions of the amiRNA precursor were also accumulated at high frequency. Some of these had previously been found to be accumulated at low frequency in the products of ath-miR319a precursor processing and some of them were accompanied by 3′-tailing variant. Potential targets of the undesired small RNAs were discovered in petunia and other Solanaceae plants. The findings draw attention to the potential occurrence of undesired target silencing induced by such additional small RNAs when amiRNA technology is used. No appreciable production of secondary small RNAs occurred, despite the fact that amiRchs1 was designed to have perfect complementarity to its CHS-J target. This confirmed that perfect pairing between an amiRNA and its targets is not the trigger for secondary small RNA production. In conjunction with the observation that amiRNAs with perfect complementarity to their target genes show high efficiency and specificity in gene silencing, this finding has an important bearing on future applications of amiRNAs in gene

  4. Undesired small RNAs originate from an artificial microRNA precursor in transgenic petunia (Petunia hybrida).

    PubMed

    Guo, Yulong; Han, Yao; Ma, Jing; Wang, Huiping; Sang, Xianchun; Li, Mingyang

    2014-01-01

    Although artificial microRNA (amiRNA) technology has been used frequently in gene silencing in plants, little research has been devoted to investigating the accuracy of amiRNA precursor processing. In this work, amiRNAchs1 (amiRchs1), based on the Arabidopsis miR319a precursor, was expressed in order to suppress the expression of CHS genes in petunia. The transgenic plants showed the CHS gene-silencing phenotype. A modified 5' RACE technique was used to map small-RNA-directed cleavage sites and to detect processing intermediates of the amiRchs1 precursor. The results showed that the target CHS mRNAs were cut at the expected sites and that the amiRchs1 precursor was processed from loop to base. The accumulation of small RNAs in amiRchs1 transgenic petunia petals was analyzed using the deep-sequencing technique. The results showed that, alongside the accumulation of the desired artificial microRNAs, additional small RNAs that originated from other regions of the amiRNA precursor were also accumulated at high frequency. Some of these had previously been found to be accumulated at low frequency in the products of ath-miR319a precursor processing and some of them were accompanied by 3'-tailing variant. Potential targets of the undesired small RNAs were discovered in petunia and other Solanaceae plants. The findings draw attention to the potential occurrence of undesired target silencing induced by such additional small RNAs when amiRNA technology is used. No appreciable production of secondary small RNAs occurred, despite the fact that amiRchs1 was designed to have perfect complementarity to its CHS-J target. This confirmed that perfect pairing between an amiRNA and its targets is not the trigger for secondary small RNA production. In conjunction with the observation that amiRNAs with perfect complementarity to their target genes show high efficiency and specificity in gene silencing, this finding has an important bearing on future applications of amiRNAs in gene

  5. 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

  6. DNA methylation and small interference RNAs participate in the regulation of MADS-box genes involved in dormancy in sweet cherry (Prunus avium L.).

    PubMed

    Rothkegel, Karin; Sánchez, Evelyn; Montes, Christian; Greve, Macarena; Tapia, Sebastián; Bravo, Soraya; Prieto, Humberto; Almeida, Andréa Miyasaka

    2017-12-01

    Epigenetic modifications can yield information about connections between genotype, phenotype variation and environmental conditions. Bud dormancy release in temperate perennial fruit trees depends on internal and environmental signals such as cold accumulation and photoperiod. Previous investigations have noted the participation of epigenetic mechanisms in the control of this physiological process. We examined whether epigenetic modifications were modulated in MADS-box genes, potential candidates for the regulation of bud dormancy and flowering in sweet cherry (Prunus avium L.). We identified and cloned two MADS-box genes homologous to the already-characterized dormancy regulators DORMANCY-ASSOCIATED MADS-box (DAM3 and DAM5) from Prunus persica (L.) Batsch. Bisulfite sequencing of the identified genes (PavMADS1 and PavMADS2), Methylated DNA Immunoprecipitation and small RNA deep sequencing were performed to analyze the presence of DNA methylations that could be guided by non-coding RNAs in the floral buds exposed to differential chilling hours. The results obtained reveal an increase in the level of DNA methylation and abundance of matching small interference RNAs (siRNAs) in the promoter of PavMADS1 when the chilling requirement is complete. For the first intron and 5' UTR of PavMADS1, de novo DNA methylation could be associated with the increase in the abundance of 24-nt siRNA matching the promoter area. Also, in the second large intron of PavMADS1, maintenance DNA methylation in all cytosine contexts is associated with the presence of homologous siRNAs in that zone. For PavMADS2, only maintenance methylation was present in the CG context, and no matching siRNAs were detected. Silencing of PavMADS1 and PavMADS2 coincided with an increase in Flowering Locus T expression during dormancy. In conclusion, DNA methylations and siRNAs appear to be involved in the silencing of PavMADS1 during cold accumulation and dormancy release in sweet cherry. © The Author 2017

  7. MicroRNAs associated with small bowel neuroendocrine tumours and their metastases.

    PubMed

    Miller, Helen C; Frampton, Adam E; Malczewska, Anna; Ottaviani, Silvia; Stronach, Euan A; Flora, Rashpal; Kaemmerer, Daniel; Schwach, Gert; Pfragner, Roswitha; Faiz, Omar; Kos-Kudła, Beata; Hanna, George B; Stebbing, Justin; Castellano, Leandro; Frilling, Andrea

    2016-09-01

    Novel molecular analytes are needed in small bowel neuroendocrine tumours (SBNETs) to better determine disease aggressiveness and predict treatment response. In this study, we aimed to profile the global miRNome of SBNETs, and identify microRNAs (miRNAs) involved in tumour progression for use as potential biomarkers. Two independent miRNA profiling experiments were performed (n=90), including primary SBNETs (n=28), adjacent normal small bowel (NSB; n=14), matched lymph node (LN) metastases (n=24), normal LNs (n=7), normal liver (n=2) and liver metastases (n=15). We then evaluated potentially targeted genes by performing integrated computational analyses. We discovered 39 miRNAs significantly deregulated in SBNETs compared with adjacent NSB. The most upregulated (miR-204-5p, miR-7-5p and miR-375) were confirmed by qRT-PCR. Two miRNAs (miR-1 and miR-143-3p) were significantly downregulated in LN and liver metastases compared with primary tumours. Furthermore, we identified upregulated gene targets for miR-1 and miR-143-3p in an existing SBNET dataset, which could contribute to disease progression, and show that these miRNAs directly regulate FOSB and NUAK2 oncogenes. Our study represents the largest global miRNA profiling of SBNETs using matched primary tumour and metastatic samples. We revealed novel miRNAs deregulated during SBNET disease progression, and important miRNA-mRNA interactions. These miRNAs have the potential to act as biomarkers for patient stratification and may also be able to guide treatment decisions. Further experiments to define molecular mechanisms and validate these miRNAs in larger tissue cohorts and in biofluids are now warranted. © 2016 Society for Endocrinology.

  8. Small RNAs: artificial piRNAs for transcriptional silencing.

    PubMed

    Hirano, Takamasa; Siomi, Haruhiko

    2015-03-30

    Technologies have been developed in animal germ cells that produce artificial piRNAs from transgenes in piRNA clusters to silence target genes by cleaving their transcripts. A new study provides a simple way to generate artificial piRNAs to direct de novo DNA methylation in mice. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. 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

  10. FASTmiR: an RNA-based sensor for in vitro quantification and live-cell localization of small RNAs

    PubMed Central

    Huang, Kun; Doyle, Francis; Wurz, Zachary E.; Tenenbaum, Scott A.; Hammond, Reza K.

    2017-01-01

    Abstract Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA–mRNA interactions combined with a fluorophore-binding sequence ‘Spinach’, a GFP-like RNA aptamer for which the RNA–fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo. PMID:28586459

  11. Adenovirus virus-associated RNAII-derived small RNAs are efficiently incorporated into the rna-induced silencing complex and associate with polyribosomes.

    PubMed

    Xu, Ning; Segerman, Bo; Zhou, Xiaofu; Akusjärvi, Göran

    2007-10-01

    Adenovirus type 5 encodes two highly structured short RNAs, the virus-associated (VA) RNAI and RNAII. Both are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC). We show here, by cloning of small RNAs, that approximately 80% of Ago2-containing RISC immunopurified from late-infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). Most surprisingly, VA RNAII, which is expressed at 20-fold lower levels compared to that of VA RNAI, appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. The mivaRNAs are derived from the 3' strand of the terminal stems of the VA RNAs, with the major fraction of VA RNAII starting at position 138. The small RNAs derived from VA RNAI were more heterogeneous in size, with the two predominant small RNAs starting at positions 137 and 138. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially, they function as miRNAs, regulating translation of cellular mRNAs. In support of this hypothesis, we detected a fraction of the VA RNAII-derived mivaRNAs on polyribosomes.

  12. Northern Blot Detection of Virus-Derived Small Interfering RNAs in Caenorhabditis elegans Using Nonradioactive Oligo Probes.

    PubMed

    Long, Tianyun; Lu, Rui

    2017-01-01

    Northern blot analysis has been widely used as a tool for detection and characterization of specific RNA molecules. When coupled with radioactive probe northern blot allows for robust detection and characterization of small RNA molecules of trace amount. Here, we describe the detection and size characterization of virus-derived small interfering RNAs (vsiRNAs) in C. elegans using nonradioactive DNA oligo probes in northern blotting. Our protocol allows for the detection and characterization of not only primary vsiRNAs but also secondary vsiRNAs, a class of single-stranded vsiRNAs that has distinct migration pattern, and can be easily adapted to the detection of vsiRNAs in other organisms.

  13. C. elegans microRNAs.

    PubMed

    Vella, Monica C; Slack, Frank J

    2005-09-21

    MicroRNAs (miRNAs) are small, non-coding regulatory RNAs found in many phyla that control such diverse events as development, metabolism, cell fate and cell death. They have also been implicated in human cancers. The C. elegans genome encodes hundreds of miRNAs, including the founding members of the miRNA family lin-4 and let-7. Despite the abundance of C. elegans miRNAs, few miRNA targets are known and little is known about the mechanism by which they function. However, C. elegans research continues to push the boundaries of discovery in this area. lin-4 and let-7 are the best understood miRNAs. They control the timing of adult cell fate determination in hypodermal cells by binding to partially complementary sites in the mRNA of key developmental regulators to repress protein expression. For example, lin-4 is predicted to bind to seven sites in the lin-14 3' untranslated region (UTR) to repress LIN-14, while let-7 is predicted to bind two let-7 complementary sites in the lin-41 3' UTR to down-regulate LIN-41. Two other miRNAs, lsy-6 and mir-273, control left-right asymmetry in neural development, and also target key developmental regulators for repression. Approximately one third of the C. elegans miRNAs are differentially expressed during development indicating a major role for miRNAs in C. elegans development. Given the remarkable conservation of developmental mechanism across phylogeny, many of the principles of miRNAs discovered in C. elegans are likely to be applicable to higher animals.

  14. Small-interfering RNAs from natural antisense transcripts derived from a cellulose synthase gene modulate cell wall biosynthesis in barley

    PubMed Central

    Held, Michael A.; Penning, Bryan; Brandt, Amanda S.; Kessans, Sarah A.; Yong, Weidong; Scofield, Steven R.; Carpita, Nicholas C.

    2008-01-01

    Small-interfering RNAs (siRNAs) from natural cis-antisense pairs derived from the 3′-coding region of the barley (Hordeum vulgare) CesA6 cellulose synthase gene substantially increase in abundance during leaf elongation. Strand-specific RT-PCR confirmed the presence of an antisense transcript of HvCesA6 that extends ≥1230 bp from the 3′ end of the CesA-coding sequence. The increases in abundance of the CesA6 antisense transcript and the 21-nt and 24-nt siRNAs derived from the transcript are coincident with the down-regulation of primary wall CesAs, several Csl genes, and GT8 glycosyl transferase genes, and are correlated with the reduction in rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Virus induced gene silencing using unique target sequences derived from HvCesA genes attenuated expression not only of the HvCesA6 gene, but also of numerous nontarget Csls and the distantly related GT8 genes and reduced the incorporation of D-14C-Glc into cellulose and into mixed-linkage (1 → 3),(1 → 4)-β-D-glucans of the developing leaves. Unique target sequences for CslF and CslH conversely silenced the same genes and lowered rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Our results indicate that the expression of individual members of the CesA/Csl superfamily and glycosyl transferases share common regulatory control points, and siRNAs from natural cis-antisense pairs derived from the CesA/Csl superfamily could function in this global regulation of cell-wall synthesis. PMID:19075248

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

    PubMed Central

    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

    2014-01-01

    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. PMID:25225396

  16. Identification and Characterization of MicroRNAs in Small Brown Planthopper (Laodephax striatellus) by Next-Generation Sequencing

    PubMed Central

    Lou, Yonggen; Cheng, Jia'an; Zhang, Hengmu; Xu, Jian-Hong

    2014-01-01

    MicroRNAs (miRNAs) are endogenous non-coding small RNAs that regulate gene expression at the post-transcriptional level and are thought to play critical roles in many metabolic activities in eukaryotes. The small brown planthopper (Laodephax striatellus Fallén), one of the most destructive agricultural pests, causes great damage to crops including rice, wheat, and maize. However, information about the genome of L. striatellus is limited. In this study, a small RNA library was constructed from a mixed L. striatellus population and sequenced by Solexa sequencing technology. A total of 501 mature miRNAs were identified, including 227 conserved and 274 novel miRNAs belonging to 125 and 250 families, respectively. Sixty-nine conserved miRNAs that are included in 38 families are predicted to have an RNA secondary structure typically found in miRNAs. Many miRNAs were validated by stem-loop RT-PCR. Comparison with the miRNAs in 84 animal species from miRBase showed that the conserved miRNA families we identified are highly conserved in the Arthropoda phylum. Furthermore, miRanda predicted 2701 target genes for 378 miRNAs, which could be categorized into 52 functional groups annotated by gene ontology. The function of miRNA target genes was found to be very similar between conserved and novel miRNAs. This study of miRNAs in L. striatellus will provide new information and enhance the understanding of the role of miRNAs in the regulation of L. striatellus metabolism and development. PMID:25057821

  17. Herpes simplex virus 1 microRNAs expressed abundantly during latent infection are not essential for latency in mouse trigeminal ganglia

    PubMed Central

    Kramer, Martha F.; Jurak, Igor; Pesola, Jean M.; Boissel, Sandrine; Knipe, David M.; Coen, Donald M.

    2013-01-01

    Several herpes simplex virus 1 microRNAs are encoded within or near the latency associated transcript (LAT) locus, and are expressed abundantly during latency. Some of these microRNAs can repress the expression of important viral proteins and are hypothesized to play important roles in establishing and/or maintaining latent infections. We found that in lytically infected cells and in acutely infected mouse ganglia, expression of LAT-encoded microRNAs was weak and unaffected by a deletion that includes the LAT promoter. In mouse ganglia latently infected with wild type virus, the microRNAs accumulated to high levels, but deletions of the LAT promoter markedly reduced expression of LAT-encoded microRNAs and also miR-H6, which is encoded upstream of LAT and can repress expression of ICP4. Because these LAT deletion mutants establish and maintain latent infections, these microRNAs are not essential for latency, at least in mouse trigeminal ganglia, but may help promote it. PMID:21782205

  18. MicroRNAs in the pineal gland: miR-483 regulates melatonin synthesis by targeting arylalkylamine N-acetyltransferase.

    PubMed

    Clokie, Samuel J H; Lau, Pierre; Kim, Hyun Hee; Coon, Steven L; Klein, David C

    2012-07-20

    MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ~75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3"-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.

  19. MicroRNAs and cancer: a meeting summary of the eponymous Keystone Conference.

    PubMed

    Godshalk, Sirie E; Melnik-Martinez, Katya V; Pasquinelli, Amy E; Slack, Frank J

    2010-02-16

    This report summarizes the information presented at the 2009 Keystone Conference on MicroRNAs and Cancer, held in Keystone, Colorado, USA, June 10th to 15th 2009. Soon after microRNAs (miRNAs) emerged as an abundant new class of non-coding RNAs (ncRNAs), evidence started to mount supporting important roles for these regulatory RNAs in human health and disease. Mis-regulation of specific miRNA pathways has been linked to diverse cancers. The recent Keystone meeting highlighted progress in understanding the role of miRNAs in normal development and oncogenesis. Recurring themes included the complexities associated with miRNA biogenesis, target recognition, elucidation of genetic networks where miRNAs play pivotal roles often within feedback loops, and the promise of small RNAs as diagnostics and therapeutics in combating cancer.

  20. Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants

    PubMed Central

    Khraiwesh, Basel; Zhu, Jian-Kang; Zhu, Jianhua

    2011-01-01

    Small, non-coding RNAs are a distinct class of regulatory RNAs in plants and animals that control a variety of biological processes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved through a series of pathways. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs control the expression of cognate target genes by binding to reverse complementary sequences, resulting in cleavage or translational inhibition of the target RNAs. siRNAs have a similar structure, function, and biogenesis as miRNAs but are derived from long double-stranded RNAs and can often direct DNA methylation at target sequences. Besides their roles in growth and development and maintenance of genome integrity, small RNAs are also important components in plant stress responses. One way in which plants respond to environmental stress is by modifying their gene expression through the activity of small RNAs. Thus, understanding how small RNAs regulate gene expression will enable researchers to explore the role of small RNAs in biotic and abiotic stress responses. This review focuses on the regulatory roles of plant small RNAs in the adaptive response to stresses. PMID:21605713

  1. Evolutionary patterns of Escherichia coli small RNAs and their regulatory interactions.

    PubMed

    Peer, Asaf; Margalit, Hanah

    2014-07-01

    Most bacterial small RNAs (sRNAs) are post-transcriptional regulators of gene expression, exerting their regulatory function by base-pairing with their target mRNAs. While it has become evident that sRNAs play central regulatory roles in the cell, little is known about their evolution and the evolution of their regulatory interactions. Here we used the prokaryotic phylogenetic tree to reconstruct the evolutionary history of Escherichia coli sRNAs and their binding sites on target mRNAs. We discovered that sRNAs currently present in E. coli mainly accumulated inside the Enterobacteriales order, succeeding the appearance of other types of noncoding RNAs and concurrently with the evolution of a variant of the Hfq protein exhibiting a longer C-terminal region. Our analysis of the evolutionary ages of sRNA-mRNA interactions revealed that while all sRNAs were evolutionarily older than most of their known binding sites on mRNA targets, for quite a few sRNAs there was at least one binding site that coappeared with or preceded them. It is conceivable that the establishment of these first interactions forced selective pressure on the sRNAs, after which additional targets were acquired by fitting a binding site to the active region of the sRNA. This conjecture is supported by the appearance of many binding sites on target mRNAs only after the sRNA gain, despite the prior presence of the target gene in ancestral genomes. Our results suggest a selective mechanism that maintained the sRNAs across the phylogenetic tree, and shed light on the evolution of E. coli post-transcriptional regulatory network. © 2014 Peer and Margalit; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  2. Adenovirus Virus-Associated RNAII-Derived Small RNAs Are Efficiently Incorporated into the RNA-Induced Silencing Complex and Associate with Polyribosomes▿ §

    PubMed Central

    Xu, Ning; Segerman, Bo; Zhou, Xiaofu; Akusjärvi, Göran

    2007-01-01

    Adenovirus type 5 encodes two highly structured short RNAs, the virus-associated (VA) RNAI and RNAII. Both are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC). We show here, by cloning of small RNAs, that approximately 80% of Ago2-containing RISC immunopurified from late-infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). Most surprisingly, VA RNAII, which is expressed at 20-fold lower levels compared to that of VA RNAI, appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. The mivaRNAs are derived from the 3′ strand of the terminal stems of the VA RNAs, with the major fraction of VA RNAII starting at position 138. The small RNAs derived from VA RNAI were more heterogeneous in size, with the two predominant small RNAs starting at positions 137 and 138. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially, they function as miRNAs, regulating translation of cellular mRNAs. In support of this hypothesis, we detected a fraction of the VA RNAII-derived mivaRNAs on polyribosomes. PMID:17652395

  3. 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

  4. 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

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

    PubMed

    Nakanishi, Kotaro

    2016-09-01

    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. © 2016 The Authors. WIREs RNA published by Wiley Periodicals, Inc.

  6. Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus

    PubMed Central

    2011-01-01

    Background In response to infection, viral genomes are processed by Dicer-like (DCL) ribonuclease proteins into viral small RNAs (vsRNAs) of discrete sizes. vsRNAs are then used as guides for silencing the viral genome. The profile of vsRNAs produced during the infection process has been extensively studied for some groups of viruses. However, nothing is known about the vsRNAs produced during infections of members of the economically important family Luteoviridae, a group of phloem-restricted viruses. Here, we report the characterization of a population of vsRNAs from cotton plants infected with Cotton leafroll dwarf virus (CLRDV), a member of the genus Polerovirus, family Luteoviridae. Results Deep sequencing of small RNAs (sRNAs) from leaves of CLRDV-infected cotton plants revealed that the vsRNAs were 21- to 24-nucleotides (nt) long and that their sequences matched the viral genome, with higher frequencies of matches in the 3- region. There were equivalent amounts of sense and antisense vsRNAs, and the 22-nt class of small RNAs was predominant. During infection, cotton Dcl transcripts appeared to be up-regulated, while Dcl2 appeared to be down-regulated. Conclusions This is the first report on the profile of sRNAs in a plant infected with a virus from the family Luteoviridae. Our sequence data strongly suggest that virus-derived double-stranded RNA functions as one of the main precursors of vsRNAs. Judging by the profiled size classes, all cotton DCLs might be working to silence the virus. The possible causes for the unexpectedly high accumulation of 22-nt vsRNAs are discussed. CLRDV is the causal agent of Cotton blue disease, which occurs worldwide. Our results are an important contribution for understanding the molecular mechanisms involved in this and related diseases. PMID:21864377

  7. Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus.

    PubMed

    Silva, Tatiane F; Romanel, Elisson A C; Andrade, Roberto R S; Farinelli, Laurent; Østerås, Magne; Deluen, Cécile; Corrêa, Régis L; Schrago, Carlos E G; Vaslin, Maite F S

    2011-08-24

    In response to infection, viral genomes are processed by Dicer-like (DCL) ribonuclease proteins into viral small RNAs (vsRNAs) of discrete sizes. vsRNAs are then used as guides for silencing the viral genome. The profile of vsRNAs produced during the infection process has been extensively studied for some groups of viruses. However, nothing is known about the vsRNAs produced during infections of members of the economically important family Luteoviridae, a group of phloem-restricted viruses. Here, we report the characterization of a population of vsRNAs from cotton plants infected with Cotton leafroll dwarf virus (CLRDV), a member of the genus Polerovirus, family Luteoviridae. Deep sequencing of small RNAs (sRNAs) from leaves of CLRDV-infected cotton plants revealed that the vsRNAs were 21- to 24-nucleotides (nt) long and that their sequences matched the viral genome, with higher frequencies of matches in the 3- region. There were equivalent amounts of sense and antisense vsRNAs, and the 22-nt class of small RNAs was predominant. During infection, cotton Dcl transcripts appeared to be up-regulated, while Dcl2 appeared to be down-regulated. This is the first report on the profile of sRNAs in a plant infected with a virus from the family Luteoviridae. Our sequence data strongly suggest that virus-derived double-stranded RNA functions as one of the main precursors of vsRNAs. Judging by the profiled size classes, all cotton DCLs might be working to silence the virus. The possible causes for the unexpectedly high accumulation of 22-nt vsRNAs are discussed. CLRDV is the causal agent of Cotton blue disease, which occurs worldwide. Our results are an important contribution for understanding the molecular mechanisms involved in this and related diseases.

  8. 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

    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 second

  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. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  10. Analysis of plant-derived miRNAs in animal small RNA datasets

    PubMed Central

    2012-01-01

    Background Plants contain significant quantities of small RNAs (sRNAs) derived from various sRNA biogenesis pathways. Many of these sRNAs play regulatory roles in plants. Previous analysis revealed that numerous sRNAs in corn, rice and soybean seeds have high sequence similarity to animal genes. However, exogenous RNA is considered to be unstable within the gastrointestinal tract of many animals, thus limiting potential for any adverse effects from consumption of dietary RNA. A recent paper reported that putative plant miRNAs were detected in animal plasma and serum, presumably acquired through ingestion, and may have a functional impact in the consuming organisms. Results To address the question of how common this phenomenon could be, we searched for plant miRNAs sequences in public sRNA datasets from various tissues of mammals, chicken and insects. Our analyses revealed that plant miRNAs were present in the animal sRNA datasets, and significantly miR168 was extremely over-represented. Furthermore, all or nearly all (>96%) miR168 sequences were monocot derived for most datasets, including datasets for two insects reared on dicot plants in their respective experiments. To investigate if plant-derived miRNAs, including miR168, could accumulate and move systemically in insects, we conducted insect feeding studies for three insects including corn rootworm, which has been shown to be responsive to plant-produced long double-stranded RNAs. Conclusions Our analyses suggest that the observed plant miRNAs in animal sRNA datasets can originate in the process of sequencing, and that accumulation of plant miRNAs via dietary exposure is not universal in animals. PMID:22873950

  11. 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

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

    PubMed

    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.

  13. 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.

  14. 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. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  15. 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.

  16. Evolutionary Dynamics of Small RNAs in 27 Escherichia coli and Shigella Genomes

    PubMed Central

    Skippington, Elizabeth; Ragan, Mark A.

    2012-01-01

    Small RNAs (sRNAs) are widespread in bacteria and play critical roles in regulating physiological processes. They are best characterized in Escherichia coli K-12 MG1655, where 83 sRNAs constitute nearly 2% of the gene complement. Most sRNAs act by base pairing with a target mRNA, modulating its translation and/or stability; many of these RNAs share only limited complementarity to their mRNA target, and require the chaperone Hfq to facilitate base pairing. Little is known about the evolutionary dynamics of bacterial sRNAs. Here, we apply phylogenetic and network analyses to investigate the evolutionary processes and principles that govern sRNA gene distribution in 27 E. coli and Shigella genomes. We identify core (encoded in all 27 genomes) and variable sRNAs; more than two-thirds of the E. coli K-12 MG1655 sRNAs are core, whereas the others show patterns of presence and absence that are principally due to genetic loss, not duplication or lateral genetic transfer. We present evidence that variable sRNAs are less tightly integrated into cellular genetic regulatory networks than are the core sRNAs, and that Hfq facilitates posttranscriptional cross talk between the E. coli–Shigella core and variable genomes. Finally, we present evidence that more than 80% of genes targeted by Hfq-associated core sRNAs have been transferred within the E. coli–Shigella clade, and that most of these genes have been transferred intact. These results suggest that Hfq and sRNAs help integrate laterally acquired genes into established regulatory networks. PMID:22223756

  17. Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes.

    PubMed

    Lemcke, Heiko; Peukert, Janine; Voronina, Natalia; Skorska, Anna; Steinhoff, Gustav; David, Robert

    2016-09-01

    Small antisense RNAs like miRNA and siRNA are of crucial importance in cardiac physiology, pathology and, moreover, can be applied as therapeutic agents for the treatment of cardiovascular diseases. Identification of novel strategies for miRNA/siRNA therapy requires a comprehensive understanding of the underlying mechanisms. Emerging data suggest that small RNAs are transferred between cells via gap junctions and provoke gene regulatory effects in the recipient cell. To elucidate the role of miRNA/siRNA as signalling molecules, suitable tools are required that will allow the analysis of these small RNAs at the cellular level. In the present study, we applied 3 dimensional fluorescence recovery after photo bleaching microscopy (3D-FRAP) to visualise and quantify the gap junctional exchange of small RNAs between neonatal cardiomyocytes in real time. Cardiomyocytes were transfected with labelled miRNA and subjected to FRAP microscopy. Interestingly, we observed recovery rates of 21% already after 13min, indicating strong intercellular shuttling of miRNA, which was significantly reduced when connexin43 was knocked down. Flow cytometry analysis confirmed our FRAP results. Furthermore, using an EGFP/siRNA reporter construct we demonstrated that the intercellular transfer does not affect proper functioning of small RNAs, leading to marker gene silencing in the recipient cell. Our results show that 3D-FRAP microscopy is a straightforward, non-invasive live cell imaging technique to evaluate the GJ-dependent shuttling of small RNAs with high spatio-temporal resolution. Moreover, the data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation where small RNAs act as signalling molecules within the intercellular network. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. 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.

  19. Identification of Small RNAs in Desulfovibrio vulgaris Hildenborough

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Burns, Andrew; Joachimiak, Marcin; Deutschbauer, Adam

    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 possessmore » 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.« less

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed

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

    2017-01-04

    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.

  2. 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

  3. 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.

  4. 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. Copyright © 2016 Elsevier GmbH. All rights reserved.

  5. Uptake of dietary milk microRNAs by adult humans: Rules for the game of hide and seek

    USDA-ARS?s Scientific Manuscript database

    Milk producers recently used a social media campaign to build public confidence in the health benefits of their product; however, it is not clear why they did not tout the abundant microRNAs (miRNAs), small non-coding RNA molecules that function in RNA silencing and post-transcriptional regulation o...

  6. Improved silencing properties using small internally segmented interfering RNAs

    PubMed Central

    Bramsen, Jesper B.; Laursen, Maria B.; Damgaard, Christian K.; Lena, Suzy W.; Ravindra Babu, B.; Wengel, Jesper; Kjems, Jørgen

    2007-01-01

    RNA interference is mediated by small interfering RNAs (siRNAs) that upon incorporation into the RNA-induced silencing complex (RISC) can target complementary mRNA for degradation. Standard siRNA design usually feature a 19–27 base pair contiguous double-stranded region that is believed to be important for RISC incorporation. Here, we describe a novel siRNA design composed of an intact antisense strand complemented with two shorter 10–12 nt sense strands. This three-stranded construct, termed small internally segmented interfering RNA (sisiRNA), is highly functional demonstrating that an intact sense strand is not a prerequisite for RNA interference. Moreover, when using the sisiRNA design only the antisense strand is functional in activated RISC thereby completely eliminating unintended mRNA targeting by the sense strand. Interestingly, the sisiRNA design supports the function of chemically modified antisense strands, which are non-functional within the context of standard siRNA designs. This suggests that the sisiRNA design has a clear potential of improving the pharmacokinetic properties of siRNA in vivo. PMID:17726057

  7. 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

    Abstract 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

  8. Conversations between kingdoms: small RNAs.

    PubMed

    Weiberg, Arne; Bellinger, Marschal; Jin, Hailing

    2015-04-01

    Humans, animals, and plants are constantly under attack from pathogens and pests, resulting in severe consequences on global human health and crop production. Small RNA (sRNA)-mediated RNA interference (RNAi) is a conserved regulatory mechanism that is involved in almost all eukaryotic cellular processes, including host immunity and pathogen virulence. Recent evidence supports the significant contribution of sRNAs and RNAi to the communication between hosts and some eukaryotic pathogens, pests, parasites, or symbiotic microorganisms. Mobile silencing signals—most likely sRNAs—are capable of translocating from the host to its interacting organism, and vice versa. In this review, we will provide an overview of sRNA communications between different kingdoms, with a primary focus on the advances in plant-pathogen interaction systems. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Identifying MicroRNAs and Transcript Targets in Jatropha Seeds

    PubMed Central

    Galli, Vanessa; Guzman, Frank; de Oliveira, Luiz F. V.; Loss-Morais, Guilherme; Körbes, Ana P.; Silva, Sérgio D. A.; Margis-Pinheiro, Márcia M. A. N.; Margis, Rogério

    2014-01-01

    MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs) as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs. PMID:24551031

  10. Deep sequencing of small RNA libraries from human prostate epithelial and stromal cells reveal distinct pattern of microRNAs primarily predicted to target growth factors.

    PubMed

    Singh, Savita; Zheng, Yun; Jagadeeswaran, Guru; Ebron, Jey Sabith; Sikand, Kavleen; Gupta, Sanjay; Sunker, Ramanjulu; Shukla, Girish C

    2016-02-28

    Complex epithelial and stromal cell interactions are required during the development and progression of prostate cancer. Regulatory small non-coding microRNAs (miRNAs) participate in the spatiotemporal regulation of messenger RNA (mRNA) and regulation of translation affecting a large number of genes involved in prostate carcinogenesis. In this study, through deep-sequencing of size fractionated small RNA libraries we profiled the miRNAs of prostate epithelial (PrEC) and stromal (PrSC) cells. Over 50 million reads were obtained for PrEC in which 860,468 were unique sequences. Similarly, nearly 76 million reads for PrSC were obtained in which over 1 million were unique reads. Expression of many miRNAs of broadly conserved and poorly conserved miRNA families were identified. Sixteen highly expressed miRNAs with significant change in expression in PrSC than PrEC were further analyzed in silico. ConsensusPathDB showed the target genes of these miRNAs were significantly involved in adherence junction, cell adhesion, EGRF, TGF-β and androgen signaling. Let-7 family of tumor-suppressor miRNAs expression was highly pervasive in both, PrEC and PrSC cells. In addition, we have also identified several miRNAs that are unique to PrEC or PrSC cells and their predicted putative targets are a group of transcription factors. This study provides perspective on the miRNA expression in PrEC and PrSC, and reveals a global trend in miRNA interactome. We conclude that the most abundant miRNAs are potential regulators of development and differentiation of the prostate gland by targeting a set of growth factors. Additionally, high level expression of the most members of let-7 family miRNAs suggests their role in the fine tuning of the growth and proliferation of prostate epithelial and stromal cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  11. Small RNA populations revealed by blocking rRNA fragments in Drosophila melanogaster reproductive tissues

    PubMed Central

    Dalmay, Tamas

    2018-01-01

    RNA interference (RNAi) is a complex and highly conserved regulatory mechanism mediated via small RNAs (sRNAs). Recent technical advances in high throughput sequencing have enabled an increasingly detailed analysis of sRNA abundances and profiles in specific body parts and tissues. This enables investigations of the localized roles of microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, variation in the proportions of non-coding RNAs in the samples being compared can hinder these analyses. Specific tissues may vary significantly in the proportions of fragments of longer non-coding RNAs (such as ribosomal RNA or transfer RNA) present, potentially reflecting tissue-specific differences in biological functions. For example, in Drosophila, some tissues contain a highly abundant 30nt rRNA fragment (the 2S rRNA) as well as abundant 5’ and 3’ terminal rRNA fragments. These can pose difficulties for the construction of sRNA libraries as they can swamp the sequencing space and obscure sRNA abundances. Here we addressed this problem and present a modified “rRNA blocking” protocol for the construction of high-definition (HD) adapter sRNA libraries, in D. melanogaster reproductive tissues. The results showed that 2S rRNAs targeted by blocking oligos were reduced from >80% to < 0.01% total reads. In addition, the use of multiple rRNA blocking oligos to bind the most abundant rRNA fragments allowed us to reveal the underlying sRNA populations at increased resolution. Side-by-side comparisons of sequencing libraries of blocked and non-blocked samples revealed that rRNA blocking did not change the miRNA populations present, but instead enhanced their abundances. We suggest that this rRNA blocking procedure offers the potential to improve the in-depth analysis of differentially expressed sRNAs within and across different tissues. PMID:29474379

  12. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.

    PubMed

    Song, Erwei; Zhu, Pengcheng; Lee, Sang-Kyung; Chowdhury, Dipanjan; Kussman, Steven; Dykxhoorn, Derek M; Feng, Yi; Palliser, Deborah; Weiner, David B; Shankar, Premlata; Marasco, Wayne A; Lieberman, Judy

    2005-06-01

    Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.

  13. 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

  14. 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

  15. Detection of Plant miRNAs Abundance in Human Breast Milk.

    PubMed

    Lukasik, Anna; Brzozowska, Iwona; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr

    2017-12-23

    Breast milk is a natural food and important component of infant nutrition. Apart from the alimentary substances, breast milk contains many important bioactive compounds, including endogenous microRNA molecules (miRNAs). These regulatory molecules were identified in various mammalian biological fluids and were shown to be mostly packed in exosomes. Recently, it was revealed that plant food-derived miRNAs are stably present in human blood and regulate the expression of specific human genes. Since then, the scientific community has focused its efforts on contradicting or confirming this discovery. With the same intention, qRT-PCR experiments were performed to evaluate the presence of five plant food-derived miRNAs (miR166a, miR156a, miR157a, miR172a and miR168a) in breast milk (whole milk and exosomes) from healthy volunteers. In whole milk samples, all examined miRNAs were identified, while only two of these miRNAs were confirmed to be present in exosomes. The plant miRNA concentration in the samples ranged from 4 to 700 fM. Complementary bioinformatics analysis suggests that the evaluated plant miRNAs may potentially influence several crucial biological pathways in the infant organism.

  16. Roles of small RNAs in plant disease resistance.

    PubMed

    Yang, Li; Huang, Hai

    2014-10-01

    The interaction between plants and pathogens represents a dynamic competition between a robust immune system and efficient infectious strategies. Plant innate immunity is composed of complex and highly regulated molecular networks, which can be triggered by the perception of either conserved or race-specific pathogenic molecular signatures. Small RNAs are emerging as versatile regulators of plant development, growth and response to biotic and abiotic stresses. They act in different tiers of plant immunity, including the pathogen-associated molecular pattern-triggered and the effector-triggered immunity. On the other hand, pathogens have evolved effector molecules to suppress or hijack the host small RNA pathways. This leads to an arms race between plants and pathogens at the level of small RNA-mediated defense. Here, we review recent advances in small RNA-mediated defense responses and discuss the challenging questions in this area. © 2014 Institute of Botany, Chinese Academy of Sciences.

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

    PubMed

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

    2016-09-14

    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.

  18. 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

  19. Prediction of bacterial small RNAs in the RsmA (CsrA) and ToxT pathways: a machine learning approach.

    PubMed

    Fakhry, Carl Tony; Kulkarni, Prajna; Chen, Ping; Kulkarni, Rahul; Zarringhalam, Kourosh

    2017-08-22

    Small RNAs (sRNAs) constitute an important class of post-transcriptional regulators that control critical cellular processes in bacteria. Recent research using high-throughput transcriptomic approaches has led to a dramatic increase in the discovery of bacterial sRNAs. However, it is generally believed that the currently identified sRNAs constitute a limited subset of the bacterial sRNA repertoire. In several cases, sRNAs belonging to a specific class are already known and the challenge is to identify additional sRNAs belonging to the same class. In such cases, machine-learning approaches can be used to predict novel sRNAs in a given class. In this work, we develop novel bioinformatics approaches that integrate sequence and structure-based features to train machine-learning models for the discovery of bacterial sRNAs. We show that features derived from recurrent structural motifs in the ensemble of low energy secondary structures can distinguish the RNA classes with high accuracy. We apply this approach to predict new members in two broad classes of bacterial small RNAs: 1) sRNAs that bind to the RNA-binding protein RsmA/CsrA in diverse bacterial species and 2) sRNAs regulated by the master regulator of virulence, ToxT, in Vibrio cholerae. The involvement of sRNAs in bacterial adaptation to changing environments is an increasingly recurring theme in current research in microbiology. It is likely that future research, combining experimental and computational approaches, will discover many more examples of sRNAs as components of critical regulatory pathways in bacteria. We have developed a novel approach for prediction of small RNA regulators in important bacterial pathways. This approach can be applied to specific classes of sRNAs for which several members have been identified and the challenge is to identify additional sRNAs.

  20. 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.

  1. Identification and characterization of microRNAs in white and brown alpaca skin

    PubMed Central

    2012-01-01

    Background MicroRNAs (miRNAs) are small, non-coding 21–25 nt RNA molecules that play an important role in regulating gene expression. Little is known about the expression profiles and functions of miRNAs in skin and their role in pigmentation. Alpacas have more than 22 natural coat colors, more than any other fiber producing species. To better understand the role of miRNAs in control of coat color we performed a comprehensive analysis of miRNA expression profiles in skin of white versus brown alpacas. Results Two small RNA libraries from white alpaca (WA) and brown alpaca (BA) skin were sequenced with the aid of Illumina sequencing technology. 272 and 267 conserved miRNAs were obtained from the WA and BA skin libraries, respectively. Of these conserved miRNAs, 35 and 13 were more abundant in WA and BA skin, respectively. The targets of these miRNAs were predicted and grouped based on Gene Ontology and KEGG pathway analysis. Many predicted target genes for these miRNAs are involved in the melanogenesis pathway controlling pigmentation. In addition to the conserved miRNAs, we also obtained 22 potentially novel miRNAs from the WA and BA skin libraries. Conclusion This study represents the first comprehensive survey of miRNAs expressed in skin of animals of different coat colors by deep sequencing analysis. We discovered a collection of miRNAs that are differentially expressed in WA and BA skin. The results suggest important potential functions of miRNAs in coat color regulation. PMID:23067000

  2. 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

  3. Effects of Acute Aerobic Exercise on Rats Serum Extracellular Vesicles Diameter, Concentration and Small RNAs Content.

    PubMed

    Oliveira, Getúlio P; Porto, William F; Palu, Cintia C; Pereira, Lydyane M; Petriz, Bernardo; Almeida, Jeeser A; Viana, Juliane; Filho, Nezio N A; Franco, Octavio L; Pereira, Rinaldo W

    2018-01-01

    Physical exercise stimulates organs, mainly the skeletal muscle, to release a broad range of molecules, recently dubbed exerkines. Among them, RNAs, such as miRNAs, piRNAs, and tRNAs loaded in extracellular vesicles (EVs) have the potential to play a significant role in the way muscle and other organs communicate to translate exercise into health. Low, moderate and high intensity treadmill protocols were applied to rat groups, aiming to investigate the impact of exercise on serum EVs and their associated small RNA molecules. Transmission electron microscopy, resistive pulse sensing, and western blotting were used to investigate EVs morphology, size distribution, concentration and EVs marker proteins. Small RNA libraries from EVs RNA were sequenced. Exercise did not change EVs size, while increased EVs concentration. Twelve miRNAs were found differentially expressed after exercise: rno-miR-128-3p, 103-3p, 330-5p, 148a-3p, 191a-5p, 10b-5p, 93-5p, 25-3p, 142-5p, 3068-3p, 142-3p, and 410-3p. No piRNA was found differentially expressed, and one tRNA, trna8336, was found down-regulated after exercise. The differentially expressed miRNAs were predicted to target genes involved in the MAPK pathway. A single bout of exercise impacts EVs and their small RNA load, reinforcing the need for a more detailed investigation into EVs and their load as mediators of health-promoting exercise.

  4. Computational investigation of small RNAs in the establishment of root nodules and arbuscular mycorrhiza in leguminous plants.

    PubMed

    Jin, Danfeng; Meng, Xianwen; Wang, Yue; Wang, Jingjing; Zhao, Yuhua; Chen, Ming

    2018-01-03

    Many small RNAs have been confirmed to play important roles in the development of root nodules and arbuscular mycorrhiza. In this study, we carried out the identification of certain small RNAs in leguminous plants (Medicago truncatula, soybean, peanut and common bean), such as miRNAs, tRFs and srRNAs, as well as the computational investigation of their regulations. Thirty miRNAs were predicted to be involved in establishing root nodules and mycorrhiza, and 12 of them were novel in common bean and peanut. The generation of tRFs in M. truncatula was not associated with tRNA gene frequencies and codon usage. Six tRFs exhibited different expressions in mycorrhiza and root nodules. Moreover, srRNA 5.8S in M. truncatula was generated from the regions with relatively low conservation at the rRNA 3' terminal. The protein-protein interactions between the proteins encoded by the target genes of miRNAs, tRFs and srRNAs were computed. The regulation of these three types of sRNAs in the symbiosis between leguminous plants and microorganisms is not a single regulation of certain signaling or metabolic pathways but a global regulation for the plants to own growth or specific events in symbiosis.

  5. The small RNA profile in latex from Hevea brasiliensis trees is affected by tapping panel dryness.

    PubMed

    Gébelin, Virginie; Leclercq, Julie; Kuswanhadi; Argout, Xavier; Chaidamsari, Tetty; Hu, Songnian; Tang, Chaorong; Sarah, Gautier; Yang, Meng; Montoro, Pascal

    2013-10-01

    Natural rubber is harvested by tapping Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. Harvesting stress can lead to tapping panel dryness (TPD). MicroRNAs (miRNAs) are induced by abiotic stress and regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs. This study set out to sequence miRNAs expressed in latex cells and to identify TPD-related putative targets. Deep sequencing of small RNAs was carried out on latex from trees affected by TPD using Solexa technology. The most abundant small RNA class size was 21 nucleotides for TPD trees compared with 24 nucleotides in healthy trees. By combining the LeARN pipeline, data from the Plant MicroRNA database and Hevea EST sequences, we identified 19 additional conserved and four putative species-specific miRNA families not found in previous studies on rubber. The relative transcript abundance of the Hbpre-MIR159b gene increased with TPD. This study revealed a small RNA-specific signature of TPD-affected trees. Both RNA degradation and a shift in miRNA biogenesis are suggested to explain the general decline in small RNAs and, particularly, in miRNAs.

  6. Automatic extraction and processing of small RNAs on a multi-well/multi-channel (M&M) chip.

    PubMed

    Zhong, Runtao; Flack, Kenneth; Zhong, Wenwan

    2012-12-07

    The study of the regulatory roles in small RNAs can be accelerated by techniques that permit simple, low-cost, and rapid extraction of small RNAs from a small number of cells. In order to ensure highly specific and sensitive detection, the extracted RNAs should be free of the background nucleic acids and present stably in a small volume. To meet these criteria, we designed a multi-well/multi-channel (M&M) chip to carry out automatic and selective isolation of small RNAs via solid-phase extraction (SPE), followed by reverse-transcription (RT) to convert them to the more stable cDNAs in a final volume of 2 μL. Droplets containing buffers for RNA binding, washing, and elution were trapped in microwells, which were connected by one channel, and suspended in mineral oil. The silica magnetic particles (SMPs) for SPE were moved along the channel from well to well, i.e. in between droplets, by a fixed magnet and a translation stage, allowing the nucleic acid fragments to bind to the SMPs, be washed, and then be eluted for RT reaction within 15 minutes. RNAs shorter than 63 nt were selectively enriched from cell lysates, with recovery comparable to that of a commercial kit. Physical separation of the droplets on our M&M chip allowed the usage of multiple channels for parallel processing of multiple samples. It also permitted smooth integration with on-chip RT-PCR, which simultaneously detected the target microRNA, mir-191, expressed in fewer than 10 cancer cells. Our results have demonstrated that the M&M chip device is a valuable and cost-saving platform for studying small RNA expression patterns in a limited number of cells with reasonable sample throughput.

  7. Comparison of Dengue Virus Type 2-Specific Small RNAs from RNA Interference-Competent and –Incompetent Mosquito Cells

    PubMed Central

    Scott, Jaclyn C.; Brackney, Doug E.; Campbell, Corey L.; Bondu-Hawkins, Virginie; Hjelle, Brian; Ebel, Greg D.; Olson, Ken E.; Blair, Carol D.

    2010-01-01

    The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts. PMID:21049014

  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. © 2014 Institute of Botany, Chinese Academy of Sciences.

  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. © FASEB.

  10. Live-cell imaging of endogenous mRNAs with a small molecule.

    PubMed

    Sato, Shin-ichi; Watanabe, Mizuki; Katsuda, Yousuke; Murata, Asako; Wang, Dan Ohtan; Uesugi, Motonari

    2015-02-02

    Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non-engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene-specific RNA aptamer, combined with a cell-permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live-cell imaging of the endogenous mRNA of β-actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin-2, cortactin, and cytoplasmic FMR1-interacting protein 2. The RNA-imaging technology and its further optimization might permit live-cell imaging of any RNA molecules. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. MicroRNAs: regulators of gene expression and cell differentiation

    PubMed Central

    Shivdasani, Ramesh A.

    2006-01-01

    The existence and roles of a class of abundant regulatory RNA molecules have recently come into sharp focus. Micro-RNAs (miRNAs) are small (approximately 22 bases), non–protein-coding RNAs that recognize target sequences of imperfect complementarity in cognate mRNAs and either destabilize them or inhibit protein translation. Although mechanisms of miRNA biogenesis have been elucidated in some detail, there is limited appreciation of their biological functions. Reported examples typically focus on miRNA regulation of a single tissue-restricted transcript, often one encoding a transcription factor, that controls a specific aspect of development, cell differentiation, or physiology. However, computational algorithms predict up to hundreds of putative targets for individual miRNAs, single transcripts may be regulated by multiple miRNAs, and miRNAs may either eliminate target gene expression or serve to finetune transcript and protein levels. Theoretical considerations and early experimental results hence suggest diverse roles for miRNAs as a class. One appealing possibility, that miRNAs eliminate low-level expression of unwanted genes and hence refine unilineage gene expression, may be especially amenable to evaluation in models of hematopoiesis. This review summarizes current understanding of miRNA mechanisms, outlines some of the important outstanding questions, and describes studies that attempt to define miRNA functions in hematopoiesis. PMID:16882713

  12. High-throughput sequencing identification and characterization of potentially adhesion-related small RNAs in Streptococcus mutans.

    PubMed

    Zhu, Wenhui; Liu, Shanshan; Liu, Jia; Zhou, Yan; Lin, Huancai

    2018-05-01

    Adherence capacity is one of the principal virulence factors of Streptococcus mutans, and adhesion virulence factors are controlled by small RNAs (sRNAs) at the post-transcriptional level in various bacteria. Here, we aimed to identify and decipher putative adhesion-related sRNAs in clinical strains of S. mutans. RNA deep-sequencing was performed to identify potential sRNAs under different adhesion conditions. The expression of sRNAs was analysed by quantitative real-time PCR (qRT-PCR), and bioinformatic methods were used to predict the functional characteristics of sRNAs. A total of 736 differentially expressed candidate sRNAs were predicted, and these included 352 sRNAs located on the antisense to mRNA (AM) and 384 sRNAs in intergenic regions (IGRs). The top 7 differentially expressed sRNAs were successfully validated by qRT-PCR in UA159, and 2 of these were further confirmed in 100 clinical isolates. Moreover, the sequences of two sRNAs were conserved in other Streptococcus species, indicating a conserved role in such closely related species. A good correlation between the expression of sRNAs and the adhesion of 100 clinical strains was observed, which, combined with GO and KEGG, provides a perspective for the comprehension of sRNA function annotation. This study revealed a multitude of novel putative adhesion-related sRNAs in S. mutans and contributed to a better understanding of information concerning the transcriptional regulation of adhesion in S. mutans.

  13. 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

  14. MicroRNA-like RNAs from the same miRNA precursors play a role in cassava chilling responses.

    PubMed

    Zeng, Changying; Xia, Jing; Chen, Xin; Zhou, Yufei; Peng, Ming; Zhang, Weixiong

    2017-12-07

    MicroRNAs (miRNAs) are known to play important roles in various cellular processes and stress responses. MiRNAs can be identified by analyzing reads from high-throughput deep sequencing. The reads realigned to miRNA precursors besides canonical miRNAs were initially considered as sequencing noise and ignored from further analysis. Here we reported a small-RNA species of phased and half-phased miRNA-like RNAs different from canonical miRNAs from cassava miRNA precursors detected under four distinct chilling conditions. They can form abundant multiple small RNAs arranged along precursors in a tandem and phased or half-phased fashion. Some of these miRNA-like RNAs were experimentally confirmed by re-amplification and re-sequencing, and have a similar qRT-PCR detection ratio as their cognate canonical miRNAs. The target genes of those phased and half-phased miRNA-like RNAs function in process of cell growth metabolism and play roles in protein kinase. Half-phased miR171d.3 was confirmed to have cleavage activities on its target gene P-glycoprotein 11, a broad substrate efflux pump across cellular membranes, which is thought to provide protection for tropical cassava during sharp temperature decease. Our results showed that the RNAs from miRNA precursors are miRNA-like small RNAs that are viable negative gene regulators and may have potential functions in cassava chilling responses.

  15. In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce.

    PubMed

    Yakovlev, Igor A; Fossdal, Carl G

    2017-01-01

    Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI) temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs) and other small non-coding RNAs (sRNAs) play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C). We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21-22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21-22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM) depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be involved in epigenetic

  16. Crosstalk between Long Noncoding RNAs and MicroRNAs in Health and Disease.

    PubMed

    Bayoumi, Ahmed S; Sayed, Amer; Broskova, Zuzana; Teoh, Jian-Peng; Wilson, James; Su, Huabo; Tang, Yao-Liang; Kim, Il-Man

    2016-03-11

    Protein-coding genes account for only a small part of the human genome; in fact, the vast majority of transcripts are comprised of non-coding RNAs (ncRNAs) including long ncRNAs (lncRNAs) and small ncRNAs, microRNAs (miRs). Accumulating evidence indicates that ncRNAs could play critical roles in regulating many cellular processes which are often implicated in health and disease. For example, ncRNAs are aberrantly expressed in cancers, heart diseases, and many other diseases. LncRNAs and miRs are therefore novel and promising targets to be developed into biomarkers for diagnosis and prognosis as well as treatment options. The interaction between lncRNAs and miRs as well as its pathophysiological significance have recently been reported. Mechanistically, it is believed that lncRNAs exert "sponge-like" effects on various miRs, which subsequently inhibits miR-mediated functions. This crosstalk between two types of ncRNAs frequently contributes to the pathogenesis of the disease. In this review, we provide a summary of the recent studies highlighting the interaction between these ncRNAs and the effects of this interaction on disease pathogenesis and regulation.

  17. Crosstalk between Long Noncoding RNAs and MicroRNAs in Health and Disease

    PubMed Central

    Bayoumi, Ahmed S.; Sayed, Amer; Broskova, Zuzana; Teoh, Jian-Peng; Wilson, James; Su, Huabo; Tang, Yao-Liang; Kim, Il-man

    2016-01-01

    Protein-coding genes account for only a small part of the human genome; in fact, the vast majority of transcripts are comprised of non-coding RNAs (ncRNAs) including long ncRNAs (lncRNAs) and small ncRNAs, microRNAs (miRs). Accumulating evidence indicates that ncRNAs could play critical roles in regulating many cellular processes which are often implicated in health and disease. For example, ncRNAs are aberrantly expressed in cancers, heart diseases, and many other diseases. LncRNAs and miRs are therefore novel and promising targets to be developed into biomarkers for diagnosis and prognosis as well as treatment options. The interaction between lncRNAs and miRs as well as its pathophysiological significance have recently been reported. Mechanistically, it is believed that lncRNAs exert “sponge-like” effects on various miRs, which subsequently inhibits miR-mediated functions. This crosstalk between two types of ncRNAs frequently contributes to the pathogenesis of the disease. In this review, we provide a summary of the recent studies highlighting the interaction between these ncRNAs and the effects of this interaction on disease pathogenesis and regulation. PMID:26978351

  18. Elucidating the Small Regulatory RNA Repertoire of the Sea Anemone Anemonia viridis Based on Whole Genome and Small RNA Sequencing

    PubMed Central

    Patel, Hardip; Forêt, Sylvain; Karlsen, Bård Ove; Jørgensen, Tor Erik; Hall-Spencer, Jason M

    2018-01-01

    Abstract Cnidarians harbor a variety of small regulatory RNAs that include microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), but detailed information is limited. Here, we report the identification and expression of novel miRNAs and putative piRNAs, as well as their genomic loci, in the symbiotic sea anemone Anemonia viridis. We generated a draft assembly of the A. viridis genome with putative size of 313 Mb that appeared to be composed of about 36% repeats, including known transposable elements. We detected approximately equal fractions of DNA transposons and retrotransposons. Deep sequencing of small RNA libraries constructed from A. viridis adults sampled at a natural CO2 gradient off Vulcano Island, Italy, identified 70 distinct miRNAs. Eight were homologous to previously reported miRNAs in cnidarians, whereas 62 appeared novel. Nine miRNAs were recognized as differentially expressed along the natural seawater pH gradient. We found a highly abundant and diverse population of piRNAs, with a substantial fraction showing ping–pong signatures. We identified nearly 22% putative piRNAs potentially targeting transposable elements within the A. viridis genome. The A. viridis genome appeared similar in size to that of other hexacorals with a very high divergence of transposable elements resembling that of the sea anemone genus Exaiptasia. The genome encodes and expresses a high number of small regulatory RNAs, which include novel miRNAs and piRNAs. Differentially expressed small RNAs along the seawater pH gradient indicated regulatory gene responses to environmental stressors. PMID:29385567

  19. 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

  20. The effects of potato virus Y-derived virus small interfering RNAs of three biologically distinct strains on potato (Solanum tuberosum) transcriptome.

    PubMed

    Moyo, Lindani; Ramesh, Shunmugiah V; Kappagantu, Madhu; Mitter, Neena; Sathuvalli, Vidyasagar; Pappu, Hanu R

    2017-07-17

    Potato virus Y (PVY) is one of the most economically important pathogen of potato that is present as biologically distinct strains. The virus-derived small interfering RNAs (vsiRNAs) from potato cv. Russet Burbank individually infected with PVY-N, PVY-NTN and PVY-O strains were recently characterized. Plant defense RNA-silencing mechanisms deployed against viruses produce vsiRNAs to degrade homologous viral transcripts. Based on sequence complementarity, the vsiRNAs can potentially degrade host RNA transcripts raising the prospect of vsiRNAs as pathogenicity determinants in virus-host interactions. This study investigated the global effects of PVY vsiRNAs on the host potato transcriptome. The strain-specific vsiRNAs of PVY, expressed in high copy number, were analyzed in silico for their proclivity to target potato coding and non-coding RNAs using psRobot and psRNATarget algorithms. Functional annotation of target coding transcripts was carried out to predict physiological effects of the vsiRNAs on the potato cv. Russet Burbank. The downregulation of selected target coding transcripts was further validated using qRT-PCR. The vsiRNAs derived from biologically distinct strains of PVY displayed diversity in terms of absolute number, copy number and hotspots for siRNAs on their respective genomes. The vsiRNAs populations were derived with a high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN, and P1, 3' UTR and NIa for PVY-O genomic regions. The number of vsiRNAs that displayed interaction with potato coding transcripts and number of putative coding target transcripts were comparable between PVY-N and PVY-O, and were relatively higher for PVY-NTN. The most abundant target non-coding RNA transcripts for the strain specific PVY-derived vsiRNAs were found to be MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5. Functional annotation and qRT-PCR validation suggested that the vsiRNAs target genes involved in plant hormone signaling, genetic

  1. Microbiota Small RNAs in Inflammatory Bowel Disease.

    PubMed

    Filip, Anca T; Balacescu, Ovidiu; Marian, Catalin; Anghel, Andrei

    2016-12-01

    MiRNAs are a class of potential gene regulators of critical importance in Inflammatory Bowel Disease (IBD). This review aims to present the connection between gut microbiota, probiotics administration and microRNA (miRNA) expression in IBD. It also brings into question cross-kingdom RNAi (RNA interference). Not only that gut host cells garden the intestinal microbiome via miRNA, but also strong evidence supports the idea that different species of bacteria have an impact on the intestinal immune response by modulating miRNA expression. Cross-kingdom RNAi refers to RNA silencing signals that travel between two unrelated, interacting organisms. RNAs communication between prokaryotes and eukaryotes (bacteria and nematodes) via RNAs transfer has been proved. Some authors also support the idea that non-coding RNAs are being transferred by bacterial pathogens to the host cells as part of the intracellular infection process. Further studies are required in order to clarify whether the mechanism by which bacteria modulate miRNA expression concerns RNAs transfer. These findings may lead to a different approach to IBD therapy in the future.

  2. Deep sequencing of small RNA libraries reveals dynamic expression patterns of microRNAs in multiple developmental stages of Bactrocera dorsalis.

    PubMed

    Huang, Y; Dou, W; Liu, B; Wei, D; Liao, C Y; Smagghe, G; Wang, J-J

    2014-10-01

    In eukaryotes, microRNAs (miRNAs) are small, conserved, noncoding RNAs that have emerged as critical regulators of gene expression. The oriental fruit fly Bactrocera dorsalis is one of the most economically important fruit fly pests in East Asia and the Pacific. Although transcriptome analyses have greatly enriched our knowledge of its structural genes, little is known about post-transcriptional regulation by miRNAs in this dipteran species. In this study, small RNA libraries corresponding to four B. dorsalis developmental stages (eggs, larvae, pupae and adults) were constructed and sequenced. Approximately 30.7 million reads of 18-30 nucleotides were obtained, with 123 known miRNAs and 60 novel miRNAs identified amongst these libraries. More than half of the miRNAs were stage-specific during the four developmental stages. A set of miRNAs was found to be up- or down-regulated during development by comparison of their reads at different developmental stages. Moreover, a small part of miRNAs owned both miR-#-3p and miR-#-5p types, with enormously variable miR-#-3p/miR-#-5p ratios in the same library and amongst different developmental stages for each miRNA. Taking these findings together, the current study has uncovered a number of miRNAs and provided insights into their possible involvement in developmental regulation by expression profiling of miRNAs. Further analyses of the expression and function of these miRNAs could increase our understanding of regulatory networks in this insect and lead to novel approaches for its control. © 2014 The Royal Entomological Society.

  3. Small RNA profiling reveals important roles for miRNAs in Arabidopsis response to Bacillus velezensis FZB42.

    PubMed

    Xie, Shanshan; Jiang, Haiyang; Xu, Zhilan; Xu, Qianqian; Cheng, Beijiu

    2017-09-20

    Bacillus velezensis FZB42 (previously classified as Bacillus amyloliquefaciens FZB42) has been confirmed to successfully colonize plant roots and enhance defense response against pathogen infection. This study indicated that FZB42 inoculation enhanced Arabidopsis defense response against Pseudomonas syringae DC3000 through inducing the expression of PR1, PDF1.2 and stomata closure. To further clarify the induced defense response at miRNA level, sRNA libraries from Arabidopsis roots inoculated with FZB42 and control were constructed and sequenced. The reads of 21nt and 24nt in length were the most abundant groups in FZB42-treated library and control library, respectively. 234 known miRNAs and 16 novel miRNAs were identified. Among them, 11 known miRNAs and 4 novel miRNAs were differentially expressed after FZB42 inoculation. Moreover cis-elements (TC-rich repeats, TCA-element and CGTCA-motif) associated with plant defense were also found in the promoters of these miRNAs. Additionally, 141 mRNAs were predicted as potential targets of these differentially expressed miRNAs. GO annotations of the target genes indicated their potential roles in polyamine biosynthetic process and intracellular protein transport biological process, which may contribute to increased defense response. Our findings indicated that Bacillus velezensis FZB42 inoculation altered the expression of Arabidopsis miRNAs and their target genes, which were associated with defense response. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. An expanding universe of noncoding RNAs.

    PubMed

    Storz, Gisela

    2002-05-17

    Noncoding RNAs (ncRNAs) have been found to have roles in a great variety of processes, including transcriptional regulation, chromosome replication, RNA processing and modification, messenger RNA stability and translation, and even protein degradation and translocation. Recent studies indicate that ncRNAs are far more abundant and important than initially imagined. These findings raise several fundamental questions: How many ncRNAs are encoded by a genome? Given the absence of a diagnostic open reading frame, how can these genes be identified? How can all the functions of ncRNAs be elucidated?

  5. Identification of novel microRNAs in Hevea brasiliensis and computational prediction of their targets

    PubMed Central

    2012-01-01

    Background Plants respond to external stimuli through fine regulation of gene expression partially ensured by small RNAs. Of these, microRNAs (miRNAs) play a crucial role. They negatively regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs (mRNAs). In Hevea brasiliensis, environmental and harvesting stresses are known to affect natural rubber production. This study set out to identify abiotic stress-related miRNAs in Hevea using next-generation sequencing and bioinformatic analysis. Results Deep sequencing of small RNAs was carried out on plantlets subjected to severe abiotic stress using the Solexa technique. By combining the LeARN pipeline, data from the Plant microRNA database (PMRD) and Hevea EST sequences, we identified 48 conserved miRNA families already characterized in other plant species, and 10 putatively novel miRNA families. The results showed the most abundant size for miRNAs to be 24 nucleotides, except for seven families. Several MIR genes produced both 20-22 nucleotides and 23-27 nucleotides. The two miRNA class sizes were detected for both conserved and putative novel miRNA families, suggesting their functional duality. The EST databases were scanned with conserved and novel miRNA sequences. MiRNA targets were computationally predicted and analysed. The predicted targets involved in "responses to stimuli" and to "antioxidant" and "transcription activities" are presented. Conclusions Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs when the complete genome is not yet available. Our study provided additional information for evolutionary studies and revealed potentially specific regulation of the control of redox status in Hevea. PMID:22330773

  6. Virus-encoded miRNAs in Ebola virus disease.

    PubMed

    Duy, Janice; Honko, Anna N; Altamura, Louis A; Bixler, Sandra L; Wollen-Roberts, Suzanne; Wauquier, Nadia; O'Hearn, Aileen; Mucker, Eric M; Johnson, Joshua C; Shamblin, Joshua D; Zelko, Justine; Botto, Miriam A; Bangura, James; Coomber, Moinya; Pitt, M Louise; Gonzalez, Jean-Paul; Schoepp, Randal J; Goff, Arthur J; Minogue, Timothy D

    2018-04-24

    Ebola virus (EBOV) is a negative-strand RNA virus that replicates in the cytoplasm and causes an often-fatal hemorrhagic fever. EBOV, like other viruses, can reportedly encode its own microRNAs (miRNAs) to subvert host immune defenses. miRNAs are short noncoding RNAs that can regulate gene expression by hybridizing to multiple mRNAs, and viral miRNAs can enhance viral replication and infectivity by regulating host or viral genes. To date, only one EBOV miRNA has been examined in human infection. Here, we assayed mouse, rhesus macaque, cynomolgus macaque, and human samples infected with three EBOV variants for twelve computationally predicted viral miRNAs using RT-qPCR. Ten miRNAs aligned to EBOV variants and were detectable in the four species during disease with several viral miRNAs showing presymptomatic amplification in animal models. miRNA abundances in both the mouse and nonhuman primate models mirrored the human cohort, with miR-1-5p, miR-1-3p, and miR-T3-3p consistently at the highest levels. These striking similarities in the most abundant miRNAs during infection with different EBOV variants and hosts indicate that these miRNAs are potential valuable diagnostic markers and key effectors of EBOV pathogenesis.

  7. Apple miRNAs and tasiRNAs with novel regulatory networks

    PubMed Central

    2012-01-01

    Background MicroRNAs (miRNAs) and their regulatory functions have been extensively characterized in model species but whether apple has evolved similar or unique regulatory features remains unknown. Results We performed deep small RNA-seq and identified 23 conserved, 10 less-conserved and 42 apple-specific miRNAs or families with distinct expression patterns. The identified miRNAs target 118 genes representing a wide range of enzymatic and regulatory activities. Apple also conserves two TAS gene families with similar but unique trans-acting small interfering RNA (tasiRNA) biogenesis profiles and target specificities. Importantly, we found that miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. These miRNA target sites are differentially conserved among MYBs, which is largely influenced by the location and conservation of the encoded amino acid residues in MYB factors. Finally, we found that 10 of the 19 miR828-targeted MYBs undergo small interfering RNA (siRNA) biogenesis at the 3' cleaved, highly divergent transcript regions, generating over 100 sequence-distinct siRNAs that potentially target over 70 diverse genes as confirmed by degradome analysis. Conclusions Our work identified and characterized apple miRNAs, their expression patterns, targets and regulatory functions. We also discovered that three miRNAs and the ensuing siRNAs exploit both conserved and divergent sequence features of MYB genes to initiate distinct regulatory networks targeting a multitude of genes inside and outside the MYB family. PMID:22704043

  8. Detection and quantification of extracellular microRNAs in murine biofluids

    PubMed Central

    2014-01-01

    Background MicroRNAs (miRNAs) are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma. As such, there has been heightened interest in the utility of extracellular miRNAs as minimally invasive biomarkers for diagnosis and monitoring of a wide range of human pathologies. However, quantification of extracellular miRNAs is subject to a number of specific challenges, including the relatively low RNA content of biofluids, the possibility of contamination with serum proteins (including RNases and PCR inhibitors), hemolysis, platelet contamination/activation, a lack of well-established reference miRNAs and the biochemical properties of miRNAs themselves. Protocols for the detection and quantification of miRNAs in biofluids are therefore of high interest. Results The following protocol was validated by quantifying miRNA abundance in C57 (wild-type) and dystrophin-deficient (mdx) mice. Important differences in miRNA abundance were observed depending on whether blood was taken from the jugular or tail vein. Furthermore, efficiency of miRNA recovery was reduced when sample volumes greater than 50 μl were used. Conclusions Here we describe robust and novel procedures to harvest murine serum/plasma, extract biofluid RNA, amplify specific miRNAs by RT-qPCR and analyze the resulting data, enabling the determination of relative and absolute miRNA abundance in extracellular biofluids with high accuracy, specificity and sensitivity. PMID:24629058

  9. Next-generation small RNA sequencing for microRNAs profiling in the honey bee Apis mellifera.

    PubMed

    Chen, X; Yu, X; Cai, Y; Zheng, H; Yu, D; Liu, G; Zhou, Q; Hu, S; Hu, F

    2010-12-01

    MicroRNAs (miRNAs) are key regulators in various physiological and pathological processes via post-transcriptional regulation of gene expression. The honey bee (Apis mellifera) is a key model for highly social species, and its complex social behaviour can be interpreted theoretically as changes in gene regulation, in which miRNAs are thought to be involved. We used the SOLiD sequencing system to identify the repertoire of miRNAs in the honey bee by sequencing a mixed small RNA library from different developmental stages. We obtained a total of 36,796,459 raw sequences; of which 5,491,100 short sequences were fragments of mRNA and other noncoding RNAs (ncRNA), and 1,759,346 reads mapped to the known miRNAs. We predicted 267 novel honey bee miRNAs representing 380,182 short reads, including eight miRNAs of other insects in 14,107,583 genome-mapped sequences. We verified 50 of them using stem-loop reverse-transcription PCR (RT-PCR), in which 35 yielded PCR products. Cross-species analyses showed 81 novel miRNAs with homologues in other insects, suggesting that they were authentic miRNAs and have similar functions. The results of this study provide a basis for studies of the miRNA-modulating networks in development and some intriguing phenomena such as caste differentiation in A. mellifera. © 2010 The Authors. Insect Molecular Biology © 2010 The Royal Entomological Society.

  10. Comparative Analysis of Fruit Ripening-Related miRNAs and Their Targets in Blueberry Using Small RNA and Degradome Sequencing

    PubMed Central

    Hou, Yanming; Zhai, Lulu; Li, Xuyan; Xue, Yu; Wang, Jingjing; Yang, Pengjie; Cao, Chunmei; Li, Hongxue; Cui, Yuhai; Bian, Shaomin

    2017-01-01

    MicroRNAs (miRNAs) play vital roles in the regulation of fruit development and ripening. Blueberry is an important small berry fruit crop with economical and nutritional value. However, nothing is known about the miRNAs and their targets involved in blueberry fruit ripening. In this study, using high-throughput sequencing of small RNAs, 84 known miRNAs belonging to 28 families and 16 novel miRNAs were identified in white fruit (WF) and blue fruit (BF) libraries, which represent fruit ripening onset and in progress, respectively. Among them, 41 miRNAs were shown to be differentially expressed during fruit maturation, and 16 miRNAs representing 16 families were further chosen to validate the sRNA sequencing data by stem-loop qRT-PCR. Meanwhile, 178 targets were identified for 41 known and 7 novel miRNAs in WF and BF libraries using degradome sequencing, and targets of miR160 were validated using RLM-RACE (RNA Ligase-Mediated (RLM)-Rapid Amplification of cDNA Ends) approach. Moreover, the expression patterns of 6 miRNAs and their targets were examined during fruit development and ripening. Finally, integrative analysis of miRNAs and their targets revealed a complex miRNA-mRNA regulatory network involving a wide variety of biological processes. The findings will facilitate future investigations of the miRNA-mediated mechanisms that regulate fruit development and ripening in blueberry. PMID:29257112

  11. Circular RNAs in Cancer – Lessons Learned From microRNAs

    PubMed Central

    Dragomir, Mihnea; Calin, George A.

    2018-01-01

    Circular RNAs (circRNA) are RNA molecules built from fragments of linear pre-messenger RNAs and other linear RNA species through a process termed “back-splicing” in which the 3′ and 5′ ends are joined together giving rise to a covalently uninterrupted loop. circRNAs are not new members of the RNA world; they were first discovered in the early 1990s. The novelty is their abundance in the mammalian cells, as recently thousands of circRNAs were discovered and annotated. The biogenesis of circRNAs is a partially characterized process, regulated by three different mechanisms: exon skipping, intron pairing, and RNA-binding proteins. On the other hand, the function of circRNAs remains largely unknown and only a handful of singular reports describe in detail the biological roles of some circular transcripts. In a very short period of time, numerous circRNAs were associated with various cancer types and were also identified in bodily fluids with the potential of being disease-specific biomarkers. In this review, we briefly describe the biogenesis and function of circRNAs and present the circular transcripts that were more than once reported in literature to be associated with cancer. Finally, we point out some of the difficulties encountered in the study of circRNAs in cancer, as we consider that taking these into account could accelerate and improve our understanding of the biologic and translational use of circRNAs in human diseases. PMID:29911069

  12. The LncRNA Connectivity Map: Using LncRNA Signatures to Connect Small Molecules, LncRNAs, and Diseases.

    PubMed

    Yang, Haixiu; Shang, Desi; Xu, Yanjun; Zhang, Chunlong; Feng, Li; Sun, Zeguo; Shi, Xinrui; Zhang, Yunpeng; Han, Junwei; Su, Fei; Li, Chunquan; Li, Xia

    2017-07-27

    Well characterized the connections among diseases, long non-coding RNAs (lncRNAs) and drugs are important for elucidating the key roles of lncRNAs in biological mechanisms in various biological states. In this study, we constructed a database called LNCmap (LncRNA Connectivity Map), available at http://www.bio-bigdata.com/LNCmap/ , to establish the correlations among diseases, physiological processes, and the action of small molecule therapeutics by attempting to describe all biological states in terms of lncRNA signatures. By reannotating the microarray data from the Connectivity Map database, the LNCmap obtained 237 lncRNA signatures of 5916 instances corresponding to 1262 small molecular drugs. We provided a user-friendly interface for the convenient browsing, retrieval and download of the database, including detailed information and the associations of drugs and corresponding affected lncRNAs. Additionally, we developed two enrichment analysis methods for users to identify candidate drugs for a particular disease by inputting the corresponding lncRNA expression profiles or an associated lncRNA list and then comparing them to the lncRNA signatures in our database. Overall, LNCmap could significantly improve our understanding of the biological roles of lncRNAs and provide a unique resource to reveal the connections among drugs, lncRNAs and diseases.

  13. MALDI-MS SCREENING FOR PSEUDOURIDINE IN MIXTURES OF SMALL RNAS BY CHEMICAL DERIVATIZATION, RNASE DIGESTION AND SIGNATURE PRODUCTS

    PubMed Central

    Durairaj, Anita; Limbach, Patrick A.

    2010-01-01

    We have developed a method to screen for pseudouridines in complex mixtures of small RNAs using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS). First, the unfractionated crude mixture of tRNAs is digested to completion with an endoribonuclease, such as RNase T1, and the digestion products are examined using MALDI-MS. Individual RNAs are identified by their signature digestion products, which arise through the detection of unique mass values after nuclease digestion. Next, the endonuclease digest is derivatized using N-cyclohexyl-N’-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CMCT), which selectively modifies all pseudouridine, thiouridine and 2-methylthio-6-isopentenyladenosine nucleosides. MALDI-MS determination of the CMCT-derivatized endonuclease digest reveals the presence of pseudouridine through a 252 Da mass increase over the underivatized digest. Proof-of-concept experiments were conducted using a mixture of Escherichia coli transfer RNAs and endoribonucleases T1 and A. More than 80% of the expected pseudouridines from this mixture were detected using this screening approach, even on a unfractionated sample of tRNAs. This approach should be particularly useful in the identification of putative pseudouridine synthases through detection of their target RNAs and can provide insight into specific small RNAs that may contain pseudouridine. PMID:18973194

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

    NASA Astrophysics Data System (ADS)

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

    2017-04-01

    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.

  15. A transgenic resource for conditional competitive inhibition of conserved Drosophila microRNAs

    PubMed Central

    Fulga, Tudor A.; McNeill, Elizabeth M.; Binari, Richard; Yelick, Julia; Blanche, Alexandra; Booker, Matthew; Steinkraus, Bruno R.; Schnall-Levin, Michael; Zhao, Yong; DeLuca, Todd; Bejarano, Fernando; Han, Zhe; Lai, Eric C.; Wall, Dennis P.; Perrimon, Norbert; Van Vactor, David

    2015-01-01

    Although the impact of microRNAs (miRNAs) in development and disease is well established, understanding the function of individual miRNAs remains challenging. Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. However, the application of these loss-of-function strategies has been limited. Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. Ubiquitous miRNA sponge delivery and consequent systemic miRNA inhibition uncovers a relatively small number of miRNA families underlying viability and gross morphogenesis, with false discovery rates in the 4–8% range. In contrast, tissue-specific silencing of muscle-enriched miRNAs reveals a surprisingly large number of novel miRNA contributions to the maintenance of adult indirect flight muscle structure and function. A strong correlation between miRNA abundance and physiological relevance is not observed, underscoring the importance of unbiased screens when assessing the contributions of miRNAs to complex biological processes. PMID:26081261

  16. Incorporation of osteogenic and angiogenic small interfering RNAs into chitosan sponge for bone tissue engineering

    PubMed Central

    Jia, Sen; Yang, Xinjie; Song, Wen; Wang, Lei; Fang, Kaixiu; Hu, Zhiqiang; Yang, Zihui; Shan, Chun; Lei, Delin; Lu, Bin

    2014-01-01

    Engineered bone substitutes are being extensively explored in response to growing demand. However, the angiogenesis that occurs during bone formation is often overlooked in scaffold design. In this novel study, we incorporated two small interfering RNAs (siRNAs), ie, small interfering RNA targets casein kinase 2 interaction protein 1 (siCkip-1) and small interfering RNA targets soluble VEGF receptor 1 (siFlt-1), which can promote osteogenesis and angiogenesis, into a chitosan sponge. This scaffold could maintain siRNAs for over 2 weeks in neutral phosphate-buffered saline and degraded rapidly in the presence of lysozyme. The chitosan sponge with siCkip-1 and siFlt-1 in vitro bioactivity was investigated using mesenchymal stem cells. Target genes were significantly suppressed, and osteocalcin, alkaline phosphatase, and vascular endothelial growth factor were significantly upregulated. Alizarin Red staining revealed that mineralization of the extracellular matrix was markedly enhanced by dual transfection. Further analysis by immunofluorescence confirmed that the siRNA-modified scaffold simultaneously improved the expression of osteocalcin and von Willebrand factor. In vivo testing in a skull critical-size defect model showed marked bone regeneration in rats treated with siCkip-1 and siFlt-1. In conclusion, chitosan sponge containing osteogenic and angiogenic siRNAs may be used as a scaffold for bone regeneration. The dual siRNA concept may also be useful in the biofunctionalization of other materials. PMID:25429217

  17. Guardian small RNAs and sex determination.

    PubMed

    Katsuma, Susumu; Kawamoto, Munetaka; Kiuchi, Takashi

    2014-01-01

    The W chromosome of the silkworm Bombyx mori has been known to determine femaleness for more than 80 years. However, the feminizing gene has not been molecularly identified, because the B. mori W chromosome is almost fully occupied by a large number of transposable elements. The W chromosome-derived feminizing factor of B. mori was recently shown to be a female-specific PIWI-interacting RNA (piRNA). piRNAs are small RNAs that potentially repress invading "non-self" elements (e.g., transposons and virus-like elements) by associating with PIWI proteins. Our results revealed that female-specific piRNA precursors, which we named Fem, are transcribed from the sex-determining region of the W chromosome at the early embryonic stage and are processed into a single mature piRNA (Fem piRNA). Fem piRNA forms a complex with Siwi (silkworm Piwi), which cleaves a protein-coding mRNA transcribed from the Z chromosome. RNA interference of this Z-linked gene, which we named Masc, revealed that this gene encodes a protein required for masculinization and dosage compensation. Fem and Masc both participate in the ping-pong cycle of the piRNA amplification loop by associating with the 2 B. mori PIWI proteins Siwi and BmAgo3 (silkworm Ago3), respectively, indicating that the piRNA-mediated interaction between the 2 sex chromosomes is the primary signal for the B. mori sex determination cascade. Fem is a non-transposable repetitive sequence on the W chromosome, whereas Masc is a single-copy protein-coding gene. It is of great interest how the piRNA system recognizes "self "Masc mRNA as "non-self" RNA.

  18. omiRas: a Web server for differential expression analysis of miRNAs derived from small RNA-Seq data.

    PubMed

    Müller, Sören; Rycak, Lukas; Winter, Peter; Kahl, Günter; Koch, Ina; Rotter, Björn

    2013-10-15

    Small RNA deep sequencing is widely used to characterize non-coding RNAs (ncRNAs) differentially expressed between two conditions, e.g. healthy and diseased individuals and to reveal insights into molecular mechanisms underlying condition-specific phenotypic traits. The ncRNAome is composed of a multitude of RNAs, such as transfer RNA, small nucleolar RNA and microRNA (miRNA), to name few. Here we present omiRas, a Web server for the annotation, comparison and visualization of interaction networks of ncRNAs derived from next-generation sequencing experiments of two different conditions. The Web tool allows the user to submit raw sequencing data and results are presented as: (i) static annotation results including length distribution, mapping statistics, alignments and quantification tables for each library as well as lists of differentially expressed ncRNAs between conditions and (ii) an interactive network visualization of user-selected miRNAs and their target genes based on the combination of several miRNA-mRNA interaction databases. The omiRas Web server is implemented in Python, PostgreSQL, R and can be accessed at: http://tools.genxpro.net/omiras/.

  19. Maintaining good miRNAs in the body keeps the doctor away?: Perspectives on the relationship between food-derived natural products and microRNAs in relation to exosomes/extracellular vesicles.

    PubMed

    Otsuka, Kurataka; Yamamoto, Yusuke; Matsuoka, Ryosuke; Ochiya, Takahiro

    2018-01-01

    During the last decade, it has been uncovered that microRNAs (miRNAs), a class of small non-coding RNAs, are related to many diseases including cancers. With an increase in reports describing the dysregulation of miRNAs in various tumor types, it has become abundantly clear that miRNAs play significant roles in the formation and progression of cancers. Intriguingly, miRNAs are present in body fluids because they are packed in exosomes/extracellular vesicles and released from all types of cells. The miRNAs in the fluids are measured in a relatively simple way and the profile of miRNAs is likely to be an indicator of health condition. In recent years, various studies have demonstrated that some naturally occurring compounds can control tumor-suppressive and oncogenic miRNAs in a positive manner, suggesting that food-derived compounds could maintain the expression levels of miRNAs and help maintain good health. Therefore, our daily food and compounds in food are of great interest. In addition, exogenous diet-derived miRNAs have been indicated to function in the regulation of target mammalian transcripts in the body. These findings highlight the possibility of diet for good health through the regulation of miRNAs, and we also discuss the perspective of food application and health promotion. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Origins and Mechanisms of miRNAs and siRNAs.

    PubMed

    Carthew, Richard W; Sontheimer, Erik J

    2009-02-20

    Over the last decade, approximately 20-30 nucleotide RNA molecules have emerged as critical regulators in the expression and function of eukaryotic genomes. Two primary categories of these small RNAs--short interfering RNAs (siRNAs) and microRNAs (miRNAs)--act in both somatic and germline lineages in a broad range of eukaryotic species to regulate endogenous genes and to defend the genome from invasive nucleic acids. Recent advances have revealed unexpected diversity in their biogenesis pathways and the regulatory mechanisms that they access. Our understanding of siRNA- and miRNA-based regulation has direct implications for fundamental biology as well as disease etiology and treatment.

  1. 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.

  2. Global Small RNA Chaperone Hfq and Regulatory Small RNAs Are Important Virulence Regulators in Erwinia amylovora

    PubMed Central

    Zeng, Quan; McNally, R. Ryan

    2013-01-01

    Hfq is a global small RNA (sRNA) chaperone that interacts with Hfq-regulated sRNAs and functions in the posttranscriptional regulation of gene expression. In this work, we identified Hfq to be a virulence regulator in the Gram-negative fire blight pathogen Erwinia amylovora. Deletion of hfq in E. amylovora Ea1189 significantly reduced bacterial virulence in both immature pear fruits and apple shoots. Analysis of virulence determinants in strain Ea1189Δhfq showed that Hfq exerts pleiotropic regulation of amylovoran exopolysaccharide production, biofilm formation, motility, and the type III secretion system (T3SS). Further characterization of biofilm regulation by Hfq demonstrated that Hfq limits bacterial attachment to solid surfaces while promoting biofilm maturation. Characterization of T3SS regulation by Hfq revealed that Hfq positively regulates the translocation and secretion of the major type III effector DspE and negatively controls the secretion of the putative translocator HrpK and the type III effector Eop1. Lastly, 10 Hfq-regulated sRNAs were identified using a computational method, and two of these sRNAs, RprA and RyhA, were found to be required for the full virulence of E. amylovora. PMID:23378513

  3. A role for neuronal piRNAs in the epigenetic control of memory-related synaptic plasticity.

    PubMed

    Rajasethupathy, Priyamvada; Antonov, Igor; Sheridan, Robert; Frey, Sebastian; Sander, Chris; Tuschl, Thomas; Kandel, Eric R

    2012-04-27

    Small RNA-mediated gene regulation during development causes long-lasting changes in cellular phenotypes. To determine whether small RNAs of the adult brain can regulate memory storage, a process that requires stable and long-lasting changes in the functional state of neurons, we generated small RNA libraries from the Aplysia CNS. In these libraries, we discovered an unexpectedly abundant expression of a 28 nucleotide sized class of piRNAs in brain, which had been thought to be germline specific. These piRNAs have unique biogenesis patterns, predominant nuclear localization, and robust sensitivity to serotonin, a modulatory transmitter that is important for memory. We find that the Piwi/piRNA complex facilitates serotonin-dependent methylation of a conserved CpG island in the promoter of CREB2, the major inhibitory constraint of memory in Aplysia, leading to enhanced long-term synaptic facilitation. These findings provide a small RNA-mediated gene regulatory mechanism for establishing stable long-term changes in neurons for the persistence of memory. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. High-Throughput Sequencing and Characterization of the Small RNA Transcriptome Reveal Features of Novel and Conserved MicroRNAs in Panax ginseng

    PubMed Central

    Ma, Yimian; Yuan, Lichai; Lu, Shanfa

    2012-01-01

    microRNAs (miRNAs) play vital regulatory roles in many organisms through direct cleavage of transcripts, translational repression, or chromatin modification. Identification of miRNAs has been carried out in various plant species. However, no information is available for miRNAs from Panax ginseng, an economically significant medicinal plant species. Using the next generation high-throughput sequencing technology, we obtained 13,326,328 small RNA reads from the roots, stems, leaves and flowers of P. ginseng. Analysis of these small RNAs revealed the existence of a large, diverse and highly complicated small RNA population in P. ginseng. We identified 73 conserved miRNAs, which could be grouped into 33 families, and 28 non-conserved ones belonging to 9 families. Characterization of P. ginseng miRNA precursors revealed many features, such as production of two miRNAs from distinct regions of a precursor, clusters of two precursors in a transcript, and generation of miRNAs from both sense and antisense transcripts. It suggests the complexity of miRNA production in P. gingseng. Using a computational approach, we predicted for the conserved and non-conserved miRNA families 99 and 31 target genes, respectively, of which eight were experimentally validated. Among all predicted targets, only about 20% are conserved among various plant species, whereas the others appear to be non-conserved, indicating the diversity of miRNA functions. Consistently, many miRNAs exhibited tissue-specific expression patterns. Moreover, we identified five dehydration- and ten heat-responsive miRNAs and found the existence of a crosstalk among some of the stress-responsive miRNAs. Our results provide the first clue to the elucidation of miRNA functions in P. ginseng. PMID:22962612

  5. 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.

  6. A Ruler Protein in a Complex for Antiviral Defense Determines the Length of Small Interfering CRISPR RNAs

    PubMed Central

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

    2013-01-01

    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. PMID:23935102

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

    PubMed

    Krishna, Srikar; Nair, Aparna; Cheedipudi, Sirisha; Poduval, Deepak; Dhawan, Jyotsna; Palakodeti, Dasaradhi; Ghanekar, Yashoda

    2013-01-07

    Small non-coding RNAs such as miRNAs, piRNAs and endo-siRNAs fine-tune gene expression through post-transcriptional regulation, modulating important processes in development, differentiation, homeostasis and regeneration. Using deep sequencing, we have profiled small non-coding RNAs in Hydra magnipapillata and investigated changes in small RNA expression pattern during head regeneration. Our results reveal a unique repertoire of small RNAs in hydra. We have identified 126 miRNA loci; 123 of these miRNAs are unique to hydra. Less than 50% are conserved across two different strains of Hydra vulgaris tested in this study, indicating a highly diverse nature of hydra miRNAs in contrast to bilaterian miRNAs. We also identified siRNAs derived from precursors with perfect stem-loop structure and that arise from inverted repeats. piRNAs were the most abundant small RNAs in hydra, mapping to transposable elements, the annotated transcriptome and unique non-coding regions on the genome. piRNAs that map to transposable elements and the annotated transcriptome display a ping-pong signature. Further, we have identified several miRNAs and piRNAs whose expression is regulated during hydra head regeneration. Our study defines different classes of small RNAs in this cnidarian model system, which may play a role in orchestrating gene expression essential for hydra regeneration.

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

    PubMed Central

    Krishna, Srikar; Nair, Aparna; Cheedipudi, Sirisha; Poduval, Deepak; Dhawan, Jyotsna; Palakodeti, Dasaradhi; Ghanekar, Yashoda

    2013-01-01

    Small non-coding RNAs such as miRNAs, piRNAs and endo-siRNAs fine-tune gene expression through post-transcriptional regulation, modulating important processes in development, differentiation, homeostasis and regeneration. Using deep sequencing, we have profiled small non-coding RNAs in Hydra magnipapillata and investigated changes in small RNA expression pattern during head regeneration. Our results reveal a unique repertoire of small RNAs in hydra. We have identified 126 miRNA loci; 123 of these miRNAs are unique to hydra. Less than 50% are conserved across two different strains of Hydra vulgaris tested in this study, indicating a highly diverse nature of hydra miRNAs in contrast to bilaterian miRNAs. We also identified siRNAs derived from precursors with perfect stem–loop structure and that arise from inverted repeats. piRNAs were the most abundant small RNAs in hydra, mapping to transposable elements, the annotated transcriptome and unique non-coding regions on the genome. piRNAs that map to transposable elements and the annotated transcriptome display a ping–pong signature. Further, we have identified several miRNAs and piRNAs whose expression is regulated during hydra head regeneration. Our study defines different classes of small RNAs in this cnidarian model system, which may play a role in orchestrating gene expression essential for hydra regeneration. PMID:23166307

  9. A unique set of 6 circulating microRNAs for early detection of non-small cell lung cancer.

    PubMed

    Halvorsen, Ann Rita; Bjaanæs, Maria; LeBlanc, Marissa; Holm, Are M; Bolstad, Nils; Rubio, Luis; Peñalver, Juan Carlos; Cervera, José; Mojarrieta, Julia Cruz; López-Guerrero, Jose Antonio; Brustugun, Odd Terje; Helland, Åslaug

    2016-06-14

    Circulating microRNAs are promising biomarkers for diagnosis, predication and prognostication of diseases. Lung cancer is the cancer disease accountable for most cancer deaths, largely due to being diagnosed at late stages. Therefore, diagnosing lung cancer patients at an early stage is crucial for improving the outcome. The purpose of this study was to identify circulating microRNAs for detection of early stage lung cancer, capable of discriminating lung cancer patients from those with chronic obstructive pulmonary disease (COPD) and healthy volunteers. We identified 7 microRNAs separating lung cancer patients from controls. By using RT-qPCR, we validated 6 microRNAs (miR-429, miR-205, miR-200b, miR-203, miR-125b and miR-34b) with a significantly higher abundance in serum from NSCLC patients. Furthermore, the 6 miRNAs were validated in a different dataset, revealing an area under the receiver operating characteristic curve of 0.89 for stage I-IV and 0.88 for stage I/II. We profiled the expression of 754 unique microRNAs by TaqMan Low Density Arrays, and analyzed serum from 38 patients with NSCLC, 16 patients suffering from COPD and 16 healthy volunteers from Norway, to explore their potential as diagnostic biomarkers. For validation, we analyzed serum collected from high-risk individuals enrolled in the Valencia branch of the International Early Lung Cancer Action Program screening trial (n=107) in addition to 51 lung cancer patients. Considering the accessibility and stability of circulating miRNAs, these 6 microRNAs are promising biomarkers as a supplement in future screening studies.

  10. Human intron-encoded Alu RNAs are processed and packaged into Wdr79-associated nucleoplasmic box H/ACA RNPs

    PubMed Central

    Jády, Beáta E.; Ketele, Amandine; Kiss, Tamás

    2012-01-01

    Alu repetitive sequences are the most abundant short interspersed DNA elements in the human genome. Full-length Alu elements are composed of two tandem sequence monomers, the left and right Alu arms, both derived from the 7SL signal recognition particle RNA. Since Alu elements are common in protein-coding genes, they are frequently transcribed into pre-mRNAs. Here, we demonstrate that the right arms of nascent Alu transcripts synthesized within pre-mRNA introns are processed into metabolically stable small RNAs. The intron-encoded Alu RNAs, termed AluACA RNAs, are structurally highly reminiscent of box H/ACA small Cajal body (CB) RNAs (scaRNAs). They are composed of two hairpin units followed by the essential H (AnAnnA) and ACA box motifs. The mature AluACA RNAs associate with the four H/ACA core proteins: dyskerin, Nop10, Nhp2, and Gar1. Moreover, the 3′ hairpin of AluACA RNAs carries two closely spaced CB localization motifs, CAB boxes (UGAG), which bind Wdr79 in a cumulative fashion. In contrast to canonical H/ACA scaRNPs, which concentrate in CBs, the AluACA RNPs accumulate in the nucleoplasm. Identification of 348 human AluACA RNAs demonstrates that intron-encoded AluACA RNAs represent a novel, large subgroup of H/ACA RNAs, which are apparently confined to human or primate cells. PMID:22892240

  11. High-throughput sequencing of small RNAs from pollen and silk and characterization of miRNAs as candidate factors involved in pollen-silk interactions in maize.

    PubMed

    Li, Xiao Ming; Sang, Ya Lin; Zhao, Xiang Yu; Zhang, Xian Sheng

    2013-01-01

    In angiosperms, successful pollen-pistil interactions are the prerequisite and guarantee of subsequent fertilization and seed production. Recent profile analyses have helped elucidate molecular mechanisms underlying these processes at both transcriptomic and proteomic levels, but the involvement of miRNAs in pollen-pistil interactions is still speculative. In this study, we sequenced four small RNA libraries derived from mature pollen, in vitro germinated pollen, mature silks, and pollinated silks of maize (Zea mays L.). We identified 161 known miRNAs belonging to 27 families and 82 novel miRNAs. Of these, 40 conserved and 16 novel miRNAs showed different expression levels between mature and germinated pollen, and 30 conserved and eight novel miRNAs were differentially expressed between mature and pollinated silks. As candidates for factors associated with pollen-silk (pistil) interactions, expression patterns of the two sets of differentially expressed miRNAs were confirmed by stem-loop real-time RT-PCR. Transcript levels of 22 predicted target genes were also validated using real-time RT-PCR; most of these exhibited expression patterns contrasting with those of their corresponding miRNAs. In addition, GO analysis of target genes of differentially expressed miRNAs revealed that functional categories related to auxin signal transduction and gene expression regulation were overrepresented. These results suggest that miRNA-mediated auxin signal transduction and transcriptional regulation have roles in pollen-silk interactions. The results of our study provide novel information for understanding miRNA regulatory roles in pollen-pistil interactions.

  12. Revealing stable processing products from ribosome-associated small RNAs by deep-sequencing data analysis.

    PubMed

    Zywicki, Marek; Bakowska-Zywicka, Kamilla; Polacek, Norbert

    2012-05-01

    The exploration of the non-protein-coding RNA (ncRNA) transcriptome is currently focused on profiling of microRNA expression and detection of novel ncRNA transcription units. However, recent studies suggest that RNA processing can be a multi-layer process leading to the generation of ncRNAs of diverse functions from a single primary transcript. Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases. Thus the correct assessment of widespread RNA processing events is one of the major obstacles in transcriptome research. Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data. The major features include efficient handling of non-unique reads, detection of novel stable ncRNA transcripts and processing products and annotation of known transcripts based on multiple sources of information. To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae. By employing the APART pipeline, we were able to detect and confirm by independent experimental methods multiple novel stable RNA molecules differentially processed from well known ncRNAs, like rRNAs, tRNAs or snoRNAs, in a stress-dependent manner.

  13. Deep sequencing of small RNA repertoires in mice reveals metabolic disorders-associated hepatic miRNAs.

    PubMed

    Liang, Tingming; Liu, Chang; Ye, Zhenchao

    2013-01-01

    Obesity and associated metabolic disorders contribute importantly to the metabolic syndrome. On the other hand, microRNAs (miRNAs) are a class of small non-coding RNAs that repress target gene expression by inducing mRNA degradation and/or translation repression. Dysregulation of specific miRNAs in obesity may influence energy metabolism and cause insulin resistance, which leads to dyslipidemia, steatosis hepatis and type 2 diabetes. In the present study, we comprehensively analyzed and validated dysregulated miRNAs in ob/ob mouse liver, as well as miRNA groups based on miRNA gene cluster and gene family by using deep sequencing miRNA datasets. We found that over 13.8% of the total analyzed miRNAs were dysregulated, of which 37 miRNA species showed significantly differential expression. Further RT-qPCR analysis in some selected miRNAs validated the similar expression patterns observed in deep sequencing. Interestingly, we found that miRNA gene cluster and family always showed consistent dysregulation patterns in ob/ob mouse liver, although they had various enrichment levels. Functional enrichment analysis revealed the versatile physiological roles (over six signal pathways and five human diseases) of these miRNAs. Biological studies indicated that overexpression of miR-126 or inhibition of miR-24 in AML-12 cells attenuated free fatty acids-induced fat accumulation. Taken together, our data strongly suggest that obesity and metabolic disturbance are tightly associated with functional miRNAs. We also identified hepatic miRNA candidates serving as potential biomarkers for the diagnose of the metabolic syndrome.

  14. microRNAs in High and Low Responders to Resistance Training in Breast Cancer Survivors.

    PubMed

    Hagstrom, Amanda D; Denham, Joshua

    2018-06-01

    Accounting for one in three cancer diagnoses, breast cancer is the second most commonly diagnosed cancer in women. Exercise has a well-accepted role in the multi-disciplinary approach to rehabilitating breast cancer survivors. Despite the many known benefits of resistance training on women recovering from breast cancer, the molecular mechanisms are poorly understood. MicroRNAs are small non-coding RNAs that have crucial roles in growth and development. Here, we analysed the abundance of 9 miRNAs, with known roles in muscle physiology and some linked to cancer, in serum samples from 24 breast cancer survivors before and after a 16-week resistance training or usual care intervention. The resistance training group completed supervised thrice-weekly training. miRNA abundance was assessed before and after the intervention period using qPCR. There were no statistically significant changes in any of the miRNAs between groups after the intervention period (all p>0.05). After assessing miRNA abundance in context with high and low responders to resistance training, we observed that relative to low responders, high responders exhibited increased miR-133a-3p and a borderline statistically significant increase in miR-370-3p. Findings from our controlled study indicate the diverse interindividual miRNA responses to resistance training and reveal a discordant regulation between high and low responders. © Georg Thieme Verlag KG Stuttgart · New York.

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

    PubMed Central

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

    2007-01-01

    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 α-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 α-proteobacteria with their eukaryotic hosts. PMID:17971083

  16. Role of transposon-derived small RNAs in the interplay between genomes and parasitic DNA in rice.

    PubMed

    Nosaka, Misuzu; Itoh, Jun-Ichi; Nagato, Yasuo; Ono, Akemi; Ishiwata, Aiko; Sato, Yutaka

    2012-09-01

    RNA silencing is a defense system against "genomic parasites" such as transposable elements (TE), which are potentially harmful to host genomes. In plants, transcripts from TEs induce production of double-stranded RNAs (dsRNAs) and are processed into small RNAs (small interfering RNAs, siRNAs) that suppress TEs by RNA-directed DNA methylation. Thus, the majority of TEs are epigenetically silenced. On the other hand, most of the eukaryotic genome is composed of TEs and their remnants, suggesting that TEs have evolved countermeasures against host-mediated silencing. Under some circumstances, TEs can become active and increase in copy number. Knowledge is accumulating on the mechanisms of TE silencing by the host; however, the mechanisms by which TEs counteract silencing are poorly understood. Here, we show that a class of TEs in rice produces a microRNA (miRNA) to suppress host silencing. Members of the microRNA820 (miR820) gene family are located within CACTA DNA transposons in rice and target a de novo DNA methyltransferase gene, OsDRM2, one of the components of epigenetic silencing. We confirmed that miR820 negatively regulates the expression of OsDRM2. In addition, we found that expression levels of various TEs are increased quite sensitively in response to decreased OsDRM2 expression and DNA methylation at TE loci. Furthermore, we found that the nucleotide sequence of miR820 and its recognition site within the target gene in some Oryza species have co-evolved to maintain their base-pairing ability. The co-evolution of these sequences provides evidence for the functionality of this regulation. Our results demonstrate how parasitic elements in the genome escape the host's defense machinery. Furthermore, our analysis of the regulation of OsDRM2 by miR820 sheds light on the action of transposon-derived small RNAs, not only as a defense mechanism for host genomes but also as a regulator of interactions between hosts and their parasitic elements.

  17. 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

  18. Monitoring the spatiotemporal activities of miRNAs in small animal models using molecular imaging modalities.

    PubMed

    Baril, Patrick; Ezzine, Safia; Pichon, Chantal

    2015-03-04

    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.

  19. Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria

    NASA Astrophysics Data System (ADS)

    Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel

    2017-10-01

    Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

  20. Small RNA Transcriptome of Hibiscus Syriacus Provides Insights into the Potential Influence of microRNAs in Flower Development and Terpene Synthesis.

    PubMed

    Kim, Taewook; Park, June Hyun; Lee, Sang-Gil; Kim, Soyoung; Kim, Jihyun; Lee, Jungho; Shin, Chanseok

    2017-08-01

    MicroRNAs (miRNAs) are essential small RNA molecules that regulate the expression of target mRNAs in plants and animals. Here, we aimed to identify miRNAs and their putative targets in Hibiscus syriacus , the national flower of South Korea. We employed high-throughput sequencing of small RNAs obtained from four different tissues ( i.e. , leaf, root, flower, and ovary) and identified 33 conserved and 30 novel miRNA families, many of which showed differential tissue-specific expressions. In addition, we computationally predicted novel targets of miRNAs and validated some of them using 5' rapid amplification of cDNA ends analysis. One of the validated novel targets of miR477 was a terpene synthase, the primary gene involved in the formation of disease-resistant terpene metabolites such as sterols and phytoalexins. In addition, a predicted target of conserved miRNAs, miR396, is SHORT VEGETATIVE PHASE , which is involved in flower initiation and is duplicated in H. syriacus . Collectively, this study provides the first reliable draft of the H. syriacus miRNA transcriptome that should constitute a basis for understanding the biological roles of miRNAs in H. syriacus.

  1. The UEA sRNA Workbench (version 4.4): a comprehensive suite of tools for analyzing miRNAs and sRNAs.

    PubMed

    Stocks, Matthew B; Mohorianu, Irina; Beckers, Matthew; Paicu, Claudia; Moxon, Simon; Thody, Joshua; Dalmay, Tamas; Moulton, Vincent

    2018-05-02

    RNA interference, a highly conserved regulatory mechanism, is mediated via small RNAs. Recent technical advances enabled the analysis of larger, complex datasets and the investigation of microRNAs and the less known small interfering RNAs. However, the size and intricacy of current data requires a comprehensive set of tools, able to discriminate the patterns from the low-level, noise-like, variation; numerous and varied suggestions from the community represent an invaluable source of ideas for future tools, the ability of the community to contribute to this software is essential. We present a new version of the UEA sRNA Workbench, reconfigured to allow an easy insertion of new tools/workflows. In its released form, it comprises of a suite of tools in a user-friendly environment, with enhanced capabilities for a comprehensive processing of sRNA-seq data e.g. tools for an accurate prediction of sRNA loci (CoLIde) and miRNA loci (miRCat2), as well as workflows to guide the users through common steps such as quality checking of the input data, normalization of abundances or detection of differential expression represent the first step in sRNA-seq analyses. The UEA sRNA Workbench is available at: http://srna-workbench.cmp.uea.ac.uk The source code is available at: https://github.com/sRNAworkbenchuea/UEA_sRNA_Workbench. v.moulton@uea.ac.uk.

  2. Diversity of miRNAs, siRNAs, and piRNAs across 25 Drosophila cell lines

    PubMed Central

    Wen, Jiayu; Mohammed, Jaaved; Bortolamiol-Becet, Diane; Tsai, Harrison; Robine, Nicolas; Westholm, Jakub O.; Ladewig, Erik; Dai, Qi; Okamura, Katsutomo; Flynt, Alex S.; Zhang, Dayu; Andrews, Justen; Cherbas, Lucy; Kaufman, Thomas C.; Cherbas, Peter; Siepel, Adam; Lai, Eric C.

    2014-01-01

    We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage. PMID:24985917

  3. PolyU tail of rho-independent terminator of bacterial small RNAs is essential for Hfq action.

    PubMed

    Otaka, Hironori; Ishikawa, Hirokazu; Morita, Teppei; Aiba, Hiroji

    2011-08-09

    Major bacterial small RNAs (sRNAs) regulate the translation and stability of target mRNAs through base pairing with the help of the RNA chaperone Hfq. The Hfq-dependent sRNAs consist of three basic elements, mRNA base-pairing region, Hfq-binding site, and rho-independent terminator. Although the base-pairing region and the terminator are well documented in many sRNAs, the Hfq-binding site is less well-defined except that Hfq binds RNA with a preference for AU-rich sequences. Here, we performed mutational and biochemical studies to define the sRNA site required for Hfq action using SgrS as a model sRNA. We found that shortening terminator polyU tail eliminates the ability of SgrS to bind to Hfq and to silence ptsG mRNA. We also demonstrate that the SgrS terminator can be replaced with any foreign rho-independent terminators possessing a polyU tail longer than 8 without losing the ability to silence ptsG mRNA in an Hfq-dependent manner. Moreover, we found that shortening the terminator polyU tail of several other sRNAs also eliminates the ability to bind to Hfq and to regulate target mRNAs. We conclude that the polyU tail of sRNAs is essential for Hfq action in general. The data also indicate that the terminator polyU tail plays a role in Hfq-dependent stabilization of sRNAs.

  4. PolyU tail of rho-independent terminator of bacterial small RNAs is essential for Hfq action

    PubMed Central

    Otaka, Hironori; Ishikawa, Hirokazu; Morita, Teppei; Aiba, Hiroji

    2011-01-01

    Major bacterial small RNAs (sRNAs) regulate the translation and stability of target mRNAs through base pairing with the help of the RNA chaperone Hfq. The Hfq-dependent sRNAs consist of three basic elements, mRNA base-pairing region, Hfq-binding site, and rho-independent terminator. Although the base-pairing region and the terminator are well documented in many sRNAs, the Hfq-binding site is less well-defined except that Hfq binds RNA with a preference for AU-rich sequences. Here, we performed mutational and biochemical studies to define the sRNA site required for Hfq action using SgrS as a model sRNA. We found that shortening terminator polyU tail eliminates the ability of SgrS to bind to Hfq and to silence ptsG mRNA. We also demonstrate that the SgrS terminator can be replaced with any foreign rho-independent terminators possessing a polyU tail longer than 8 without losing the ability to silence ptsG mRNA in an Hfq-dependent manner. Moreover, we found that shortening the terminator polyU tail of several other sRNAs also eliminates the ability to bind to Hfq and to regulate target mRNAs. We conclude that the polyU tail of sRNAs is essential for Hfq action in general. The data also indicate that the terminator polyU tail plays a role in Hfq-dependent stabilization of sRNAs. PMID:21788484

  5. Identification and validation of sRNAs in Edwardsiella tarda S08.

    PubMed

    Sun, Yuying; Zhang, Jiquan; Qin, Lei; Yan, Cui; Zhang, Xiaojun; Liu, Dandan

    2017-01-01

    Bacterial small non-coding RNAs (sRNAs) are known as novel regulators involved in virulence, stress responsibility, and so on. Recently, a lot of new researches have highlighted the critical roles of sRNAs in fine-tune gene regulation in both prokaryotes and eukaryotes. Edwardsiella tarda (E. tarda) is a gram-negative, intracellular pathogen that causes edwardsiellosis in fish. Thus far, no sRNA has been reported in E. tarda. The present study represents the first attempt to identify sRNAs in E. tarda S08. Ten sRNAs were validated by RNA sequencing and quantitative PCR (qPCR). ET_sRNA_1 and ET_sRNA_2 were homolous to tmRNA and GcvB, respectively. However, the other candidate sRNAs have not been reported till now. The cellular abundance of 10 validated sRNA was detected by qPCR at different growth phases to monitor their biosynthesis. Nine candidate sRNAs were expressed in the late-stage of exponential growth and stationary stages of growth (36~60 h). And the expression of the nine sRNAs was growth phase-dependent. But ET_sRNA_10 was almost expressed all the time and reached the highest peak at 48 h. Their targets were predicted by TargetRNA2 and each sRNA target contains some genes that directly or indirectly relate to virulence. These results preliminary showed that sRNAs probably play a regulatory role of virulence in E. tarda.

  6. Transcription start site associated RNAs (TSSaRNAs) are ubiquitous in all domains of life.

    PubMed

    Zaramela, Livia S; Vêncio, Ricardo Z N; ten-Caten, Felipe; Baliga, Nitin S; Koide, Tie

    2014-01-01

    A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs) are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq) and a primary-transcript enriched (dRNA-seq) strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome). Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

  7. Interaction between Host MicroRNAs and the Gut Microbiota in Colorectal Cancer.

    PubMed

    Yuan, Ce; Burns, Michael B; Subramanian, Subbaya; Blekhman, Ran

    2018-01-01

    Although variation in gut microbiome composition has been linked with colorectal cancer (CRC), the factors that mediate the interactions between CRC tumors and the microbiome are poorly understood. MicroRNAs (miRNAs) are known to regulate CRC progression and are associated with patient survival outcomes. In addition, recent studies suggested that host miRNAs can also regulate bacterial growth and influence the composition of the gut microbiome. Here, we investigated the association between miRNA expression and microbiome composition in human CRC tumor and normal tissues. We identified 76 miRNAs as differentially expressed (DE) in tissue from CRC tumors and normal tissue, including the known oncogenic miRNAs miR-182, miR-503, and mir-17~92 cluster. These DE miRNAs were correlated with the relative abundances of several bacterial taxa, including Firmicutes , Bacteroidetes , and Proteobacteria . Bacteria correlated with DE miRNAs were enriched with distinct predicted metabolic categories. Additionally, we found that miRNAs that correlated with CRC-associated bacteria are predicted to regulate targets that are relevant for host-microbiome interactions and highlight a possible role for miRNA-driven glycan production in the recruitment of pathogenic microbial taxa. Our work characterized a global relationship between microbial community composition and miRNA expression in human CRC tissues. IMPORTANCE Recent studies have found an association between colorectal cancer (CRC) and the gut microbiota. One potential mechanism by which the microbiota can influence host physiology is through affecting gene expression in host cells. MicroRNAs (miRNAs) are small noncoding RNA molecules that can regulate gene expression and have important roles in cancer development. Here, we investigated the link between the gut microbiota and the expression of miRNA in CRC. We found that dozens of miRNAs are differentially regulated in CRC tumors and adjacent normal colon and that these miRNAs

  8. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Potential and use of bacterial small RNAs to combat drug resistance: a systematic review

    PubMed Central

    Liu, Xiaodong; Zhang, Lin; Wong, Sunny Hei; Chan, Matthew TV; Wu, William KK

    2017-01-01

    Background Over the decades, new antibacterial agents have been developed in an attempt to combat drug resistance, but they remain unsuccessful. Recently, a novel class of bacterial gene expression regulators, bacterial small RNAs (sRNAs), has received increasing attention toward their involvement in antibiotic resistance. This systematic review aimed to discuss the potential of these small molecules as antibacterial drug targets. Methods Two investigators performed a comprehensive search of MEDLINE, EmBase, and ISI Web of Knowledge from inception to October 2016, without restriction on language. We included all in vitro and in vivo studies investigating the role of bacterial sRNA in antibiotic resistance. Risk of bias of the included studies was assessed by a modified guideline of Systematic Review Center for Laboratory Animal Experimentation (SYRCLE). Results Initial search yielded 432 articles. After exclusion of non-original articles, 20 were included in this review. Of these, all studies examined bacterial-type strains only. There were neither relevant in vivo nor clinical studies. The SYRCLE scores ranged from to 5 to 7, with an average of 5.9. This implies a moderate risk of bias. sRNAs influenced the antibiotics susceptibility through modulation of gene expression relevant to efflux pumps, cell wall synthesis, and membrane proteins. Conclusion Preclinical studies on bacterial-type strains suggest that modulation of sRNAs could enhance bacterial susceptibility to antibiotics. Further studies on clinical isolates and in vivo models are needed to elucidate the therapeutic value of sRNA modulation on treatment of multidrug-resistant bacterial infection. PMID:29290689

  10. 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'. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  11. Exercise alters mouse sperm small noncoding RNAs and induces a transgenerational modification of male offspring conditioned fear and anxiety

    PubMed Central

    Short, A K; Yeshurun, S; Powell, R; Perreau, V M; Fox, A; Kim, J H; Pang, T Y; Hannan, A J

    2017-01-01

    There is growing evidence that the preconceptual lifestyle and other environmental exposures of a father can significantly alter the physiological and behavioral phenotypes of their children. We and others have shown that paternal preconception stress, regardless of whether the stress was experienced during early-life or adulthood, results in offspring with altered anxiety and depression-related behaviors, attributed to hypothalamic–pituitary–adrenal axis dysregulation. The transgenerational response to paternal preconceptual stress is believed to be mediated by sperm-borne small noncoding RNAs, specifically microRNAs. As physical activity confers physical and mental health benefits for the individual, we used a model of voluntary wheel-running and investigated the transgenerational response to paternal exercise. We found that male offspring of runners had suppressed reinstatement of juvenile fear memory, and reduced anxiety in the light–dark apparatus during adulthood. No changes in these affective behaviors were observed in female offspring. We were surprised to find that running had a limited impact on sperm-borne microRNAs. The levels of three unique microRNAs (miR-19b, miR-455 and miR-133a) were found to be altered in the sperm of runners. In addition, we discovered that the levels of two species of tRNA-derived RNAs (tDRs)—tRNA-Gly and tRNA-Pro—were also altered by running. Taken together, we believe this is the first evidence that paternal exercise is associated with an anxiolytic behavioral phenotype of male offspring and altered levels of small noncoding RNAs in sperm. These small noncoding RNAs are known to have an impact on post-transcriptional gene regulation and can thus change the developmental trajectory of offspring brains and associated affective behaviors. PMID:28463242

  12. The Accumulation of miRNAs Differentially Modulated by Drought Stress Is Affected by Grafting in Grapevine1[OPEN

    PubMed Central

    Vitali, Marco; Vitulo, Nicola; Incarbone, Marco

    2017-01-01

    Grapevine (Vitis vinifera) is routinely grafted, and rootstocks inducing drought tolerance represent a source for adapting vineyards to climate change in temperate areas. Our goal was to investigate drought stress effects on microRNA (miRNA) abundance in a drought-resistant grapevine rootstock, M4 (Vitis vinifera × Vitis berlandieri), compared with a commercial cultivar, Cabernet Sauvignon, using their autografts and reciprocal grafts. RNA extracted from roots and leaves of droughted and irrigated plants of different graft combinations was used to prepare cDNA libraries for small RNA sequencing and to analyze miRNAs by quantitative real-time polymerase chain reaction (RT-qPCR). Measurements of leaf water potential, leaf gas exchange, and root hydraulic conductance attested that, under irrigation, M4 reduced water loss in comparison with cultivar Cabernet Sauvignon mostly through nonhydraulic, root-specific mechanisms. Under drought, stomatal conductance decreased at similar levels in the two genotypes. Small RNA sequencing allowed the identification of 70 conserved miRNAs and the prediction of 28 novel miRNAs. Different accumulation trends of miRNAs, observed upon drought and in different genotypes and organs, were confirmed by RT-qPCR. Corresponding target transcripts, predicted in silico and validated by RT-qPCR, often showed opposite expression profiles than the related miRNAs. Drought effects on miRNA abundance differed between the two genotypes. Furthermore, the concentration of drought-responsive miRNAs in each genotype was affected by reciprocal grafting, suggesting either the movement of signals inducing miRNA expression in the graft partner or, possibly, miRNA transport between scion and rootstock. These results open new perspectives in the selection of rootstocks for improving grapevine adaptation to drought. PMID:28235889

  13. Analysis of Antisense Expression by Whole Genome Tiling Microarrays and siRNAs Suggests Mis-Annotation of Arabidopsis Orphan Protein-Coding Genes

    PubMed Central

    Richardson, Casey R.; Luo, Qing-Jun; Gontcharova, Viktoria; Jiang, Ying-Wen; Samanta, Manoj; Youn, Eunseog; Rock, Christopher D.

    2010-01-01

    Background MicroRNAs (miRNAs) and trans-acting small-interfering RNAs (tasi-RNAs) are small (20–22 nt long) RNAs (smRNAs) generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs) are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery. Principal Findings We explored rice (Oryza sativa) sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans) and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis ‘orphan’ hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM) was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the “ancient” (deeply conserved) class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for “new” rapidly-evolving MIRNA genes. Conclusions Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non

  14. Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes

    PubMed Central

    Nicolás, Francisco E.; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M.

    2003-01-01

    Transformation of Mucor circinelloides with self-replicative plasmids containing a wild-type copy of the carotenogenic gene carB causes silencing of the carB function in 3% of transformants. Genomic analyses revealed a relationship between silenced phenotype and number of copies of plasmids. This phenotype results from a reduction of the steady-state levels of carB mRNA, a reduction that is not due to differences in the level of transcription, indicating that silencing is post-transcriptional. Small sense and antisense RNAs have been found to be associated with gene silencing in M.circinelloides. Two size classes of small antisense RNAs, differentially accumulated during the vegetative growth of silenced transformants, have been detected: a long 25-nucleotide RNA and a short 21-nucleotide RNA. Secondary sense and antisense RNAs corresponding to sequences of the endogenous gene downstream of the initial triggering molecule have also been detected, revealing the existence of spreading of RNA targeting in fungi. These findings, together with the self-replicative nature of the triggering molecules, make M.circinelloides a suitable organism for investigating some unresolved questions in RNA silencing. PMID:12881432

  15. Small RNA-seq during acute maximal exercise reveal RNAs involved in vascular inflammation and cardiometabolic health: brief report.

    PubMed

    Shah, Ravi; Yeri, Ashish; Das, Avash; Courtright-Lim, Amanda; Ziegler, Olivia; Gervino, Ernest; Ocel, Jeffrey; Quintero-Pinzon, Pablo; Wooster, Luke; Bailey, Cole Shields; Tanriverdi, Kahraman; Beaulieu, Lea M; Freedman, Jane E; Ghiran, Ionita; Lewis, Gregory D; Van Keuren-Jensen, Kendall; Das, Saumya

    2017-12-01

    Exercise improves cardiometabolic and vascular function, although the mechanisms remain unclear. Our objective was to demonstrate the diversity of circulating extracellular RNA (ex-RNA) release during acute exercise in humans and its relevance to exercise-mediated benefits on vascular inflammation. We performed plasma small RNA sequencing in 26 individuals undergoing symptom-limited maximal treadmill exercise, with replication of our top candidate miRNA in a separate cohort of 59 individuals undergoing bicycle ergometry. We found changes in miRNAs and other ex-RNAs with exercise (e.g., Y RNAs and tRNAs) implicated in cardiovascular disease. In two independent cohorts of acute maximal exercise, we identified miR-181b-5p as a key ex-RNA increased in plasma after exercise, with validation in a separate cohort. In a mouse model of acute exercise, we found significant increases in miR-181b-5p expression in skeletal muscle after acute exercise in young (but not older) mice. Previous work revealed a strong role for miR-181b-5p in vascular inflammation in obesity, insulin resistance, sepsis, and cardiovascular disease. We conclude that circulating ex-RNAs were altered in plasma after acute exercise target pathways involved in inflammation, including miR-181b-5p. Further investigation into the role of known (e.g., miRNA) and novel (e.g., Y RNAs) RNAs is warranted to uncover new mechanisms of vascular inflammation on exercise-mediated benefits on health. NEW & NOTEWORTHY How exercise provides benefits to cardiometabolic health remains unclear. We performed RNA sequencing in plasma during exercise to identify the landscape of small noncoding circulating transcriptional changes. Our results suggest a link between inflammation and exercise, providing rich data on circulating noncoding RNAs for future studies by the scientific community. Copyright © 2017 the American Physiological Society.

  16. Role of the Box C/D Motif in Localization of Small Nucleolar RNAs to Coiled Bodies and Nucleoli

    PubMed Central

    Narayanan, Aarthi; Speckmann, Wayne; Terns, Rebecca; Terns, Michael P.

    1999-01-01

    Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C′, box D, and the 3′ terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs. PMID:10397754

  17. Small interfering RNAs based on huntingtin trinucleotide repeats are highly toxic to cancer cells.

    PubMed

    Murmann, Andrea E; Gao, Quan Q; Putzbach, William E; Patel, Monal; Bartom, Elizabeth T; Law, Calvin Y; Bridgeman, Bryan; Chen, Siquan; McMahon, Kaylin M; Thaxton, C Shad; Peter, Marcus E

    2018-03-01

    Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington's disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA-sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complementary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the six members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR-based siRNAs induce cell death in vitro in all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR-based siRNAs as a novel form of anticancer reagents. © 2018 The Authors.

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

    PubMed

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

    2016-03-01

    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. © 2016 Bandyra et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  19. 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

  20. Standing your Ground to Exoribonucleases: Function of Flavivirus Long Non-coding RNAs

    PubMed Central

    Charley, Phillida A.; Wilusz, Jeffrey

    2015-01-01

    Members of the Flaviviridae (e.g. Dengue virus, West Nile virus, and Hepatitis C virus) contain a positive-sense RNA genome that encodes a large polyprotein. It is now also clear most if not all of these viruses also produce an abundant subgenomic long non-coding RNA. These non-coding RNAs, which are called subgenomicflavivirus RNAs (sfRNAs) or Xrn1-resistant RNAs (xrRNAs), are stable decay intermediates generated from the viral genomic RNA through the stalling of the cellular exoribonuclease Xrn1 at highly structured regions. Several functions of these flavivirus long non-coding RNAs have been revealed in recent years. The generation of these sfRNAs/xrRNAs from viral transcripts results in the repression of Xrn1 and the dysregulation of cellular mRNA stability. The abundant sfRNAs also serve directly as a decoy for important cellular protein regulators of the interferon and RNA interference antiviral pathways. Thus the generation of long non-coding RNAs from flaviviruses, hepaciviruses and pestiviruses likely disrupts aspects of innate immunity and may directly contribute to viral replication, cytopathology and pathogenesis. PMID:26368052

  1. MicroRNAs: A novel potential biomarker for diagnosis and therapy in patients with non-small cell lung cancer.

    PubMed

    Zhou, Qun; Huang, Shao-Xin; Zhang, Feng; Li, Shu-Jun; Liu, Cong; Xi, Yong-Yong; Wang, Liang; Wang, Xin; He, Qi-Qiang; Sun, Cheng-Cao; Li, De-Jia

    2017-12-01

    Lung cancer is still one of the most serious causes of cancer-related deaths all over the world. MicroRNAs (miRNAs) are defined as small non-coding RNAs which could play a pivotal role in post-transcriptional regulation of gene expression. Increasing evidence demonstrated dysregulation of miRNA expression associates with the development and progression of NSCLC. To emphasize a variety of tissue-specific miRNAs, circulating miRNAs and miRNA-derived exosomes could be used as potential diagnostic and therapeutic biomarkers in NSCLC patients. In the current review, we paid attention to the significant discoveries of preclinical and clinical studies, which performed on tissue-specific miRNA, circulating miRNA and exosomal miRNA. The related studies were obtained through a systematic search of Pubmed, Web of Science, Embase. A variety of tissue-specific miRNAs and circulating miRNAs with high sensitivity and specificity which could be used as potential diagnostic and therapeutic biomarkers in NSCLC patients. In addition, we emphasize that the miRNA-derived exosomes become novel diagnostic biomarkers potentially in these patients with NSCLC. MiRNAs have emerged as non-coding RNAs, which have potential to be candidates for the diagnosis and therapy of NSCLC. © 2017 John Wiley & Sons Ltd.

  2. The small non-coding RNA response to virus infection in the Leishmania vector Lutzomyia longipalpis.

    PubMed

    Ferreira, Flávia Viana; Aguiar, Eric Roberto Guimarães Rocha; Olmo, Roenick Proveti; de Oliveira, Karla Pollyanna Vieira; Silva, Emanuele Guimarães; Sant'Anna, Maurício Roberto Viana; Gontijo, Nelder de Figueiredo; Kroon, Erna Geessien; Imler, Jean Luc; Marques, João Trindade

    2018-06-01

    Sandflies are well known vectors for Leishmania but also transmit a number of arthropod-borne viruses (arboviruses). Few studies have addressed the interaction between sandflies and arboviruses. RNA interference (RNAi) mechanisms utilize small non-coding RNAs to regulate different aspects of host-pathogen interactions. The small interfering RNA (siRNA) pathway is a broad antiviral mechanism in insects. In addition, at least in mosquitoes, another RNAi mechanism mediated by PIWI interacting RNAs (piRNAs) is activated by viral infection. Finally, endogenous microRNAs (miRNA) may also regulate host immune responses. Here, we analyzed the small non-coding RNA response to Vesicular stomatitis virus (VSV) infection in the sandfly Lutzoymia longipalpis. We detected abundant production of virus-derived siRNAs after VSV infection in adult sandflies. However, there was no production of virus-derived piRNAs and only mild changes in the expression of vector miRNAs in response to infection. We also observed abundant production of virus-derived siRNAs against two other viruses in Lutzomyia Lulo cells. Together, our results suggest that the siRNA but not the piRNA pathway mediates an antiviral response in sandflies. In agreement with this hypothesis, pre-treatment of cells with dsRNA against VSV was able to inhibit viral replication while knock-down of the central siRNA component, Argonaute-2, led to increased virus levels. Our work begins to elucidate the role of RNAi mechanisms in the interaction between L. longipalpis and viruses and should also open the way for studies with other sandfly-borne pathogens.

  3. Dynamic localisation of mature microRNAs in Human nucleoli is influenced by exogenous genetic materials.

    PubMed

    Li, Zhou Fang; Liang, Yi Min; Lau, Pui Ngan; Shen, Wei; Wang, Dai Kui; Cheung, Wing Tai; Xue, Chun Jason; Poon, Lit Man; Lam, Yun Wah

    2013-01-01

    Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines. The nucleolar association of these miRNAs is resistant to various cellular stresses, but highly sensitive to the presence of exogenous nucleic acids. Introduction of both single- and double-stranded DNA as well as double stranded RNA rapidly induce the redistribution of nucleolar miRNAs to the cytoplasm. A similar change in subcellular distribution is also observed in cells infected with the influenza A virus. The partition of miRNAs between the nucleolus and the cytoplasm is affected by Leptomycin B, suggesting a role of Exportin-1 in the intracellular shuttling of miRNAs. This study reveals a previously unknown aspect of miRNA biology, and suggests a possible link between these small noncoding RNAs and the cellular management of foreign genetic materials.

  4. Application of small RNA sequencing to identify microRNAs in acute kidney injury and fibrosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pellegrini, Kathryn L.

    Establishing a microRNA (miRNA) expression profile in affected tissues provides an important foundation for the discovery of miRNAs involved in the development or progression of pathologic conditions. We conducted small RNA sequencing to generate a temporal profile of miRNA expression in the kidneys using a mouse model of folic acid-induced (250 mg/kg i.p.) kidney injury and fibrosis. From the 103 miRNAs that were differentially expressed over the time course (> 2-fold, p < 0.05), we chose to further investigate miR-18a-5p, which is expressed during the acute stage of the injury; miR-132-3p, which is upregulated during transition between acute and fibroticmore » injury; and miR-146b-5p, which is highly expressed at the peak of fibrosis. Using qRT-PCR, we confirmed the increased expression of these candidate miRNAs in the folic acid model as well as in other established mouse models of acute injury (ischemia/reperfusion injury) and fibrosis (unilateral ureteral obstruction). In situ hybridization confirmed high expression of miR-18a-5p, miR-132-3p and miR-146b-5p throughout the kidney cortex in mice and humans with severe kidney injury or fibrosis. When primary human proximal tubular epithelial cells were treated with model nephrotoxicants such as cadmium chloride (CdCl{sub 2}), arsenic trioxide, aristolochic acid (AA), potassium dichromate (K{sub 2}Cr{sub 2}O{sub 7}) and cisplatin, miRNA-132-3p was upregulated 4.3-fold after AA treatment and 1.5-fold after K{sub 2}Cr{sub 2}O{sub 7} and CdCl{sub 2} treatment. These results demonstrate the application of temporal small RNA sequencing to identify miR-18a, miR-132 and miR-146b as differentially expressed miRNAs during distinct phases of kidney injury and fibrosis progression. - Highlights: • We used small RNA sequencing to identify differentially expressed miRNAs in kidney. • Distinct patterns were found for acute injury and fibrotic stages in the kidney. • Upregulation of miR-18a, -132 and -146b was confirmed

  5. 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

  6. The RNAs of RNA-directed DNA methylation

    PubMed Central

    Wendte, Jered M.; Pikaard, Craig S.

    2016-01-01

    Summary RNA-directed chromatin modification that includes cytosine methylation silences transposable elements in both plants and mammals, contributing to genome defense and stability. In Arabidopsis thaliana, most RNA-directed DNA methylation (RdDM) is guided by small RNAs derived from double-stranded precursors synthesized at cytosine-methylated loci by nuclear multisubunit RNA Polymerase IV (Pol IV), in close partnership with the RNA-dependent RNA polymerase, RDR2. These small RNAs help keep transposons transcriptionally inactive. However, if transposons escape silencing, and are transcribed by multisubunit RNA polymerase II (Pol II), their mRNAs can be recognized and degraded, generating small RNAs that can also guide initial DNA methylation, thereby enabling subsequent Pol IV-RDR2 recruitment. In both pathways, the small RNAs find their target sites by interacting with longer noncoding RNAs synthesized by multisubunit RNA Polymerase V (Pol V). Despite a decade of progress, numerous questions remain concerning the initiation, synthesis, processing, size and features of the RNAs that drive RdDM. Here, we review recent insights, questions and controversies concerning RNAs produced by Pols IV and V, and their functions in RdDM. We also provide new data concerning Pol V transcript 5’ and 3’ ends. PMID:27521981

  7. Novel meiotic miRNAs and indications for a role of phasiRNAs in meiosis

    USDA-ARS?s Scientific Manuscript database

    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 miRNAs directing post-transcriptional regulation of specific target genes, mostly by mRNA cleavage. We used manually isolate...

  8. 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. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  9. Exploring the trans-acting short interfering RNAs (ta-siRNAs) technology for virus control in plants

    USDA-ARS?s Scientific Manuscript database

    Small ribonucleic acid (RNAs) (~20-24nt) processed from double-stranded RNA in plants can trigger degradation of the target mRNAs in cytoplasm or de novo DNA methylation in nucleus leading to gene silencing. Trans-acting short-interfering RNAs (ta-siRNAs) have been shown to enhance the target mRNA d...

  10. A Panel of MicroRNAs as Diagnostic Biomarkers for the Identification of Prostate Cancer.

    PubMed

    Daniel, Rhonda; Wu, Qianni; Williams, Vernell; Clark, Gene; Guruli, Georgi; Zehner, Zendra

    2017-06-16

    Prostate cancer is the most common non-cutaneous cancer among men; yet, current diagnostic methods are insufficient, and more reliable diagnostic markers need to be developed. One answer that can bridge this gap may lie in microRNAs. These small RNA molecules impact protein expression at the translational level, regulating important cellular pathways, the dysregulation of which can exert tumorigenic effects contributing to cancer. In this study, high throughput sequencing of small RNAs extracted from blood from 28 prostate cancer patients at initial stages of diagnosis and prior to treatment was used to identify microRNAs that could be utilized as diagnostic biomarkers for prostate cancer compared to 12 healthy controls. In addition, a group of four microRNAs (miR-1468-3p, miR-146a-5p, miR-1538 and miR-197-3p) was identified as normalization standards for subsequent qRT-PCR confirmation. qRT-PCR analysis corroborated microRNA sequencing results for the seven top dysregulated microRNAs. The abundance of four microRNAs (miR-127-3p, miR-204-5p, miR-329-3p and miR-487b-3p) was upregulated in blood, whereas the levels of three microRNAs (miR-32-5p, miR-20a-5p and miR-454-3p) were downregulated. Data analysis of the receiver operating curves for these selected microRNAs exhibited a better correlation with prostate cancer than PSA (prostate-specific antigen), the current gold standard for prostate cancer detection. In summary, a panel of seven microRNAs is proposed, many of which have prostate-specific targets, which may represent a significant improvement over current testing methods.

  11. The domain structure and distribution of Alu elements in long noncoding RNAs and mRNAs

    PubMed Central

    Kim, Eugene Z.; Wespiser, Adam R.; Caffrey, Daniel R.

    2016-01-01

    Approximately 75% of the human genome is transcribed and many of these spliced transcripts contain primate-specific Alu elements, the most abundant mobile element in the human genome. The majority of exonized Alu elements are located in long noncoding RNAs (lncRNAs) and the untranslated regions of mRNA, with some performing molecular functions. To further assess the potential for Alu elements to be repurposed as functional RNA domains, we investigated the distribution and evolution of Alu elements in spliced transcripts. Our analysis revealed that Alu elements are underrepresented in mRNAs and lncRNAs, suggesting that most exonized Alu elements arising in the population are rare or deleterious to RNA function. When mRNAs and lncRNAs retain exonized Alu elements, they have a clear preference for Alu dimers, left monomers, and right monomers. mRNAs often acquire Alu elements when their genes are duplicated within Alu-rich regions. In lncRNAs, reverse-oriented Alu elements are significantly enriched and are not restricted to the 3′ and 5′ ends. Both lncRNAs and mRNAs primarily contain the Alu J and S subfamilies that were amplified relatively early in primate evolution. Alu J subfamilies are typically overrepresented in lncRNAs, whereas the Alu S dimer is overrepresented in mRNAs. The sequences of Alu dimers tend to be constrained in both lncRNAs and mRNAs, whereas the left and right monomers are constrained within particular Alu subfamilies and classes of RNA. Collectively, these findings suggest that Alu-containing RNAs are capable of forming stable structures and that some of these Alu domains might have novel biological functions. PMID:26654912

  12. Profiling of EBV-Encoded microRNAs in EBV-Associated Hemophagocytic Lymphohistiocytosis.

    PubMed

    Zhou, Chen; Xie, Zhengde; Gao, Liwei; Liu, Chunyan; Ai, Junhong; Zhang, Li; Shen, Kunling

    2015-10-01

    Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a life-threatening complication of EBV infection. MicroRNAs (miRNAs) were small non-coding RNA, and EBV could encode miRNAs that are involved in the progression of infection. However, the profiles of EBV-miRNAs in EBV-HLH were unknown. Here, we aimed to profile the expression of EBV-miRNAs in children with EBV-HLH by analyzing 44 known EBV-miRNAs, encoded within the BamHI fragment H rightward open reading frame 1 (BHRF1) and the BamHI-A region rightward transcript (BART), in plasma and cellular targets by real-time quantitative PCR. The study included 15 children with EBV-HLH, 15 children with infectious mononucleosis (IM), and 15 healthy controls. CD8(+) T cells were found to be the cellular target of EBV infection in EBV-HLH, while CD19(+) B cells were infected with EBV in IM. We also found the greater levels of several miRNAs encoded by BART in EBV-HLH, compared to those in IM and healthy controls, whereas the levels of BHRF1 miRNAs were lower than those in IM. The profile and pattern of EBV-miRNAs in EBV-HLH indicated that EBV could display type II latency in EBV-HLH. Importantly, the level of plasma miR-BART16-1 continued decreasing during the whole chemotherapy, suggesting that plasma miR-BART16-1 could be a potential biomarker for monitoring EBV-HLH progression. The pathogenesis of EBV-HLH might be attributed to the abundance of EBV-miRNAs in EBV-HLH. These findings help elucidate the roles of EBV miRNAs in EBV-HLH, enabling the understanding of the basis of this disease and providing clues for its treatment.

  13. Genome-wide identification of microRNAs in pomegranate (Punica granatum L.) by high-throughput sequencing.

    PubMed

    Saminathan, Thangasamy; Bodunrin, Abiodun; Singh, Nripendra V; Devarajan, Ramajayam; Nimmakayala, Padma; Jeff, Moersfelder; Aradhya, Mallikarjuna; Reddy, Umesh K

    2016-05-26

    MicroRNAs (miRNAs), a class of small non-coding endogenous RNAs that regulate gene expression post-transcriptionally, play multiple key roles in plant growth and development and in biotic and abiotic stress response. Knowledge and roles of miRNAs in pomegranate fruit development have not been explored. Pomegranate, which accumulates a large amount of anthocyanins in skin and arils, is valuable to human health, mainly because of its antioxidant properties. In this study, we developed a small RNA library from pooled RNA samples from young seedlings to mature fruits and identified both conserved and pomegranate-specific miRNA from 29,948,480 high-quality reads. For the pool of 15- to 30-nt small RNAs, ~50 % were 24 nt. The miR157 family was the most abundant, followed by miR156, miR166, and miR168, with variants within each family. The base bias at the first position from the 5' end had a strong preference for U for most 18- to 26-nt sRNAs but a preference for A for 18-nt sRNAs. In addition, for all 24-nt sRNAs, the nucleotide U was preferred (97 %) in the first position. Stem-loop RT-qPCR was used to validate the expression of the predominant miRNAs and novel miRNAs in leaves, male and female flowers, and multiple fruit developmental stages; miR156, miR156a, miR159a, miR159b, and miR319b were upregulated during the later stages of fruit development. Higher expression of miR156 in later fruit developmental may positively regulate anthocyanin biosynthesis by reducing SPL transcription factor. Novel miRNAs showed variation in expression among different tissues. These novel miRNAs targeted different transcription factors and hormone related regulators. Gene ontology and KEGG pathway analyses revealed predominant metabolic processes and catalytic activities, important for fruit development. In addition, KEGG pathway analyses revealed the involvement of miRNAs in ascorbate and linolenic acid, starch and sucrose metabolism; RNA transport; plant hormone signaling pathways

  14. Detecting and characterizing circular RNAs

    PubMed Central

    Jeck, William R.; Sharpless, Norman E.

    2014-01-01

    Circular RNA transcripts were first identified in the early 1990s but knowledge of these species has remained limited, as their study has been difficult through traditional methods of RNA analysis. Now, novel bioinformatic approaches coupled with biochemical enrichment strategies and deep sequencing have allowed comprehensive studies of circular RNA species. Recent studies have revealed thousands of endogenous circular RNAs (circRNAs) in mammalian cells, some of which are highly abundant and evolutionarily conserved. Evidence is emerging that some circRNAs might regulate microRNA (miRNA) function, and roles in transcriptional control have also been suggested. Therefore, study of this class of non-coding RNAs has potential implications for therapeutic and research applications. We believe the key future challenge to the field will be to understand the regulation and function of these unusual molecules. PMID:24811520

  15. Analysis of sucrose-induced small RNAs in Streptococcus mutans in the presence of different sucrose concentrations.

    PubMed

    Liu, Shan Shan; Zhu, Wen Hui; Zhi, Qing Hui; Liu, Jia; Wang, Yan; Lin, Huan Cai

    2017-07-01

    Streptococcus mutans (S. mutans) is the major pathogen contributing to dental caries. Sucrose is an important carbohydrate source for S. mutans and is crucial for dental caries. Small RNAs (sRNAs) are key post-transcriptional regulators of stress adaptation and virulence in bacteria. Here, for the first time, we created three replicate RNA libraries exposed to either 1 or 5% sucrose. The expression levels of sRNAs and target genes (gtfB, gtfC, and spaP) related to virulence were assessed. In addition, some phenotypic traits were evaluated. We obtained 2125 sRNA candidates with at least 100 average reads in 1% sucrose or 5% sucrose. Of these candidates, 2 were upregulated and 20 were downregulated in 1% sucrose. Six of these 22 differentially expressed sRNAs were validated by qRT-PCR. The expression level of target gene gtfB was higher in 1% sucrose. The adherence ratio of S. mutans was higher in 1% sucrose than in 5% sucrose. The synthesis of water-insoluble glucans (WIGs) was significantly higher in 5% sucrose than in 1% sucrose. These data suggest that a series of sRNAs can be induced in response to sucrose, and that some sRNAs might be involved in the regulation of phenotypes, providing new insight into the prevention of caries.

  16. Biology and clinical relevance of noncoding sno/scaRNAs.

    PubMed

    Cao, Thuy; Rajasingh, Sheeja; Samanta, Saheli; Dawn, Buddhadeb; Bittel, Douglas C; Rajasingh, Johnson

    2018-02-01

    Small nucleolar RNAs (snoRNAs) are a group of noncoding RNAs that perform various biological functions, including biochemical modifications of other RNAs, precursors of miRNA, splicing, and telomerase activity. The small Cajal body-associated RNAs (scaRNAs) are a subset of the snoRNA family and collect in the Cajal body where they perform their canonical function to biochemically modify spliceosomal RNAs prior to maturation. Failure of sno/scaRNAs have been implicated in pathology such as congenital heart anomalies, neuromuscular disorders, and various malignancies. Thus, understanding of sno/scaRNAs demonstrates the clinical value. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Integrated genomic analysis of recurrence-associated small non-coding RNAs in oesophageal cancer.

    PubMed

    Jang, Hee-Jin; Lee, Hyun-Sung; Burt, Bryan M; Lee, Geon Kook; Yoon, Kyong-Ah; Park, Yun-Yong; Sohn, Bo Hwa; Kim, Sang Bae; Kim, Moon Soo; Lee, Jong Mog; Joo, Jungnam; Kim, Sang Cheol; Yun, Ju Sik; Na, Kook Joo; Choi, Yoon-La; Park, Jong-Lyul; Kim, Seon-Young; Lee, Yong Sun; Han, Leng; Liang, Han; Mak, Duncan; Burks, Jared K; Zo, Jae Ill; Sugarbaker, David J; Shim, Young Mog; Lee, Ju-Seog

    2017-02-01

    Oesophageal squamous cell carcinoma (ESCC) is a heterogeneous disease with variable outcomes that are challenging to predict. A better understanding of the biology of ESCC recurrence is needed to improve patient care. Our goal was to identify small non-coding RNAs (sncRNAs) that could predict the likelihood of recurrence after surgical resection and to uncover potential molecular mechanisms that dictate clinical heterogeneity. We developed a robust prediction model for recurrence based on the analysis of the expression profile data of sncRNAs from 108 fresh frozen ESCC specimens as a discovery set and assessment of the associations between sncRNAs and recurrence-free survival (RFS). We also evaluated the mechanistic and therapeutic implications of sncRNA obtained through integrated analysis from multiple datasets. We developed a risk assessment score (RAS) for recurrence with three sncRNAs (microRNA (miR)-223, miR-1269a and nc886) whose expression was significantly associated with RFS in the discovery cohort (n=108). RAS was validated in an independent cohort of 512 patients. In multivariable analysis, RAS was an independent predictor of recurrence (HR, 2.27; 95% CI, 1.26 to 4.09; p=0.007). This signature implies the expression of ΔNp63 and multiple alterations of driver genes like PIK3CA. We suggested therapeutic potentials of immune checkpoint inhibitors in low-risk patients, and Polo-like kinase inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and histone deacetylase inhibitors in high-risk patients. We developed an easy-to-use prognostic model with three sncRNAs as robust prognostic markers for postoperative recurrence of ESCC. We anticipate that such a stratified and systematic, tumour-specific biological approach will potentially contribute to significant improvement in ESCC treatment. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  18. 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.

  19. Dynamic Localisation of Mature MicroRNAs in Human Nucleoli is Influenced by Exogenous Genetic Materials

    PubMed Central

    Li, Zhou Fang; Liang, Yi Min; Lau, Pui Ngan; Shen, Wei; Wang, Dai Kui; Cheung, Wing Tai; Xue, Chun Jason; Poon, Lit Man; Lam, Yun Wah

    2013-01-01

    Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines. The nucleolar association of these miRNAs is resistant to various cellular stresses, but highly sensitive to the presence of exogenous nucleic acids. Introduction of both single- and double-stranded DNA as well as double stranded RNA rapidly induce the redistribution of nucleolar miRNAs to the cytoplasm. A similar change in subcellular distribution is also observed in cells infected with the influenza A virus. The partition of miRNAs between the nucleolus and the cytoplasm is affected by Leptomycin B, suggesting a role of Exportin-1 in the intracellular shuttling of miRNAs. This study reveals a previously unknown aspect of miRNA biology, and suggests a possible link between these small noncoding RNAs and the cellular management of foreign genetic materials. PMID:23940654

  20. 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

  1. Identification and Characterization of Liver MicroRNAs of the Chinese Tree Shrew via Deep Sequencing.

    PubMed

    Feng, Yue; Feng, Yue-Mei; Feng, Yang; Lu, Caixia; Liu, Li; Sun, Xiaomei; Dai, Jiejie; Xia, Xueshan

    2015-10-01

    Chinese tree shrew (Tupaia belangeri chinensis) is a small animal that possess many features, which are valuable in biomedical research, as experimental models. Currently, there are numerous attempts to utilize tree shrews as models for hepatitis C virus (HCV) infection. This study aimed to construct a liver microRNA (miRNA) data of the tree shrew. Three second filial generation tree shrews were used in this study. Total RNA was extracted from each liver of the tree shrew and equal quality mixed, then reverse-transcribed to complementary DNA (cDNA). The cDNAs were amplified by polymerase chain reaction and subjected to high-throughput sequencing. A total of 2060 conserved miRNAs were identified through alignment with the mature miRNAs in miRBase 20.0 database. The gene ontology and Kyoto encyclopedia of genes and genomes analyses of the target genes of the miRNAs revealed several candidate miRNAs, genes and pathways that may involve in the process of HCV infection. The abundance of miR-122 and Let-7 families and their other characteristics provided us more evidences for the utilization of this animal, as a potential model for HCV infection and other related biomedical research. Moreover, 80 novel microRNAs were predicted using the software Mireap. The top 3 abundant miRNAs were validated in other tree samples, based on stem-loop quantitative reverse transcription-polymerase chain reaction. According to the liver microRNA data of Chinese tree shrew, characteristics of the miR-122 and Let-7 families further highlight the suitability of tree shrew as the animal model in HCV research.

  2. Small mammal abundance in Mediterranean post-fire habitats: a role for predators?

    NASA Astrophysics Data System (ADS)

    Torre, I.; Díaz, M.

    2004-05-01

    We studied patterns of small mammal abundance and species richness in post-fire habitats by sampling 33 plots (225 m 2 each) representing different stages of vegetation recovery after fire. Small mammal abundance was estimated by live trapping during early spring 1999 and vegetation structure was sampled by visual estimation at the same plots. Recently-burnt areas were characterised by shrubby and herbaceous vegetation with low structural variability, and unburnt areas were characterised by well developed forest cover with high structural complexity. Small mammal abundance and species richness decreased with time elapsed since the last fire (from 5 to at least 50 years), and these differences were associated to the decreasing cover of short shrubs as the post-fire succession of plant communities advanced. However, relationships between vegetation structure and small mammals differed among areas burned in different times, with weak or negative relationship in recently burnt areas and positive and stronger relationship in unburnt areas. Furthermore, the abundance of small mammals was larger than expected from vegetation structure in plots burned recently whereas the contrary pattern was found in unburned areas. We hypothesised that the pattern observed could be related to the responses of small mammal predators to changes in vegetation and landscape structure promoted by fire. Fire-related fragmentation could have promoted the isolation of forest predators (owls and carnivores) in unburned forest patches, a fact that could have produced a higher predation pressure for small mammals. Conversely, small mammal populations would have been enhanced in early post-fire stages by lower predator numbers combined with better predator protection in areas covered by resprouting woody vegetation.

  3. Prohormone convertase and autocrine growth factor mRNAs are coexpressed in small cell lung carcinoma.

    PubMed

    Rounseville, M P; Davis, T P

    2000-08-01

    A hallmark of small cell lung carcinoma (SCLC) is the expression of autocrine growth factors such as neurotensin and gastrin-releasing peptide, which bind to cellular receptors and stimulate cell division. The biological activity of autocrine growth factors requires the concurrent expression of prohormone convertases that cleave the growth factors to their active form, suggesting the expression of these genes is linked in SCLCs. RNase protection assays were used to detect the expression of autocrine growth factor and prohormone convertase mRNAs in a panel of lung cancer cell lines. These mRNAs are coexpressed in SCLC and lung carcinoid cell lines, but not in normal lung epithelium or in non-small cell lung cancers. These findings, together with earlier results from our laboratory, suggest the expression of prohormone convertases has an important role in the development and maintenance of the SCLC phenotype and that autocrine growth factor and prohormone convertase genes respond to a common transcriptional activator in SCLC.

  4. Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

    PubMed

    Autour, Alexis; C Y Jeng, Sunny; D Cawte, Adam; Abdolahzadeh, Amir; Galli, Angela; Panchapakesan, Shanker S S; Rueda, David; Ryckelynck, Michael; Unrau, Peter J

    2018-02-13

    Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

  5. Visualization of membrane RNAs

    PubMed Central

    JANAS, TADEUSZ; YARUS, MICHAEL

    2003-01-01

    Using fluorescence microscopy, we show that previously isolated membrane-binding RNAs coat artificial phospholipid membranes relatively uniformly, except for a frequent tendency to concentrate at bends, membrane junctions, and other unusual sites. Membrane RNAs can also be visualized as single molecules or isolated complexes by atomic force microscopy (AFM) of free RNAs on mica. Finally, RNAs can be seen within membranes by AFM of RNA-liposomes immobilized on hydrophobic mica surfaces. Monomer RNAs appear globular, as expected for small RNAs. When mixed under conditions in which RNAs bind bilayers, RNA 9 and RNA 10 combine to yield about 80% of RNAs as mainly linear oligomers of ≈2–8 molecules. Once inserted in membranes, the RNAs oligomerize further, yielding larger, irregular ropelike structures that prefer the edges of altered lipid patches. These properties can be interpreted in terms of RNA–RNA loop interactions, and the RNA effects on membranes can be explained in terms of an RNA preference for irregular lipid conformations. The RNA-bilayer system poses new opportunities for combining the properties of membranes and RNA in contemporary cells, and also in the ribocytes of an RNA world. PMID:14561885

  6. Association of Genetic Variants of Small Non-Coding RNAs with Survival in Colorectal Cancer

    PubMed Central

    Pao, Jiunn-Bey; Lu, Te-Ling; Ting, Wen-Chien; Chen, Lu-Min; Bao, Bo-Ying

    2018-01-01

    Background: Single nucleotide polymorphisms (SNPs) of small non-coding RNAs (sncRNAs) can influence sncRNA function and target gene expression to mediate the risk of certain diseases. The aim of the present study was to evaluate the prognostic relevance of sncRNA SNPs for colorectal cancer, which has not been well characterized to date. Methods: We comprehensively examined 31 common SNPs of sncRNAs, and assessed the impact of these variants on survival in a cohort of 188 patients with colorectal cancer. Results: Three SNPs were significantly associated with survival of patients with colorectal cancer after correction for multiple testing, and two of the SNPs (hsa-mir-196a-2 rs11614913 and U85 rs714775) remained significant in multivariate analyses. Additional in silico analysis provided further evidence of this association, since the expression levels of the target genes of the hsa-miR-196a (HOXA7, HOXB8, and AKT1) were significantly correlated with colorectal cancer progression. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that hsa-miR-196a is associated with well-known oncogenic pathways, including cellular protein modification process, mitotic cell cycle, adherens junction, and extracellular matrix receptor interaction pathways. Conclusion: Our results suggest that SNPs of sncRNAs could play a critical role in cancer progression, and that hsa-miR-196a might be a valuable biomarker or therapeutic target for colorectal cancer patients. PMID:29483812

  7. Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor

    PubMed Central

    Enuka, Yehoshua; Lauriola, Mattia; Feldman, Morris E.; Sas-Chen, Aldema; Ulitsky, Igor; Yarden, Yosef

    2016-01-01

    Circular RNAs (circRNAs) are widespread circles of non-coding RNAs with largely unknown function. Because stimulation of mammary cells with the epidermal growth factor (EGF) leads to dynamic changes in the abundance of coding and non-coding RNA molecules, and culminates in the acquisition of a robust migratory phenotype, this cellular model might disclose functions of circRNAs. Here we show that circRNAs of EGF-stimulated mammary cells are stably expressed, while mRNAs and microRNAs change within minutes. In general, the circRNAs we detected are relatively long-lived and weakly expressed. Interestingly, they are almost ubiquitously co-expressed with the corresponding linear transcripts, and the respective, shared promoter regions are more active compared to genes producing linear isoforms with no detectable circRNAs. These findings imply that altered abundance of circRNAs, unlike changes in the levels of other RNAs, might not play critical roles in signaling cascades and downstream transcriptional networks that rapidly commit cells to specific outcomes. PMID:26657629

  8. The Silkworm (Bombyx mori) microRNAs and Their Expressions in Multiple Developmental Stages

    PubMed Central

    Luo, Qibin; Cai, Yimei; Lin, Wen-chang; Chen, Huan; Yang, Yue; Hu, Songnian; Yu, Jun

    2008-01-01

    Background MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species. Methodology/Principal Findings We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5′ and/or 3′ ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis. Conclusions/Significance Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental

  9. Noncoding RNAs in DNA Repair and Genome Integrity

    PubMed Central

    Wan, Guohui; Liu, Yunhua; Han, Cecil; Zhang, Xinna

    2014-01-01

    Abstract Significance: The well-studied sequences in the human genome are those of protein-coding genes, which account for only 1%–2% of the total genome. However, with the advent of high-throughput transcriptome sequencing technology, we now know that about 90% of our genome is extensively transcribed and that the vast majority of them are transcribed into noncoding RNAs (ncRNAs). It is of great interest and importance to decipher the functions of these ncRNAs in humans. Recent Advances: In the last decade, it has become apparent that ncRNAs play a crucial role in regulating gene expression in normal development, in stress responses to internal and environmental stimuli, and in human diseases. Critical Issues: In addition to those constitutively expressed structural RNA, such as ribosomal and transfer RNAs, regulatory ncRNAs can be classified as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs), and long noncoding RNAs (lncRNAs). However, little is known about the biological features and functional roles of these ncRNAs in DNA repair and genome instability, although a number of miRNAs and lncRNAs are regulated in the DNA damage response. Future Directions: A major goal of modern biology is to identify and characterize the full profile of ncRNAs with regard to normal physiological functions and roles in human disorders. Clinically relevant ncRNAs will also be evaluated and targeted in therapeutic applications. Antioxid. Redox Signal. 20, 655–677. PMID:23879367

  10. Functions of MicroRNAs in Cardiovascular Biology and Disease

    PubMed Central

    Hata, Akiko

    2015-01-01

    In 1993, lin-4 was discovered as a critical modulator of temporal development in Caenorhabditis elegans and, most notably, as the first in the class of small, single-stranded noncoding RNAs now defined as microRNAs (miRNAs). Another eight years elapsed before miRNA expression was detected in mammalian cells. Since then, explosive advancements in the field of miRNA biology have elucidated the basic mechanism of miRNA biogenesis, regulation, and gene-regulatory function. The discovery of this new class of small RNAs has augmented the complexity of gene-regulatory programs as well as the understanding of developmental and pathological processes in the cardiovascular system. Indeed, the contributions of miRNAs in cardiovascular development and function have been widely explored, revealing the extensive role of these small regulatory RNAs in cardiovascular physiology. PMID:23157557

  11. A glance at subgenomic flavivirus RNAs and microRNAs in flavivirus infections.

    PubMed

    Bavia, Lorena; Mosimann, Ana Luiza Pamplona; Aoki, Mateus Nóbrega; Duarte Dos Santos, Claudia Nunes

    2016-05-28

    The family Flaviviridae comprises a wide variety of viruses that are distributed worldwide, some of which are associated with high rates of morbidity and mortality. There are neither vaccines nor antivirals for most flavivirus infections, reinforcing the importance of research on different aspects of the viral life cycle. During infection, cytoplasmic accumulation of RNA fragments mainly originating from the 3' UTRs, which have been designated subgenomic flavivirus RNAs (sfRNAs), has been detected. It has been shown that eukaryotic exoribonucleases are involved in viral sfRNA production. Additionally, viral and human small RNAs (sRNAs) have also been found in flavivirus-infected cells, especially microRNAs (miRNAs). miRNAs were first described in eukaryotic cells and in a mature and functional state present as single-stranded 18-24 nt RNA fragments. Their main function is the repression of translation through base pairing with cellular mRNAs, besides other functions, such as mRNA degradation. Canonical miRNA biogenesis involves Drosha and Dicer, however miRNA can also be generated by alternative pathways. In the case of flaviviruses, alternative pathways have been suggested. Both sfRNAs and miRNAs are involved in viral infection and host cell response modulation, representing interesting targets of antiviral strategies. In this review, we focus on the generation and function of viral sfRNAs, sRNAs and miRNAs in West Nile, dengue, Japanese encephalitis, Murray Valley encephalitis and yellow fever infections, as well as their roles in viral replication, translation and cell immune response evasion. We also give an overview regarding other flaviviruses and the generation of cellular miRNAs during infection.

  12. Effective inhibition of HIV-1 production by short hairpin RNAs and small interfering RNAs targeting a highly conserved site in HIV-1 Gag RNA is optimized by evaluating alternative length formats.

    PubMed

    Scarborough, Robert J; Adams, Kelsey L; Daher, Aïcha; Gatignol, Anne

    2015-09-01

    We have previously identified a target site in HIV-1 RNA that was particularly accessible to a ribozyme and a short hairpin RNA (shRNA). To design small interfering RNAs (siRNAs) targeting this site, we evaluated the effects of siRNAs with different lengths on HIV-1 production. The potency and efficacy of these siRNAs were dependent on the length of their intended sense strand with trends for symmetrical and asymmetrical formats that were similar. Although a typical canonical format with a 21-nucleotide (nt) sense strand was effective at inhibiting HIV-1 production, Dicer substrate siRNAs (dsiRNAs) with the longest lengths (27 to 29 nucleotides) were the most effective. Induction of double-stranded RNA immune responses and effects on cell viability were not detected in cells transfected with different siRNAs, suggesting that the differences observed were not related to indirect effects on HIV-1 production. For the corresponding shRNA designs, a different trend in potency and efficacy against HIV-1 production was observed, with the most effective shRNAs having stem lengths from 20 to 27 bp. Our results highlight the importance of evaluating different designs to identify the best siRNA and shRNA formats for any particular target site and provide a set of highly effective molecules for further development as drug and gene therapies for HIV-1 infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Characterisation of microRNAs from apple (Malus domestica 'Royal Gala') vascular tissue and phloem sap.

    PubMed

    Varkonyi-Gasic, Erika; Gould, Nick; Sandanayaka, Manoharie; Sutherland, Paul; MacDiarmid, Robin M

    2010-08-04

    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. 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. 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.

  14. 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

  15. 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

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

    2012-04-01

    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.

  16. 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

  17. Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions

    PubMed Central

    Kuligina, Elena V.; Bariakin, Dmitry N.; Kozlov, Vadim V.; Richter, Vladimir A.; Semenov, Dmitry V.

    2017-01-01

    Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000g and 160,000g, and vesicle-depleted plasma supernatant of healthy donors and non-small cell lung cancer (NSCLC) patients. It was determined that 16,000g blood plasma vesicles were enriched with cell-free mitochondria and with a set of mitochondrial RNAs. The variable RNA set of blood plasma 160,000g pellets reflected the prominent contribution of U1, U5, and U6 small nuclear RNAs' fragments and at the same time was characterized by a remarkable depletion of small nucleolar RNAs. Besides microRNAs, the variety of fragments of mRNAs and snoRNAs dominated in the set of circulating RNAs differentially expressed in blood fractions of NSCLC patients. Taken together, our data emphasize that not only extracellular microRNAs but also circulating fragments of messenger and small nuclear/nucleolar RNAs represent prominent classes of circulating regulatory ncRNAs as well as promising circulating biomarkers for the development of disease diagnostic approaches. PMID:28127559

  18. Regulatory RNAs derived from transfer RNA?

    PubMed

    Pederson, Thoru

    2010-10-01

    Four recent studies suggest that cleavages of transfer RNAs generate products with microRNA-like features, with some evidence of function. If their regulatory functions were to be confirmed, these newly revealed RNAs would add to the expanding repertoire of small noncoding RNAs and would also provide new perspectives on the coevolution of transfer RNA and messenger RNA.

  19. Identification of novel drought-responsive microRNAs and trans-acting siRNAs from Sorghum bicolor (L.) Moench by high-throughput sequencing analysis

    PubMed Central

    Katiyar, Amit; Smita, Shuchi; Muthusamy, Senthilkumar K.; Chinnusamy, Viswanathan; Pandey, Dev M.; Bansal, Kailash C.

    2015-01-01

    Small non-coding RNAs (sRNAs) namely microRNAs (miRNAs) and trans-acting small interfering RNAs (tasi-RNAs) play a crucial role in post-transcriptional regulation of gene expression and thus the control plant development and stress responses. In order to identify drought-responsive miRNAs and tasi-RNAs in sorghum, we constructed small RNA libraries from a drought tolerant (M35-1) and susceptible (C43) sorghum genotypes grown under control and drought stress conditions, and sequenced by Illumina Genome Analyzer IIx. Ninety seven conserved and 526 novel miRNAs representing 472 unique miRNA families were identified from sorghum. Ninety-six unique miRNAs were found to be regulated by drought stress, of which 32 were up- and 49 were down-regulated (fold change ≥ 2 or ≤ −2) at least in one genotype, while the remaining 15 miRNAs showed contrasting drought-regulated expression pattern between genotypes. A maximum of 17 and 18 miRNAs was differentially regulated under drought stress condition in the sensitive and tolerant genotypes, respectively. These results suggest that genotype dependent stress responsive regulation of miRNAs may contribute, at least in part, to the differential drought tolerance of sorghum genotypes. We also identified two miR390-directed TAS3 gene homologs and the auxin response factors as tasi-RNA targets. We predicted more than 1300 unique target genes for the novel and conserved miRNAs. These target genes were predicted to be involved in different cellular, metabolic, response to stimulus, biological regulation, and developmental processes. Genome-wide identification of stress-responsive miRNAs, tasi-RNAs and their targets identified in this study will be useful in unraveling the molecular mechanisms underlying drought stress responses and genetic improvement of biomass production and stress tolerance in sorghum. PMID:26236318

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

    USDA-ARS?s Scientific Manuscript database

    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...

  1. Characteristics of siRNAs derived from Southern rice black-streaked dwarf virus in infected rice and their potential role in host gene regulation.

    PubMed

    Xu, Donglin; Zhou, Guohui

    2017-02-10

    Virus-derived siRNAs (vsiRNAs)-mediated RNA silencing plays important roles in interaction between plant viruses and their hosts. Southern rice black-streaked dwarf virus (SRBSDV) is a newly emerged devastating rice reovirus with ten dsRNA genomic segments. The characteristics of SRBSDV-derived siRNAs and their biological implications in SRBSDV-rice interaction remain unexplored. VsiRNAs profiling from SRBSDV-infected rice samples was done via small RNA deep sequencing. The putative rice targets of abundantly expressed vsiRNAs were bioinformatically predicted and subjected to functional annotation. Differential expression analysis of rice targets and RNA silencing components between infected and healthy samples was done using RT-qPCR. The vsiRNA was barely detectable at 14 days post infection (dpi) but abundantly present along with elevated expression level of the viral genome at 28 dpi. From the 28-dpi sample, 70,878 reads of 18 ~ 30-nt vsiRNAs were recognized (which mostly were 21-nt and 22-nt), covering 75 ~ 91% of the length of the ten genomic segments respectively. 86% of the vsiRNAs had a <50% GC content and 79% of them were 5'-uridylated or adenylated. The production of vsiRNAs had no strand polarity but varied among segment origins. Each segment had a few hotspot regions where vsiRNAs of high abundance were produced. 151 most abundant vsiRNAs were predicted to target 844 rice genes, including several types of host resistance or pathogenesis related genes encoding F-box/LRR proteins, receptor-like protein kinases, universal stress proteins, tobamovirus multiplication proteins, and RNA silencing components OsDCL2a and OsAGO17 respectively, some of which showed down regulation in infected plants in RT-qPCR. GO and KEGG classification showed that a majority of the predicted targets were related to cell parts and cellular processes and involved in carbohydrate metabolism, translation, and signal transduction. The silencing component genes OsDCL2a, Os

  2. Utility of MicroRNAs and siRNAs in Cervical Carcinogenesis

    PubMed Central

    Díaz-González, Sacnite del Mar; Benítez-Boijseauneau, Odelia; Gómez-Cerón, Claudia; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Peralta-Zaragoza, Oscar

    2015-01-01

    MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3′-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer. PMID:25874209

  3. Utility of microRNAs and siRNAs in cervical carcinogenesis.

    PubMed

    Díaz-González, Sacnite del Mar; Deas, Jessica; Benítez-Boijseauneau, Odelia; Gómez-Cerón, Claudia; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Peralta-Zaragoza, Oscar

    2015-01-01

    MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3'-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer.

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

    PubMed

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

    2015-07-01

    Endogenous small non-coding RNAs (sRNAs), including microRNAs, PIWI-interacting RNAs and small interfering RNAs, play important gene regulatory roles in animals and plants by pairing to the protein-coding and non-coding transcripts. However, computationally assigning these various sRNAs to their regulatory target genes remains technically challenging. Recently, a high-throughput degradome sequencing method was applied to identify biologically relevant sRNA cleavage sites. In this study, an integrated web-based tool, StarScan (sRNA target Scan), was developed for scanning sRNA targets using degradome sequencing data from 20 species. Given a sRNA sequence from plants or animals, our web server performs an ultrafast and exhaustive search for potential sRNA-target interactions in annotated and unannotated genomic regions. The interactions between small RNAs and target transcripts were further evaluated using a novel tool, alignScore. A novel tool, degradomeBinomTest, was developed to quantify the abundance of degradome fragments located at the 9-11th nucleotide from the sRNA 5' end. This is the first web server for discovering potential sRNA-mediated RNA cleavage events in plants and animals, which affords mechanistic insights into the regulatory roles of sRNAs. The StarScan web server is available at http://mirlab.sysu.edu.cn/starscan/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Reverse transcription polymerase chain reaction protocols for cloning small circular RNAs.

    PubMed

    Navarro, B; Daròs, J A; Flores, R

    1998-07-01

    A protocol is described for general application for cloning small circular RNAs which requires only minimal amounts of template (approximately 50 ng) of unknown sequence. Both cDNA strands are synthesized with a 26-mer primer whose six 3'-terminal positions are totally degenerate in two consecutive reactions catalyzed by reverse transcriptase and DNA polymerase, respectively. The cDNAs are then PCR-amplified, using a 20-mer primer with the non-degenerate sequence of the previous primer, cloned and sequenced. This information permits the synthesis of one or more pairs of specific and adjacent primers for obtaining full-length cDNA clones by a protocol which is also described.

  6. Mobile microRNAs hit the target.

    PubMed

    Gursanscky, Nial R; Searle, Iain R; Carroll, Bernard J

    2011-11-01

    MicroRNAs (miRNAs) are negative regulators of gene expression in eukaryotic organisms, whereas small interfering RNAs (siRNAs) guide host-cell defence against viruses, transposons and transgenes. A key issue in plant biology is whether miRNAs act only in cells in which they are formed, or if, like siRNAs, they also function after passive diffusion or active transportation into other cells. Recent reports show that miRNAs are indeed able to move between plant cells to direct developmental programming of gene expression. In both leaf and root development, miRNAs establish intercellular gradients of gene expression that are essential for cell and tissue differentiation. Gradients in gene expression also play crucial roles in animal development, and there is strong evidence for intercellular movement of miRNAs in animals. Thus, intercellular movement of miRNAs may be crucial to animal developmental biology as well as plants. © 2011 John Wiley & Sons A/S.

  7. Genome-wide maps of m6A circRNAs identify widespread and cell-type-specific methylation patterns that are distinct from mRNAs

    PubMed Central

    Zhou, Chan; Molinie, Benoit; Daneshvar, Kaveh; Pondick, Joshua V.; Wang, Jinkai; Van Wittenberghe, Nicholas O.; Xing, Yi; Giallourakis, Cosmas C.; Mullen, Alan C.

    2017-01-01

    Summary N6-methyladenosine (m6A) is the most abundant internal modification of mRNAs and is implicated in all aspects of post-transcriptional RNA metabolism. However, little is known about m6A modifications to circular (circ) RNAs. We developed a computational pipeline (AutoCirc) that together with depletion of ribosomal RNA and m6A immunoprecipitation defined thousands of m6A-circRNAs, with cell-type-specific expression. The presence of m6A-circRNAs is corroborated by interaction between circRNAs and YTHDF1/YTHDF2, proteins that read m6A sites in mRNAs, and by reduced m6A levels upon depletion of METTL3, the m6A writer. Despite sharing m6A readers and writers, m6A-circRNAs are frequently derived from exons that are not methylated in mRNAs, while mRNAs that are methylated on the same exons that compose m6A-circRNAs exhibit less stability, in a process regulated by YTHDF2. These results expand our understanding of the breadth of m6A modifications and uncover regulation of circRNAs through m6A modification. PMID:28854373

  8. Functional Roles of microRNAs in Agronomically Important Plants—Potential as Targets for Crop Improvement and Protection

    PubMed Central

    Djami-Tchatchou, Arnaud T.; Sanan-Mishra, Neeti; Ntushelo, Khayalethu; Dubery, Ian A.

    2017-01-01

    MicroRNAs (miRNAs) are a class of small non-coding RNAs that have recently emerged as important regulators of gene expression, mainly through cleavage and/or translation inhibition of the target mRNAs during or after transcription. miRNAs play important roles by regulating a multitude of biological processes in plants which include maintenance of genome integrity, development, metabolism, and adaptive responses toward environmental stresses. The increasing population of the world and their food demands requires focused efforts for the improvement of crop plants to ensure sustainable food production. Manipulation of mRNA transcript abundance via miRNA control provides a unique strategy for modulating differential plant gene expression and miRNAs are thus emerging as the next generation targets for genetic engineering for improvement of the agronomic properties of crops. However, a deeper understanding of its potential and the mechanisms involved will facilitate the design of suitable strategies to obtain the desirable traits with minimum trade-offs in the modified crops. In this regard, this review highlights the diverse roles of conserved and newly identified miRNAs in various food and industrial crops and recent advances made in the uses of miRNAs to improve plants of agronomically importance so as to significantly enhance crop yields and increase tolerance to various environmental stress agents of biotic—or abiotic origin. PMID:28382044

  9. Small non-coding RNA profiling in human biofluids and surrogate tissues from healthy individuals: description of the diverse and most represented species.

    PubMed

    Ferrero, Giulio; Cordero, Francesca; Tarallo, Sonia; Arigoni, Maddalena; Riccardo, Federica; Gallo, Gaetano; Ronco, Guglielmo; Allasia, Marco; Kulkarni, Neha; Matullo, Giuseppe; Vineis, Paolo; Calogero, Raffaele A; Pardini, Barbara; Naccarati, Alessio

    2018-01-09

    The role of non-coding RNAs in different biological processes and diseases is continuously expanding. Next-generation sequencing together with the parallel improvement of bioinformatics analyses allows the accurate detection and quantification of an increasing number of RNA species. With the aim of exploring new potential biomarkers for disease classification, a clear overview of the expression levels of common/unique small RNA species among different biospecimens is necessary. However, except for miRNAs in plasma, there are no substantial indications about the pattern of expression of various small RNAs in multiple specimens among healthy humans. By analysing small RNA-sequencing data from 243 samples, we have identified and compared the most abundantly and uniformly expressed miRNAs and non-miRNA species of comparable size with the library preparation in four different specimens (plasma exosomes, stool, urine, and cervical scrapes). Eleven miRNAs were commonly detected among all different specimens while 231 miRNAs were globally unique across them. Classification analysis using these miRNAs provided an accuracy of 99.6% to recognize the sample types. piRNAs and tRNAs were the most represented non-miRNA small RNAs detected in all specimen types that were analysed, particularly in urine samples. With the present data, the most uniformly expressed small RNAs in each sample type were also identified. A signature of small RNAs for each specimen could represent a reference gene set in validation studies by RT-qPCR. Overall, the data reported hereby provide an insight of the constitution of the human miRNome and of other small non-coding RNAs in various specimens of healthy individuals.

  10. Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs

    PubMed Central

    Jima, Dereje D.; Zhang, Jenny; Jacobs, Cassandra; Richards, Kristy L.; Dunphy, Cherie H.; Choi, William W. L.; Yan Au, Wing; Srivastava, Gopesh; Czader, Magdalena B.; Rizzieri, David A.; Lagoo, Anand S.; Lugar, Patricia L.; Mann, Karen P.; Flowers, Christopher R.; Bernal-Mizrachi, Leon; Naresh, Kikkeri N.; Evens, Andrew M.; Gordon, Leo I.; Luftig, Micah; Friedman, Daphne R.; Weinberg, J. Brice; Thompson, Michael A.; Gill, Javed I.; Liu, Qingquan; How, Tam; Grubor, Vladimir; Gao, Yuan; Patel, Amee; Wu, Han; Zhu, Jun; Blobe, Gerard C.; Lipsky, Peter E.; Chadburn, Amy

    2010-01-01

    A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions. PMID:20733160

  11. 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

  12. Evidence for plant-derived xenomiRs based on a large-scale analysis of public small RNA sequencing data from human samples.

    PubMed

    Zhao, Qi; Liu, Yuanning; Zhang, Ning; Hu, Menghan; Zhang, Hao; Joshi, Trupti; Xu, Dong

    2018-01-01

    In recent years, an increasing number of studies have reported the presence of plant miRNAs in human samples, which resulted in a hypothesis asserting the existence of plant-derived exogenous microRNA (xenomiR). However, this hypothesis is not widely accepted in the scientific community due to possible sample contamination and the small sample size with lack of rigorous statistical analysis. This study provides a systematic statistical test that can validate (or invalidate) the plant-derived xenomiR hypothesis by analyzing 388 small RNA sequencing data from human samples in 11 types of body fluids/tissues. A total of 166 types of plant miRNAs were found in at least one human sample, of which 14 plant miRNAs represented more than 80% of the total plant miRNAs abundance in human samples. Plant miRNA profiles were characterized to be tissue-specific in different human samples. Meanwhile, the plant miRNAs identified from microbiome have an insignificant abundance compared to those from humans, while plant miRNA profiles in human samples were significantly different from those in plants, suggesting that sample contamination is an unlikely reason for all the plant miRNAs detected in human samples. This study also provides a set of testable synthetic miRNAs with isotopes that can be detected in situ after being fed to animals.

  13. Circular RNAs: An emerging type of RNA in cancer.

    PubMed

    Hou, Li-Dan; Zhang, Jing

    2017-03-01

    Circular RNAs (circRNAs), a novel type of widespread and diverse endogenous non-coding RNAs (ncRNAs), which are different from the linear RNAs, form a covalently closed continuous loop without 5' or 3' polarities. The majority of circRNAs are abundant, conserved and stable across different species, and exhibit tissue/developmental-stage-specific characteristics. They are generated primarily through a type of alternative RNA splicing called "back-splicing," in which a downstream splice donor is joined to an upstream splice acceptor through splice skipping or direct splice. Recent studies have discovered circRNAs function as microRNA sponges, binding with RNA-associated proteins to form RNA-protein complexes and then regulating gene transcription and translation into polypeptides. Emerging evidence indicates that circRNAs play important roles in the regulation of the development and progression of multiple cancers by serving as potential diagnostic and predictive biomarkers involved in tumor growth and invasion and providing new strategies for cancer diagnosis and targeted therapy. In this review, we briefly delineate the diversity and characteristics of circRNAs and discuss the highlights of the biogenesis of circRNAs and their potential functions in tumor.

  14. Identification of heavy-ion radiation-induced microRNAs in rice

    NASA Astrophysics Data System (ADS)

    Zhang, Meng; Liang, Shujian; Hang, Xiaoming; Xiang, Yingxia; Cheng, Zhenlong; Li, Wenjian; Shi, Jinming; Huang, Lei; Sun, Yeqing

    2011-03-01

    MicroRNAs (miRNAs) are a family of small non-coding RNAs, which play significant roles in regulating development and stress responses in plant. As an excellent model organism for studying the effects of environmental stress, rice has been used to assess the damage of the space radiation environment for decades. Heavy-ions radiation show higher relative biological effectiveness compared to other cosmic-rays radiation. To identify the specific miRNAs that underlie biological effects of heavy-ion radiation, the germinated seeds of rice were exposed to 1 Gy, 10 Gy and 20 Gy dose of 12C heavy-ion radiation, respectively. Analysis of phenotype indicated that 20 Gy dose of heavy-ion radiation was the semi-lethal dose of rice seedling. The microarray of μparaflo™ chip was employed to monitor the expression profiles of miRNAs in rice (Oryza sativa) under 20 Gy dose of radiation stress. miR164a, miR164c, miR164d and miR156a-j were identified as heavy-ion radiation-induced miRNAs. miR164 and miR156 family were increased in all three exposed samples by using quantitative real-time PCR (qRT-RCP). As targets of miR156 and miR164, SQUAMOSA PROMOTER BINDING-LIKE (SPL) transcription factors and NAM/ATAF/CUC (NAC) transcription factors expression were down-regulated correlating with an up-regulated level of the regulated miRNAs. Since SPL transcription factors and NAC transcription factors regulated growth and development of plant, we used 2-dimension electrophoresis (2-DE) gel to analyze changes of functional proteins in 20 Gy exposed samples. It was evident that both the height and survival rates of seedlings were markedly decreased. The abundance of some developmentally regulated proteins was also changed. To our knowledge, this study is the first to report heavy-ion radiation stress responsive miRNAs in plant. Moreover, our findings are important to understand the molecular mechanism of space biology.

  15. Analysis of RDR1/RDR2/RDR6-independent small RNAs in Arabidopsis thaliana improves MIRNA annotations and reveals unexplained types of short interfering RNA loci.

    PubMed

    Polydore, Seth; Axtell, Michael J

    2018-06-01

    Plant small RNAs (sRNAs) modulate key physiological mechanisms through post-transcriptional and transcriptional silencing of gene expression. Small RNAs fall into two major categories: those are reliant on RNA-dependent RNA polymerases (RDRs) for biogenesis and those that are not. Known RDR1/2/6-dependent sRNAs include phased and repeat-associated short interfering RNAs, while known RDR1/2/6-independent sRNAs are primarily microRNAs (miRNA) and other hairpin-derived sRNAs. In this study we produced and analyzed sRNA-seq libraries from rdr1/rdr2/rdr6 triple mutant plants. We found 58 previously annotated miRNA loci that were reliant on RDR1, -2, or -6 function, casting doubt on their classification. We also found 38 RDR1/2/6-independent sRNA loci that are not MIRNAs or otherwise hairpin-derived, and did not fit into other known paradigms for sRNA biogenesis. These 38 sRNA-producing loci have as-yet-undescribed biogenesis mechanisms, and are frequently located in the vicinity of protein-coding genes. Altogether, our analysis suggests that these 38 loci represent one or more undescribed types of sRNA in Arabidopsis thaliana. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

  16. Visualized and precise design of artificial small RNAs for regulating T7 RNA polymerase and enhancing recombinant protein folding in Escherichia coli.

    PubMed

    Zhao, Yujia; Fan, Jingjing; Li, Jinlin; Li, Jun; Zhou, Xiaohong; Li, Chun

    2016-12-01

    Small non-coding RNAs (sRNAs) have received much attention in recent years due to their unique biological properties, which can efficiently and specifically tune target gene expressions in bacteria. Inspired by natural sRNAs, recent works have proposed the use of artificial sRNAs (asRNAs) as genetic tools to regulate desired gene that has been applied in several fields, such as metabolic engineering and bacterial physiology studies. However, the rational design of asRNAs is still a challenge. In this study, we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs. T7 expression system was one of the most useful recombinant protein expression systems. However, it was deeply limited by the formation of inclusion body. To settle this problem, we designed a series of asRNAs to inhibit the T7 RNA polymerase (Gene1) expression to balance the rate between transcription and folding of recombinant protein. Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E. coli , the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.

  17. PIWI proteins and PIWI-interacting RNAs function in Hydra somatic stem cells

    PubMed Central

    Juliano, Celina E.; Reich, Adrian; Liu, Na; Götzfried, Jessica; Zhong, Mei; Uman, Selen; Reenan, Robert A.; Wessel, Gary M.; Steele, Robert E.; Lin, Haifan

    2014-01-01

    PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) are found in animal germlines and are essential for fertility, but their functions outside of the gonad are not well understood. The cnidarian Hydra is a simple metazoan with well-characterized stem/progenitor cells that provides a unique model for analysis of PIWI function. Here we report that Hydra has two PIWI proteins, Hydra PIWI (Hywi) and Hydra PIWI-like (Hyli), both of which are expressed in all Hydra stem/progenitor cells, but not in terminally differentiated cells. We identified ∼15 million piRNAs associated with Hywi and/or Hyli and found that they exhibit the ping-pong signature of piRNA biogenesis. Hydra PIWI proteins are strictly cytoplasmic and thus likely act as posttranscriptional regulators. To explore this function, we generated a Hydra transcriptome for piRNA mapping. piRNAs map to transposons with a 25- to 35-fold enrichment compared with the abundance of transposon transcripts. By sequencing the small RNAs specific to the interstitial, ectodermal, and endodermal lineages, we found that the targeting of transposons appears to be largely restricted to the interstitial lineage. We also identified putative nontransposon targets of the pathway unique to each lineage. Finally we demonstrate that hywi function is essential in the somatic epithelial lineages. This comprehensive analysis of the PIWI–piRNA pathway in the somatic stem/progenitor cells of a nonbilaterian animal suggests that this pathway originated with broader stem cell functionality. PMID:24367095

  18. PIWI proteins and PIWI-interacting RNAs function in Hydra somatic stem cells.

    PubMed

    Juliano, Celina E; Reich, Adrian; Liu, Na; Götzfried, Jessica; Zhong, Mei; Uman, Selen; Reenan, Robert A; Wessel, Gary M; Steele, Robert E; Lin, Haifan

    2014-01-07

    PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) are found in animal germlines and are essential for fertility, but their functions outside of the gonad are not well understood. The cnidarian Hydra is a simple metazoan with well-characterized stem/progenitor cells that provides a unique model for analysis of PIWI function. Here we report that Hydra has two PIWI proteins, Hydra PIWI (Hywi) and Hydra PIWI-like (Hyli), both of which are expressed in all Hydra stem/progenitor cells, but not in terminally differentiated cells. We identified ∼15 million piRNAs associated with Hywi and/or Hyli and found that they exhibit the ping-pong signature of piRNA biogenesis. Hydra PIWI proteins are strictly cytoplasmic and thus likely act as posttranscriptional regulators. To explore this function, we generated a Hydra transcriptome for piRNA mapping. piRNAs map to transposons with a 25- to 35-fold enrichment compared with the abundance of transposon transcripts. By sequencing the small RNAs specific to the interstitial, ectodermal, and endodermal lineages, we found that the targeting of transposons appears to be largely restricted to the interstitial lineage. We also identified putative nontransposon targets of the pathway unique to each lineage. Finally we demonstrate that hywi function is essential in the somatic epithelial lineages. This comprehensive analysis of the PIWI-piRNA pathway in the somatic stem/progenitor cells of a nonbilaterian animal suggests that this pathway originated with broader stem cell functionality.

  19. Identification of Submergence-Responsive MicroRNAs and Their Targets Reveals Complex MiRNA-Mediated Regulatory Networks in Lotus (Nelumbo nucifera Gaertn)

    PubMed Central

    Jin, Qijiang; Xu, Yingchun; Mattson, Neil; Li, Xin; Wang, Bei; Zhang, Xiao; Jiang, Hongwei; Liu, Xiaojing; Wang, Yanjie; Yao, Dongrui

    2017-01-01

    MicroRNAs (miRNAs) are endogenous non-coding RNAs with important regulatory functions in plant development and stress responses. However, their population abundance in lotus (Nelumbo nucifera Gaertn) has so far been poorly described, particularly in response to stresses. In this work, submergence-related miRNAs and their target genes were systematically identified, compared, and validated at the transcriptome-wide level using high-throughput sequencing data of small RNA, Mrna, and the degradome. A total of 128 known and 20 novel miRNAs were differentially expressed upon submergence. We identified 629 target transcripts for these submergence-responsive miRNAs. Based on the miRNA expression profiles and GO and KEGG annotation of miRNA target genes, we suggest possible molecular responses and physiological changes of lotus in response to submergence. Several metabolic, physiological and morphological adaptations-related miRNAs, i.e., NNU_far-miR159, NNU_gma-miR393h, and NNU_aly-miR319c-3p, were found to play important regulatory roles in lotus response to submergence. This work will contribute to a better understanding of miRNA-regulated adaption responses of lotus to submergence stress. PMID:28149304

  20. The role of microRNAs in synaptic development and function

    PubMed Central

    Corbin, Rachel; Olsson-Carter, Katherine; Slack, Frank

    2015-01-01

    MicroRNAs control gene expression by inhibiting translation or promoting degradation of their target mRNAs. Since the discovery of the first microRNAs, lin-4 and let-7, in C. elegans, hundreds of microRNAs have been identified as key regulators of cell fate determination, lifespan, and cancer in species ranging from plants to humans. However, while microRNAs have been shown to be particularly abundant in the brain, their role in the development and activity of the nervous system is still largely unknown. In this review, we describe recent advances in our understanding of microRNA function at synapses, the specialized structures required for communication between neurons and their targets. We also propose how these advances might inform the molecular model of memory. PMID:19335998

  1. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Regulatory RNAs

    PubMed Central

    Vazquez-Anderson, Jorge; Contreras, Lydia M

    2013-01-01

    RNAs have many important functional properties, including that they are independently controllable and highly tunable. As a result of these advantageous properties, their use in a myriad of sophisticated devices has been widely explored. Yet, the exploitation of RNAs for synthetic applications is highly dependent on the ability to characterize the many new molecules that continue to be discovered by large-scale sequencing and high-throughput screening techniques. In this review, we present an exhaustive survey of the most recent synthetic bacterial riboswitches and small RNAs while emphasizing their virtues in gene expression management. We also explore the use of these RNA components as building blocks in the RNA synthetic biology toolbox and discuss examples of synthetic RNA components used to rewire bacterial regulatory circuitry. We anticipate that this field will expand its catalog of smart devices by mimicking and manipulating natural RNA mechanisms and functions. PMID:24356572

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  4. miRNAs in brain development

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Petri, Rebecca; Malmevik, Josephine; Fasching, Liana

    2014-02-01

    MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. In the brain, a large number of miRNAs are expressed and there is a growing body of evidence demonstrating that miRNAs are essential for brain development and neuronal function. Conditional knockout studies of the core components in the miRNA biogenesis pathway, such as Dicer and DGCR8, have demonstrated a crucial role for miRNAs during the development of the central nervous system. Furthermore, mice deleted for specific miRNAs and miRNA-clusters demonstrate diverse functional roles for different miRNAs during the development of different brain structures. miRNAs havemore » been proposed to regulate cellular functions such as differentiation, proliferation and fate-determination of neural progenitors. In this review we summarise the findings from recent studies that highlight the importance of miRNAs in brain development with a focus on the mouse model. We also discuss the technical limitations of current miRNA studies that still limit our understanding of this family of non-coding RNAs and propose the use of novel and refined technologies that are needed in order to fully determine the impact of specific miRNAs in brain development. - Highlights: • miRNAs are essential for brain development and neuronal function. • KO of Dicer is embryonically lethal. • Conditional Dicer KO results in defective proliferation or increased apoptosis. • KO of individual miRNAs or miRNA families is necessary to determine function.« less

  5. Global assessment of small RNAs reveals a non-coding transcript involved in biofilm formation and attachment in Acinetobacter baumannii ATCC 17978

    PubMed Central

    Pérez, Astrid; Gómez, Manuel J.; Gayoso, Carmen; Vallejo, Juan A.; Ohneck, Emily J.; Valle, Jaione; Actis, Luis A.; Beceiro, Alejandro; Bou, Germán

    2017-01-01

    Many strains of Acinetobacter baumannii have been described as being able to form biofilm. Small non-coding RNAs (sRNAs) control gene expression in many regulatory circuits in bacteria. The aim of the present work was to provide a global description of the sRNAs produced both by planktonic and biofilm-associated (sessile) cells of A. baumannii ATCC 17978, and to compare the corresponding gene expression profiles to identify sRNAs molecules associated to biofilm formation and virulence. sRNA was extracted from both planktonic and sessile cells and reverse transcribed. cDNA was subjected to 454-pyrosequencing using the GS-FLX Titanium chemistry. The global analysis of the small RNA transcriptome revealed different sRNA expression patterns in planktonic and biofilm associated cells, with some of the transcripts only expressed or repressed in sessile bacteria. A total of 255 sRNAs were detected, with 185 of them differentially expressed in the different types of cells. A total of 9 sRNAs were expressed only in biofilm cells, while the expression of other 21 coding regions were repressed only in biofilm cells. Strikingly, the expression level of the sRNA 13573 was 120 times higher in biofilms than in planktonic cells, an observation that prompted us to further investigate the biological role of this non-coding transcript. Analyses of an isogenic mutant and over-expressing strains revealed that the sRNA 13573 gene is involved in biofilm formation and attachment to A549 human alveolar epithelial cells. The present work serves as a basis for future studies examining the complex regulatory network that regulate biofilm biogenesis and attachment to eukaryotic cells in A. baumannii ATCC 17978. PMID:28763494

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schuler, Simon C.; Vaz, Zachary A.; Santrich, Orlando J. Katime

    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-typemore » 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.« less

  7. Discovery of replicating circular RNAs by RNA-seq and computational algorithms.

    PubMed

    Zhang, Zhixiang; Qi, Shuishui; Tang, Nan; Zhang, Xinxin; Chen, Shanshan; Zhu, Pengfei; Ma, Lin; Cheng, Jinping; Xu, Yun; Lu, Meiguang; Wang, Hongqing; Ding, Shou-Wei; Li, Shifang; Wu, Qingfa

    2014-12-01

    Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs.

  8. Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms

    PubMed Central

    Tang, Nan; Zhang, Xinxin; Chen, Shanshan; Zhu, Pengfei; Ma, Lin; Cheng, Jinping; Xu, Yun; Lu, Meiguang; Wang, Hongqing; Ding, Shou-Wei; Li, Shifang; Wu, Qingfa

    2014-01-01

    Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs. PMID:25503469

  9. Quantitative Characteristics of Gene Regulation by Small RNA

    PubMed Central

    Levine, Erel; Zhang, Zhongge; Kuhlman, Thomas; Hwa, Terence

    2007-01-01

    An increasing number of small RNAs (sRNAs) have been shown to regulate critical pathways in prokaryotes and eukaryotes. In bacteria, regulation by trans-encoded sRNAs is predominantly found in the coordination of intricate stress responses. The mechanisms by which sRNAs modulate expression of its targets are diverse. In common to most is the possibility that interference with the translation of mRNA targets may also alter the abundance of functional sRNAs. Aiming to understand the unique role played by sRNAs in gene regulation, we studied examples from two distinct classes of bacterial sRNAs in Escherichia coli using a quantitative approach combining experiment and theory. Our results demonstrate that sRNA provides a novel mode of gene regulation, with characteristics distinct from those of protein-mediated gene regulation. These include a threshold-linear response with a tunable threshold, a robust noise resistance characteristic, and a built-in capability for hierarchical cross-talk. Knowledge of these special features of sRNA-mediated regulation may be crucial toward understanding the subtle functions that sRNAs can play in coordinating various stress-relief pathways. Our results may also help guide the design of synthetic genetic circuits that have properties difficult to attain with protein regulators alone. PMID:17713988

  10. MicroRNAs and cancer.

    PubMed

    Cowland, Jack B; Hother, Christoffer; Grønbaek, Kirsten

    2007-10-01

    MicroRNAs (miRNAs) are a recently discovered group of small RNA molecules involved in the regulation of gene expression. Analogously to mRNAs, the non-protein-encoding pri-miRNAs are synthesized by RNA polymerase II and post-transcriptionally modified by addition of a 5'-cap and a 3'-poly (A) tail. Subsequently, the pri-miRNA undergoes a number of processing steps in the nucleus and cytoplasm, and ends up as a mature approximately 22 nt miRNA, which can exert its function by binding to the 3'-untranslated region of a subset of mRNAs. Binding of the miRNA to the mRNA results in a reduced translation rate and/or increased degradation of the mRNA. In this way a large number of cellular pathways, such as cellular proliferation, differentiation, and apoptosis, are regulated by mi-RNAs. As corruption of these pathways is the hallmark of many cancers, dysregulation of miRNA biogenesis or expression levels may lead to tumorigenesis. The mechanisms that alter the expression of miRNAs are similar to those that change the expression levels of mRNAs of tumor suppressor- and oncogenes, i.e. gross genomic aberrations, epigenetic changes, and minor mutations affecting the expression level, processing, or target-interaction potential of the miRNA. Furthermore, expression profiling of miRNAs has been found to be useful for classification of different tumor types. Taken together, miRNAs can be classified as onco-miRs or tumor suppressor-miRs, and may turn out to be potential targets for cancer therapy.

  11. 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

  12. The Fragmented Mitochondrial Ribosomal RNAs of Plasmodium falciparum

    PubMed Central

    Feagin, Jean E.; Harrell, Maria Isabel; Lee, Jung C.; Coe, Kevin J.; Sands, Bryan H.; Cannone, Jamie J.; Tami, Germaine; Schnare, Murray N.; Gutell, Robin R.

    2012-01-01

    Background The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis. Principal Findings The identification of 14 additional small mitochondrial transcripts from P. falcipaurm and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome. Significance All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered. PMID:22761677

  13. Long (27-nucleotides) small inhibitory RNAs targeting E6 protein eradicate effectively the cervical cancer cells harboring human papilloma virus.

    PubMed

    Cho, Jun Sik; Lee, Shin-Wha; Kim, Yong-Man; Kim, Dongho; Kim, Dae-Yeon; Kim, Young-Tak

    2015-05-01

    This study was to identify small inhibitory RNAs (siRNAs) that are effective in inhibiting growth of cervical cancer cell lines harboring human papilloma virus (HPV) and to examine how siRNAs interact with interferon beta (IFN-β) and thimerosal. The HPV18-positive HeLa and C-4I cell lines were used. Four types of siRNAs were designed according to their target (both E6 and E7 vs. E6 only) and sizes (21- vs. 27-nucleotides); Ex-18E6/21, Ex-18E6/27, Sp-18E6/21, and Sp-18E6/27. Each siRNA-transfected cells were cultured with or without IFN-b and thimerosal and their viability was measured. The viabilities of HPV18-positive tumor cells were reduced by 21- and 27-nucleotide siRNAs in proportion to the siRNA concentrations. Of the two types of siRNAs, the 27-nucleotide siRNA constructs showed greater inhibitory efficacy. Sp-18E6 siRNAs, which selectively downregulates E6 protein only, were more effective than the E6- and E7-targeting Ex-18E6 siRNAs. siRNAs and IFN-β showed the synergistic effect to inhibit HeLa cell survival and the effect was proportional to both siRNA and IFN-β concentrations. Thimerosal in the presence of siRNA exerted a dose-dependent inhibition of C-4I cell survival. Finally, co-treatment with siRNA, IFN-β, and thimerosal induced the most profound decrease in the viability of both cell lines. Long (27-nucleotides) siRNAs targeting E6-E7 mRNAs effectively reduce the viability of HPV18-positive cervical cancer cells and show the synergistic effect in combination with IFN-b and thimerosal. It is necessary to find the rational design of siRNAs and effective co-factors to eradicate particular cervical cancer.

  14. Detection of piRNAs in whitespotted bamboo shark liver.

    PubMed

    Yang, Lingrong; Ge, Yinghua; Cheng, Dandan; Nie, Zuoming; Lv, Zhengbing

    2016-09-15

    Piwi-interacting RNAs (piRNAs) are 26 to 31-nt small non-coding RNAs that have been reported mostly in germ-line cells and cancer cells. However, the presence of piRNAs in the whitespotted bamboo shark liver has not yet been reported. In a previous study of microRNAs in shark liver, some piRNAs were detected from small RNAs sequenced by Solexa technology. A total of 4857 piRNAs were predicted and found in shark liver. We further selected 17 piRNAs with high and significantly differential expression between normal and regenerative liver tissues for subsequent verification by Northern blotting. Ten piRNAs were further identified, and six of these were matched to known piRNAs in piRNABank. The actual expression of six known and four novel piRNAs was validated by qRT-PCR. In addition, a total of 401 target genes of the 10 piRNAs were predicted by miRanda. Through GO and pathway function analyses, only five piRNAs could be annotated with eighteen GO annotations. The results indicated that the identified piRNAs are involved in many important biological responses, including immune inflammation, cell-specific differentiation and development, and angiogenesis. This manuscript provides the first identification of piRNAs in the liver of whitespotted bamboo shark using Solexa technology as well as further elucidation of the regulatory role of piRNAs in whitespotted bamboo shark liver. These findings may provide a useful resource and may facilitate the development of therapeutic strategies against liver damage. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Signs of embryo-maternal communication: miRNAs in the maternal serum of pregnant pigs.

    PubMed

    Reliszko, Z P; Gajewski, Z; Kaczmarek, M M

    2017-09-01

    Circulating miRNAs were proposed to be indicators of normal or complicated pregnancies. Based on this knowledge and our recent transcriptomic approach showing expression of miRNAs in the porcine endometrium, conceptuses and uterine extracellular vesicles during pregnancy, we have hypothesized that signs of ongoing local embryo-maternal crosstalk involving miRNAs can be detected in the circulation of pregnant gilts as early as a few days after maternal recognition of pregnancy. By applying several molecular biology techniques that differ in dynamic range and precision in maternal serum of Day 16 pregnant pigs, we were able to show for the first time increased levels of several miRNAs, previously reported to be expressed in either conceptuses and extracellular vesicles (miR-26a and miR-125b) or pregnant endometrium (miR-23b). Our results clearly showed that real-time RT-PCR and digital PCR are the most reliable methods, being able to detect small-fold changes of low-abundant circulating miRNAs. Further validation in a separate group of gilts confirmed an increase in miR-23b and miR-125b levels. In silico analyses identified pregnancy-related biological processes and pathways affected by these miRNAs. Target prediction analysis revealed hundreds of porcine transcripts with conserved sites for these miRNAs, which were classified into signaling pathways relevant to pregnancy. We conclude that a unique set of miRNAs can already be observed in the circulation of pigs during the first weeks of pregnancy, as a result of the initiation of embryo-maternal communication. © 2017 Society for Reproduction and Fertility.

  16. Extraordinary Structured Noncoding RNAs Revealed by Bacterial Metagenome Analysis

    PubMed Central

    Weinberg, Zasha; Perreault, Jonathan; Meyer, Michelle M.; Breaker, Ronald R.

    2012-01-01

    Estimates of the total number of bacterial species1-3 suggest that existing DNA sequence databases carry only a tiny fraction of the total amount of DNA sequence space represented by this division of life. Indeed, environmental DNA samples have been shown to encode many previously unknown classes of proteins4 and RNAs5. Bioinformatics searches6-10 of genomic DNA from bacteria commonly identify novel noncoding RNAs (ncRNAs)10-12 such as riboswitches13,14. In rare instances, RNAs that exhibit more extensive sequence and structural conservation across a wide range of bacteria are encountered15,16. Given that large structured RNAs are known to carry out complex biochemical functions such as protein synthesis and RNA processing reactions, identifying more RNAs of great size and intricate structure is likely to reveal additional biochemical functions that can be achieved by RNA. We applied an updated computational pipeline17 to discover ncRNAs that rival the known large ribozymes in size and structural complexity or that are among the most abundant RNAs in bacteria that encode them. These RNAs would have been difficult or impossible to detect without examining environmental DNA sequences, suggesting that numerous RNAs with extraordinary size, structural complexity, or other exceptional characteristics remain to be discovered in unexplored sequence space. PMID:19956260

  17. Identification of genes that function in the biogenesis and localization of small nucleolar RNAs in Saccharomyces cerevisiae.

    PubMed

    Qiu, Hui; Eifert, Julia; Wacheul, Ludivine; Thiry, Marc; Berger, Adam C; Jakovljevic, Jelena; Woolford, John L; Corbett, Anita H; Lafontaine, Denis L J; Terns, Rebecca M; Terns, Michael P

    2008-06-01

    Small nucleolar RNAs (snoRNAs) orchestrate the modification and cleavage of pre-rRNA and are essential for ribosome biogenesis. Recent data suggest that after nucleoplasmic synthesis, snoRNAs transiently localize to the Cajal body (in plant and animal cells) or the homologous nucleolar body (in budding yeast) for maturation and assembly into snoRNPs prior to accumulation in their primary functional site, the nucleolus. However, little is known about the trans-acting factors important for the intranuclear trafficking and nucleolar localization of snoRNAs. Here, we describe a large-scale genetic screen to identify proteins important for snoRNA transport in Saccharomyces cerevisiae. We performed fluorescence in situ hybridization analysis to visualize U3 snoRNA localization in a collection of temperature-sensitive yeast mutants. We have identified Nop4, Prp21, Tao3, Sec14, and Htl1 as proteins important for the proper localization of U3 snoRNA. Mutations in genes encoding these proteins lead to specific defects in the targeting or retention of the snoRNA to either the nucleolar body or the nucleolus. Additional characterization of the mutants revealed impairment in specific steps of U3 snoRNA processing, demonstrating that snoRNA maturation and trafficking are linked processes.

  18. MicroRNAs regulate osteogenesis and chondrogenesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dong, Shiwu, E-mail: shiwudong@gmail.com; Yang, Bo; Guo, Hongfeng

    Highlights: Black-Right-Pointing-Pointer To focus on the role of miRNAs in chondrogenesis and osteogenesis. Black-Right-Pointing-Pointer Involved in the regulation of miRNAs in osteoarthritis. Black-Right-Pointing-Pointer To speculate some therapeutic targets for bone diseases. -- Abstract: MicroRNAs (miRNAs) are a class of small molecules and non-coding single strand RNAs that regulate gene expression at the post-transcriptional level by binding to specific sequences within target genes. miRNAs have been recognized as important regulatory factors in organism development and disease expression. Some miRNAs regulate the proliferation and differentiation of osteoblasts, osteoclasts and chondrocytes, eventually influencing metabolism and bone formation. miRNAs are expected to provide potentialmore » gene therapy targets for the clinical treatment of metabolic bone diseases and bone injuries. Here, we review the recent research progress on the regulation of miRNAs in bone biology, with a particular focus on the miRNA-mediated control mechanisms of bone and cartilage formation.« less

  19. Small interfering RNAs from bidirectional transcripts of GhMML3_A12 regulate cotton fiber development.

    PubMed

    Wan, Qun; Guan, Xueying; Yang, Nannan; Wu, Huaitong; Pan, Mengqiao; Liu, Bingliang; Fang, Lei; Yang, Shouping; Hu, Yan; Ye, Wenxue; Zhang, Hua; Ma, Peiyong; Chen, Jiedan; Wang, Qiong; Mei, Gaofu; Cai, Caiping; Yang, Donglei; Wang, Jiawei; Guo, Wangzhen; Zhang, Wenhua; Chen, Xiaoya; Zhang, Tianzhen

    2016-06-01

    Natural antisense transcripts (NATs) are commonly observed in eukaryotic genomes, but only a limited number of such genes have been identified as being involved in gene regulation in plants. In this research, we investigated the function of small RNA derived from a NAT in fiber cell development. Using a map-based cloning strategy for the first time in tetraploid cotton, we cloned a naked seed mutant gene (N1 ) encoding a MYBMIXTA-like transcription factor 3 (MML3)/GhMYB25-like in chromosome A12, GhMML3_A12, that is associated with fuzz fiber development. The extremely low expression of GhMML3_A12 in N1 is associated with NAT production, driven by its 3' antisense promoter, as indicated by the promoter-driven histochemical staining assay. In addition, small RNA deep sequencing analysis suggested that the bidirectional transcriptions of GhMML3_A12 form double-stranded RNAs and generate 21-22 nt small RNAs. Therefore, in a fiber-specific manner, small RNA derived from the GhMML3_A12 locus can mediate GhMML3_A12 mRNA self-cleavage and result in the production of naked seeds followed by lint fiber inhibition in N1 plants. The present research reports the first observation of gene-mediated NATs and siRNA directly controlling fiber development in cotton. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  20. Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon

    PubMed Central

    2013-01-01

    Background The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using parallel analysis of RNA ends (PARE) data is lacking in B. distachyon. Results B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset for analyzing small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. Conclusions B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants. PMID:24367943

  1. A DNA logic gate based on strand displacement reaction and rolling circle amplification, responding to multiple low-abundance DNA fragment input signals, and its application in detecting miRNAs.

    PubMed

    Chen, Yuqi; Song, Yanyan; Wu, Fan; Liu, Wenting; Fu, Boshi; Feng, Bingkun; Zhou, Xiang

    2015-04-25

    A conveniently amplified DNA AND logic gate platform was designed for the highly sensitive detection of low-abundance DNA fragment inputs based on strand displacement reaction and rolling circle amplification strategy. Compared with others, this system can detect miRNAs in biological samples. The success of this strategy demonstrates the potential of DNA logic gates in disease diagnosis.

  2. Conservation of a microRNA cluster in parasitic nematodes and profiling of miRNAs in excretory-secretory products and microvesicles of Haemonchus contortus

    PubMed Central

    Gu, Henry Y.; Marks, Neil D.; Winter, Alan D.; Weir, William; Tzelos, Thomas; McNeilly, Tom N.; Britton, Collette

    2017-01-01

    microRNAs are small non-coding RNAs that are important regulators of gene expression in a range of animals, including nematodes. We have analysed a cluster of four miRNAs from the pathogenic nematode species Haemonchus contortus that are closely linked in the genome. We find that the cluster is conserved only in clade V parasitic nematodes and in some ascarids, but not in other clade III species nor in clade V free-living nematodes. Members of the cluster are present in parasite excretory-secretory products and can be detected in the abomasum and draining lymph nodes of infected sheep, indicating their release in vitro and in vivo. As observed for other parasitic nematodes, H. contortus adult worms release extracellular vesicles (EV). Small RNA libraries were prepared from vesicle-enriched and vesicle-depleted supernatants from both adult worms and L4 stage larvae. Comparison of the miRNA species in the different fractions indicated that specific miRNAs are packaged within vesicles, while others are more abundant in vesicle-depleted supernatant. Hierarchical clustering analysis indicated that the gut is the likely source of vesicle-associated miRNAs in the L4 stage, but not in the adult worm. These findings add to the growing body of work demonstrating that miRNAs released from parasitic helminths may play an important role in host-parasite interactions. PMID:29145392

  3. Hepatitis A virus-encoded miRNAs attenuate the accumulation of viral genomic RNAs in infected cells.

    PubMed

    Shi, Jiandong; Sun, Jing; Wu, Meini; Hu, Ningzhu; Hu, Yunzhang

    2016-06-01

    The establishment of persistent infection with hepatitis A virus (HAV) is the common result of most HAV/cell culture systems. Previous observations show that the synthesis of viral RNAs is reduced during infection. However, the underlying mechanism is poorly understood. We characterized three HAV-encoded miRNAs in our previous study. In this study, we aim to investigate the impact of these miRNAs on the accumulation of viral RNAs. The results indicated that the synthesis of viral genomic RNAs was dramatically reduced (more than 75 % reduction, P < 0.05) when transfected with one or two viral miRNA mimics. Conversely, they were significantly increased (more than 3.3-fold addition, P < 0.05) when transfected with one or two viral miRNA inhibitors. The luciferase reporter assay of miRNA targets showed that viral miRNAs were fully complementary to specific sites of the viral plus or minus strand RNA and strongly inhibited their expressions. Further data showed that the relative abundance of viral genomic RNA fragments that contain miRNA targets was also dramatically reduced (more than 80 % reduction, P < 0.05) when viral miRNAs were overexpressed with miRNA mimics. In contrast, they were significantly increased (approximately 2-fold addition, P < 0.05) when viral miRNAs were inhibited with miRNA inhibitors. In conclusion, these data suggest a possible mechanism for the reduction of viral RNA synthesis during HAV infection. Thus, we propose that it is likely that RNA virus-derived miRNA could serve as a self-mediated feedback regulator during infection.

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

    PubMed Central

    Dalmay, Tamas; Powell, Penny P.

    2013-01-01

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

  5. 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.

  6. EBV‐encoded miRNAs target ATM‐mediated response in nasopharyngeal carcinoma

    PubMed Central

    Lung, Raymond W‐M; Hau, Pok‐Man; Yu, Ken H‐O; Yip, Kevin Y; Tong, Joanna H‐M; Chak, Wing‐Po; Chan, Anthony W‐H; Lam, Ka‐Hei; Lo, Angela Kwok‐Fung; Tin, Edith K‐Y; Chau, Shuk‐Ling; Pang, Jesse C‐S; Kwan, Johnny S‐H; Busson, Pierre; Young, Lawrence S; Yap, Lee‐Fah; Tsao, Sai‐Wah

    2018-01-01

    Abstract Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein–Barr virus (EBV) infection. In NPC, miR‐BARTs, the EBV‐encoded miRNAs derived from BamH1‐A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV‐encoded miRNAs in a panel of NPC patient‐derived xenografts and an EBV‐positive NPC cell line by small RNA sequencing. Among the 40 miR‐BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV‐miRNAs, BART5‐5p, BART7‐3p, BART9‐3p, and BART14‐3p could negatively regulate the expression of a key DNA double‐strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'‐UTR. Notably, the expression of these four miR‐BARTs represented more than 10% of all EBV‐encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT‐PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5‐5p, BART7‐3p, BART9‐3p, and BART14‐3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR‐BARTs in EBV‐positive NPC cells, we further demonstrated the novel function of miR‐BARTs in inhibiting Zta‐induced lytic reactivation. These findings imply that the four viral miRNAs work co‐operatively to modulate ATM activity in response to DNA damage and to maintain viral latency

  7. EBV-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma.

    PubMed

    Lung, Raymond W-M; Hau, Pok-Man; Yu, Ken H-O; Yip, Kevin Y; Tong, Joanna H-M; Chak, Wing-Po; Chan, Anthony W-H; Lam, Ka-Hei; Lo, Angela Kwok-Fung; Tin, Edith K-Y; Chau, Shuk-Ling; Pang, Jesse C-S; Kwan, Johnny S-H; Busson, Pierre; Young, Lawrence S; Yap, Lee-Fah; Tsao, Sai-Wah; To, Ka-Fai; Lo, Kwok-Wai

    2018-04-01

    Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein-Barr virus (EBV) infection. In NPC, miR-BARTs, the EBV-encoded miRNAs derived from BamH1-A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV-encoded miRNAs in a panel of NPC patient-derived xenografts and an EBV-positive NPC cell line by small RNA sequencing. Among the 40 miR-BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV-miRNAs, BART5-5p, BART7-3p, BART9-3p, and BART14-3p could negatively regulate the expression of a key DNA double-strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'-UTR. Notably, the expression of these four miR-BARTs represented more than 10% of all EBV-encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT-PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5-5p, BART7-3p, BART9-3p, and BART14-3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR-BARTs in EBV-positive NPC cells, we further demonstrated the novel function of miR-BARTs in inhibiting Zta-induced lytic reactivation. These findings imply that the four viral miRNAs work co-operatively to modulate ATM activity in response to DNA damage and to maintain viral latency, contributing to the tumorigenesis of NPC. © 2017 The Authors

  8. Unproductively spliced ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans

    PubMed Central

    Mitrovich, Quinn M.; Anderson, Philip

    2000-01-01

    Messenger RNA surveillance, the selective and rapid degradation of mRNAs containing premature stop codons, occurs in all eukaryotes tested. The biological role of this decay pathway, however, is not well understood. To identify natural substrates of mRNA surveillance, we used a cDNA-based representational difference analysis to identify mRNAs whose abundance increases in Caenorhabditis elegans smg(−) mutants, which are deficient for mRNA surveillance. Alternatively spliced mRNAs of genes encoding ribosomal proteins L3, L7a, L10a, and L12 are abundant natural targets of mRNA surveillance. Each of these genes expresses two distinct mRNAs. A productively spliced mRNA, whose abundance does not change in smg(−) mutants, encodes a normal, full-length, ribosomal protein. An unproductively spliced mRNA, whose abundance increases dramatically in smg(−) mutants, contains premature stop codons because of incomplete removal of an alternatively spliced intron. In transgenic animals expressing elevated quantities of RPL-12, a greater proportion of endogenous rpl-12 transcript is spliced unproductively. Thus, RPL-12 appears to autoregulate its own splicing, with unproductively spliced mRNAs being degraded by mRNA surveillance. We demonstrate further that alternative splicing of rpl introns is conserved among widely diverged nematodes. Our results suggest that one important role of mRNA surveillance is to eliminate unproductive by-products of gene regulation. PMID:10970881

  9. The role of miRNAs in endometrial cancer.

    PubMed

    Vasilatou, Diamantina; Sioulas, Vasileios D; Pappa, Vasiliki; Papageorgiou, Sotirios G; Vlahos, Nikolaos F

    2015-01-01

    miRNAs are small noncoding RNAs that regulate gene expression at the post-transcriptional level. Since their discovery, miRNAs have been associated with every cell function including malignant transformation and metastasis. Endometrial cancer is the most common gynecologic malignancy. However, improvement should be made in interobserver agreement on histological typing and individualized therapeutic approaches. This article summarizes the role of miRNAs in endometrial cancer pathogenesis and treatment.

  10. Conservation and divergence of microRNAs in Populus

    PubMed Central

    Barakat, Abdelali; Wall, Phillip K; DiLoreto, Scott; dePamphilis, Claude W; Carlson, John E

    2007-01-01

    Background MicroRNAs (miRNAs) are small RNAs (sRNA) ~21 nucleotides in length that negatively control gene expression by cleaving or inhibiting the translation of target gene transcripts. miRNAs have been extensively analyzed in Arabidopsis and rice and partially investigated in other non-model plant species. To date, 109 and 62 miRNA families have been identified in Arabidopsis and rice respectively. However, only 33 miRNAs have been identified from the genome of the model tree species (Populus trichocarpa), of which 11 are Populus specific. The low number of miRNA families previously identified in Populus, compared with the number of families identified in Arabidopsis and rice, suggests that many miRNAs still remain to be discovered in Populus. In this study, we analyzed expressed small RNAs from leaves and vegetative buds of Populus using high throughput pyrosequencing. Results Analysis of almost eighty thousand small RNA reads allowed us to identify 123 new sequences belonging to previously identified miRNA families as well as 48 new miRNA families that could be Populus-specific. Comparison of the organization of miRNA families in Populus, Arabidopsis and rice showed that miRNA family sizes were generally expanded in Populus. The putative targets of non-conserved miRNA include both previously identified targets as well as several new putative target genes involved in development, resistance to stress, and other cellular processes. Moreover, almost half of the genes predicted to be targeted by non-conserved miRNAs appear to be Populus-specific. Comparative analyses showed that genes targeted by conserved and non-conserved miRNAs are biased mainly towards development, electron transport and signal transduction processes. Similar results were found for non-conserved miRNAs from Arabidopsis. Conclusion Our results suggest that while there is a conserved set of miRNAs among plant species, a large fraction of miRNAs vary among species. The non-conserved miRNAs may

  11. EVLncRNAs: a manually curated database for long non-coding RNAs validated by low-throughput experiments.

    PubMed

    Zhou, Bailing; Zhao, Huiying; Yu, Jiafeng; Guo, Chengang; Dou, Xianghua; Song, Feng; Hu, Guodong; Cao, Zanxia; Qu, Yuanxu; Yang, Yuedong; Zhou, Yaoqi; Wang, Jihua

    2018-01-04

    Long non-coding RNAs (lncRNAs) play important functional roles in various biological processes. Early databases were utilized to deposit all lncRNA candidates produced by high-throughput experimental and/or computational techniques to facilitate classification, assessment and validation. As more lncRNAs are validated by low-throughput experiments, several databases were established for experimentally validated lncRNAs. However, these databases are small in scale (with a few hundreds of lncRNAs only) and specific in their focuses (plants, diseases or interactions). Thus, it is highly desirable to have a comprehensive dataset for experimentally validated lncRNAs as a central repository for all of their structures, functions and phenotypes. Here, we established EVLncRNAs by curating lncRNAs validated by low-throughput experiments (up to 1 May 2016) and integrating specific databases (lncRNAdb, LncRANDisease, Lnc2Cancer and PLNIncRBase) with additional functional and disease-specific information not covered previously. The current version of EVLncRNAs contains 1543 lncRNAs from 77 species that is 2.9 times larger than the current largest database for experimentally validated lncRNAs. Seventy-four percent lncRNA entries are partially or completely new, comparing to all existing experimentally validated databases. The established database allows users to browse, search and download as well as to submit experimentally validated lncRNAs. The database is available at http://biophy.dzu.edu.cn/EVLncRNAs. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. EVLncRNAs: a manually curated database for long non-coding RNAs validated by low-throughput experiments

    PubMed Central

    Zhao, Huiying; Yu, Jiafeng; Guo, Chengang; Dou, Xianghua; Song, Feng; Hu, Guodong; Cao, Zanxia; Qu, Yuanxu

    2018-01-01

    Abstract Long non-coding RNAs (lncRNAs) play important functional roles in various biological processes. Early databases were utilized to deposit all lncRNA candidates produced by high-throughput experimental and/or computational techniques to facilitate classification, assessment and validation. As more lncRNAs are validated by low-throughput experiments, several databases were established for experimentally validated lncRNAs. However, these databases are small in scale (with a few hundreds of lncRNAs only) and specific in their focuses (plants, diseases or interactions). Thus, it is highly desirable to have a comprehensive dataset for experimentally validated lncRNAs as a central repository for all of their structures, functions and phenotypes. Here, we established EVLncRNAs by curating lncRNAs validated by low-throughput experiments (up to 1 May 2016) and integrating specific databases (lncRNAdb, LncRANDisease, Lnc2Cancer and PLNIncRBase) with additional functional and disease-specific information not covered previously. The current version of EVLncRNAs contains 1543 lncRNAs from 77 species that is 2.9 times larger than the current largest database for experimentally validated lncRNAs. Seventy-four percent lncRNA entries are partially or completely new, comparing to all existing experimentally validated databases. The established database allows users to browse, search and download as well as to submit experimentally validated lncRNAs. The database is available at http://biophy.dzu.edu.cn/EVLncRNAs. PMID:28985416

  13. The use of high-throughput small RNA sequencing reveals differentially expressed microRNAs in response to aster yellows phytoplasma-infection in Vitis vinifera cv. ‘Chardonnay’

    PubMed Central

    Solofoharivelo, Marie-Chrystine; Souza-Richards, Rose; Stephan, Dirk; Murray, Shane; Burger, Johan T.

    2017-01-01

    Phytoplasmas are cell wall-less plant pathogenic bacteria responsible for major crop losses throughout the world. In grapevine they cause grapevine yellows, a detrimental disease associated with a variety of symptoms. The high economic impact of this disease has sparked considerable interest among researchers to understand molecular mechanisms related to pathogenesis. Increasing evidence exist that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation during plant development and responses to biotic and abiotic stresses. Thus, we aimed to dissect complex high-throughput small RNA sequencing data for the genome-wide identification of known and novel differentially expressed miRNAs, using read libraries constructed from healthy and phytoplasma-infected Chardonnay leaf material. Furthermore, we utilised computational resources to predict putative miRNA targets to explore the involvement of possible pathogen response pathways. We identified multiple known miRNA sequence variants (isomiRs), likely generated through post-transcriptional modifications. Sequences of 13 known, canonical miRNAs were shown to be differentially expressed. A total of 175 novel miRNA precursor sequences, each derived from a unique genomic location, were predicted, of which 23 were differentially expressed. A homology search revealed that some of these novel miRNAs shared high sequence similarity with conserved miRNAs from other plant species, as well as known grapevine miRNAs. The relative expression of randomly selected known and novel miRNAs was determined with real-time RT-qPCR analysis, thereby validating the trend of expression seen in the normalised small RNA sequencing read count data. Among the putative miRNA targets, we identified genes involved in plant morphology, hormone signalling, nutrient homeostasis, as well as plant stress. Our results may assist in understanding the role that miRNA pathways play

  14. Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC

    PubMed Central

    Castanotto, Daniela; Sakurai, Kumi; Lingeman, Robert; Li, Haitang; Shively, Louise; Aagaard, Lars; Soifer, Harris; Gatignol, Anne; Riggs, Arthur; Rossi, John J.

    2007-01-01

    Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs. PMID:17660190

  15. Compartmentalized, functional role of angiogenin during spotted fever group rickettsia-induced endothelial barrier dysfunction: evidence of possible mediation by host tRNA-derived small noncoding RNAs

    PubMed Central

    2013-01-01

    Background Microvascular endothelial barrier dysfunction is the central enigma in spotted fever group (SFG) rickettsioses. Angiogenin (ANG) is one of the earliest identified angiogenic factors, of which some are relevant to the phosphorylation of VE-cadherins that serve as endothelial adherens proteins. Although exogenous ANG is known to translocate into the nucleus of growing endothelial cells (ECs) where it plays a functional role, nuclear ANG is not detected in quiescent ECs. Besides its nuclear role, ANG is thought to play a cytoplasmic role, owing to its RNase activity that cleaves tRNA to produce small RNAs. Recently, such tRNA-derived RNA fragments (tRFs) have been shown to be induced under stress conditions. All these observations raise an intriguing hypothesis about a novel cytoplasmic role of ANG, which is induced upon infection with Rickettsia and generates tRFs that may play roles in SFG rickettsioses. Methods C3H/HeN mice were infected intravenously with a sublethal dose of R. conorii. At days 1, 3, and 5 post infection (p.i.), liver, lung and brain were collected for immunofluorescence (IF) studies of R. conorii and angiogenin (ANG). Human umbilical vein endothelial cells (HUVECs) were infected with R. conorii for 24, 48, and 72 hrs before incubation with 1μg/ml recombinant human ANG (rANG) in normal medium for 2 hrs. HUVEC samples were subjected to IF, exogenous ANG translocation, endothelial permeability, and immunoprecipitation phosphorylation assays. To identify small non-coding RNAs (sncRNAs) upon rickettsial infection, RNAs from pulverized mouse lung tissues and HUVECs were subjected to library preparation and deep sequencing analysis using an Illumina 2000 instrument. Identified sncRNAs were confirmed by Northern hybridization, and their target mRNAs were predicted in silico using BLAST and RNA hybrid programs. Results In the present study, we have demonstrated endothelial up-regulation of ANG, co-localized with SFG rickettsial infection in

  16. Genome-wide identification of conserved and novel microRNAs in one bud and two tender leaves of tea plant (Camellia sinensis) by small RNA sequencing, microarray-based hybridization and genome survey scaffold sequences.

    PubMed

    Jeyaraj, Anburaj; Zhang, Xiao; Hou, Yan; Shangguan, Mingzhu; Gajjeraman, Prabu; Li, Yeyun; Wei, Chaoling

    2017-11-21

    MicroRNAs (miRNAs) are important for plant growth and responses to environmental stresses via post-transcriptional regulation of gene expression. Tea, which is primarily produced from one bud and two tender leaves of the tea plant (Camellia sinensis), is one of the most popular non-alcoholic beverages worldwide owing to its abundance of secondary metabolites. A large number of miRNAs have been identified in various plants, including non-model species. However, due to the lack of reference genome sequences and/or information of tea plant genome survey scaffold sequences, discovery of miRNAs has been limited in C. sinensis. Using small RNA sequencing, combined with our recently obtained genome survey data, we have identified and analyzed 175 conserved and 83 novel miRNAs mainly in one bud and two tender leaves of the tea plant. Among these, 93 conserved and 18 novel miRNAs were validated using miRNA microarray hybridization. In addition, the expression pattern of 11 conserved and 8 novel miRNAs were validated by stem-loop-qRT-PCR. A total of 716 potential target genes of identified miRNAs were predicted. Further, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that most of the target genes were primarily involved in stress response and enzymes related to phenylpropanoid biosynthesis. The predicted targets of 4 conserved miRNAs were further validated by 5'RLM-RACE. A negative correlation between expression profiles of 3 out of 4 conserved miRNAs (csn-miR160a-5p, csn-miR164a, csn-miR828 and csn-miR858a) and their targets (ARF17, NAC100, WER and MYB12 transcription factor) were observed. In summary, the present study is one of few such studies on miRNA detection and identification in the tea plant. The predicted target genes of majority of miRNAs encoded enzymes, transcription factors, and functional proteins. The miRNA-target transcription factor gene interactions may provide important clues about the regulatory

  17. Recipe for a Busy Bee: MicroRNAs in Honey Bee Caste Determination

    PubMed Central

    Skogerboe, Geir; Dai, Shuanjin; Li, Wenfeng; Li, Zhiguo; Liu, Fang; Ni, Ruifeng; Guo, Yu; Chen, Shenglu; Zhang, Shaowu; Chen, Runsheng

    2013-01-01

    Social caste determination in the honey bee is assumed to be determined by the dietary status of the young larvae and translated into physiological and epigenetic changes through nutrient-sensing pathways. We have employed Illumina/Solexa sequencing to examine the small RNA content in the bee larval food, and show that worker jelly is enriched in miRNA complexity and abundance relative to royal jelly. The miRNA levels in worker jelly were 7–215 fold higher than in royal jelly, and both jellies showed dynamic changes in miRNA content during the 4th to 6th day of larval development. Adding specific miRNAs to royal jelly elicited significant changes in queen larval mRNA expression and morphological characters of the emerging adult queen bee. We propose that miRNAs in the nurse bee secretions constitute an additional element in the regulatory control of caste determination in the honey bee. PMID:24349106

  18. Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli

    PubMed Central

    Kim, Sang Hyon; Spensley, Mark; Choi, Seung Kook; Calixto, Cristiane P. G.; Pendle, Ali F.; Koroleva, Olga; Shaw, Peter J.; Brown, John W. S.

    2010-01-01

    Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants. PMID:20081206

  19. Systematic discovery and characterization of fly microRNAs using 12 Drosophila genomes

    PubMed Central

    Stark, Alexander; Kheradpour, Pouya; Parts, Leopold; Brennecke, Julius; Hodges, Emily; Hannon, Gregory J.; Kellis, Manolis

    2007-01-01

    MicroRNAs (miRNAs) are short regulatory RNAs that inhibit target genes by complementary binding in 3′ untranslated regions (3′ UTRs). They are one of the most abundant classes of regulators, targeting a large fraction of all genes, making their comprehensive study a requirement for understanding regulation and development. Here we use 12 Drosophila genomes to define structural and evolutionary signatures of miRNA hairpins, which we use for their de novo discovery. We predict >41 novel miRNA genes, which encompass many unique families, and 28 of which are validated experimentally. We also define signals for the precise start position of mature miRNAs, which suggest corrections of previously known miRNAs, often leading to drastic changes in their predicted target spectrum. We show that miRNA discovery power scales with the number and divergence of species compared, suggesting that such approaches can be successful in human as dozens of mammalian genomes become available. Interestingly, for some miRNAs sense and anti-sense hairpins score highly and mature miRNAs from both strands can indeed be found in vivo. Similarly, miRNAs with weak 5′ end predictions show increased in vivo processing of multiple alternate 5′ ends and have fewer predicted targets. Lastly, we show that several miRNA star sequences score highly and are likely functional. For mir-10 in particular, both arms show abundant processing, and both show highly conserved target sites in Hox genes, suggesting a possible cooperation of the two arms, and their role as a master Hox regulator. PMID:17989255

  20. High-Throughput Sequencing Identifies MicroRNAs from Posterior Intestine of Loach (Misgurnus anguillicaudatus) and Their Response to Intestinal Air-Breathing Inhibition.

    PubMed

    Huang, Songqian; Cao, Xiaojuan; Tian, Xianchang; Wang, Weimin

    2016-01-01

    MicroRNAs (miRNAs) exert important roles in animal growth, immunity, and development, and regulate gene expression at the post-transcriptional level. Knowledges about the diversities of miRNAs and their roles in accessory air-breathing organs (ABOs) of fish remain unknown. In this work, we used high-throughput sequencing to identify known and novel miRNAs from the posterior intestine, an important ABO, in loach (Misgurnus anguillicaudatus) under normal and intestinal air-breathing inhibited conditions. A total of 204 known and 84 novel miRNAs were identified, while 47 miRNAs were differentially expressed between the two small RNA libraries (i.e. between the normal and intestinal air-breathing inhibited group). Potential miRNA target genes were predicted by combining our transcriptome data of the posterior intestine of the loach under the same conditions, and then annotated using COG, GO, KEGG, Swissprot and Nr databases. The regulatory networks of miRNAs and their target genes were analyzed. The abundances of nine known miRNAs were validated by qRT-PCR. The relative expression profiles of six known miRNAs and their eight corresponding target genes, and two novel potential miRNAs were also detected. Histological characteristics of the posterior intestines in both normal and air-breathing inhibited group were further analyzed. This study contributes to our understanding on the functions and molecular regulatory mechanisms of miRNAs in accessory air-breathing organs of fish.

  1. Detection and Reconstruction of Circular RNAs from Transcriptomic Data.

    PubMed

    Zheng, Yi; Zhao, Fangqing

    2018-01-01

    Recent studies have shown that circular RNAs (circRNAs) are a novel class of abundant, stable, and ubiquitous noncoding RNA molecules in eukaryotic organisms. Comprehensive detection and reconstruction of circRNAs from high-throughput transcriptome data is an initial step to study their biogenesis and function. Several tools have been developed to deal with this issue, but they require many steps and are difficult to use. To solve this problem, we provide a protocol for researchers to detect and reconstruct circRNA by employing CIRI2, CIRI-AS, and CIRI-full. This protocol can not only simplify the usage of above tools but also integrate their results.

  2. Bioavailability of transgenic microRNAs in genetically modified plants

    USDA-ARS?s Scientific Manuscript database

    Transgenic expression of small RNAs is a prevalent approach in agrobiotechnology for the global enhancement of plant foods. Meanwhile, emerging studies have, on the one hand, emphasized the potential of transgenic microRNAs (miRNAs) as novel dietary therapeutics and, on the other, suggested potentia...

  3. Diversity of Antisense and Other Non-Coding RNAs in Archaea Revealed by Comparative Small RNA Sequencing in Four Pyrobaculum Species

    PubMed Central

    Bernick, David L.; Dennis, Patrick P.; Lui, Lauren M.; Lowe, Todd M.

    2012-01-01

    A great diversity of small, non-coding RNA (ncRNA) molecules with roles in gene regulation and RNA processing have been intensely studied in eukaryotic and bacterial model organisms, yet our knowledge of possible parallel roles for small RNAs (sRNA) in archaea is limited. We employed RNA-seq to identify novel sRNA across multiple species of the hyperthermophilic genus Pyrobaculum, known for unusual RNA gene characteristics. By comparing transcriptional data collected in parallel among four species, we were able to identify conserved RNA genes fitting into known and novel families. Among our findings, we highlight three novel cis-antisense sRNAs encoded opposite to key regulatory (ferric uptake regulator), metabolic (triose-phosphate isomerase), and core transcriptional apparatus genes (transcription factor B). We also found a large increase in the number of conserved C/D box sRNA genes over what had been previously recognized; many of these genes are encoded antisense to protein coding genes. The conserved opposition to orthologous genes across the Pyrobaculum genus suggests similarities to other cis-antisense regulatory systems. Furthermore, the genus-specific nature of these sRNAs indicates they are relatively recent, stable adaptations. PMID:22783241

  4. Circular RNAs: A novel type of biomarker and genetic tools in cancer

    PubMed Central

    Han, Yi-Neng; Xia, Sheng-Qiang; Zhang, Yuan-Yuan; Zheng, Jun-Hua; Li, Wei

    2017-01-01

    Circular RNAs (circRNAs) are a novel type of universal and diverse endogenous noncoding RNAs (ncRNAs) and they form a covalently closed continuous loop without 5′ or 3′ tails unlike linear RNAs. Most circRNAs are presented with characteristics of abundance, stability, conservatism, and often exhibiting tissue/developmental-stage-specific expression. CircRNAs are generated either from exons or introns by back splicing or lariat introns. CircRNAs play important roles as miRNA sponges, gene transcription and expression regulators, RNA-binding protein (RBP) sponges and protein/peptide translators. Emerging evidence revealed the function of circRNAs in cancer and may potentially serve as a required novel biomarker and therapeutic target for cancer treatment. In this review, we discuss about the origins, characteristics and functions of circRNA and how they work as miRNA sponges, gene transcription and expression regulators, RBP sponges in cancer as well as current research methods of circRNAs, providing evidence for the significance of circRNAs in cancer diagnosis and clinical treatment. PMID:28969093

  5. De novo characterization of microRNAs in oriental fruit moth Grapholita molesta and selection of reference genes for normalization of microRNA expression

    PubMed Central

    Zhang, Jing; Zhang, Qingwen; Liu, Xiaoxia; Li, Zhen

    2017-01-01

    MicroRNAs (miRNAs) are a group of endogenous non-coding small RNAs that have critical regulatory functions in almost all known biological processes at the post-transcriptional level in a variety of organisms. The oriental fruit moth Grapholita molesta is one of the most serious pests in orchards worldwide and threatens the production of Rosacea fruits. In this study, a de novo small RNA library constructed from mixed stages of G. molesta was sequenced through Illumina sequencing platform and a total of 536 mature miRNAs consisting of 291 conserved and 245 novel miRNAs were identified. Most of the conserved and novel miRNAs were detected with moderate abundance. The miRNAs in the same cluster normally showed correlated expressional profiles. A comparative analysis of the 79 conserved miRNA families within 31 arthropod species indicated that these miRNA families were more conserved among insects and within orders of closer phylogenetic relationships. The KEGG pathway analysis and network prediction of target genes indicated that the complex composed of miRNAs, clock genes and developmental regulation genes may play vital roles to regulate the developmental circadian rhythm of G. molesta. Furthermore, based on the sRNA library of G. molesta, suitable reference genes were selected and validated for study of miRNA transcriptional profile in G. molesta under two biotic and six abiotic experimental conditions. This study systematically documented the miRNA profile in G. molesta, which could lay a foundation for further understanding of the regulatory roles of miRNAs in the development and metabolism in this pest and might also suggest clues to the development of genetic-based techniques for agricultural pest control. PMID:28158242

  6. Genome organization and characteristics of soybean microRNAs

    PubMed Central

    2012-01-01

    Background microRNAs (miRNAs) are key regulators of gene expression and play important roles in many aspects of plant biology. The role(s) of miRNAs in nitrogen-fixing root nodules of leguminous plants such as soybean is not well understood. We examined a library of small RNAs from Bradyrhizobium japonicum-inoculated soybean roots and identified novel miRNAs. In order to enhance our understanding of miRNA evolution, diversification and function, we classified all known soybean miRNAs based on their phylogenetic conservation (conserved, legume- and soybean-specific miRNAs) and examined their genome organization, family characteristics and target diversity. We predicted targets of these miRNAs and experimentally validated several of them. We also examined organ-specific expression of selected miRNAs and their targets. Results We identified 120 previously unknown miRNA genes from soybean including 5 novel miRNA families. In the soybean genome, genes encoding miRNAs are primarily intergenic and a small percentage were intragenic or less than 1000 bp from a protein-coding gene, suggesting potential co-regulation between the miRNA and its parent gene. Difference in number and orientation of tandemly duplicated miRNA genes between orthologous genomic loci indicated continuous evolution and diversification. Conserved miRNA families are often larger in size and produce less diverse mature miRNAs than legume- and soybean-specific families. In addition, the majority of conserved and legume-specific miRNA families produce 21 nt long mature miRNAs with distinct nucleotide distribution and regulate a more conserved set of target mRNAs compared to soybean-specific families. A set of nodule-specific target mRNAs and their cognate regulatory miRNAs had inverse expression between root and nodule tissues suggesting that spatial restriction of target gene transcripts by miRNAs might govern nodule-specific gene expression in soybean. Conclusions Genome organization of soybean miRNAs

  7. Small RNA biology is systems biology.

    PubMed

    Jost, Daniel; Nowojewski, Andrzej; Levine, Erel

    2011-01-01

    During the last decade small regulatory RNA (srRNA) emerged as central players in the regulation of gene expression in all kingdoms of life. Multiple pathways for srRNA biogenesis and diverse mechanisms of gene regulation may indicate that srRNA regulation evolved independently multiple times. However, small RNA pathways share numerous properties, including the ability of a single srRNA to regulate multiple targets. Some of the mechanisms of gene regulation by srRNAs have significant effect on the abundance of free srRNAs that are ready to interact with new targets. This results in indirect interactions among seemingly unrelated genes, as well as in a crosstalk between different srRNA pathways. Here we briefly review and compare the major srRNA pathways, and argue that the impact of srRNA is always at the system level. We demonstrate how a simple mathematical model can ease the discussion of governing principles. To demonstrate these points we review a few examples from bacteria and animals.

  8. 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.

  9. Metazoan tRNA introns generate stable circular RNAs in vivo

    PubMed Central

    Lu, Zhipeng; Filonov, Grigory S.; Noto, John J.; Schmidt, Casey A.; Hatkevich, Talia L.; Wen, Ying; Jaffrey, Samie R.; Matera, A. Gregory

    2015-01-01

    We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating “designer” circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science. PMID:26194134

  10. microRNAs of parasites: current status and future perspectives

    USDA-ARS?s Scientific Manuscript database

    MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs regulating gene expression in eukaryotes at the post-transcriptional level. The complex life cycles of parasites may require the ability to respond to environmental and developmental signals through miRNA-mediated gene expression. Ov...

  11. Expression of Small RNA in Aphis gossypii and Its Potential Role in the Resistance Interaction with Melon

    PubMed Central

    Sattar, Sampurna; Anstead, James A.; Sunkar, Ramanjulu; Thompson, Gary A.

    2012-01-01

    Background The regulatory role of small RNAs (sRNAs) in various biological processes is an active area of investigation; however, there has been limited information available on the role of sRNAs in plant-insect interactions. This study was designed to identify sRNAs in cotton-melon aphid (Aphis gossypii) during the Vat-mediated resistance interaction with melon (Cucumis melo). Methodology/Principal Findings The role of miRNAs was investigated in response to aphid herbivory, during both resistant and susceptible interactions. sRNA libraries made from A. gossypii tissues feeding on Vat+ and Vat− plants revealed an unexpected abundance of 27 nt long sRNA sequences in the aphids feeding on Vat+ plants. Eighty-one conserved microRNAs (miRNAs), twelve aphid-specific miRNAs, and nine novel candidate miRNAs were also identified. Plant miRNAs found in the aphid libraries were most likely ingested during phloem feeding. The presence of novel miRNAs was verified by qPCR experiments in both resistant Vat+ and susceptible Vat− interactions. The comparative analyses revealed that novel miRNAs were differentially regulated during the resistant and susceptible interactions. Gene targets predicted for the miRNAs identified in this study by in silico analyses revealed their involvement in morphogenesis and anatomical structure determination, signal transduction pathways, cell differentiation and catabolic processes. Conclusion/Significance In this study, conserved and novel miRNAs were reported in A. gossypii. Deep sequencing data showed differences in the abundance of miRNAs and piRNA-like sequences in A. gossypii. Quantitative RT-PCR revealed that A. gossypii miRNAs were differentially regulated during resistant and susceptible interactions. Aphids can also ingest plant miRNAs during phloem feeding that are stable in the insect. PMID:23173035

  12. DNA rearrangements directed by non-coding RNAs in ciliates

    PubMed Central

    Mochizuki, Kazufumi

    2013-01-01

    Extensive programmed rearrangement of DNA, including DNA elimination, chromosome fragmentation, and DNA descrambling, takes place in the newly developed macronucleus during the sexual reproduction of ciliated protozoa. Recent studies have revealed that two distant classes of ciliates use distinct types of non-coding RNAs to regulate such DNA rearrangement events. DNA elimination in Tetrahymena is regulated by small non-coding RNAs that are produced and utilized in an RNAi-related process. It has been proposed that the small RNAs produced from the micronuclear genome are used to identify eliminated DNA sequences by whole-genome comparison between the parental macronucleus and the micronucleus. In contrast, DNA descrambling in Oxytricha is guided by long non-coding RNAs that are produced from the parental macronuclear genome. These long RNAs are proposed to act as templates for the direct descrambling events that occur in the developing macronucleus. Both cases provide useful examples to study epigenetic chromatin regulation by non-coding RNAs. PMID:21956937

  13. Accumulation of 24 nucleotide transgene-derived siRNAs is associated with crinivirus immunity in transgenic plants.

    PubMed

    Qiao, Wenjie; Zarzyńska-Nowak, Aleksandra; Nerva, Luca; Kuo, Yen-Wen; Falk, Bryce W

    2018-04-28

    RNA silencing is a conserved antiviral defense mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing-mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harboring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RdRp sequence exhibited immunity to systemic LIYV infection. Deep-sequencing analysis was performed to characterize virus-derived siRNAs (vsiRNAs) generated upon systemic LIYV infection in non-transgenic N. benthamiana plants as well as transgene-derived siRNAs (t-siRNAs) derived from the immune transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t-siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants except a significant increase of t-siRNAs of 24 nt in length, which was consistent with small RNA northern blot results that showed the abundance of t-siRNAs of 21-, 22-, and 24- nt in length. The accumulated 24-nt sequences haven't yet been reported in transgenic plants partially resistant to criniviruses, thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24-nt t-siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24 nt t-siRNAs is associated with crinivirus immunity in transgenic plants. This article is protected by copyright. All rights reserved. © 2018 BSPP and John Wiley & Sons Ltd.

  14. MicroRNAs in Palatogenesis and Cleft Palate

    PubMed Central

    Schoen, Christian; Aschrafi, Armaz; Thonissen, Michelle; Poelmans, Geert; Von den Hoff, Johannes W.; Carels, Carine E. L.

    2017-01-01

    Palatogenesis requires a precise spatiotemporal regulation of gene expression, which is controlled by an intricate network of transcription factors and their corresponding DNA motifs. Even minor perturbations of this network may cause cleft palate, the most common congenital craniofacial defect in humans. MicroRNAs (miRNAs), a class of small regulatory non-coding RNAs, have elicited strong interest as key regulators of embryological development, and as etiological factors in disease. MiRNAs function as post-transcriptional repressors of gene expression and are therefore able to fine-tune gene regulatory networks. Several miRNAs are already identified to be involved in congenital diseases. Recent evidence from research in zebrafish and mice indicates that miRNAs are key factors in both normal palatogenesis and cleft palate formation. Here, we provide an overview of recently identified molecular mechanisms underlying palatogenesis involving specific miRNAs, and discuss how dysregulation of these miRNAs may result in cleft palate. PMID:28420997

  15. Isolation and Identification of miRNAs in Jatropha curcas

    PubMed Central

    Wang, Chun Ming; Liu, Peng; Sun, Fei; Li, Lei; Liu, Peng; Ye, Jian; Yue, Gen Hua

    2012-01-01

    MicroRNAs (miRNAs) are small noncoding RNAs that play crucial regulatory roles by targeting mRNAs for silencing. To identify miRNAs in Jatropha curcas L, a bioenergy crop, cDNA clones from two small RNA libraries of leaves and seeds were sequenced and analyzed using bioinformatic tools. Fifty-two putative miRNAs were found from the two libraries, among them six were identical to known miRNAs and 46 were novel. Differential expression patterns of 15 miRNAs in root, stem, leave, fruit and seed were detected using quantitative real-time PCR. Ten miRNAs were highly expressed in fruit or seed, implying that they may be involved in seed development or fatty acids synthesis in seed. Moreover, 28 targets of the isolated miRNAs were predicted from a jatropha cDNA library database. The miRNA target genes were predicted to encode a broad range of proteins. Sixteen targets had clear BLASTX hits to the Uniprot database and were associated with genes belonging to the three major gene ontology categories of biological process, cellular component, and molecular function. Four targets were identified for JcumiR004. By silencing JcumiR004 primary miRNA, expressions of the four target genes were up-regulated and oil composition were modulated significantly, indicating diverse functions of JcumiR004. PMID:22419887

  16. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Identification of MicroRNAs in the Coral Stylophora pistillata

    PubMed Central

    Liew, Yi Jin; Aranda, Manuel; Carr, Adrian; Baumgarten, Sebastian; Zoccola, Didier; Tambutté, Sylvie; Allemand, Denis; Micklem, Gos; Voolstra, Christian R.

    2014-01-01

    Coral reefs are major contributors to marine biodiversity. However, they are in rapid decline due to global environmental changes such as rising sea surface temperatures, ocean acidification, and pollution. Genomic and transcriptomic analyses have broadened our understanding of coral biology, but a study of the microRNA (miRNA) repertoire of corals is missing. miRNAs constitute a class of small non-coding RNAs of ∼22 nt in size that play crucial roles in development, metabolism, and stress response in plants and animals alike. In this study, we examined the coral Stylophora pistillata for the presence of miRNAs and the corresponding core protein machinery required for their processing and function. Based on small RNA sequencing, we present evidence for 31 bona fide microRNAs, 5 of which (miR-100, miR-2022, miR-2023, miR-2030, and miR-2036) are conserved in other metazoans. Homologues of Argonaute, Piwi, Dicer, Drosha, Pasha, and HEN1 were identified in the transcriptome of S. pistillata based on strong sequence conservation with known RNAi proteins, with additional support derived from phylogenetic trees. Examination of putative miRNA gene targets indicates potential roles in development, metabolism, immunity, and biomineralisation for several of the microRNAs. Here, we present first evidence of a functional RNAi machinery and five conserved miRNAs in S. pistillata, implying that miRNAs play a role in organismal biology of scleractinian corals. Analysis of predicted miRNA target genes in S. pistillata suggests potential roles of miRNAs in symbiosis and coral calcification. Given the importance of miRNAs in regulating gene expression in other metazoans, further expression analyses of small non-coding RNAs in transcriptional studies of corals should be informative about miRNA-affected processes and pathways. PMID:24658574

  18. Identification of microRNAs in the Toxigenic Dinoflagellate Alexandrium catenella by High-Throughput Illumina Sequencing and Bioinformatic Analysis

    PubMed Central

    Geng, Huili; Sui, Zhenghong; Zhang, Shu; Du, Qingwei; Ren, Yuanyuan; Liu, Yuan; Kong, Fanna; Zhong, Jie; Ma, Qingxia

    2015-01-01

    Micro-ribonucleic acids (miRNAs) are a large group of endogenous, tiny, non-coding RNAs consisting of 19–25 nucleotides that regulate gene expression at either the transcriptional or post-transcriptional level by mediating gene silencing in eukaryotes. They are considered to be important regulators that affect growth, development, and response to various stresses in plants. Alexandrium catenella is an important marine toxic phytoplankton species that can cause harmful algal blooms (HABs). To date, identification and function analysis of miRNAs in A. catenella remain largely unexamined. In this study, high-throughput sequencing was performed on A. catenella to identify and quantitatively profile the repertoire of small RNAs from two different growth phases. A total of 38,092,056 and 32,969,156 raw reads were obtained from the two small RNA libraries, respectively. In total, 88 mature miRNAs belonging to 32 miRNA families were identified. Significant differences were found in the member number, expression level of various families, and expression abundance of each member within a family. A total of 15 potentially novel miRNAs were identified. Comparative profiling showed that 12 known miRNAs exhibited differential expression between the lag phase and the logarithmic phase. Real-time quantitative RT-PCR (qPCR) was performed to confirm the expression of two differentially expressed miRNAs that were one up-regulated novel miRNA (aca-miR-3p-456915), and one down-regulated conserved miRNA (tae-miR159a). The expression trend of the qPCR assay was generally consistent with the deep sequencing result. Target predictions of the 12 differentially expressed miRNAs resulted in 1813target genes. Gene ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG) annotations revealed that some miRNAs were associated with growth and developmental processes of the alga. These results provide insights into the roles that miRNAs play in the growth of

  19. Simultaneous sequencing of coding and noncoding RNA reveals a human transcriptome dominated by a small number of highly expressed noncoding genes.

    PubMed

    Boivin, Vincent; Deschamps-Francoeur, Gabrielle; Couture, Sonia; Nottingham, Ryan M; Bouchard-Bourelle, Philia; Lambowitz, Alan M; Scott, Michelle S; Abou-Elela, Sherif

    2018-07-01

    Comparing the abundance of one RNA molecule to another is crucial for understanding cellular functions but most sequencing techniques can target only specific subsets of RNA. In this study, we used a new fragmented ribodepleted TGIRT sequencing method that uses a thermostable group II intron reverse transcriptase (TGIRT) to generate a portrait of the human transcriptome depicting the quantitative relationship of all classes of nonribosomal RNA longer than 60 nt. Comparison between different sequencing methods indicated that FRT is more accurate in ranking both mRNA and noncoding RNA than viral reverse transcriptase-based sequencing methods, even those that specifically target these species. Measurements of RNA abundance in different cell lines using this method correlate with biochemical estimates, confirming tRNA as the most abundant nonribosomal RNA biotype. However, the single most abundant transcript is 7SL RNA, a component of the signal recognition particle. S tructured n on c oding RNAs (sncRNAs) associated with the same biological process are expressed at similar levels, with the exception of RNAs with multiple functions like U1 snRNA. In general, sncRNAs forming RNPs are hundreds to thousands of times more abundant than their mRNA counterparts. Surprisingly, only 50 sncRNA genes produce half of the non-rRNA transcripts detected in two different cell lines. Together the results indicate that the human transcriptome is dominated by a small number of highly expressed sncRNAs specializing in functions related to translation and splicing. © 2018 Boivin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  20. A structured viroid RNA serves as a substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RNA-induced silencing complex-mediated degradation.

    PubMed

    Itaya, Asuka; Zhong, Xuehua; Bundschuh, Ralf; Qi, Yijun; Wang, Ying; Takeda, Ryuta; Harris, Ann R; Molina, Carlos; Nelson, Richard S; Ding, Biao

    2007-03-01

    RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.

  1. A Structured Viroid RNA Serves as a Substrate for Dicer-Like Cleavage To Produce Biologically Active Small RNAs but Is Resistant to RNA-Induced Silencing Complex-Mediated Degradation▿

    PubMed Central

    Itaya, Asuka; Zhong, Xuehua; Bundschuh, Ralf; Qi, Yijun; Wang, Ying; Takeda, Ryuta; Harris, Ann R.; Molina, Carlos; Nelson, Richard S.; Ding, Biao

    2007-01-01

    RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures. PMID:17202210

  2. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems

    PubMed Central

    Caplen, Natasha J.; Parrish, Susan; Imani, Farhad; Fire, Andrew; Morgan, Richard A.

    2001-01-01

    Short interfering RNAs (siRNAs) are double-stranded RNAs of ≈21–25 nucleotides that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates. siRNAs have a characteristic structure, with 5′-phosphate/3′-hydroxyl ends and a 2-base 3′ overhang on each strand of the duplex. In this study, we present data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice. In each case, the interference by siRNAs was superior to the inhibition of gene expression mediated by single-stranded antisense oligonucleotides. The siRNAs seem to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells. These observations may open a path toward the use of siRNAs as a reverse genetic and therapeutic tool in mammalian cells. PMID:11481446

  3. MicroRNAs Expression Profiles in Cardiovascular Diseases

    PubMed Central

    Bronze-da-Rocha, Elsa

    2014-01-01

    The current search for new markers of cardiovascular diseases (CVDs) is explained by the high morbidity and mortality still observed in developed and developing countries due to cardiovascular events. Recently, microRNAs (miRNAs or miRs) have emerged as potential new biomarkers and are small sequences of RNAs that regulate gene expression at posttranscriptional level by inhibiting translation or inducing degradation of the target mRNAs. Circulating miRNAs are involved in the regulation of signaling pathways associated to aging and can be used as novel diagnostic markers for acute and chronic diseases such as cardiovascular pathologies. This review summarizes the biogenesis, maturation, and stability of miRNAs and their use as potential biomarkers for coronary artery disease (CAD), myocardial infarction (MI), and heart failure (HF). PMID:25013816

  4. Cerebellar neurodegeneration in the absence of microRNAs

    PubMed Central

    Schaefer, Anne; O'Carroll, Dónal; Tan, Chan Lek; Hillman, Dean; Sugimori, Mutsuyuki; Llinas, Rodolfo; Greengard, Paul

    2007-01-01

    Genome-encoded microRNAs (miRNAs) are potent regulators of gene expression. The significance of miRNAs in various biological processes has been suggested by studies showing an important role of these small RNAs in regulation of cell differentiation. However, the role of miRNAs in regulation of differentiated cell physiology is not well established. Mature neurons express a large number of distinct miRNAs, but the role of miRNAs in postmitotic neurons has not been examined. Here, we provide evidence for an essential role of miRNAs in survival of differentiated neurons. We show that conditional Purkinje cell–specific ablation of the key miRNA-generating enzyme Dicer leads to Purkinje cell death. Deficiency in Dicer is associated with progressive loss of miRNAs, followed by cerebellar degeneration and development of ataxia. The progressive neurodegeneration in the absence of Dicer raises the possibility of an involvement of miRNAs in neurodegenerative disorders. PMID:17606634

  5. In Vitro Assembly of Human H/ACA Small Nucleolar RNPs Reveals Unique Features of U17 and Telomerase RNAs

    PubMed Central

    Dragon, François; Pogačić, Vanda; Filipowicz, Witold

    2000-01-01

    The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. They usually fold into a two-domain hairpin-hinge-hairpin-tail structure, with the conserved motifs H and ACA located in the hinge and tail, respectively. Synthetic RNA transcripts and extracts from HeLa cells were used to reconstitute human U17 and other H/ACA ribonucleoproteins (RNPs) in vitro. Competition and UV cross-linking experiments showed that proteins of about 60, 29, 23, and 14 kDa interact specifically with U17 RNA. Except for U17, RNPs could be reconstituted only with full-length H/ACA snoRNAs. For U17, the 3′-terminal stem-loop followed by box ACA (U17/3′st) was sufficient to form an RNP, and U17/3′st could compete other full-length H/ACA snoRNAs for assembly. The H/ACA-like domain that constitutes the 3′ moiety of human telomerase RNA (hTR), and its 3′-terminal stem-loop (hTR/3′st), also could form an RNP by binding H/ACA proteins. Hence, the 3′-terminal stem-loops of U17 and hTR have some specific features that distinguish them from other H/ACA RNAs. Antibodies that specifically recognize the human GAR1 (hGAR1) protein could immunoprecipitate H/ACA snoRNAs and hTR from HeLa cell extracts, which demonstrates that hGAR1 is a component of H/ACA snoRNPs and telomerase in vivo. Moreover, we show that in vitro-reconstituted RNPs contain hGAR1 and that binding of hGAR1 does not appear to be a prerequisite for the assembly of the other H/ACA proteins. PMID:10757788

  6. 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

  7. Characterization of acid‑tolerance‑associated small RNAs in clinical isolates of Streptococcus mutans: Potential biomarkers for caries prevention.

    PubMed

    Zhu, Wenhui; Liu, Shanshan; Zhuang, Peilin; Liu, Jia; Wang, Yan; Lin, Huancai

    2017-12-01

    Streptococcus mutans is a cariogenic bacterium that contributes to dental caries due to its ability to produce lactic acid, which acidifies the local environment. The potential of S. mutans to respond to environmental stress and tolerate low pH is essential for its survival and predominance in caries lesions. Small noncoding RNAs (sRNAs) have been reported to be involved in bacterial stress and virulence. Few studies have investigated the sRNAs of S. mutans and the function of these sRNAs remains to be elucidated. In the present study, the association between sRNA133474 and acid tolerance, including potential underlying mechanisms, were investigated within clinical strains of S. mutans. From pediatric dental plaques, 20 strains of S. mutans were isolated. An acid killing assay was performed to analyze acid tolerance of S. mutans. Expression patterns of sRNA133474 were investigated during various growth phases under various acidic conditions via reverse transcription‑quantitative polymerase chain reaction. RNA predator and Kyoto Encyclopedia of Genes and Genomes analyses were performed to predict target mRNAs of sRNA133474 and to examine the involvement of putative pathways of target mRNAs, respectively. The results of the present study demonstrated that sRNA133474 activity was growth phase‑dependent, and two distinct expression patterns were identified in 10 clinical strains. At pH 5.5 and 7.5 the expression levels of sRNA133474 were significantly different, and high‑acid tolerant strains exhibited reduced expression levels of sRNA133474 compared with low‑acid tolerant strains. A correlation between sRNA133474 expression levels and acid tolerance was observed in 20 clinical isolates of S. mutans (r=‑0.6298, P<0.01). Finally, five target mRNAs (liaS, liaR, comE, covR and ciaR) involved in the two‑component system (TCS) were selected for further evaluation; the expression levels of three target mRNAs (liaR, ciaR and covR) were negatively

  8. Birth, coming of age and death: The intriguing life of long noncoding RNAs.

    PubMed

    Samudyata; Castelo-Branco, Gonçalo; Bonetti, Alessandro

    2018-07-01

    Mammalian genomes are pervasively transcribed, with long noncoding RNAs being the most abundant fraction. Recent studies have highlighted the central role played by these transcripts in several physiological and pathological processes. Despite several metabolic features shared between coding and noncoding transcripts, these two classes of RNAs exhibit multiple differences regarding their biogenesis and processing. Here we review such distinctions, focusing on the unique features of specific long noncoding RNAs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Circular RNAs: Regulators of Cancer-Related Signaling Pathways and Potential Diagnostic Biomarkers for Human Cancers

    PubMed Central

    Yang, Zuozhang; Xie, Lin; Han, Lei; Qu, Xin; Yang, Yihao; Zhang, Ya; He, Zewei; Wang, Yu; Li, Jing

    2017-01-01

    Circular RNAs (circRNAs) are newly discovered endogenous non-coding RNAs featuring structural stability, high abundance, and tissue-specific expression. CircRNAs are prevalent and conserved in mammalian cells. They are involved in cellular processes and regulate gene expression at the transcriptional or post-transcriptional level by interacting with microRNAs (miRNAs) and other molecules. Recent studies have shown that circRNAs play an important role in the progression of various human diseases including atherosclerosis, nervous system disorders, diabetes, and cancer. In this review, we summarize the advances on endogenous circRNAs in eukaryotic cells and elucidate their diagnostic and prognostic significance in human cancers. Especially, we highlight the involvement of circRNAs in signal transduction pathways as well as their clinical potential to serve as biomarkers. PMID:28839467

  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. A complex dominance hierarchy is controlled by polymorphism of small RNAs and their targets.

    PubMed

    Yasuda, Shinsuke; Wada, Yuko; Kakizaki, Tomohiro; Tarutani, Yoshiaki; Miura-Uno, Eiko; Murase, Kohji; Fujii, Sota; Hioki, Tomoya; Shimoda, Taiki; Takada, Yoshinobu; Shiba, Hiroshi; Takasaki-Yasuda, Takeshi; Suzuki, Go; Watanabe, Masao; Takayama, Seiji

    2016-12-22

    In diploid organisms, phenotypic traits are often biased by effects known as Mendelian dominant-recessive interactions between inherited alleles. Phenotypic expression of SP11 alleles, which encodes the male determinants of self-incompatibility in Brassica rapa, is governed by a complex dominance hierarchy 1-3 . Here, we show that a single polymorphic 24 nucleotide small RNA, named SP11 methylation inducer 2 (Smi2), controls the linear dominance hierarchy of the four SP11 alleles (S 44 > S 60 > S 40 > S 29 ). In all dominant-recessive interactions, small RNA variants derived from the linked region of dominant SP11 alleles exhibited high sequence similarity to the promoter regions of recessive SP11 alleles and acted in trans to epigenetically silence their expression. Together with our previous study 4 , we propose a new model: sequence similarity between polymorphic small RNAs and their target regulates mono-allelic gene expression, which explains the entire five-phased linear dominance hierarchy of the SP11 phenotypic expression in Brassica.

  12. Detailed Abundances of Stars with Small Planets Discovered by Kepler. I. The First Sample

    NASA Astrophysics Data System (ADS)

    Schuler, Simon C.; Vaz, Zachary A.; Katime Santrich, Orlando J.; Cunha, Katia; Smith, Verne V.; King, Jeremy R.; Teske, Johanna K.; Ghezzi, Luan; Howell, Steve B.; Isaacson, Howard

    2015-12-01

    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⊕, 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. Some of the data presented herein were obtained at the W.M. Keck Observatory, which is operated as a scientific partnership among the California

  13. Arabidopsis m6A demethylase activity modulates viral infection of a plant virus and the m6A abundance in its genomic RNAs

    PubMed Central

    Martínez-Pérez, Mireya; Aparicio, Frederic; López-Gresa, Maria Pilar; Bellés, Jose María; Sánchez-Navarro, Jesus A.

    2017-01-01

    N6-methyladenosine (m6A) is an internal, reversible nucleotide modification that constitutes an important regulatory mechanism in RNA biology. Unlike mammals and yeast, no component of the m6A cellular machinery has been described in plants at present. m6A has been identified in the genomic RNAs of diverse mammalian viruses and, additionally, viral infection was found to be modulated by the abundance of m6A in viral RNAs. Here we show that the Arabidopsis thaliana protein atALKBH9B (At2g17970) is a demethylase that removes m6A from single-stranded RNA molecules in vitro. atALKBH9B accumulates in cytoplasmic granules, which colocalize with siRNA bodies and associate with P bodies, suggesting that atALKBH9B m6A demethylase activity could be linked to mRNA silencing and/or mRNA decay processes. Moreover, we identified the presence of m6A in the genomes of two members of the Bromoviridae family, alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV). The demethylation activity of atALKBH9B affected the infectivity of AMV but not of CMV, correlating with the ability of atALKBH9B to interact (or not) with their coat proteins. Suppression of atALKBH9B increased the relative abundance of m6A in the AMV genome, impairing the systemic invasion of the plant, while not having any effect on CMV infection. Our findings suggest that, as recently found in animal viruses, m6A modification may represent a plant regulatory strategy to control cytoplasmic-replicating RNA viruses. PMID:28923956

  14. The evolution of microRNAs in plants

    PubMed Central

    Cui, Jie; You, Chenjiang; Chen, Xuemei

    2016-01-01

    MicroRNAs (miRNAs) are a central player in post-transcriptional regulation of gene expression and are involved in numerous biological processes in eukaryotes. Knowledge of the origins and divergence of miRNAs paves the way for a better understanding of the complexity of the regulatory networks that they participate in. The biogenesis, degradation, and regulatory activities of miRNAs are relatively better understood, but the evolutionary history of miRNAs still needs more exploration. Inverted duplication of target genes, random hairpin sequences and small transposable elements constitute three main models that explain the origination of miRNA genes (MIR). Both inter- and intra-species divergence of miRNAs exhibits functional adaptation and adaptation to changing environments in evolution. Here we summarize recent progress in studies on the evolution of MIR and related genes. PMID:27886593

  15. SiLEncing SLE: the power and promise of small noncoding RNAs.

    PubMed

    Rigby, Robert J; Vinuesa, Carola G

    2008-09-01

    In this study, we outline the evidence suggesting that defects in the RNA silencing machinery can lead to the prototypic systemic autoimmune disease, systemic lupus erythematosus, and describe the potential for RNA interference to provide novel therapeutic agents. Over the last year, a class of small noncoding RNAs--microRNAs--have been shown to play key roles in immune regulation including T-cell selection in the thymus, B cell affinity maturation and selection in germinal centres, and development of regulatory T cells, suggesting that the microRNA machinery may be crucial in the maintenance of immunological tolerance. Two RNA silencing mechanisms have been shown to be involved in lupus pathogenesis: failed Roquin-mediated repression of inducible costimulatory receptors messenger RNA through miR-101 in roquin(san/san) mice and decreased expression of pro-apoptotic molecule and phosphatase and tensin homologue on chromosome 10 in mice transgenic for the miR-17-92 cluster, leading to lymphoproliferation and other lupus manisfestations. MicroRNA array experiments performed on peripheral blood mononuclear cells have revealed different expression profiles in systemic lupus erythematosus patients. RNA interference has also been used ex vivo to silence dysregulated T-cell molecules in cells from systemic lupus erythematosus patients. Dysregulation of the RNA silencing machinery has been implicated in systemic lupus erythematosus pathogenesis. Although microRNA profiling may prove to be a useful diagnostic and prognostic tool for a notoriously heterogeneous disease, manipulation of RNA interference emerges as a powerful and potentially specific means to correct dysregulated gene expression in systemic lupus erythematosus patients.

  16. Identification and characterization of MicroRNAs expressed in chicken skeletal muscle

    USDA-ARS?s Scientific Manuscript database

    MicroRNAs (miRNAs, miRs) encompass a class of small noncoding RNAs that negatively regulate gene expression. MicroRNAs play an essential role in skeletal muscle, determining the proper development and maintenance of this tissue. In comparison to other organs and tissues, the full set of muscle miRNA...

  17. Small RNA analysis in Petunia hybrida identifies unusual tissue-specific expression patterns of conserved miRNAs and of a 24mer RNA

    PubMed Central

    Tedder, Philip; Zubko, Elena; Westhead, David R.; Meyer, Peter

    2009-01-01

    Two pools of small RNAs were cloned from inflorescences of Petunia hybrida using a 5′-ligation dependent and a 5′-ligation independent approach. The two libraries were integrated into a public website that allows the screening of individual sequences against 359,769 unique clones. The library contains 15 clones with 100% identity and 53 clones with one mismatch to miRNAs described for other plant species. For two conserved miRNAs, miR159 and miR390, we find clear differences in tissue-specific distribution, compared with other species. This shows that evolutionary conservation of miRNA sequences does not necessarily include a conservation of the miRNA expression profile. Almost 60% of all clones in the database are 24-nucleotide clones. In accordance with the role of 24mers in marking repetitive regions, we find them distributed across retroviral and transposable element sequences but other 24mers map to promoter regions and to different transcript regions. For one target region we observe tissue-specific variation of matching 24mers, which demonstrates that, as for 21mers, 24mer concentrations are not necessarily identical in different tissues. Asymmetric distribution of a putative novel miRNA in the two libraries suggests that the cloning method can be selective for the representation of certain small RNAs in a collection. PMID:19369427

  18. NGS of Virus-Derived Small RNAs as a Diagnostic Method Used to Determine Viromes of Hungarian Vineyards

    PubMed Central

    Czotter, Nikoletta; Molnar, Janos; Szabó, Emese; Demian, Emese; Kontra, Levente; Baksa, Ivett; Szittya, Gyorgy; Kocsis, Laszlo; Deak, Tamas; Bisztray, Gyorgy; Tusnady, Gabor E.; Burgyan, Jozsef; Varallyay, Eva

    2018-01-01

    As virus diseases cannot be controlled by traditional plant protection methods, the risk of their spread have to be minimized on vegetatively propagated plants, such as grapevine. Metagenomic approaches used for virus diagnostics offer a unique opportunity to reveal the presence of all viral pathogens in the investigated plant, which is why their application can reduce the risk of using infected material for a new plantation. Here we used a special branch, deep sequencing of virus-derived small RNAs, of this high-throughput method for virus diagnostics, and determined viromes of vineyards in Hungary. With NGS of virus-derived small RNAs we could detect not only the viruses tested routinely, but also new ones, which had never been described in Hungary before. Virus presence did not correlate with the age of the plantation, moreover phylogenetic analysis of the identified virus isolates suggests that infections are mostly caused by the use of infected propagating material. Our results, validated by other molecular methods, raised further questions to be answered before this method can be introduced as a routine, reliable test for grapevine virus diagnostics. PMID:25741336

  19. MicroRNAs in CAG trinucleotide repeat expansion disorders: an integrated review of the literature.

    PubMed

    Dumitrescu, Laura; Popescu, Bogdan O

    2015-01-01

    MicroRNAs are small RNAs involved in gene silencing. They play important roles in transcriptional regulation and are selectively and abundantly expressed in the central nervous system. A considerable amount of the human genome is comprised of tandem repeating nucleotide streams. Several diseases are caused by above-threshold expansion of certain trinucleotide repeats occurring in a protein-coding or non-coding region. Though monogenic, CAG trinucleotide repeat expansion disorders have a complex pathogenesis, various combinations of multiple coexisting pathways resulting in one common final consequence: selective neurodegeneration. Mutant protein and mutant transcript gain of toxic function are considered to be the core pathogenic mechanisms. The profile of microRNAs in CAG trinucleotide repeat disorders is scarcely described, however microRNA dysregulation has been identified in these diseases and microRNA-related intereference with gene expression is considered to be involved in their pathogenesis. Better understanding of microRNAs functions and means of manipulation promises to offer further insights into the pathogenic pathways of CAG repeat expansion disorders, to point out new potential targets for drug intervention and to provide some of the much needed etiopathogenic therapeutic agents. A number of disease-modifying microRNA silencing strategies are under development, but several implementation impediments still have to be resolved. CAG targeting seems feasible and efficient in animal models and is an appealing approach for clinical practice. Preliminary human trials are just beginning.

  20. HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages.

    PubMed

    Vongrad, Valentina; Imig, Jochen; Mohammadi, Pejman; Kishore, Shivendra; Jaskiewicz, Lukasz; Hall, Jonathan; Günthard, Huldrych F; Beerenwinkel, Niko; Metzner, Karin J

    2015-01-01

    MiRNAs and other small noncoding RNAs (sncRNAs) are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs) have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi) pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM). The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs. PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

  1. Staphylococcus aureus Regulatory RNAs as Potential Biomarkers for Bloodstream Infections

    PubMed Central

    Bordeau, Valérie; Cady, Anne; Revest, Matthieu; Rostan, Octavie; Sassi, Mohamed; Tattevin, Pierre; Donnio, Pierre-Yves

    2016-01-01

    Staphylococcus aureus is a commensal bacterium and pathogen. Identifying biomarkers for the transition from colonization to disease caused by this organism would be useful. Several S. aureus small RNAs (sRNAs) regulate virulence. We investigated presence and expression of 8 sRNAs in 83 S. aureus strains from 42 patients with sepsis or septic shock and 41 asymptomatic colonized carriers. Small pathogenicity island sRNAs sprB and sprC were clade specific. Six sRNAs had variable expression not correlated with clinical status. Expression of RNAIII was lower in strains from septic shock patients than in strains from colonized patients. When RNAIII was associated with expression of sprD, colonizing strains could be discriminated from strains in patients with bloodstream infections, including patients with sepsis and septic shock. Isolates associated with colonization might have sRNAs with target expression different from those of disease isolates. Monitoring expression of RNAIII and sprD could help determine severity of bloodstream infections. PMID:27224202

  2. Staphylococcus aureus Regulatory RNAs as Potential Biomarkers for Bloodstream Infections.

    PubMed

    Bordeau, Valérie; Cady, Anne; Revest, Matthieu; Rostan, Octavie; Sassi, Mohamed; Tattevin, Pierre; Donnio, Pierre-Yves; Felden, Brice

    2016-09-01

    Staphylococcus aureus is a commensal bacterium and pathogen. Identifying biomarkers for the transition from colonization to disease caused by this organism would be useful. Several S. aureus small RNAs (sRNAs) regulate virulence. We investigated presence and expression of 8 sRNAs in 83 S. aureus strains from 42 patients with sepsis or septic shock and 41 asymptomatic colonized carriers. Small pathogenicity island sRNAs sprB and sprC were clade specific. Six sRNAs had variable expression not correlated with clinical status. Expression of RNAIII was lower in strains from septic shock patients than in strains from colonized patients. When RNAIII was associated with expression of sprD, colonizing strains could be discriminated from strains in patients with bloodstream infections, including patients with sepsis and septic shock. Isolates associated with colonization might have sRNAs with target expression different from those of disease isolates. Monitoring expression of RNAIII and sprD could help determine severity of bloodstream infections.

  3. The emergence of noncoding RNAs as Heracles in autophagy.

    PubMed

    Zhang, Jian; Wang, Peiyuan; Wan, Lin; Xu, Shouping; Pang, Da

    2017-06-03

    Macroautophagy/autophagy is a catabolic process that is widely found in nature. Over the past few decades, mounting evidence has indicated that noncoding RNAs, ranging from small noncoding RNAs to long noncoding RNAs (lncRNAs) and even circular RNAs (circRNAs), mediate the transcriptional and post-transcriptional regulation of autophagy-related genes by participating in autophagy regulatory networks. The differential expression of noncoding RNAs affects autophagy levels at different physiological and pathological stages, including embryonic proliferation and differentiation, cellular senescence, and even diseases such as cancer. We summarize the current knowledge regarding noncoding RNA dysregulation in autophagy and investigate the molecular regulatory mechanisms underlying noncoding RNA involvement in autophagy regulatory networks. Then, we integrate public resources to predict autophagy-related noncoding RNAs across species and discuss strategies for and the challenges of identifying autophagy-related noncoding RNAs. This article will deepen our understanding of the relationship between noncoding RNAs and autophagy, and provide new insights to specifically target noncoding RNAs in autophagy-associated therapeutic strategies.

  4. Characterisation and expression of microRNAs in developing wings of the neotropical butterfly Heliconius melpomene

    PubMed Central

    2011-01-01

    Background Heliconius butterflies are an excellent system for studies of adaptive convergent and divergent phenotypic traits. Wing colour patterns are used as signals to both predators and potential mates and are inherited in a Mendelian manner. The underlying genetic mechanisms of pattern formation have been studied for many years and shed light on broad issues, such as the repeatability of evolution. In Heliconius melpomene, the yellow hindwing bar is controlled by the HmYb locus. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that have key roles in many biological processes, including development. miRNAs could act as regulators of genes involved in wing development, patterning and pigmentation. For this reason we characterised miRNAs in developing butterfly wings and examined differences in their expression between colour pattern races. Results We sequenced small RNA libraries from two colour pattern races and detected 142 Heliconius miRNAs with homology to others found in miRBase. Several highly abundant miRNAs were differentially represented in the libraries between colour pattern races. These candidates were tested further using Northern blots, showing that differences in expression were primarily due to developmental stage rather than colour pattern. Assembly of sequenced reads to the HmYb region identified hme-miR-193 and hme-miR-2788; located 2380 bp apart in an intergenic region. These two miRNAs are expressed in wings and show an upregulation between 24 and 72 hours post-pupation, indicating a potential role in butterfly wing development. A search for miRNAs in all available H. melpomene BAC sequences (~ 2.5 Mb) did not reveal any other miRNAs and no novel miRNAs were predicted. Conclusions Here we describe the first butterfly miRNAs and characterise their expression in developing wings. Some show differences in expression across developing pupal stages and may have important functions in butterfly wing development

  5. Characterisation and expression of microRNAs in developing wings of the neotropical butterfly Heliconius melpomene.

    PubMed

    Surridge, Alison K; Lopez-Gomollon, Sara; Moxon, Simon; Maroja, Luana S; Rathjen, Tina; Nadeau, Nicola J; Dalmay, Tamas; Jiggins, Chris D

    2011-01-26

    Heliconius butterflies are an excellent system for studies of adaptive convergent and divergent phenotypic traits. Wing colour patterns are used as signals to both predators and potential mates and are inherited in a Mendelian manner. The underlying genetic mechanisms of pattern formation have been studied for many years and shed light on broad issues, such as the repeatability of evolution. In Heliconius melpomene, the yellow hindwing bar is controlled by the HmYb locus. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that have key roles in many biological processes, including development. miRNAs could act as regulators of genes involved in wing development, patterning and pigmentation. For this reason we characterised miRNAs in developing butterfly wings and examined differences in their expression between colour pattern races. We sequenced small RNA libraries from two colour pattern races and detected 142 Heliconius miRNAs with homology to others found in miRBase. Several highly abundant miRNAs were differentially represented in the libraries between colour pattern races. These candidates were tested further using Northern blots, showing that differences in expression were primarily due to developmental stage rather than colour pattern. Assembly of sequenced reads to the HmYb region identified hme-miR-193 and hme-miR-2788; located 2380 bp apart in an intergenic region. These two miRNAs are expressed in wings and show an upregulation between 24 and 72 hours post-pupation, indicating a potential role in butterfly wing development. A search for miRNAs in all available H. melpomene BAC sequences (~2.5 Mb) did not reveal any other miRNAs and no novel miRNAs were predicted. Here we describe the first butterfly miRNAs and characterise their expression in developing wings. Some show differences in expression across developing pupal stages and may have important functions in butterfly wing development. Two miRNAs were located in the Hm

  6. Analyzing the interactions of mRNAs, miRNAs, lncRNAs and circRNAs to predict competing endogenous RNA networks in glioblastoma.

    PubMed

    Yuan, Yang; Jiaoming, Li; Xiang, Wang; Yanhui, Liu; Shu, Jiang; Maling, Gou; Qing, Mao

    2018-05-01

    Cross-talk between competitive endogenous RNAs (ceRNAs) may play a critical role in revealing potential mechanisms of tumor development and physiology. Glioblastoma is the most common type of malignant primary brain tumor, and the mechanisms of tumor genesis and development in glioblastoma are unclear. Here, to investigate the role of non-coding RNAs and the ceRNA network in glioblastoma, we performed paired-end RNA sequencing and microarray analyses to obtain the expression profiles of mRNAs, lncRNAs, circRNAs and miRNAs. We identified that the expression of 501 lncRNAs, 1999 mRNAs, 2038 circRNAs and 143 miRNAs were often altered between glioblastoma and matched normal brain tissue. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed on these differentially expressed mRNAs and miRNA-mediated target genes of lncRNAs and circRNAs. Furthermore, we used a multi-step computational framework and several bioinformatics methods to construct a ceRNA network combining mRNAs, miRNAs, lncRNAs and circRNA, based on co-expression analysis between the differentially expressed RNAs. We identified that plenty of lncRNAs, CircRNAs and their downstream target genes in the ceRNA network are related to glutamatergic synapse, suggesting that glutamate metabolism is involved in glioma biological functions. Our results will accelerate the understanding of tumorigenesis, cancer progression and even therapeutic targeting in glioblastoma.

  7. MicroRNAs in neuronal function and dysfunction

    PubMed Central

    Im, Heh-In; Kenny, Paul J.

    2012-01-01

    MicroRNAs (miRNAs) are small noncoding RNA transcripts expressed throughout the brain that can regulate neuronal gene expression at the post-transcriptional level. Here, we provide an overview of the role for miRNAs in brain development and function, and review evidence suggesting that dysfunction in miRNA signaling contributes to neurodevelopment disorders such as Rett and fragile X syndromes, as well as complex behavioral disorders including schizophrenia, depression and drug addiction. A better understanding of how miRNAs influence the development of neuropsychiatric disorders may reveal fundamental insights into the causes of these devastating illnesses and offer novel targets for therapeutic development. PMID:22436491

  8. La protein and its associated small nuclear and nucleolar precursor RNAs.

    PubMed

    Maraia, Richard J; Intine, Robert V

    2002-01-01

    After transcription by RNA polymerase (pol) III, nascent Pol III transcripts pass through RNA processing, modification, and transport machineries as part of their posttranscriptional maturation process. The first factor to interact with Pol III transcripts is La protein, which binds principally via its conserved N-terminal domain (NTD), to the UUU-OH motif that results from transcription termination. This review includes a sequence Logo of the most conserved region of La and its refined modeling as an RNA recognition motif (RRM). La protects RNAs from 3' exonucleolytic digestion and also contributes to their nuclear retention. The variety of modifications found on La-associated RNAs is reviewed in detail and considered in the contexts of how La may bind the termini of structured RNAs without interfering with recognition by modification enzymes, and its ability to chaperone RNAs through multiple parts of their maturation pathways. The CTD of human La recognizes the 5' end region of nascent RNA in a manner that is sensitive to serine 366 phosphorylation. Although the CTD can control pre-tRNA cleavage by RNase P, a rate-limiting step in tRNASerUGA maturation, the extent to which it acts in the maturation pathway(s) of other transcripts is unknown but considered here. Evidence that a fraction of La resides in the nucleolus together with recent findings that several Pol III transcripts pass through the nucleolus is also reviewed. An imminent goal is to understand how the bipartite RNA binding, intracellular trafficking, and signal transduction activities of La are integrated with the maturation pathways of the various RNAs with which it associates.

  9. Metazoan tRNA introns generate stable circular RNAs in vivo.

    PubMed

    Lu, Zhipeng; Filonov, Grigory S; Noto, John J; Schmidt, Casey A; Hatkevich, Talia L; Wen, Ying; Jaffrey, Samie R; Matera, A Gregory

    2015-09-01

    We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating "designer" circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science. © 2015 Lu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  10. Global Analysis of Small RNA Dynamics during Seed Development of Picea glauca and Arabidopsis thaliana Populations Reveals Insights on their Evolutionary Trajectories

    PubMed Central

    Liu, Yang; El-Kassaby, Yousry A.

    2017-01-01

    While DNA methylation carries genetic signals and is instrumental in the evolution of organismal complexity, small RNAs (sRNAs), ~18–24 ribonucleotide (nt) sequences, are crucial mediators of methylation as well as gene silencing. However, scant study deals with sRNA evolution via featuring their expression dynamics coupled with species of different evolutionary time. Here we report an atlas of sRNAs and microRNAs (miRNAs, single-stranded sRNAs) produced over time at seed-set of two major spermatophytes represented by populations of Picea glauca and Arabidopsis thaliana with different seed-set duration. We applied diverse profiling methods to examine sRNA and miRNA features, including size distribution, sequence conservation and reproduction-specific regulation, as well as to predict their putative targets. The top 27 most abundant miRNAs were highly overlapped between the two species (e.g., miR166,−319 and−396), but in P. glauca, they were less abundant and significantly less correlated with seed-set phases. The most abundant sRNAs in libraries were deeply conserved miRNAs in the plant kingdom for Arabidopsis but long sRNAs (24-nt) for P. glauca. We also found significant difference in normalized expression between populations for population-specific sRNAs but not for lineage-specific ones. Moreover, lineage-specific sRNAs were enriched in the 21-nt size class. This pattern is consistent in both species and alludes to a specific type of sRNAs (e.g., miRNA, tasiRNA) being selected for. In addition, we deemed 24 and 9 sRNAs in P. glauca and Arabidopsis, respectively, as sRNA candidates targeting known adaptive genes. Temperature had significant influence on selected gene and miRNA expression at seed development in both species. This study increases our integrated understanding of sRNA evolution and its potential link to genomic architecture (e.g., sRNA derivation from genome and sRNA-mediated genomic events) and organismal complexity (e.g., association between

  11. A MicroRNA Superfamily Regulates Nucleotide Binding Site–Leucine-Rich Repeats and Other mRNAs[W][OA

    PubMed Central

    Shivaprasad, Padubidri V.; Chen, Ho-Ming; Patel, Kanu; Bond, Donna M.; Santos, Bruno A.C.M.; Baulcombe, David C.

    2012-01-01

    Analysis of tomato (Solanum lycopersicum) small RNA data sets revealed the presence of a regulatory cascade affecting disease resistance. The initiators of the cascade are microRNA members of an unusually diverse superfamily in which miR482 and miR2118 are prominent members. Members of this superfamily are variable in sequence and abundance in different species, but all variants target the coding sequence for the P-loop motif in the mRNA sequences for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. We confirm, using transient expression in Nicotiana benthamiana, that miR482 targets mRNAs for NBS-LRR disease resistance proteins with coiled-coil domains at their N terminus. The targeting causes mRNA decay and production of secondary siRNAs in a manner that depends on RNA-dependent RNA polymerase 6. At least one of these secondary siRNAs targets other mRNAs of a defense-related protein. The miR482-mediated silencing cascade is suppressed in plants infected with viruses or bacteria so that expression of mRNAs with miR482 or secondary siRNA target sequences is increased. We propose that this process allows pathogen-inducible expression of NBS-LRR proteins and that it contributes to a novel layer of defense against pathogen attack. PMID:22408077

  12. High-resolution analysis of the human retina miRNome reveals isomiR variations and novel microRNAs

    PubMed Central

    Karali, Marianthi; Persico, Maria; Mutarelli, Margherita; Carissimo, Annamaria; Pizzo, Mariateresa; Singh Marwah, Veer; Ambrosio, Concetta; Pinelli, Michele; Carrella, Diego; Ferrari, Stefano; Ponzin, Diego; Nigro, Vincenzo; di Bernardo, Diego; Banfi, Sandro

    2016-01-01

    MicroRNAs play a fundamental role in retinal development and function. To characterise the miRNome of the human retina, we carried out deep sequencing analysis on sixteen individuals. We established the catalogue of retina-expressed miRNAs, determined their relative abundance and found that a small number of miRNAs accounts for almost 90% of the retina miRNome. We discovered more than 3000 miRNA variants (isomiRs), encompassing a wide range of sequence variations, which include seed modifications that are predicted to have an impact on miRNA action. We demonstrated that a seed-modifying isomiR of the retina-enriched miR-124-3p was endowed with different targeting properties with respect to the corresponding canonical form. Moreover, we identified 51 putative novel, retina-specific miRNAs and experimentally validated the expression for nine of them. Finally, a parallel analysis of the human Retinal Pigment Epithelium (RPE)/choroid, two tissues that are known to be crucial for retina homeostasis, yielded notably distinct miRNA enrichment patterns compared to the retina. The generated data are accessible through an ad hoc database. This study is the first to reveal the complexity of the human retina miRNome at nucleotide resolution and constitutes a unique resource to assess the contribution of miRNAs to the pathophysiology of the human retina. PMID:26819412

  13. Differences in Relative Levels of 88 microRNAs in Various Regions of the Normal Adult Human Brain.

    PubMed

    Filatova, Elena V; Alieva, Anelya; Shadrina, Maria I; Slominsky, Petr A

    2017-08-16

    Since the discovery of microRNAs (miRNAs) in the 1990s, our knowledge about their biology has grown considerably. The increasing number of studies addressing the role of miRNAs in development and in various diseases emphasizes the need for a comprehensive catalogue of accurate sequence, expression and conservation information regarding the large number of miRNAs proposed recently in all organs and tissues. The objective of this study was to provide data on the levels of miRNA expression in 15 tissues of the normal human brain. We conducted an analysis of the relative levels of 88 of the most abundantly expressed and best characterized miRNA derived postmortem from well-characterized samples of various regions of the brains from five normal individuals. The cluster analysis revealed some differences in the relative levels of these miRNAs among the brain regions studied. Such diversity can be explained by different functioning of these brain regions. We hope that the data from the current study are a resource that will be useful to our colleagues in this exciting field, as more hypotheses will be generated and tested with regard to small noncoding RNA in the human brain in healthy and disease states. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. Evolutionary conserved microRNAs are ubiquitously expressed compared to tick-specific miRNAs in the cattle tick Rhipicephalus (Boophilus) microplus

    PubMed Central

    2011-01-01

    Background MicroRNAs (miRNAs) are small non-coding RNAs that act as regulators of gene expression in eukaryotes modulating a large diversity of biological processes. The discovery of miRNAs has provided new opportunities to understand the biology of a number of species. The cattle tick, Rhipicephalus (Boophilus) microplus, causes significant economic losses in cattle production worldwide and this drives us to further understand their biology so that effective control measures can be developed. To be able to provide new insights into the biology of cattle ticks and to expand the repertoire of tick miRNAs we utilized Illumina technology to sequence the small RNA transcriptomes derived from various life stages and selected organs of R. microplus. Results To discover and profile cattle tick miRNAs we employed two complementary approaches, one aiming to find evolutionary conserved miRNAs and another focused on the discovery of novel cattle-tick specific miRNAs. We found 51 evolutionary conserved R. microplus miRNA loci, with 36 of these previously found in the tick Ixodes scapularis. The majority of the R. microplus miRNAs are perfectly conserved throughout evolution with 11, 5 and 15 of these conserved since the Nephrozoan (640 MYA), Protostomian (620MYA) and Arthropoda (540 MYA) ancestor, respectively. We then employed a de novo computational screening for novel tick miRNAs using the draft genome of I. scapularis and genomic contigs of R. microplus as templates. This identified 36 novel R. microplus miRNA loci of which 12 were conserved in I. scapularis. Overall we found 87 R. microplus miRNA loci, of these 15 showed the expression of both miRNA and miRNA* sequences. R. microplus miRNAs showed a variety of expression profiles, with the evolutionary-conserved miRNAs mainly expressed in all life stages at various levels, while the expression of novel tick-specific miRNAs was mostly limited to particular life stages and/or tick organs. Conclusions Anciently acquired miRNAs

  15. Identification and verification of potential piRNAs from domesticated yak testis.

    PubMed

    Gong, Jishang; Zhang, Quanwei; Wang, Qi; Ma, Youji; Du, Jiaxiang; Zhang, Yong; Zhao, Xingxu

    2018-02-01

    PIWI-interacting RNAs (piRNA) are small non-coding RNA molecules expressed in animal germ cells that interact with PIWI family proteins to form RNA-protein complexes involved in epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, including reproductive stem cell self-sustainment, differentiation, meiosis and spermatogenesis. In the present study, we performed high-throughput sequencing of piRNAs in testis samples from yaks in different stages of sexual maturity. Deep sequencing of the small RNAs (18-40 nt in length) yielded 4,900,538 unique reads from a total of 53,035,635 reads. We identified yak small RNAs (18-30 nt) and performed functional characterization. Yak small RNAs showed a bimodal length distribution, with two peaks at 22 nt and >28 nt. More than 80% of the 3,106,033 putative piRNAs were mapped to 4637 piRNA-producing genomic clusters using RPKM. 6388 candidate piRNAs were identified from clean reads and the annotations were compared with the yak reference genome repeat region. Integrated network analysis suggested that some differentially expressed genes were involved in spermatogenesis through ECM-receptor interaction and PI3K-Akt signaling pathways. Our data provide novel insights into the molecular expression and regulation similarities and diversities in spermatogenesis and testicular development in yaks at different stages of sexual maturity. © 2018 The authors.

  16. Identification and verification of potential piRNAs from domesticated yak testis

    PubMed Central

    Gong, Jishang; Zhang, Quanwei; Wang, Qi; Ma, Youji; Du, Jiaxiang; Zhang, Yong

    2018-01-01

    PIWI-interacting RNAs (piRNA) are small non-coding RNA molecules expressed in animal germ cells that interact with PIWI family proteins to form RNA–protein complexes involved in epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, including reproductive stem cell self-sustainment, differentiation, meiosis and spermatogenesis. In the present study, we performed high-throughput sequencing of piRNAs in testis samples from yaks in different stages of sexual maturity. Deep sequencing of the small RNAs (18–40 nt in length) yielded 4,900,538 unique reads from a total of 53,035,635 reads. We identified yak small RNAs (18–30 nt) and performed functional characterization. Yak small RNAs showed a bimodal length distribution, with two peaks at 22 nt and >28 nt. More than 80% of the 3,106,033 putative piRNAs were mapped to 4637 piRNA-producing genomic clusters using RPKM. 6388 candidate piRNAs were identified from clean reads and the annotations were compared with the yak reference genome repeat region. Integrated network analysis suggested that some differentially expressed genes were involved in spermatogenesis through ECM–receptor interaction and PI3K-Akt signaling pathways. Our data provide novel insights into the molecular expression and regulation similarities and diversities in spermatogenesis and testicular development in yaks at different stages of sexual maturity. PMID:29101267

  17. 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. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  18. The expanding universe of noncoding RNAs.

    PubMed

    Hannon, G J; Rivas, F V; Murchison, E P; Steitz, J A

    2006-01-01

    The 71st Cold Spring Harbor Symposium on Quantitative Biology celebrated the numerous and expanding roles of regulatory RNAs in systems ranging from bacteria to mammals. It was clearly evident that noncoding RNAs are undergoing a renaissance, with reports of their involvement in nearly every cellular process. Previously known classes of longer noncoding RNAs were shown to function by every possible means-acting catalytically, sensing physiological states through adoption of complex secondary and tertiary structures, or using their primary sequences for recognition of target sites. The many recently discovered classes of small noncoding RNAs, generally less than 35 nucleotides in length, most often exert their effects by guiding regulatory complexes to targets via base-pairing. With the ability to analyze the RNA products of the genome in ever greater depth, it has become clear that the universe of noncoding RNAs may extend far beyond the boundaries we had previously imagined. Thus, as much as the Symposium highlighted exciting progress in the field, it also revealed how much farther we must go to understand fully the biological impact of noncoding RNAs.

  19. Identifying Disease Associated miRNAs Based on Protein Domains.

    PubMed

    Qin, Gui-Min; Li, Rui-Yi; Zhao, Xing-Ming

    2016-01-01

    MicroRNAs (miRNAs) are a class of small endogenous non-coding genes, acting as regulators in the post-transcriptional processes. Recently, the miRNAs are found to be widely involved in different types of diseases. Therefore, the identification of disease associated miRNAs can help understand the mechanisms that underlie the disease and identify new biomarkers. However, it is not easy to identify the miRNAs related to diseases due to its extensive involvements in various biological processes. In this work, we present a new approach to identify disease associated miRNAs based on domains, the functional and structural blocks of proteins. The results on real datasets demonstrate that our method can effectively identify disease related miRNAs with high precision.

  20. Identification of differentially expressed small RNAs and prediction of target genes in Italian Large White pigs with divergent backfat deposition.

    PubMed

    Davoli, R; Gaffo, E; Zappaterra, M; Bortoluzzi, S; Zambonelli, P

    2018-06-01

    The identification of the molecular mechanisms regulating pathways associated with the potential for fat deposition in pigs can lead to the detection of key genes and markers for the genetic improvement of fat traits. Interactions of microRNAs (miRNAs) with target RNAs regulate gene expression and modulate pathway activation in cells and tissues. In pigs, miRNA discovery is far from saturation, and the knowledge of miRNA expression in backfat tissue and particularly of the impact of miRNA variations is still fragmentary. Using RNA-seq, we characterized the small RNA (sRNA) expression profiles in Italian Large White pig backfat tissue. Comparing two groups of pigs divergent for backfat deposition, we detected 31 significant differentially expressed (DE) sRNAs: 14 up-regulated (including ssc-miR-132, ssc-miR-146b, ssc-miR-221-5p, ssc-miR-365-5p and the moRNA ssc-moR-21-5p) and 17 down-regulated (including ssc-miR-136, ssc-miR-195, ssc-miR-199a-5p and ssc-miR-335). To understand the biological impact of the observed miRNA expression variations, we used the expression correlation of DE miRNA target transcripts expressed in the same samples to define a regulatory network of 193 interactions between DE miRNAs and 40 DE target transcripts showing opposite expression profiles and being involved in specific pathways. Several miRNAs and mRNAs in the network were found to be expressed from backfat-related pig QTL. These results are informative for the complex mechanisms influencing fat traits, shed light on a new aspect of the genetic regulation of fat deposition in pigs and facilitate the prospective implementation of innovative strategies of pig genetic improvement based on genomic markers. © 2018 Stichting International Foundation for Animal Genetics.

  1. C. elegans ADARs antagonize silencing of cellular dsRNAs by the antiviral RNAi pathway.

    PubMed

    Reich, Daniel P; Tyc, Katarzyna M; Bass, Brenda L

    2018-02-01

    Cellular dsRNAs are edited by adenosine deaminases that act on RNA (ADARs). While editing can alter mRNA-coding potential, most editing occurs in noncoding sequences, the function of which is poorly understood. Using dsRNA immunoprecipitation (dsRIP) and RNA sequencing (RNA-seq), we identified 1523 regions of clustered A-to-I editing, termed editing-enriched regions (EERs), in four stages of Caenorhabditis elegans development, often with highest expression in embryos. Analyses of small RNA-seq data revealed 22- to 23-nucleotide (nt) siRNAs, reminiscent of viral siRNAs, that mapped to EERs and were abundant in adr-1;adr-2 mutant animals. Consistent with roles for these siRNAs in silencing, EER-associated genes (EAGs) were down-regulated in adr-1;adr-2 embryos, and this was dependent on associated EERs and the RNAi factor RDE-4. We observed that ADARs genetically interact with the 26G endogenous siRNA (endo-siRNA) pathway, which likely competes for RNAi components; deletion of factors required for this pathway ( rrf-3 or ergo-1 ) in adr-1;adr-2 mutant strains caused a synthetic phenotype that was rescued by deleting antiviral RNAi factors. Poly(A) + RNA-seq revealed EAG down-regulation and antiviral gene induction in adr-1;adr-2;rrf-3 embryos, and these expression changes were dependent on rde-1 and rde-4 Our data suggest that ADARs restrict antiviral silencing of cellular dsRNAs. © 2018 Reich et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Small-mammal seed predation limits the recruitment and abundance of two perennial grassland forbs.

    PubMed

    Bricker, Mary; Pearson, Dean; Maron, John

    2010-01-01

    Although post-dispersal seed predators are common and often reduce seed density, their influence on plant population abundance remains unclear. On the one hand, increasing evidence suggests that many plant populations are seed limited, implying that seed predators could reduce plant abundance. On the other hand,.it is generally uncertain whether the magnitude of seed limitation imposed by granivores is strong enough to overcome density-dependent processes that could compensate for seed loss at later stages. We examined the impact of seed predation by small mammals, primarily deer mice (Peromyscus maniculatus), on seedling recruitment and subsequent plant establishment of two perennial grassland forbs in western Montana, USA: Lupinus sericeus (Fabaceae) and Lithospermum ruderale (Boraginaceae). The experiment combined graded densities of seed addition for each species with a small-mammal exclusion treatment. Seedling recruitment and plant establishment were monitored in the experimental plots for up to three years. For both species, small-mammal exclusion increased the total number of seedlings that emerged, and these effects were still significant three years after seed addition, resulting in greater numbers of established plants inside exclosures than in control plots. We also found evidence of seed limitation, with increasing density of seeds added leading to increased numbers of seedlings. Results from seed addition and small-mammal exclusion experiments in later years also revealed significant impacts of small mammals on seedling emergence. These results suggest that granivores can have potentially important impacts in limiting forb abundance in grasslands communities.

  3. Small RNA-mediated repair of UV-induced DNA lesions by the DNA DAMAGE-BINDING PROTEIN 2 and ARGONAUTE 1

    PubMed Central

    Schalk, Catherine; Cognat, Valérie; Graindorge, Stéfanie; Vincent, Timothée; Voinnet, Olivier; Molinier, Jean

    2017-01-01

    As photosynthetic organisms, plants need to prevent irreversible UV-induced DNA lesions. Through an unbiased, genome-wide approach, we have uncovered a previously unrecognized interplay between Global Genome Repair and small interfering RNAs (siRNAs) in the recognition of DNA photoproducts, prevalently in intergenic regions. Genetic and biochemical approaches indicate that, upon UV irradiation, the DNA DAMAGE-BINDING PROTEIN 2 (DDB2) and ARGONAUTE 1 (AGO1) of Arabidopsis thaliana form a chromatin-bound complex together with 21-nt siRNAs, which likely facilitates recognition of DNA damages in an RNA/DNA complementary strand-specific manner. The biogenesis of photoproduct-associated siRNAs involves the noncanonical, concerted action of RNA POLYMERASE IV, RNA-DEPENDENT RNA POLYMERASE-2, and DICER-LIKE-4. Furthermore, the chromatin association/dissociation of the DDB2-AGO1 complex is under the control of siRNA abundance and DNA damage signaling. These findings reveal unexpected nuclear functions for DCL4 and AGO1, and shed light on the interplay between small RNAs and DNA repair recognition factors at damaged sites. PMID:28325872

  4. Zygotic amplification of secondary piRNAs during silkworm embryogenesis

    PubMed Central

    Kawaoka, Shinpei; Arai, Yuji; Kadota, Koji; Suzuki, Yutaka; Hara, Kahori; Sugano, Sumio; Shimizu, Kentaro; Tomari, Yukihide; Shimada, Toru; Katsuma, Susumu

    2011-01-01

    PIWI-interacting RNAs (piRNAs) are 23–30-nucleotide-long small RNAs that act as sequence-specific silencers of transposable elements in animal gonads. In flies, genetics and deep sequencing data have led to a hypothesis for piRNA biogenesis called the ping-pong cycle, where antisense primary piRNAs initiate an amplification loop to generate sense secondary piRNAs. However, to date, the process of the ping-pong cycle has never been monitored at work. Here, by large-scale profiling of piRNAs from silkworm ovary and embryos of different developmental stages, we demonstrate that maternally inherited antisense-biased piRNAs trigger acute amplification of secondary sense piRNA production in zygotes, at a time coinciding with zygotic transcription of sense transposon mRNAs. These results provide on-site evidence for the ping-pong cycle. PMID:21628432

  5. Identification of microRNAs and long non-coding RNAs involved in fatty acid biosynthesis in tree peony seeds.

    PubMed

    Yin, Dan-Dan; Li, Shan-Shan; Shu, Qing-Yan; Gu, Zhao-Yu; Wu, Qian; Feng, Cheng-Yong; Xu, Wen-Zhong; Wang, Liang-Sheng

    2018-08-05

    MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) act as important molecular regulators in a wide range of biological processes during plant development and seed formation, including oil production. Tree peony seeds contain >90% unsaturated fatty acids (UFAs) and high proportions of α-linolenic acid (ALA, > 40%). To dissect the non-coding RNAs (ncRNAs) pathway involved in fatty acids synthesis in tree peony seeds, we construct six small RNA libraries and six transcriptome libraries from developing seeds of two cultivars (J and S) containing different content of fatty acid compositions. After deep sequencing the RNA libraries, the ncRNA expression profiles of tree peony seeds in two cultivars were systematically and comparatively analyzed. A total of 318 known and 153 new miRNAs and 22,430 lncRNAs were identified, among which 106 conserved and 9 novel miRNAs and 2785 lncRNAs were differentially expressed between the two cultivars. In addition, potential target genes of the microRNA and lncRNAs were also predicted and annotated. Among them, 9 miRNAs and 39 lncRNAs were predicted to target lipid related genes. Results showed that all of miR414, miR156b, miR2673b, miR7826, novel-m0027-5p, TR24651|c0_g1, TR24544|c0_g15, and TR27305|c0_g1 were up-regulated and expressed at a higher level in high-ALA cultivar J when compared to low-ALA cultivar S, suggesting that these ncRNAs and target genes are possibly involved in different fatty acid synthesis and lipid metabolism through post-transcriptional regulation. These results provide a better understanding of the roles of ncRNAs during fatty acid biosynthesis and metabolism in tree peony seeds. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. De novo transcriptome sequencing reveals a considerable bias in the incidence of simple sequence repeats towards the downstream of 'Pre-miRNAs' of black pepper.

    PubMed

    Joy, Nisha; Asha, Srinivasan; Mallika, Vijayan; Soniya, Eppurathu Vasudevan

    2013-01-01

    Next generation sequencing has an advantageon transformational development of species with limited available sequence data as it helps to decode the genome and transcriptome. We carried out the de novo sequencing using illuminaHiSeq™ 2000 to generate the first leaf transcriptome of black pepper (Piper nigrum L.), an important spice variety native to South India and also grown in other tropical regions. Despite the economic and biochemical importance of pepper, a scientifically rigorous study at the molecular level is far from complete due to lack of sufficient sequence information and cytological complexity of its genome. The 55 million raw reads obtained, when assembled using Trinity program generated 2,23,386 contigs and 1,28,157 unigenes. Reports suggest that the repeat-rich genomic regions give rise to small non-coding functional RNAs. MicroRNAs (miRNAs) are the most abundant type of non-coding regulatory RNAs. In spite of the widespread research on miRNAs, little is known about the hair-pin precursors of miRNAs bearing Simple Sequence Repeats (SSRs). We used the array of transcripts generated, for the in silico prediction and detection of '43 pre-miRNA candidates bearing different types of SSR motifs'. The analysis identified 3913 different types of SSR motifs with an average of one SSR per 3.04 MB of thetranscriptome. About 0.033% of the transcriptome constituted 'pre-miRNA candidates bearing SSRs'. The abundance, type and distribution of SSR motifs studied across the hair-pin miRNA precursors, showed a significant bias in the position of SSRs towards the downstream of predicted 'pre-miRNA candidates'. The catalogue of transcripts identified, together with the demonstration of reliable existence of SSRs in the miRNA precursors, permits future opportunities for understanding the genetic mechanism of black pepper and likely functions of 'tandem repeats' in miRNAs.

  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

    Liu, Yang; El-Kassaby, Yousry A

    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. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  8. The Role of microRNAs in Bovine Infection and Immunity

    PubMed Central

    Lawless, Nathan; Vegh, Peter; O’Farrelly, Cliona; Lynn, David J.

    2014-01-01

    MicroRNAs (miRNAs) are a class of small, non-coding RNAs that are recognized as critical regulators of immune gene expression during infection. Many immunologically significant human miRNAs have been found to be conserved in agriculturally important species, including cattle. Discovering how bovine miRNAs mediate the immune defense during infection is critical to understanding the etiology of the most prevalent bovine diseases. Here, we review current knowledge of miRNAs in the bovine genome, and discuss the advances in understanding of miRNAs as regulators of immune cell function, and bovine immune response activation, regulation, and resolution. Finally, we consider the future perspectives on miRNAs in bovine viral disease, their role as potential biomarkers and in therapy. PMID:25505900

  9. Exploiting tRNAs to Boost Virulence

    PubMed Central

    Albers, Suki; Czech, Andreas

    2016-01-01

    Transfer RNAs (tRNAs) are powerful small RNA entities that are used to translate nucleotide language of genes into the amino acid language of proteins. Their near-uniform length and tertiary structure as well as their high nucleotide similarity and post-transcriptional modifications have made it difficult to characterize individual species quantitatively. However, due to the central role of the tRNA pool in protein biosynthesis as well as newly emerging roles played by tRNAs, their quantitative assessment yields important information, particularly relevant for virus research. Viruses which depend on the host protein expression machinery have evolved various strategies to optimize tRNA usage—either by adapting to the host codon usage or encoding their own tRNAs. Additionally, several viruses bear tRNA-like elements (TLE) in the 5′- and 3′-UTR of their mRNAs. There are different hypotheses concerning the manner in which such structures boost viral protein expression. Furthermore, retroviruses use special tRNAs for packaging and initiating reverse transcription of their genetic material. Since there is a strong specificity of different viruses towards certain tRNAs, different strategies for recruitment are employed. Interestingly, modifications on tRNAs strongly impact their functionality in viruses. Here, we review those intersection points between virus and tRNA research and describe methods for assessing the tRNA pool in terms of concentration, aminoacylation and modification. PMID:26797637

  10. Epigenetic Therapy in Lung Cancer - Role of microRNAs.

    PubMed

    Rothschild, Sacha I

    2013-01-01

    Lung cancer is the leading cause of cancer deaths worldwide. microRNAs (miRNAs) are a class of small non-coding RNA species that have been implicated in the control of many fundamental cellular and physiological processes such as cellular differentiation, proliferation, apoptosis, and stem cell maintenance. Some miRNAs have been categorized as "oncomiRs" as opposed to "tumor suppressor miRs." This review focuses on the role of miRNAs in the lung cancer carcinogenesis and their potential as diagnostic, prognostic, or predictive markers.

  11. MicroRNAs: A Puzzling Tool in Cancer Diagnostics and Therapy.

    PubMed

    D'Angelo, Barbara; Benedetti, Elisabetta; Cimini, Annamaria; Giordano, Antonio

    2016-11-01

    MicroRNAs (miRNAs) constitute a dominating class of small RNAs that regulate diverse cellular functions. Due the pivotal role of miRNAs in biological processes, a deregulated miRNA expression is likely involved in human cancers. MicroRNAs possess tumor suppressor capability, as well as display oncogenic characteristics. Interestingly, miRNAs exist in various biological fluids as circulating entities. Changes in the profile of circulating miRNAs are indicative of pathophysiological conditions in human cancer. This concept has led to consider circulating miRNAs valid biomarkers in cancer diagnostics. Furthermore, current research promotes the use of miRNAs as a target in cancer therapy. However, miRNAs are an evolving research field. Although miRNAs have been demonstrated to be potentially valuable tools both in cancer diagnosis and treatment, a greater effort should be made to improve our understanding of miRNAs biology. This review describes the biology of microRNAs, emphasizing on the use of miRNAs in cancer diagnostics and therapy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  12. Identification of microRNAs in Caragana intermedia by high-throughput sequencing and expression analysis of 12 microRNAs and their targets under salt stress.

    PubMed

    Zhu, Jianfeng; Li, Wanfeng; Yang, Wenhua; Qi, Liwang; Han, Suying

    2013-09-01

    142 miRNAs were identified and 38 miRNA targets were predicted, 4 of which were validated, in C. intermedia . The expression of 12 miRNAs in salt-stressed leaves was assessed by qRT-PCR. MicroRNAs (miRNAs) are endogenous small RNAs that play important roles in various biological and metabolic processes in plants. Caragana intermedia is an important ecological and economic tree species prominent in the desert environment of west and northwest China. To date, no investigation into C. intermedia miRNAs has been reported. In this study, high-throughput sequencing of small RNAs and analysis of transcriptome data were performed to identify both conserved and novel miRNAs, and also their target mRNA genes in C. intermedia. Based on sequence similarity and hairpin structure prediction, 132 putative conserved miRNAs (12 of which were confirmed to form hairpin precursors) belonging to 31 known miRNA families were identified. Ten novel miRNAs (including the miRNA* sequences of three novel miRNAs) were also discovered. Furthermore, 36 potential target genes of 17 known miRNA families and 2 potential target genes of 1 novel miRNA were predicted; 4 of these were validated by 5' RACE. The expression of 12 miRNAs was validated in different tissues, and these and five target mRNAs were assessed by qRT-PCR after salt treatment. The expression levels of seven miRNAs (cin-miR157a, cin-miR159a, cin-miR165a, cin-miR167b, cin-miR172b, cin-miR390a and cin-miR396a) were upregulated, while cin-miR398a expression was downregulated after salt treatment. The targets of cin-miR157a, cin-miR165a, cin-miR172b and cin-miR396a were downregulated and showed an approximately negative correlation with their corresponding miRNAs under salt treatment. These results would help further understanding of miRNA regulation in response to abiotic stress in C. intermedia.

  13. Argonaute identity defines the length of mature mammalian microRNAs.

    PubMed

    Juvvuna, Prasanna Kumar; Khandelia, Piyush; Lee, Li Ming; Makeyev, Eugene V

    2012-08-01

    MicroRNAs (miRNAs) are 19- to 25-nt-long non-coding RNAs that regulate gene expression by base-pairing with target mRNAs and reducing their stability or translational efficiency. Mammalian miRNAs function in association with four closely related Argonaute proteins, AGO1-4. All four proteins contain the PAZ and the MID domains interacting with the miRNA 3' and 5' termini, respectively, as well as the PIWI domain comprising an mRNA 'slicing' activity in the case of AGO2 but not AGO1, AGO3 and AGO4. However, the slicing mode of the miRNA-programmed AGO2 is rarely realized in vivo and the four Argonautes are thought to play largely overlapping roles in the mammalian miRNA pathway. Here, we show that the average length of many miRNAs is diminished during nervous system development as a result of progressive shortening of the miRNA 3' ends. We link this modification with an increase in the fractional abundance of Ago2 in the adult brain and identify a specific structural motif within the PAZ domain that enables efficient trimming of miRNAs associated with this but not the other three Argonautes. Taken together, our data suggest that mammalian Argonautes may define the length and possibly biological activity of mature mammalian miRNAs in a developmentally controlled manner.

  14. High-Throughput Sequencing Reveals Circulating miRNAs as Potential Biomarkers of Kidney Damage in Patients with Systemic Lupus Erythematosus.

    PubMed

    Navarro-Quiroz, Elkin; Pacheco-Lugo, Lisandro; Lorenzi, Hernan; Díaz-Olmos, Yirys; Almendrales, Lisneth; Rico, Edwin; Navarro, Roberto; España-Puccini, Pierine; Iglesias, Antonio; Egea, Eduardo; Aroca, Gustavo

    2016-01-01

    Renal involvement is one of the most severe manifestations of systemic lupus erythematosus (SLE). Renal biopsy is the gold standard when it comes to knowing whether a patient has lupus nephritis, and the degree of renal disease present. However, the biopsy has various complications, bleeding being the most common. Therefore, the development of alternative, non-invasive diagnostic tests for kidney disease in patients with SLE is a priority. Micro RNAs (miRNAs) are differentially expressed in various tissues, and changes in their expression have been associated with several pathological processes. The aim of this study was to identify changes in the abundance of miRNAs in plasma samples from patients with lupus nephritis that could potentially allow the diagnosis of renal damage in SLE patients. This is an observational case-control cross-sectional study, in which we characterized the differential abundance profiles of miRNAs among patients with different degrees of lupus compared with SLE patients without renal involvement and healthy control individuals. We found 89 miRNAs with changes in their abundance between lupus nephritis patients and healthy controls, and 17 miRNAs that showed significant variations between SLE patients with or without renal involvement. Validation for qPCR of a group of miRNAs on additional samples from lupus patients with or without nephritis, and from healthy individuals, showed that five miRNAs presented an average detection sensitivity of 97%, a specificity of 70.3%, a positive predictive value of 82.5%, a negative predictive value of 96% and a diagnosis efficiency of 87.9%. These results strongly suggest that miR-221-5p, miR-380-3p, miR-556-5p, miR-758-3p and miR-3074-3p are potential diagnostic biomarkers of lupus nephritis in patients with SLE. The observed differential pattern of miRNA abundance may have functional implications in the pathophysiology of SLE renal damage.

  15. Circulating RNAs as new biomarkers for detecting pancreatic cancer

    PubMed Central

    Kishikawa, Takahiro; Otsuka, Motoyuki; Ohno, Motoko; Yoshikawa, Takeshi; Takata, Akemi; Koike, Kazuhiko

    2015-01-01

    Pancreatic cancer remains difficult to treat and has a high mortality rate. It is difficult to diagnose early, mainly due to the lack of screening imaging modalities and specific biomarkers. Consequently, it is important to develop biomarkers that enable the detection of early stage tumors. Emerging evidence is accumulating that tumor cells release substantial amounts of RNA into the bloodstream that strongly resist RNases in the blood and are present at sufficient levels for quantitative analyses. These circulating RNAs are upregulated in the serum and plasma of cancer patients, including those with pancreatic cancer, compared with healthy controls. The majority of RNA biomarker studies have assessed circulating microRNAs (miRs), which are often tissue-specific. There are few reports of the tumor-specific upregulation of other types of small non-coding RNAs (ncRNAs), such as small nucleolar RNAs and Piwi-interacting RNAs. Long ncRNAs (lncRNAs), such as HOTAIR and MALAT1, in the serum/plasma of pancreatic cancer patients have also been reported as diagnostic and prognostic markers. Among tissue-derived RNAs, some miRs show increased expression even in pre-cancerous tissues, and their expression profiles may allow for the discrimination between a chronic inflammatory state and carcinoma. Additionally, some miRs and lncRNAs have been reported with significant alterations in expression according to disease progression, and they may thus represent potential candidate diagnostic or prognostic biomarkers that may be used to evaluate patients once detection methods in peripheral blood are well established. Furthermore, recent innovations in high-throughput sequencing techniques have enabled the discovery of unannotated tumor-associated ncRNAs and tumor-specific alternative splicing as novel and specific biomarkers of cancers. Although much work is required to clarify the release mechanism, origin of tumor-specific circulating RNAs, and selectivity of carrier complexes

  16. Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular.

    PubMed

    Izuogu, Osagie G; Alhasan, Abd A; Mellough, Carla; Collin, Joseph; Gallon, Richard; Hyslop, Jonathon; Mastrorosa, Francesco K; Ehrmann, Ingrid; Lako, Majlinda; Elliott, David J; Santibanez-Koref, Mauro; Jackson, Michael S

    2018-04-20

    Circular RNAs (circRNAs) are predominantly derived from protein coding genes, and some can act as microRNA sponges or transcriptional regulators. Changes in circRNA levels have been identified during human development which may be functionally important, but lineage-specific analyses are currently lacking. To address this, we performed RNAseq analysis of human embryonic stem (ES) cells differentiated for 90 days towards 3D laminated retina. A transcriptome-wide increase in circRNA expression, size, and exon count was observed, with circRNA levels reaching a plateau by day 45. Parallel statistical analyses, controlling for sample and locus specific effects, identified 239 circRNAs with expression changes distinct from the transcriptome-wide pattern, but these all also increased in abundance over time. Surprisingly, circRNAs derived from long non-coding RNAs (lncRNAs) were found to account for a significantly larger proportion of transcripts from their loci of origin than circRNAs from coding genes. The most abundant, circRMST:E12-E6, showed a > 100X increase during differentiation accompanied by an isoform switch, and accounts for > 99% of RMST transcripts in many adult tissues. The second most abundant, circFIRRE:E10-E5, accounts for > 98% of FIRRE transcripts in differentiating human ES cells, and is one of 39 FIRRE circRNAs, many of which include multiple unannotated exons. Our results suggest that during human ES cell differentiation, changes in circRNA levels are primarily globally controlled. They also suggest that RMST and FIRRE, genes with established roles in neurogenesis and topological organisation of chromosomal domains respectively, are processed as circular lncRNAs with only minor linear species.

  17. Independent activity of the homologous small regulatory RNAs AbcR1 and AbcR2 in the legume symbiont Sinorhizobium meliloti.

    PubMed

    Torres-Quesada, Omar; Millán, Vicenta; Nisa-Martínez, Rafael; Bardou, Florian; Crespi, Martín; Toro, Nicolás; Jiménez-Zurdo, José I

    2013-01-01

    The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure. Homologous sRNAs (designated AbcR1 and AbcR2) have been shown to regulate several ABC transporters in the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In Rm1021, AbcR1 and AbcR2 exhibit divergent unlinked regulation and are stabilized by the RNA chaperone Hfq. AbcR1 is transcribed in actively dividing bacteria, either in culture, rhizosphere or within the invasion zone of mature alfalfa nodules. Conversely, AbcR2 expression is induced upon entry into stationary phase and under abiotic stress. Only deletion of AbcR1 resulted into a discrete growth delay in rich medium, but both are dispensable for symbiosis. Periplasmic proteome profiling revealed down-regulation of the branched-chain amino acid binding protein LivK by AbcR1, but not by AbcR2. A double-plasmid reporter assay confirmed the predicted specific targeting of the 5'-untranslated region of the livK mRNA by AbcR1 in vivo. Our findings provide evidences of independent regulatory functions of these sRNAs, probably to fine-tune nutrient uptake in free-living and undifferentiated symbiotic rhizobia.

  18. Independent Activity of the Homologous Small Regulatory RNAs AbcR1 and AbcR2 in the Legume Symbiont Sinorhizobium meliloti

    PubMed Central

    Torres-Quesada, Omar; Millán, Vicenta; Nisa-Martínez, Rafael; Bardou, Florian; Crespi, Martín; Toro, Nicolás; Jiménez-Zurdo, José I.

    2013-01-01

    The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure. Homologous sRNAs (designated AbcR1 and AbcR2) have been shown to regulate several ABC transporters in the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In Rm1021, AbcR1 and AbcR2 exhibit divergent unlinked regulation and are stabilized by the RNA chaperone Hfq. AbcR1 is transcribed in actively dividing bacteria, either in culture, rhizosphere or within the invasion zone of mature alfalfa nodules. Conversely, AbcR2 expression is induced upon entry into stationary phase and under abiotic stress. Only deletion of AbcR1 resulted into a discrete growth delay in rich medium, but both are dispensable for symbiosis. Periplasmic proteome profiling revealed down-regulation of the branched-chain amino acid binding protein LivK by AbcR1, but not by AbcR2. A double-plasmid reporter assay confirmed the predicted specific targeting of the 5′-untranslated region of the livK mRNA by AbcR1 in vivo. Our findings provide evidences of independent regulatory functions of these sRNAs, probably to fine-tune nutrient uptake in free-living and undifferentiated symbiotic rhizobia. PMID:23869210

  19. Isolation of Exosome-Like Nanoparticles and Analysis of MicroRNAs Derived from Coconut Water Based on Small RNA High-Throughput Sequencing.

    PubMed

    Zhao, Zhehao; Yu, Siran; Li, Min; Gui, Xin; Li, Ping

    2018-03-21

    In this study, the presence of microRNAs in coconut water was identified by real-time polymerase chain reaction (PCR) based on the results of high-throughput small RNA sequencing. In addition, the differences in microRNA content between immature and mature coconut water were compared. A total of 47 known microRNAs belonging to 25 families and 14 new microRNAs were identified in coconut endosperm. Through analysis using a target gene prediction software, potential microRNA target genes were identified in the human genome. Real-time PCR showed that the level of most microRNAs was higher in mature coconut water than in immature coconut water. Then, exosome-like nanoparticles were isolated from coconut water. After ultracentrifugation, some particle structures were seen in coconut water samples using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate fluorescence staining. Subsequent scanning electron microscopy observation and dynamic light scattering analysis also revealed some exosome-like nanoparticles in coconut water, and the mean diameters of the particles detected by the two methods were 13.16 and 59.72 nm, respectively. In conclusion, there are extracellular microRNAs in coconut water, and their levels are higher in mature coconut water than in immature coconut water. Some exosome-like nanoparticles were isolated from coconut water, and the diameter of these particles was smaller than that of animal-derived exosomes.

  20. Doppler Imaging and Chemical Abundance Analysis of EK Dra: Capabilities of Small Telescopes

    NASA Astrophysics Data System (ADS)

    Kilicoglu, Tolgahan; Senavci, H. V.; Bahar, E.; Isik, E.; Montes, D.; Hussain, G. A. J.

    2018-04-01

    We investigate the chromospheric and spot activity behaviour of the young Solar-like star EK Dra via Doppler imaging and spectral synthesis methods, using mid-resolution time series spectra of the system. We also present the atmospheric parameters and detailed elemental photospheric abundances of the star. The chemical abundance pattern of EK Dra do not suggest any remarkable peculiarities except few elements. The Titanium Oxide (TiO) bandheads at 7000 - 7100 A region also give clues about the spot temperature that may be cooler than 4000 K. In addition, we also discuss the capabilities of small telescopes (40 cm in our case) and medium resolution spectrographs in terms of Doppler imaging and chemical abundance analysis.

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

    PubMed Central

    2013-01-01

    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

  2. In vivo screening of modified siRNAs for non-specific antiviral effect in a small fish model: number and localization in the strands are important

    PubMed Central

    Schyth, Brian Dall; Bramsen, Jesper Bertram; Pakula, Malgorzata Maria; Larashati, Sekar; Kjems, Jørgen; Wengel, Jesper; Lorenzen, Niels

    2012-01-01

    Small interfering RNAs (siRNAs) are promising new active compounds in gene medicine but the induction of non-specific immune responses following their delivery continues to be a serious problem. With the purpose of avoiding such effects chemically modified siRNAs are tested in screening assay but often only examining the expression of specific immunologically relevant genes in selected cell populations typically blood cells from treated animals or humans. Assays using a relevant physiological state in biological models as read-out are not common. Here we use a fish model where the innate antiviral effect of siRNAs is functionally monitored as reduced mortality in challenge studies involving an interferon sensitive virus. Modifications with locked nucleic acid (LNA), altritol nucleic acid (ANA) and hexitol nucleic acid (HNA) reduced the antiviral protection in this model indicative of altered immunogenicity. For LNA modified siRNAs, the number and localization of modifications in the single strands was found to be important and a correlation between antiviral protection and the thermal stability of siRNAs was found. The previously published sisiRNA will in some sequences, but not all, increase the antiviral effect of siRNAs. The applied fish model represents a potent tool for conducting fast but statistically and scientifically relevant evaluations of chemically optimized siRNAs with respect to non-specific antiviral effects in vivo. PMID:22287630

  3. Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing

    PubMed Central

    2012-01-01

    Background MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression. MiRNAs play essential roles in almost all plant biological processes. Currently, few miRNAs have been identified in the model food legume Phaseolus vulgaris (common bean). Recent advances in next generation sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used Illumina's sequencing by synthesis (SBS) technology to identify and characterize the miRNA population of Phaseolus vulgaris. Results Small RNA libraries were generated from roots, flowers, leaves, and seedlings of P. vulgaris. Based on similarity to previously reported plant miRNAs,114 miRNAs belonging to 33 conserved miRNA families were identified. Stem-loop precursors and target gene sequences for several conserved common bean miRNAs were determined from publicly available databases. Less conserved miRNA families and species-specific common bean miRNA isoforms were also characterized. Moreover, novel miRNAs based on the small RNAs were found and their potential precursors were predicted. In addition, new target candidates for novel and conserved miRNAs were proposed. Finally, we studied organ-specific miRNA family expression levels through miRNA read frequencies. Conclusions This work represents the first massive-scale RNA sequencing study performed in Phaseolus vulgaris to identify and characterize its miRNA population. It significantly increases the number of miRNAs, precursors, and targets identified in this agronomically important species. The miRNA expression analysis provides a foundation for understanding common bean miRNA organ-specific expression patterns. The present study offers an expanded picture of P. vulgaris miRNAs in relation to those of other legumes. PMID:22394504

  4. Role of microRNAs in herpesvirus latency and persistence.

    PubMed

    Grey, Finn

    2015-04-01

    The identification of virally encoded microRNAs (miRNAs) has had a major impact on the field of herpes virology. Given their ability to target cellular and viral transcripts, and the lack of immune response to small RNAs, miRNAs represent an ideal mechanism of gene regulation during viral latency and persistence. In this review, we discuss the role of miRNAs in virus latency and persistence, specifically focusing on herpesviruses. We cover the current knowledge on miRNAs in establishing and maintaining virus latency and promoting survival of infected cells through targeting of both viral and cellular transcripts, highlighting key publications in the field. We also discuss potential areas of future research and how novel technologies may aid in determining how miRNAs shape virus latency in the context of herpesvirus infections. © 2015 The Author.

  5. Computational and transcriptional evidence for microRNAs in the honey bee genome

    PubMed Central

    Weaver, Daniel B; Anzola, Juan M; Evans, Jay D; Reid, Jeffrey G; Reese, Justin T; Childs, Kevin L; Zdobnov, Evgeny M; Samanta, Manoj P; Miller, Jonathan; Elsik, Christine G

    2007-01-01

    Background Non-coding microRNAs (miRNAs) are key regulators of gene expression in eukaryotes. Insect miRNAs help regulate the levels of proteins involved with development, metabolism, and other life history traits. The recently sequenced honey bee genome provides an opportunity to detect novel miRNAs in both this species and others, and to begin to infer the roles of miRNAs in honey bee development. Results Three independent computational surveys of the assembled honey bee genome identified a total of 65 non-redundant candidate miRNAs, several of which appear to have previously unrecognized orthologs in the Drosophila genome. A subset of these candidate miRNAs were screened for expression by quantitative RT-PCR and/or genome tiling arrays and most predicted miRNAs were confirmed as being expressed in at least one honey bee tissue. Interestingly, the transcript abundance for several known and novel miRNAs displayed caste or age-related differences in honey bees. Genes in proximity to miRNAs in the bee genome are disproportionately associated with the Gene Ontology terms 'physiological process', 'nucleus' and 'response to stress'. Conclusion Computational approaches successfully identified miRNAs in the honey bee and indicated previously unrecognized miRNAs in the well-studied Drosophila melanogaster genome despite the 280 million year distance between these insects. Differentially transcribed miRNAs are likely to be involved in regulating honey bee development, and arguably in the extreme developmental switch between sterile worker bees and highly fertile queens. PMID:17543122

  6. MicroRNAs Change the Landscape of Cancer Resistance.

    PubMed

    Zhu, Jun; Zhu, Wei; Wu, Wei

    2018-01-01

    One of the major challenges in the cancer treatment is the development of drug resistance. It represents a major obstacle to curing cancer with constrained efficacy of both conventional chemotherapy and targeted therapies, even recent immune checkpoint blockade therapy. Deciphering the mechanisms of resistance is critical to further understanding the multifactorial pathways involved, and developing more specific targeted treatments. To date, numerous studies have reported the potential role of microRNAs (miRNAs) in the resistance to various cancer treatments. MicroRNAs are a family of small noncoding RNAs that regulate gene expression by sequence-specific targeting of mRNAs causing translational repression or mRNA degradation. More than 1200 validated human miRNAs have been identified in human genome. While one miRNA can regulate hundreds of targets, a single target can also be affected by multiple miRNAs. Evidence suggests that dysregulation of specific miRNAs may be involved in the acquisition of resistance, thereby modulating the sensitivity of cancer cells to treatment. Therefore, manipulation of miRNAs may be an attractive strategy for more effective individualized therapies through reprograming resistant network in cancer cells.

  7. Genome-Wide Identification of Destruxin A-Responsive Immunity-Related MicroRNAs in Diamondback Moth, Plutella xylostella.

    PubMed

    Shakeel, Muhammad; Xu, Xiaoxia; Xu, Jin; Li, Shuzhong; Yu, Jialin; Zhou, Xianqiang; Xu, Xiaojing; Hu, Qiongbo; Yu, Xiaoqiang; Jin, Fengliang

    2018-01-01

    Plutella xylostella , a global key pest, is one of the major lepidopteran pests of cruciferous vegetables owing to its strong ability of resistance development to a wide range of insecticides. Destruxin A, a mycotoxin of the entomopathogenic fungus, Metarhizium anisopliae , has broad-spectrum insecticidal effects and has been used as an alternative control strategy to reduce harmful effects of insecticides. However, microRNA (miRNA)-regulated reactions against destruxin A have not been elucidated yet. Therefore, here, to identify immunity-related miRNAs, we constructed four small RNA libraries from destruxin A-injected larvae of P. xylostella at three different time courses (2, 4, and 6 h) with a control, and sequenced by Illumina. Our results showed that totally 187 known and 44 novel miRNAs were identified in four libraries by bioinformatic analysis. Interestingly, among differentially expressed known miRNAs, some conserved miRNAs, such as miR-263, miR-279, miR-306, miR-2a, and miR-308, predicted to be involved in regulating immunity-related genes, were also identified. Worthy to mention, miR-306 and miR-279 were also listed as common abundantly expressed miRNA in all treatments. The Kyoto Encyclopedia of Genes and Genomes pathway analysis also indicated that differentially expressed miRNAs were involved in several immunity-related signaling pathways, including toll signaling pathway, IMD signaling pathway, JAK-STAT signaling pathway, and cell adhesion molecules signaling pathway. To the best of our knowledge, this is the first comprehensive report of destruxin A-responsive immunity-related miRNAs in P. xylostella . Our findings will improve in understanding the role of destruxin A-responsive miRNAs in the host immune system and would be useful to develop biological control strategies for controlling P. xylostella .

  8. Genome-Wide Identification of Destruxin A-Responsive Immunity-Related MicroRNAs in Diamondback Moth, Plutella xylostella

    PubMed Central

    Shakeel, Muhammad; Xu, Xiaoxia; Xu, Jin; Li, Shuzhong; Yu, Jialin; Zhou, Xianqiang; Xu, Xiaojing; Hu, Qiongbo; Yu, Xiaoqiang; Jin, Fengliang

    2018-01-01

    Plutella xylostella, a global key pest, is one of the major lepidopteran pests of cruciferous vegetables owing to its strong ability of resistance development to a wide range of insecticides. Destruxin A, a mycotoxin of the entomopathogenic fungus, Metarhizium anisopliae, has broad-spectrum insecticidal effects and has been used as an alternative control strategy to reduce harmful effects of insecticides. However, microRNA (miRNA)-regulated reactions against destruxin A have not been elucidated yet. Therefore, here, to identify immunity-related miRNAs, we constructed four small RNA libraries from destruxin A-injected larvae of P. xylostella at three different time courses (2, 4, and 6 h) with a control, and sequenced by Illumina. Our results showed that totally 187 known and 44 novel miRNAs were identified in four libraries by bioinformatic analysis. Interestingly, among differentially expressed known miRNAs, some conserved miRNAs, such as miR-263, miR-279, miR-306, miR-2a, and miR-308, predicted to be involved in regulating immunity-related genes, were also identified. Worthy to mention, miR-306 and miR-279 were also listed as common abundantly expressed miRNA in all treatments. The Kyoto Encyclopedia of Genes and Genomes pathway analysis also indicated that differentially expressed miRNAs were involved in several immunity-related signaling pathways, including toll signaling pathway, IMD signaling pathway, JAK–STAT signaling pathway, and cell adhesion molecules signaling pathway. To the best of our knowledge, this is the first comprehensive report of destruxin A-responsive immunity-related miRNAs in P. xylostella. Our findings will improve in understanding the role of destruxin A-responsive miRNAs in the host immune system and would be useful to develop biological control strategies for controlling P. xylostella. PMID:29472927

  9. 5SRNAdb: an information resource for 5S ribosomal RNAs.

    PubMed

    Szymanski, Maciej; Zielezinski, Andrzej; Barciszewski, Jan; Erdmann, Volker A; Karlowski, Wojciech M

    2016-01-04

    Ribosomal 5S RNA (5S rRNA) is the ubiquitous RNA component found in the large subunit of ribosomes in all known organisms. Due to its small size, abundance and evolutionary conservation 5S rRNA for many years now is used as a model molecule in studies on RNA structure, RNA-protein interactions and molecular phylogeny. 5SRNAdb (http://combio.pl/5srnadb/) is the first database that provides a high quality reference set of ribosomal 5S RNAs (5S rRNA) across three domains of life. Here, we give an overview of new developments in the database and associated web tools since 2002, including updates to database content, curation processes and user web interfaces. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Sequencing of small RNAs of the fern Pleopeltis minima (Polypodiaceae) offers insight into the evolution of the microrna repertoire in land plants

    PubMed Central

    Berruezo, Florencia; de Souza, Flávio S. J.; Picca, Pablo I.; Nemirovsky, Sergio I.; Martínez Tosar, Leandro; Rivero, Mercedes; Mentaberry, Alejandro N.

    2017-01-01

    MicroRNAs (miRNAs) are short, single stranded RNA molecules that regulate the stability and translation of messenger RNAs in diverse eukaryotic groups. Several miRNA genes are of ancient origin and have been maintained in the genomes of animal and plant taxa for hundreds of millions of years, playing key roles in development and physiology. In the last decade, genome and small RNA (sRNA) sequencing of several plant species have helped unveil the evolutionary history of land plants. Among these, the fern group (monilophytes) occupies a key phylogenetic position, as it represents the closest extant cousin taxon of seed plants, i.e. gymno- and angiosperms. However, in spite of their evolutionary, economic and ecological importance, no fern genome has been sequenced yet and few genomic resources are available for this group. Here, we sequenced the small RNA fraction of an epiphytic South American fern, Pleopeltis minima (Polypodiaceae), and compared it to plant miRNA databases, allowing for the identification of miRNA families that are shared by all land plants, shared by all vascular plants (tracheophytes) or shared by euphyllophytes (ferns and seed plants) only. Using the recently described transcriptome of another fern, Lygodium japonicum, we also estimated the degree of conservation of fern miRNA targets in relation to other plant groups. Our results pinpoint the origin of several miRNA families in the land plant evolutionary tree with more precision and are a resource for future genomic and functional studies of fern miRNAs. PMID:28494025

  11. Sequencing of small RNAs of the fern Pleopeltis minima (Polypodiaceae) offers insight into the evolution of the microrna repertoire in land plants.

    PubMed

    Berruezo, Florencia; de Souza, Flávio S J; Picca, Pablo I; Nemirovsky, Sergio I; Martínez Tosar, Leandro; Rivero, Mercedes; Mentaberry, Alejandro N; Zelada, Alicia M

    2017-01-01

    MicroRNAs (miRNAs) are short, single stranded RNA molecules that regulate the stability and translation of messenger RNAs in diverse eukaryotic groups. Several miRNA genes are of ancient origin and have been maintained in the genomes of animal and plant taxa for hundreds of millions of years, playing key roles in development and physiology. In the last decade, genome and small RNA (sRNA) sequencing of several plant species have helped unveil the evolutionary history of land plants. Among these, the fern group (monilophytes) occupies a key phylogenetic position, as it represents the closest extant cousin taxon of seed plants, i.e. gymno- and angiosperms. However, in spite of their evolutionary, economic and ecological importance, no fern genome has been sequenced yet and few genomic resources are available for this group. Here, we sequenced the small RNA fraction of an epiphytic South American fern, Pleopeltis minima (Polypodiaceae), and compared it to plant miRNA databases, allowing for the identification of miRNA families that are shared by all land plants, shared by all vascular plants (tracheophytes) or shared by euphyllophytes (ferns and seed plants) only. Using the recently described transcriptome of another fern, Lygodium japonicum, we also estimated the degree of conservation of fern miRNA targets in relation to other plant groups. Our results pinpoint the origin of several miRNA families in the land plant evolutionary tree with more precision and are a resource for future genomic and functional studies of fern miRNAs.

  12. Viral Infection Induces Expression of Novel Phased MicroRNAs from Conserved Cellular MicroRNA Precursors

    PubMed Central

    Zhang, Jiayao; Zhao, Shuqi; Zheng, Hong; Gao, Ge; Wei, Liping; Li, Yi

    2011-01-01

    RNA silencing, mediated by small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs), is a potent antiviral or antibacterial mechanism, besides regulating normal cellular gene expression critical for development and physiology. To gain insights into host small RNA metabolism under infections by different viruses, we used Solexa/Illumina deep sequencing to characterize the small RNA profiles of rice plants infected by two distinct viruses, Rice dwarf virus (RDV, dsRNA virus) and Rice stripe virus (RSV, a negative sense and ambisense RNA virus), respectively, as compared with those from non-infected plants. Our analyses showed that RSV infection enhanced the accumulation of some rice miRNA*s, but not their corresponding miRNAs, as well as accumulation of phased siRNAs from a particular precursor. Furthermore, RSV infection also induced the expression of novel miRNAs in a phased pattern from several conserved miRNA precursors. In comparison, no such changes in host small RNA expression was observed in RDV-infected rice plants. Significantly RSV infection elevated the expression levels of selective OsDCLs and OsAGOs, whereas RDV infection only affected the expression of certain OsRDRs. Our results provide a comparative analysis, via deep sequencing, of changes in the small RNA profiles and in the genes of RNA silencing machinery induced by different viruses in a natural and economically important crop host plant. They uncover new mechanisms and complexity of virus-host interactions that may have important implications for further studies on the evolution of cellular small RNA biogenesis that impact pathogen infection, pathogenesis, as well as organismal development. PMID:21901091

  13. Vegetation patterns and abundances of amphibians and small mammals along small streams in a northwestern California watershed

    Treesearch

    Jeffrey R. Waters; Cynthia J. Zabel; Kevin S. McKelvey; Hartwell H. Welsh

    2001-01-01

    Our goal was to describe and evaluate patterns of association between stream size and abundances of amphibians and small mammals in a northwestern California watershed. We sampled populations at 42 stream sites and eight upland sites within a 100- watershed in 1995 and 1996. Stream reaches sampled ranged from poorly defined channels that rarely flowed to 10-m-wide...

  14. Identification of serum miRNAs by nano-quantum dots microarray as diagnostic biomarkers for early detection of non-small cell lung cancer.

    PubMed

    Fan, Lihong; Qi, Huiwei; Teng, Junliang; Su, Bo; Chen, Hao; Wang, Changhui; Xia, Qing

    2016-06-01

    Circulating microRNAs (miRNAs) are potential noninvasive biomarkers for cancer detection. We used preoperative serum samples from non-small cell lung cancer (NSCLC) patients and healthy controls to investigate whether serum levels of candidate miRNAs could be used as diagnostic biomarkers in patients with resectable NSCLC and whether they were associated with clinicopathologic characteristics. We initially detected expression of 12 miRNAs using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in preoperative serum samples of 94 NSCLC patients and 58 healthy controls. We further validated our results using the fluorescence quantum dots liquid bead array for differentially expressed miRNAs in serum samples of 70 NSCLC patients and 54 healthy controls. Receiver operating characteristic (ROC) analysis was performed to select the best diagnostic miRNA cutoff value. A predictive model of miRNAs for NSCLC was derived by multivariate logistic regression. We found that five serum miRNAs (miR-16-5p, miR-17b-5p, miR-19-3p, miR-20a-5p, and miR-92-3p) were significantly downregulated in NSCLC, while miR-15b-5p was significantly upregulated (p < 0.05). Multivariate logistic regression analysis revealed that miR-15b-5p, miR-16-5p, and miR-20a-5p expression were independent diagnostic factors for the identification of patients with NSCLC after adjustment for patient's age and sex. In addition, the expression of serum miR-106-5p was higher in stage I than in stages IIa-IIIb, and no significant association was observed between expression of miRNAs and other variables including pathological type, tumor size, and lymph nodes status. Six serum miRNAs could potentially serve as noninvasive diagnostic biomarkers for resectable NSCLC. The predictive model combining miR-15b-5p, miR-16-5p, and miR-20a-5p was the best diagnostic approach.

  15. Long Noncoding RNAs: Past, Present, and Future

    PubMed Central

    Kung, Johnny T. Y.; Colognori, David; Lee, Jeannie T.

    2013-01-01

    Long noncoding RNAs (lncRNAs) have gained widespread attention in recent years as a potentially new and crucial layer of biological regulation. lncRNAs of all kinds have been implicated in a range of developmental processes and diseases, but knowledge of the mechanisms by which they act is still surprisingly limited, and claims that almost the entirety of the mammalian genome is transcribed into functional noncoding transcripts remain controversial. At the same time, a small number of well-studied lncRNAs have given us important clues about the biology of these molecules, and a few key functional and mechanistic themes have begun to emerge, although the robustness of these models and classification schemes remains to be seen. Here, we review the current state of knowledge of the lncRNA field, discussing what is known about the genomic contexts, biological functions, and mechanisms of action of lncRNAs. We also reflect on how the recent interest in lncRNAs is deeply rooted in biology’s longstanding concern with the evolution and function of genomes. PMID:23463798

  16. Plant microRNAs as novel immunomodulatory agents

    PubMed Central

    Cavalieri, Duccio; Rizzetto, Lisa; Tocci, Noemi; Rivero, Damariz; Asquini, Elisa; Si-Ammour, Azeddine; Bonechi, Elena; Ballerini, Clara; Viola, Roberto

    2016-01-01

    An increasing body of literature is addressing the immuno-modulating functions of miRNAs which include paracrine signaling via exosome-mediated intercellular miRNA. In view of the recent evidence of intake and bioavailability of dietary miRNAs in humans and animals we explored the immuno-modulating capacity of plant derived miRNAs. Here we show that transfection of synthetic miRNAs or native miRNA-enriched fractions obtained from a wide range of plant species and organs modifies dendritic cells ability to respond to inflammatory agents by limiting T cell proliferation and consequently dampening inflammation. This immuno-modulatory effect appears associated with binding of plant miRNA on TLR3 with ensuing impairment of TRIF signaling. Similarly, in vivo, plant small RNAs reduce the onset of severity of Experimental Autoimmune Encephalomyelities by limiting dendritic cell migration and dampening Th1 and Th17 responses in a Treg-independent manner. Our results indicate a potential for therapeutic use of plant miRNAs in the prevention of chronic-inflammation related diseases. PMID:27167363

  17. Hypermethylated-capped selenoprotein mRNAs in mammals

    PubMed Central

    Wurth, Laurence; Gribling-Burrer, Anne-Sophie; Verheggen, Céline; Leichter, Michael; Takeuchi, Akiko; Baudrey, Stéphanie; Martin, Franck; Krol, Alain; Bertrand, Edouard; Allmang, Christine

    2014-01-01

    Mammalian mRNAs are generated by complex and coordinated biogenesis pathways and acquire 5′-end m7G caps that play fundamental roles in processing and translation. Here we show that several selenoprotein mRNAs are not recognized efficiently by translation initiation factor eIF4E because they bear a hypermethylated cap. This cap modification is acquired via a 5′-end maturation pathway similar to that of the small nucle(ol)ar RNAs (sn- and snoRNAs). Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs. We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated. Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo. PMID:25013170

  18. Bioinformatics of prokaryotic RNAs

    PubMed Central

    Backofen, Rolf; Amman, Fabian; Costa, Fabrizio; Findeiß, Sven; Richter, Andreas S; Stadler, Peter F

    2014-01-01

    The genome of most prokaryotes gives rise to surprisingly complex transcriptomes, comprising not only protein-coding mRNAs, often organized as operons, but also harbors dozens or even hundreds of highly structured small regulatory RNAs and unexpectedly large levels of anti-sense transcripts. Comprehensive surveys of prokaryotic transcriptomes and the need to characterize also their non-coding components is heavily dependent on computational methods and workflows, many of which have been developed or at least adapted specifically for the use with bacterial and archaeal data. This review provides an overview on the state-of-the-art of RNA bioinformatics focusing on applications to prokaryotes. PMID:24755880

  19. Identification of novel non-coding small RNAs from Streptococcus pneumoniae TIGR4 using high-resolution genome tiling arrays

    PubMed Central

    2010-01-01

    Background The identification of non-coding transcripts in human, mouse, and Escherichia coli has revealed their widespread occurrence and functional importance in both eukaryotic and prokaryotic life. In prokaryotes, studies have shown that non-coding transcripts participate in a broad range of cellular functions like gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Streptococcus pneumoniae (pneumococcus), an obligate human respiratory pathogen responsible for significant worldwide morbidity and mortality. Tiling microarrays enable genome wide mRNA profiling as well as identification of novel transcripts at a high-resolution. Results Here, we describe a high-resolution transcription map of the S. pneumoniae clinical isolate TIGR4 using genomic tiling arrays. Our results indicate that approximately 66% of the genome is expressed under our experimental conditions. We identified a total of 50 non-coding small RNAs (sRNAs) from the intergenic regions, of which 36 had no predicted function. Half of the identified sRNA sequences were found to be unique to S. pneumoniae genome. We identified eight overrepresented sequence motifs among sRNA sequences that correspond to sRNAs in different functional categories. Tiling arrays also identified approximately 202 operon structures in the genome. Conclusions In summary, the pneumococcal operon structures and novel sRNAs identified in this study enhance our understanding of the complexity and extent of the pneumococcal 'expressed' genome. Furthermore, the results of this study open up new avenues of research for understanding the complex RNA regulatory network governing S. pneumoniae physiology and virulence. PMID:20525227

  20. Cnidarian microRNAs frequently regulate targets by cleavage.

    PubMed

    Moran, Yehu; Fredman, David; Praher, Daniela; Li, Xin Z; Wee, Liang Meng; Rentzsch, Fabian; Zamore, Phillip D; Technau, Ulrich; Seitz, Hervé

    2014-04-01

    In bilaterians, which comprise most of extant animals, microRNAs (miRNAs) regulate the majority of messenger RNAs (mRNAs) via base-pairing of a short sequence (the miRNA "seed") to the target, subsequently promoting translational inhibition and transcript instability. In plants, many miRNAs guide endonucleolytic cleavage of highly complementary targets. Because little is known about miRNA function in nonbilaterian animals, we investigated the repertoire and biological activity of miRNAs in the sea anemone Nematostella vectensis, a representative of Cnidaria, the sister phylum of Bilateria. Our work uncovers scores of novel miRNAs in Nematostella, increasing the total miRNA gene count to 87. Yet only a handful are conserved in corals and hydras, suggesting that microRNA gene turnover in Cnidaria greatly exceeds that of other metazoan groups. We further show that Nematostella miRNAs frequently direct the cleavage of their mRNA targets via nearly perfect complementarity. This mode of action resembles that of small interfering RNAs (siRNAs) and plant miRNAs. It appears to be common in Cnidaria, as several of the miRNA target sites are conserved among distantly related anemone species, and we also detected miRNA-directed cleavage in Hydra. Unlike in bilaterians, Nematostella miRNAs are commonly coexpressed with their target transcripts. In light of these findings, we propose that post-transcriptional regulation by miRNAs functions differently in Cnidaria and Bilateria. The similar, siRNA-like mode of action of miRNAs in Cnidaria and plants suggests that this may be an ancestral state.

  1. Small RNA sorting: matchmaking for Argonautes

    PubMed Central

    Czech, Benjamin; Hannon, Gregory J.

    2013-01-01

    Small RNAs directly or indirectly impact nearly every biological process in eukaryotic cells. To perform their myriad roles, not only must precise small RNA species be generated, but they must also be loaded into specific effector complexes called RNA-induced silencing complexes (RISCs). Argonaute proteins form the core of RISCs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. In this Review, we explore the mechanisms that pair specific small RNA strands with their partner proteins, with an eye towards the substantial progress that has been recently made in understanding the sorting of the major small RNA classes — microRNAs (miRNAs) and small interfering RNAs (siRNAs) — in plants and animals. PMID:21116305

  2. Identification of microRNA-like RNAs from Curvularia lunata associated with maize leaf spot by bioinformation analysis and deep sequencing.

    PubMed

    Liu, Tong; Hu, John; Zuo, Yuhu; Jin, Yazhong; Hou, Jumei

    2016-04-01

    Deep sequencing of small RNAs is a useful tool to identify novel small RNAs that may be involved in fungal growth and pathogenesis. In this study, we used HiSeq deep sequencing to identify 747,487 unique small RNAs from Curvularia lunata. Among these small RNAs were 1012 microRNA-like RNAs (milRNAs), which are similar to other known microRNAs, and 48 potential novel milRNAs without homologs in other organisms have been identified using the miRBase© database. We used quantitative PCR to analyze the expression of four of these milRNAs from C. lunata at different developmental stages. The analysis revealed several changes associated with germinating conidia and mycelial growth, suggesting that these milRNAs may play a role in pathogen infection and mycelial growth. A total of 8334 target mRNAs for the 1012 milRNAs that were identified, and 256 target mRNAs for the 48 novel milRNAs were predicted by computational analysis. These target mRNAs of milRNAs were also performed by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. To our knowledge, this study is the first report of C. lunata's milRNA profiles. This information will provide a better understanding of pathogen development and infection mechanism.

  3. MicroRNAs in osteosarcoma: diagnostic and therapeutic aspects.

    PubMed

    Miao, Jinglei; Wu, Song; Peng, Zhi; Tania, Mousumi; Zhang, Chaoyue

    2013-08-01

    MicroRNAs (miRNAs) are small RNA molecules, which can interfere with the expression of several genes and act as gene regulator. miRNAs have been proved as a successful diagnostic and therapeutic tool in several cancers. In this review, the differential expression of miRNAs in osteosarcoma and their possibility to be used as diagnostic and therapeutic tools have been discussed. Osteosarcoma is the most common primary bone tumor that mainly affects children and adolescents. The current treatment of osteosarcoma remains difficult, and osteosarcoma causes many deaths because of its complex pathogenesis and resistance to conventional treatments. Several studies demonstrated that the differential expression patterns of miRNAs are a promising tool for the diagnosis and treatment of osteosarcoma. Although some aspect of the mechanism of action of miRNAs in controlling osteosarcoma has been identified (e.g., targeting the Notch signaling pathway), it is far beyond to the clear understanding of miRNA targets in osteosarcoma. Identification of the specific target of miRNAs may aid molecular targets for drug development and future relief of osteosarcoma.

  4. Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation

    PubMed Central

    Tang, Weixin; Hu, Johnny H.; Liu, David R.

    2017-01-01

    Programmable sequence-specific genome editing agents such as CRISPR-Cas9 have greatly advanced our ability to manipulate the human genome. Although canonical forms of genome-editing agents and programmable transcriptional regulators are constitutively active, precise temporal and spatial control over genome editing and transcriptional regulation activities would enable the more selective and potentially safer use of these powerful technologies. Here, by incorporating ligand-responsive self-cleaving catalytic RNAs (aptazymes) into guide RNAs, we developed a set of aptazyme-embedded guide RNAs that enable small molecule-controlled nuclease-mediated genome editing and small molecule-controlled base editing, as well as small molecule-dependent transcriptional activation in mammalian cells. PMID:28656978

  5. Argonaute identity defines the length of mature mammalian microRNAs

    PubMed Central

    Juvvuna, Prasanna Kumar; Khandelia, Piyush; Lee, Li Ming; Makeyev, Eugene V.

    2012-01-01

    MicroRNAs (miRNAs) are 19- to 25-nt-long non-coding RNAs that regulate gene expression by base-pairing with target mRNAs and reducing their stability or translational efficiency. Mammalian miRNAs function in association with four closely related Argonaute proteins, AGO1–4. All four proteins contain the PAZ and the MID domains interacting with the miRNA 3′ and 5′ termini, respectively, as well as the PIWI domain comprising an mRNA ‘slicing’ activity in the case of AGO2 but not AGO1, AGO3 and AGO4. However, the slicing mode of the miRNA-programmed AGO2 is rarely realized in vivo and the four Argonautes are thought to play largely overlapping roles in the mammalian miRNA pathway. Here, we show that the average length of many miRNAs is diminished during nervous system development as a result of progressive shortening of the miRNA 3′ ends. We link this modification with an increase in the fractional abundance of Ago2 in the adult brain and identify a specific structural motif within the PAZ domain that enables efficient trimming of miRNAs associated with this but not the other three Argonautes. Taken together, our data suggest that mammalian Argonautes may define the length and possibly biological activity of mature mammalian miRNAs in a developmentally controlled manner. PMID:22505576

  6. Pathobiologic Roles of Epstein–Barr Virus-Encoded MicroRNAs in Human Lymphomas

    PubMed Central

    Navari, Mohsen; Etebari, Maryam; Ibrahimi, Mostafa; Leoncini, Lorenzo

    2018-01-01

    Epstein–Barr virus (EBV) is a human γ-herpesvirus implicated in several human malignancies, including a wide range of lymphomas. Several molecules encoded by EBV in its latent state are believed to be related to EBV-induced lymphomagenesis, among which microRNAs—small RNAs with a posttranscriptional regulating role—are of great importance. The genome of EBV encodes 44 mature microRNAs belonging to two different classes, including BamHI-A rightward transcript (BART) and Bam HI fragment H rightward open reading frame 1 (BHRF1), with different expression levels in different EBV latency types. These microRNAs might contribute to the pathogenetic effects exerted by EBV through targeting self mRNAs and host mRNAs and interfering with several important cellular mechanisms such as immunosurveillance, cell proliferation, and apoptosis. In addition, EBV microRNAs can regulate the surrounding microenvironment of the infected cells through exosomal transportation. Moreover, these small molecules could be potentially used as molecular markers. In this review, we try to present an updated and extensive view of the role of EBV-encoded miRNAs in human lymphomas. PMID:29649101

  7. The role of microRNAs in skeletal muscle health and disease

    PubMed Central

    Kirby, Tyler J.; Chaillou, Thomas; McCarthy, John J.

    2016-01-01

    Over the last decade non-coding RNAs have emerged as importance regulators of gene expression. In particular, microRNAs are a class of small RNAs of ~ 22 nucleotides that repress gene expression through a post-transcriptional mechanism. MicroRNAs have been shown to be involved in a broader range of biological processes, both physiological and pathological, including myogenesis, adaptation to exercise and various myopathies. The purpose of this review is to provide a comprehensive summary of what is currently known about the role of microRNAs in skeletal muscle health and disease. PMID:25553440

  8. Cell stress and translational inhibitors transiently increase the abundance of mammalian SINE transcripts.

    PubMed Central

    Liu, W M; Chu, W M; Choudary, P V; Schmid, C W

    1995-01-01

    The abundance of Alu RNA is transiently increased by heat shock in human cell lines. This effect is specific to Alu repeats among Pol III transcribed genes, since the abundance of 7SL, 7SK, 5S and U6 RNAs is essentially unaffected by heat shock. The rapid induction of Alu expression precedes the heat shock induction of mRNAs for the ubiquitin and HSP 70 heat shock genes. Heat shock mimetics also transiently induce Alu expression indicating that increased Alu expression is a general cell-stress response. Cycloheximide treatment rapidly and transiently increases the abundance of Alu RNA. Again, compared with other genes transcribed by Pol III, this increase is specific to Alu. However, as distinguished from the cell stress response, cycloheximide does not induce expression of HSP 70 and ubiquitin mRNAs. Puromycin also increases Alu expression, suggesting that this response is generally caused by translational inhibition. The response of mammalian SINEs to cell stress and translational inhibition is not limited to SINEs which are Alu homologues. Heat shock and cycloheximide each transiently induce Pol III directed expression of B1 and B2 RNAs in mouse cells and C-element RNA in rabbit cells. Together, these three species exemplify the known SINE composition of placental mammals, suggesting that mammalian SINEs are similarly regulated and may serve a common function. Images PMID:7784180

  9. Expanding the action of duplex RNAs into the nucleus: redirecting alternative splicing

    PubMed Central

    Liu, Jing; Hu, Jiaxin; Corey, David R.

    2012-01-01

    Double-stranded RNAs are powerful agents for silencing gene expression in the cytoplasm of mammalian cells. The potential for duplex RNAs to control expression in the nucleus has received less attention. Here, we investigate the ability of small RNAs to redirect splicing. We identify RNAs targeting an aberrant splice site that restore splicing and production of functional protein. RNAs can target sequences within exons or introns and affect the inclusion of exons within SMN2 and dystrophin, genes responsible for spinal muscular atrophy and Duchenne muscular dystrophy, respectively. Duplex RNAs recruit argonaute 2 (AGO2) to pre-mRNA transcripts and altered splicing requires AGO2 expression. AGO2 promotes transcript cleavage in the cytoplasm, but recruitment of AGO2 to pre-mRNAs does not reduce transcript levels, exposing a difference between cytoplasmic and nuclear pathways. Involvement of AGO2 in splicing, a classical nuclear process, reinforces the conclusion from studies of RNA-mediated transcriptional silencing that RNAi pathways can be adapted to function in the mammalian nucleus. These data provide a new strategy for controlling splicing and expand the reach of small RNAs within the nucleus of mammalian cells. PMID:21948593

  10. In silico identification of conserved microRNAs in large number of diverse plant species

    PubMed Central

    Sunkar, Ramanjulu; Jagadeeswaran, Guru

    2008-01-01

    Background MicroRNAs (miRNAs) are recently discovered small non-coding RNAs that play pivotal roles in gene expression, specifically at the post-transcriptional level in plants and animals. Identification of miRNAs in large number of diverse plant species is important to understand the evolution of miRNAs and miRNA-targeted gene regulations. Now-a-days, publicly available databases play a central role in the in-silico biology. Because, at least ~21 miRNA families are conserved in higher plants, a homology based search using these databases can help identify orthologs or paralogs in plants. Results We searched all publicly available nucleotide databases of genome survey sequences (GSS), high-throughput genomics sequences (HTGS), expressed sequenced tags (ESTs) and nonredundant (NR) nucleotides and identified 682 miRNAs in 155 diverse plant species. We found more than 15 conserved miRNA families in 11 plant species, 10 to14 families in 10 plant species and 5 to 9 families in 29 plant species. Nineteen conserved miRNA families were identified in important model legumes such as Medicago, Lotus and soybean. Five miRNA families – miR319, miR156/157, miR169, miR165/166 and miR394 – were found in 51, 45, 41, 40 and 40 diverse plant species, respectively. miR403 homologs were found in 16 dicots, whereas miR437 and miR444 homologs, as well as the miR396d/e variant of the miR396 family, were found only in monocots, thus providing large-scale authenticity for the dicot- and monocot-specific miRNAs. Furthermore, we provide computational and/or experimental evidence for the conservation of 6 newly found Arabidopsis miRNA homologs (miR158, miR391, miR824, miR825, miR827 and miR840) and 2 small RNAs (small-85 and small-87) in Brassica spp. Conclusion Using all publicly available nucleotide databases, 682 miRNAs were identified in 155 diverse plant species. By combining the expression analysis with the computational approach, we found that 6 miRNAs and 2 small RNAs that have

  11. Plasma exosome microRNAs are indicative of breast cancer.

    PubMed

    Hannafon, Bethany N; Trigoso, Yvonne D; Calloway, Cameron L; Zhao, Y Daniel; Lum, David H; Welm, Alana L; Zhao, Zhizhuang J; Blick, Kenneth E; Dooley, William C; Ding, W Q

    2016-09-08

    microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of

  12. miRNAs and other non-coding RNAs in posttraumatic stress disorder: A systematic review of clinical and animal studies.

    PubMed

    Schmidt, Ulrike; Keck, Martin E; Buell, Dominik R

    2015-06-01

    In the last couple of years, non-coding (nc) RNAs like micro-RNAs (miRNAs), small interference RNAs (siRNAs) and long ncRNAs (lncRNAs) have emerged as promising candidates for biomarkers and drug-targets in a variety of psychiatric disorders. In contrast to reports on ncRNAs in affective disorders, schizophrenia and anxiety disorders, manuscripts on ncRNAs in posttraumatic stress disorder (PTSD) and associated animal models are scarce. Aiming to stimulate ncRNA research in PTSD and to identify the hitherto most promising ncRNA candidates and associated pathways for psychotrauma research, we conducted the first review on ncRNAs in PTSD. We aimed to identify studies reporting on the expression, function and regulation of ncRNAs in PTSD patients and in animals exhibiting a PTSD-like syndrome. Following the PRISMA guidelines for systematic reviews, we systematically screened the PubMed database for clinical and animal studies on ncRNAs in PTSD, animal models for PTSD and animal models employing a classical fear conditioning paradigm. Using 112 different combinations of search terms, we retrieved 523 articles of which we finally included and evaluated three clinical and 12 animal studies. In addition, using the web-based tool DIANA miRPath v2.0, we searched for molecular pathways shared by the predicted targets of the here-evaluated miRNA candidates. Our findings suggest that mir-132, which has been found to be regulated in three of the here included studies, as well as miRNAs with an already established role in Alzheimer's disease (AD) seem to be particularly promising candidates for future miRNA studies in PTSD. These results are limited by the low number of human trials and by the heterogeneity of included animal studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Regulation of microRNAs in Cancer Metastasis

    PubMed Central

    Bouyssou, Juliette M.C.; Manier, Salomon; Huynh, Daisy; Issa, Samar; Roccaro, Aldo M.; Ghobrial, Irene M.

    2014-01-01

    Metastasis is a phenomenon of crucial importance in defining prognosis in patients with cancer and is often responsible for cancer-related mortality. It is known that several steps are necessary for clonal cells to disseminate from their primary tumor site and colonize distant tissues, thus originating metastatic lesions. Therefore, investigating the molecular actors regulating this process may provide helpful insights in the development of efficient therapeutic responses. Recent evidences have indicated the role of microRNAs (miRNAs) in modulating the metastatic process in solid tumors. miRNAs are small regulatory non-coding RNAs that bind specific target mRNAs, leading to translational repression. miRNAs are known to act as negative regulators of gene expression and are involved in the regulation of biological processes, including cell growth, differentiation and apoptosis, both in physiological conditions and during diseases, such as tumors. In the specific field of tumorigenesis, miRNAs play an important role in mediating oncogenesis and favoring tumor progression, as a result of their ability to modulate epithelial-to-mesenchymal transition (EMT) and other series of events facilitating the formation of metastasis. The role of miRNAs in cancer development has been widely studied and has helped elucidate events such as the change in expression of oncogenes, tumor-suppressors and cancer-related proteins. This review focuses on the mechanisms underlying the role of miRNAs as part of the metastatic process. PMID:24569228

  14. Involvement of MicroRNAs in Lung Cancer Biology and Therapy

    PubMed Central

    Liu, Xi; Sempere, Lorenzo F.; Guo, Yongli; Korc, Murray; Kauppinen, Sakari; Freemantle, Sarah J.; Dmitrovsky, Ethan

    2011-01-01

    MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression. Expression profiles of specific miRNAs have improved cancer diagnosis and classification and even provided prognostic information in many human cancers, including lung cancer. Tumor suppressive and oncogenic miRNAs were uncovered in lung carcinogenesis. The biological functions of these miRNAs in lung cancer were recently validated in well characterized cellular, murine transgenic as well as transplantable lung cancer models and in human paired normal-malignant lung tissue banks and tissue arrays. Tumor suppressive and oncogenic miRNAs that were identified in lung cancer will be reviewed here. Emphasis is placed on highlighting those functionally validated miRNAs that are not only biomarkers of lung carcinogenesis, but also candidate pharmacologic targets. How these miRNA findings advance an understanding of lung cancer biology and could improve lung cancer therapy are discussed in this article. PMID:21420030

  15. 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.

  16. miRNAs as therapeutic targets in ischemic heart disease.

    PubMed

    Frost, Robert J A; van Rooij, Eva

    2010-06-01

    Ischemic heart disease is a form of congestive heart failure that is caused by insufficient blood supply to the heart, resulting in a loss of viable tissue. In response to the injury, the non-ischemic myocardium displays signs of secondary remodeling, like interstitial fibrosis and hypertrophy of cardiac myocytes. This remodeling process further deteriorates pump function and increases susceptibility to arrhythmias. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression in a sequence-dependent manner. Recently, several groups identified miRNAs as crucial gene regulators in response to myocardial infarction (MI) and during post-MI remodeling. In this review, we discuss how modulation of these miRNAs represents a promising new therapeutic strategy to improve the clinical outcome in ischemic heart disease.

  17. microRNAs in parasites and parasite infection

    PubMed Central

    Zheng, Yadong; Cai, Xuepeng; Bradley, Janette E.

    2013-01-01

    miRNAs, a subclass of small regulatory RNAs, are present from ancient unicellular protozoans to parasitic helminths and parasitic arthropods. The miRNA-silencing mechanism appears, however, to be absent in a number of protozoan parasites. Protozoan miRNAs and components of their silencing machinery possess features different from other eukaryotes, providing some clues on the evolution of the RNA-induced silencing machinery. miRNA functions possibly associate with neoblast biology, development, physiology, infection and immunity of parasites. Parasite infection can alter host miRNA expression that can favor both parasite clearance and infection. miRNA pathways are, thus, a potential target for the therapeutic control of parasitic diseases. PMID:23392243

  18. Epigenetic Therapy in Lung Cancer – Role of microRNAs

    PubMed Central

    Rothschild, Sacha I.

    2013-01-01

    Lung cancer is the leading cause of cancer deaths worldwide. microRNAs (miRNAs) are a class of small non-coding RNA species that have been implicated in the control of many fundamental cellular and physiological processes such as cellular differentiation, proliferation, apoptosis, and stem cell maintenance. Some miRNAs have been categorized as “oncomiRs” as opposed to “tumor suppressor miRs.” This review focuses on the role of miRNAs in the lung cancer carcinogenesis and their potential as diagnostic, prognostic, or predictive markers. PMID:23802096

  19. The entangled history of animal and plant microRNAs.

    PubMed

    Reis, Rodrigo S

    2017-05-01

    MicroRNAs (miRNAs) are small RNAs (sRNAs) that regulate gene expression in development and adaptive responses to the environment. The early days in the sRNA field was one of the most exciting and promising moments in modern biology, attracting large investments to the understanding of the underlining mechanisms and their applications, such as in gene therapy. miRNAs and other sRNAs have since been extensively studied in animals and plants, and are currently well established as an important part of most gene regulatory processes in animals and as master regulators in plants. Here, this review presents the critical discoveries and early misconceptions that shaped our current understanding of RNA silencing by miRNAs in most eukaryotes, with a focus on plant miRNAs. The presentation and language used are simple to facilitate a clear comprehension by researchers and students from various backgrounds. Hence, this is a valuable teaching tool and should also draw attention to the discovery processes themselves, such that scientists from various fields can gain insights from the successful and rapidly evolving miRNA field.

  20. Characterization of microRNAs from goat (Capra hircus) by Solexa deep-sequencing technology.

    PubMed

    Ling, Y H; Ding, J P; Zhang, X D; Wang, L J; Zhang, Y H; Li, Y S; Zhang, Z J; Zhang, X R

    2013-06-13

    MicroRNAs (miRNAs) are an important class of small noncoding RNAs that are highly conserved in plants and animals. Many miRNAs are known to mediate a myriad of cell processes, including proliferation and differentiation, via the regulation of some transcription and signaling factors, which are closely related to muscle development and disease. In this study, small RNA cDNA libraries of Boer goats were constructed. In addition, we obtained the goat muscle miRNAs by using Solexa deep-sequencing technology and analyzed these miRNA characteristics by combining it with the bioinformatics technology. Based on Solexa sequencing and bioinformatics analysis, 562 species-conserved and 5 goat genome-specific miRNAs were identified, 322 of which exceeded 100 in the expression levels. The results of real-time quantitative polymerase chain reaction from 8 randomly selected miRNAs showed that the 8 miRNAs were expressed in goat muscle, and the expression patterns were consistent with the Solexa sequencing results. The identification and characterization of miRNAs in goat muscle provide important information on the role of miRNA regulation in muscle growth and development. These data will help to facilitate studies on the regulatory roles played by miRNAs during goat growth and development.

  1. A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains

    PubMed Central

    Hutchinson, John N; Ensminger, Alexander W; Clemson, Christine M; Lynch, Christopher R; Lawrence, Jeanne B; Chess, Andrew

    2007-01-01

    Background Noncoding RNA species play a diverse set of roles in the eukaryotic cell. While much recent attention has focused on smaller RNA species, larger noncoding transcripts are also thought to be highly abundant in mammalian cells. To search for large noncoding RNAs that might control gene expression or mRNA metabolism, we used Affymetrix expression arrays to identify polyadenylated RNA transcripts displaying nuclear enrichment. Results This screen identified no more than three transcripts; XIST, and two unique noncoding nuclear enriched abundant transcripts (NEAT) RNAs strikingly located less than 70 kb apart on human chromosome 11: NEAT1, a noncoding RNA from the locus encoding for TncRNA, and NEAT2 (also known as MALAT-1). While the two NEAT transcripts share no significant homology with each other, each is conserved within the mammalian lineage, suggesting significant function for these noncoding RNAs. NEAT2 is extraordinarily well conserved for a noncoding RNA, more so than even XIST. Bioinformatic analyses of publicly available mouse transcriptome data support our findings from human cells as they confirm that the murine homologs of these noncoding RNAs are also nuclear enriched. RNA FISH analyses suggest that these noncoding RNAs function in mRNA metabolism as they demonstrate an intimate association of these RNA species with SC35 nuclear speckles in both human and mouse cells. These studies show that one of these transcripts, NEAT1 localizes to the periphery of such domains, whereas the neighboring transcript, NEAT2, is part of the long-sought polyadenylated component of nuclear speckles. Conclusion Our genome-wide screens in two mammalian species reveal no more than three abundant large non-coding polyadenylated RNAs in the nucleus; the canonical large noncoding RNA XIST and NEAT1 and NEAT2. The function of these noncoding RNAs in mRNA metabolism is suggested by their high levels of conservation and their intimate association with SC35 splicing

  2. Insilico profiling of microRNAs in Korean ginseng (Panax ginseng Meyer)

    PubMed Central

    Mathiyalagan, Ramya; Subramaniyam, Sathiyamoorthy; Natarajan, Sathishkumar; Kim, Yeon Ju; Sun, Myung Suk; Kim, Se Young; Kim, Yu-Jin; Yang, Deok Chun

    2013-01-01

    MicroRNAs (miRNAs) are a class of recently discovered non-coding small RNA molecules, on average approximately 21 nucleotides in length, which underlie numerous important biological roles in gene regulation in various organisms. The miRNA database (release 18) has 18,226 miRNAs, which have been deposited from different species. Although miRNAs have been identified and validated in many plant species, no studies have been reported on discovering miRNAs in Panax ginseng Meyer, which is a traditionally known medicinal plant in oriental medicine, also known as Korean ginseng. It has triterpene ginseng saponins called ginsenosides, which are responsible for its various pharmacological activities. Predicting conserved miRNAs by homology-based analysis with available expressed sequence tag (EST) sequences can be powerful, if the species lacks whole genome sequence information. In this study by using the EST based computational approach, 69 conserved miRNAs belonging to 44 miRNA families were identified in Korean ginseng. The digital gene expression patterns of predicted conserved miRNAs were analyzed by deep sequencing using small RNA sequences of flower buds, leaves, and lateral roots. We have found that many of the identified miRNAs showed tissue specific expressions. Using the insilico method, 346 potential targets were identified for the predicted 69 conserved miRNAs by searching the ginseng EST database, and the predicted targets were mainly involved in secondary metabolic processes, responses to biotic and abiotic stress, and transcription regulator activities, as well as a variety of other metabolic processes. PMID:23717176

  3. RsmV a small non-coding regulatory RNA in Pseudomonas aeruginosa that sequesters RsmA and RsmF from target mRNAs.

    PubMed

    Janssen, Kayley H; Diaz, Manisha R; Gode, Cindy J; Wolfgang, Matthew C; Yahr, Timothy L

    2018-06-04

    The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has distinct genetic programs that favor either acute or chronic virulence gene expression. Acute virulence is associated with twitching and swimming motility, expression of a type III secretion system (T3SS), and the absence of alginate, Psl, or Pel polysaccharide production. Traits associated with chronic infection include growth as a biofilm, reduced motility, and expression of a type VI secretion system (T6SS). The Rsm post-transcriptional regulatory system plays important roles in the inverse control of phenotypes associated with acute and chronic virulence. RsmA and RsmF are RNA-binding proteins that interact with target mRNAs to control gene expression at the post-transcriptional level. Previous work found that RsmA activity is controlled by at least three small, non-coding regulatory RNAs (RsmW, RsmY, and RsmZ). In this study, we took an in-silico approach to identify additional sRNAs that might function in the sequestration of RsmA and/or RsmF and identified RsmV, a 192 nt transcript with four predicted RsmA/RsmF consensus binding sites. RsmV is capable of sequestering RsmA and RsmF in vivo to activate translation of tssA1 , a component of the T6SS, and to inhibit T3SS gene expression. Each of the predicted RsmA/RsmF consensus binding sites contribute to RsmV activity. Electrophoretic mobility shifts assays show that RsmF binds RsmV with >10-fold higher affinity than RsmY and RsmZ. Gene expression studies revealed that the temporal expression pattern of RsmV differs from RsmW, RsmY, and RsmZ. These findings suggest that each sRNA may play distinct roles in controlling RsmA and RsmF activity. IMPORTANCE The CsrA/RsmA family of RNA-binding proteins play important roles in post-transcriptional control of gene expression. The activity of CsrA/RsmA proteins is controlled by small non-coding RNAs that function as decoys to sequester CsrA/RsmA from target mRNAs. Pseudomonas aeruginosa has two Csr

  4. Circulating microRNAs as novel biomarkers of ALK-positive non-small cell lung cancer and predictors of response to crizotinib therapy

    PubMed Central

    Fu, Han-Jiang; Zheng, Xiao-Fei; Tang, Chuan-Hao; Li, Xiao-Yan; Chen, Jian; Wang, Wei-Xia; Yang, Shao-Xing; Wang, Lin; Zhao, Guan-Hua; Lv, Pan-Pan; Zhang, Min; Lei, Yang-Yang; Qin, Hai-Feng; Wang, Hong; Gao, Hong-Jun; Liu, Xiao-Qing

    2017-01-01

    Circulating microRNAs are potential diagnostic and predictive biomarkers, but have not been investigated for patients with anaplastic lymphoma kinase (ALK)-positive lung cancer. In this exploratory study, we sought to identify potential plasma biomarkers for ALK-positive non-small cell lung cancer (NSCLC). A microRNA microarray was used to select ALK-related microRNAs in ALK-positive NSCLC (n = 3), ALK-negative NSCLC (n = 3), and healthy subjects (n = 3). Plasma levels of 21 microRNAs were differentially expressed for ALK-positive and ALK-negative NSCLC, including 14 down-regulated and 7 up-regulated microRNAs. We also identified 5s rRNA as the most stable endogenous control gene using geNorm and NormFinder algorithms. Candidate microRNAs in plasma from ALK-positive (n = 41) and ALK-negative NSCLC patients (n = 32) were quantified using real-time reverse transcriptase quantitative polymerase chain reaction. The expression levels of miR-28-5p, miR-362-5p, and miR-660-5p were all down-regulated in ALK-positive NSCLC, compared with ALK-negative NSCLC. The areas under the receiver operating characteristic curves of miR-28-5p, miR-362-5p, miR-660-5p, and 3-microRNAs panel were 0.873, 0.673, 0.760, and 0.876, respectively. The positive predictive values of miR-28-5p, miR-362-5p, and miR-660-5p were 96.43%, 80.77%, and 83.87%, respectively. Increased plasma levels of miR-660-5p after crizotinib treatment predicted good tumor response (p = 0.012). The pre-crizotinib levels of miR-362-5p were significantly associated with progression-free survival (p = 0.015). Thus, in this preliminary investigation, we identified a potential panel of 3 microRNAs for distinguishing between patients with ALK-positive and ALK-negative NSCLC. We also identified miR-660-5p and miR-362-5p as potential predictors for response to crizotinib treatment. PMID:28514730

  5. siRNAs and piRNAs Collaborate for Transposon Control in the Two-Spotted Spider Mite.

    PubMed

    Mondal, Mosharrof; Mansfield, Kody; Flynt, Alex

    2018-04-20

    RNAi has revolutionized genetic research, and is being commercialized as an insect pest control technology. Mechanisms exploited for this purpose are antiviral and therefore rapidly evolving. Ideally, RNAi will also be used for non-insect pests, however, differences in RNAi biology makes this uncertain. Tetranychus urticae (two-spotted spider mite) is a destructive non-insect pest, which has a proclivity to develop pesticide resistance. Here we provide a comprehensive study of the endogenous RNAi pathways of spider mites to inform design of exogenous RNAi triggers. This effort revealed unexpected roles for small RNAs and novel genome surveillance pathways. Spider mites have an expanded RNAi machinery relative to insects, encoding RNA dependent RNA polymerase (Rdrp) and extra Piwi-class effectors. Through analyzing T. urticae transcriptome data we explored small RNA biogenesis, and discovered five siRNA loci that appear central to genome surveillance. These RNAs are expressed in the gonad, which we hypothesize to trigger production of piRNAs for control of transposable elements (TEs). This work highlights the need to investigate endogenous RNAi biology as lessons from model organisms may not hold in other species, impacting development of an RNAi strategy. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  6. Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity.

    PubMed

    Buck, Amy H; Coakley, Gillian; Simbari, Fabio; McSorley, Henry J; Quintana, Juan F; Le Bihan, Thierry; Kumar, Sujai; Abreu-Goodger, Cei; Lear, Marissa; Harcus, Yvonne; Ceroni, Alessandro; Babayan, Simon A; Blaxter, Mark; Ivens, Alasdair; Maizels, Rick M

    2014-11-25

    In mammalian systems RNA can move between cells via vesicles. Here we demonstrate that the gastrointestinal nematode Heligmosomoides polygyrus, which infects mice, secretes vesicles containing microRNAs (miRNAs) and Y RNAs as well as a nematode Argonaute protein. These vesicles are of intestinal origin and are enriched for homologues of mammalian exosome proteins. Administration of the nematode exosomes to mice suppresses Type 2 innate responses and eosinophilia induced by the allergen Alternaria. Microarray analysis of mouse cells incubated with nematode exosomes in vitro identifies Il33r and Dusp1 as suppressed genes, and Dusp1 can be repressed by nematode miRNAs based on a reporter assay. We further identify miRNAs from the filarial nematode Litomosoides sigmodontis in the serum of infected mice, suggesting that miRNA secretion into host tissues is conserved among parasitic nematodes. These results reveal exosomes as another mechanism by which helminths manipulate their hosts and provide a mechanistic framework for RNA transfer between animal species.

  7. Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity

    PubMed Central

    Buck, Amy H.; Coakley, Gillian; Simbari, Fabio; McSorley, Henry J.; Quintana, Juan F.; Le Bihan, Thierry; Kumar, Sujai; Abreu-Goodger, Cei; Lear, Marissa; Harcus, Yvonne; Ceroni, Alessandro; Babayan, Simon A.; Blaxter, Mark; Ivens, Alasdair; Maizels, Rick M.

    2014-01-01

    In mammalian systems RNA can move between cells via vesicles. Here we demonstrate that the gastrointestinal nematode Heligmosomoides polygyrus, which infects mice, secretes vesicles containing microRNAs (miRNAs) and Y RNAs as well as a nematode Argonaute protein. These vesicles are of intestinal origin and are enriched for homologues of mammalian exosome proteins. Administration of the nematode exosomes to mice suppresses Type 2 innate responses and eosinophilia induced by the allergen Alternaria. Microarray analysis of mouse cells incubated with nematode exosomes in vitro identifies Il33r and Dusp1 as suppressed genes, and Dusp1 can be repressed by nematode miRNAs based on a reporter assay. We further identify miRNAs from the filarial nematode Litomosoides sigmodontis in the serum of infected mice, suggesting that miRNA secretion into host tissues is conserved among parasitic nematodes. These results reveal exosomes as another mechanism by which helminths manipulate their hosts and provide a mechanistic framework for RNA transfer between animal species. PMID:25421927

  8. MicroRNAs in large herpesvirus DNA genomes: recent advances.

    PubMed

    Sorel, Océane; Dewals, Benjamin G

    2016-08-01

    MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that regulate gene expression. They alter mRNA translation through base-pair complementarity, leading to regulation of genes during both physiological and pathological processes. Viruses have evolved mechanisms to take advantage of the host cells to multiply and/or persist over the lifetime of the host. Herpesviridae are a large family of double-stranded DNA viruses that are associated with a number of important diseases, including lymphoproliferative diseases. Herpesviruses establish lifelong latent infections through modulation of the interface between the virus and its host. A number of reports have identified miRNAs in a very large number of human and animal herpesviruses suggesting that these short non-coding transcripts could play essential roles in herpesvirus biology. This review will specifically focus on the recent advances on the functions of herpesvirus miRNAs in infection and pathogenesis.

  9. Identification of miRNAs Involved in Stolon Formation in Tulipa edulis by High-Throughput Sequencing

    PubMed Central

    Zhu, Zaibiao; Miao, Yuanyuan; Guo, Qiaosheng; Zhu, Yunhao; Yang, Xiaohua; Sun, Yuan

    2016-01-01

    MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNAs that play an important role in transcriptional and post-transcriptional gene regulation. However, the sequence information and functions of miRNAs are still unexplored in Tulipa edulis. In this study, high-throughput sequencing was used to identify small RNAs in stolon formation stages (stage 1, 2, and 3) in T. edulis. A total of 12,890,912, 12,182,122, and 12,061,434 clean reads were obtained from stage 1, 2, and 3, respectively. Among the reads, 88 conserved miRNAs and 70 novel miRNAs were identified. Target prediction of 122 miRNAs resulted in 531 potential target genes. Nr, Swiss-Prot, GO, COG, and KEGG annotations revealed that these target genes participate in many biologic and metabolic processes. Moreover, qRT-PCR was performed to analyze the expression levels of the miRNAs and target genes in stolon formation. The results revealed that miRNAs play a key role in T. edulis stolon formation. PMID:27446103

  10. Identification of miRNAs Involved in Stolon Formation in Tulipa edulis by High-Throughput Sequencing.

    PubMed

    Zhu, Zaibiao; Miao, Yuanyuan; Guo, Qiaosheng; Zhu, Yunhao; Yang, Xiaohua; Sun, Yuan

    2016-01-01

    MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNAs that play an important role in transcriptional and post-transcriptional gene regulation. However, the sequence information and functions of miRNAs are still unexplored in Tulipa edulis. In this study, high-throughput sequencing was used to identify small RNAs in stolon formation stages (stage 1, 2, and 3) in T. edulis. A total of 12,890,912, 12,182,122, and 12,061,434 clean reads were obtained from stage 1, 2, and 3, respectively. Among the reads, 88 conserved miRNAs and 70 novel miRNAs were identified. Target prediction of 122 miRNAs resulted in 531 potential target genes. Nr, Swiss-Prot, GO, COG, and KEGG annotations revealed that these target genes participate in many biologic and metabolic processes. Moreover, qRT-PCR was performed to analyze the expression levels of the miRNAs and target genes in stolon formation. The results revealed that miRNAs play a key role in T. edulis stolon formation.

  11. The expanding roles of microRNAs in kidney pathophysiology.

    PubMed

    Metzinger-Le Meuth, Valérie; Fourdinier, Ophélie; Charnaux, Nathalie; Massy, Ziad A; Metzinger, Laurent

    2018-05-25

    MicroRNAs (miRNAs) are short single-stranded RNAs that control gene expression through base pairing with regions within the 3'-untranslated region of target mRNAs. These small non-coding RNAs are now increasingly known to be involved in kidney physiopathology. In this review we will describe how miRNAs were in recent years implicated in cellular and animal models of kidney disease but also in chronic kidney disease, haemodialysed and grafted patients, acute kidney injury patients and so on. At the moment miRNAs are considered as potential biomarkers in nephrology, but larger cohorts as well as the standardization of methods of measurement will be needed to confirm their usefulness. It will further be of the utmost importance to select specific tissues and biofluids to make miRNAs appropriate in day-to-day clinical practice. In addition, up- or down-regulating miRNAs that were described as deregulated in kidney diseases may represent innovative therapeutic methods to cure these disorders. We will enumerate in this review the most recent methods that can be used to deliver miRNAs in a specific and suitable way in kidney and other organs damaged by kidney failure, such as the cardiovascular system.

  12. Using thinning as a management tool for gypsy moth: the influence on small mammal abundance

    Treesearch

    R.M. Muzika; S.T. Grushecky; A.M. Liebhold; R.L. Smith

    2004-01-01

    Silvicultural manipulations may be used to reduce forest susceptibility or vulnerability to defoliation by the gypsy moth. The effects of this management strategy on small mammal abundance were determined by pitfall trapping small mammals 1 year before silvicultural thinnings and for 3 years following thinning in a deciduous montane forest. Sorex cinereus...

  13. High throughput sequencing reveals novel and abiotic stress-regulated microRNAs in the inflorescences of rice.

    PubMed

    Barrera-Figueroa, Blanca E; Gao, Lei; Wu, Zhigang; Zhou, Xuefeng; Zhu, Jianhua; Jin, Hailing; Liu, Renyi; Zhu, Jian-Kang

    2012-08-03

    MicroRNAs (miRNAs) are small RNA molecules that play important regulatory roles in plant development and stress responses. Identification of stress-regulated miRNAs is crucial for understanding how plants respond to environmental stimuli. Abiotic stresses are one of the major factors that limit crop growth and yield. Whereas abiotic stress-regulated miRNAs have been identified in vegetative tissues in several plants, they are not well studied in reproductive tissues such as inflorescences. We used Illumina deep sequencing technology to sequence four small RNA libraries that were constructed from the inflorescences of rice plants that were grown under control condition and drought, cold, or salt stress. We identified 227 miRNAs that belong to 127 families, including 70 miRNAs that are not present in the miRBase. We validated 62 miRNAs (including 10 novel miRNAs) using published small RNA expression data in DCL1, DCL3, and RDR2 RNAi lines and confirmed 210 targets from 86 miRNAs using published degradome data. By comparing the expression levels of miRNAs, we identified 18, 15, and 10 miRNAs that were regulated by drought, cold and salt stress conditions, respectively. In addition, we identified 80 candidate miRNAs that originated from transposable elements or repeats, especially miniature inverted-repeat elements (MITEs). We discovered novel miRNAs and stress-regulated miRNAs that may play critical roles in stress response in rice inflorescences. Transposable elements or repeats, especially MITEs, are rich sources for miRNA origination.

  14. Ambient temperature regulates the expression of a small set of sRNAs influencing plant development through NF-YA2 and YUC2.

    PubMed

    Gyula, Péter; Baksa, Ivett; Tóth, Tamás; Mohorianu, Irina; Dalmay, Tamás; Szittya, György

    2018-06-01

    Plants substantially alter their developmental program upon changes in the ambient temperature. The 21-24 nt small RNAs (sRNAs) are important gene expression regulators, which play a major role in development and adaptation. However, little is known about how the different sRNA classes respond to changes in the ambient temperature. We profiled the sRNA populations in four different tissues of Arabidopsis thaliana plants grown at 15, 21 and 27 °C. We found that only a small fraction (0.6%) of the sRNA loci are ambient temperature-controlled. We identified thermoresponsive miRNAs and identified their target genes using degradome libraries. We verified that the target of the thermoregulated miR169, NF-YA2, is also ambient temperature-regulated. NF-YA2, as the component of the conserved transcriptional regulator NF-Y complex, binds the promoter of the flowering time regulator FT and the auxin biosynthesis gene YUC2. Other differentially expressed loci include thermoresponsive phased siRNA loci that target various auxin pathway genes and tRNA fragments. Furthermore, a temperature dependent 24-nt heterochromatic siRNA locus in the promoter of YUC2 may contribute to the epigenetic regulation of auxin homeostasis. This holistic approach facilitated a better understanding of the role of different sRNA classes in ambient temperature adaptation of plants. This article is protected by copyright. All rights reserved.

  15. Analysis of high iron rice lines reveals new miRNAs that target iron transporters in roots

    PubMed Central

    Paul, Soumitra; Gayen, Dipak; Datta, Swapan K.; Datta, Karabi

    2016-01-01

    The present study highlights the molecular regulation of iron transport in soyFER1-overexpressing transgenic rice. Accumulation of iron in three different seed developmental stages, milk, dough, and mature, has been examined. The transgenic seeds of the milk stage showed significant augmentation of iron and zinc levels compared with wild-type seeds, and similar results were observed throughout the dough and mature stages. To investigate the regulation of iron transport, the role of miRNAs was studied in roots of transgenic rice. Sequencing of small RNA libraries revealed 153 known and 41 novel miRNAs in roots. Among them, 59 known and 14 novel miRNAs were found to be significantly expressed. miR166, miR399, and miR408 were identified as playing a vital role in iron uptake in roots of transgenic plants . Most importantly, four putative novel miRNAs, namely miR11, miR26, miR30, and miR31, were found to be down-regulated in roots of transgenic plants. For all these four novel miRNAs, natural resistance-associated macrophage protein 4 (NRAMP4), encoding a metal transporter, was predicted as a target gene. It is hypothesized that the NRAMP4 transporter is activated in roots of transgenic plants due to the lower abundance of its corresponding putative novel miRNAs. The relative transcript level of the NRAMP4 transcript was increased from 0.107 in the wild type to 65.24 and 55.39 in transgenic plants, which demonstrates the elevated amount of iron transport in transgenic plants. In addition, up-regulation of OsYSL15, OsFRO2, and OsIRT1 in roots also facilitates iron loading in transgenic seeds. PMID:27729476

  16. The Inescapable Influence of Noncoding RNAs in Cancer

    PubMed Central

    Adams, Brian D.; Anastasiadou, Eleni; Esteller, Manel; He, Lin; Slack, Frank J.

    2015-01-01

    This report summarizes information presented at the 2015 Keystone Symposium on “MicroRNAs and Noncoding RNAs in Cancer”. Nearly two decades after the discovery of the first microRNA (miRNA), the role of noncoding RNAs in developmental processes and the mechanisms behind their dysregulation in cancer has been steadily elucidated. Excitingly, miRNAs have begun making their way into the clinic to combat disease such a hepatitis C, and various forms of cancer. Therefore, at this Keystone meeting novel findings were presented that enhance our view on how small and long noncoding RNAs control developmental timing and oncogenic processes. Recurring themes included, 1) how miRNAs can be differentially processed, degraded, and regulated by ribonucleoprotein (RNP) complexes, 2) how particular miRNA genetic networks that control developmental process, when disrupted, can result in cancer disease, 3) the technologies available to therapeutically deliver RNA to combat diseases such as cancer, and 4) the elucidation of the mechanism of actions for long noncoding RNAs, currently a poorly understood class of noncoding RNA. During the meeting there was an emphasis on presenting unpublished findings, and the breadth of topics covered reflected how inescapable the influence of noncoding RNAs are in development and cancer. PMID:26567137

  17. Posttranscriptional regulation of lipid metabolism by non-coding RNAs and RNA binding proteins.

    PubMed

    Singh, Abhishek K; Aryal, Binod; Zhang, Xinbo; Fan, Yuhua; Price, Nathan L; Suárez, Yajaira; Fernández-Hernando, Carlos

    2017-11-29

    Alterations in lipoprotein metabolism enhance the risk of cardiometabolic disorders including type-2 diabetes and atherosclerosis, the leading cause of death in Western societies. While the transcriptional regulation of lipid metabolism has been well characterized, recent studies have uncovered the importance of microRNAs (miRNAs), long-non-coding RNAs (lncRNAs) and RNA binding proteins (RBP) in regulating the expression of lipid-related genes at the posttranscriptional level. Work from several groups has identified a number of miRNAs, including miR-33, miR-122 and miR-148a, that play a prominent role in controlling cholesterol homeostasis and lipoprotein metabolism. Importantly, dysregulation of miRNA expression has been associated with dyslipidemia, suggesting that manipulating the expression of these miRNAs could be a useful therapeutic approach to ameliorate cardiovascular disease (CVD). The role of lncRNAs in regulating lipid metabolism has recently emerged and several groups have demonstrated their regulation of lipoprotein metabolism. However, given the high abundance of lncRNAs and the poor-genetic conservation between species, much work will be needed to elucidate the specific role of lncRNAs in controlling lipoprotein metabolism. In this review article, we summarize recent findings in the field and highlight the specific contribution of lncRNAs and RBPs in regulating lipid metabolism. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. A universal small molecule, inorganic phosphate, restricts the substrate specificity of Dicer-2 in small RNA biogenesis

    PubMed Central

    Fukunaga, Ryuya; Zamore, Phillip D

    2014-01-01

    The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme. PMID:24787225

  19. Distribution and abundance of small plastic debris on beaches in the SE Pacific (Chile): a study supported by a citizen science project.

    PubMed

    Hidalgo-Ruz, Valeria; Thiel, Martin

    2013-01-01

    The accumulation of large and small plastic debris is a problem throughout the world's oceans and coastlines. Abundances and types of small plastic debris have only been reported for some isolated beaches in the SE Pacific, but these data are insufficient to evaluate the situation in this region. The citizen science project "National Sampling of Small Plastic Debris" was supported by schoolchildren from all over Chile who documented the distribution and abundance of small plastic debris on Chilean beaches. Thirty-nine schools and nearly 1000 students from continental Chile and Easter Island participated in the activity. To validate the data obtained by the students, all samples were recounted in the laboratory. The results of the present study showed that the students were able to follow the instructions and generate reliable data. The average abundance obtained was 27 small plastic pieces per m(2) for the continental coast of Chile, but the samples from Easter Island had extraordinarily higher abundances (>800 items per m(2)). The abundance of small plastic debris on the continental coast could be associated with coastal urban centers and their economic activities. The high abundance found on Easter Island can be explained mainly by the transport of plastic debris via the surface currents in the South Pacific Subtropical Gyre, resulting in the accumulation of small plastic debris on the beaches of the island. This first report of the widespread distribution and abundance of small plastic debris on Chilean beaches underscores the need to extend plastic debris research to ecological aspects of the problem and to improve waste management. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. MicroRNAs and Drug-induced Kidney Injury

    PubMed Central

    Pavkovic, Mira; Vaidya, Vishal S.

    2016-01-01

    Drug-induced kidney injury (DIKI) is a severe complication in hospitalized patients associated with higher probabilities of developing progressive chronic kidney disease or end-stage renal diseases. Furthermore, DIKI is a problem during preclinical and clinical phases of drug development leading to high rates of project terminations. Understanding the molecular perturbations caused by DIKI would pave the way for a new class of therapeutics to mitigate the damage. Yet, another approach to ameliorate DIKI is identifying sensitive and specific translational biomarkers that outperform the current diagnostic analytes like serum creatinine and facilitate early diagnosis. MicroRNAs (miRNAs), a class of non-coding RNAs, are increasingly being recognized to have a two-pronged approach towards DIKI management: 1) miRNAs have a regulatory role in gene expression and signaling pathways thereby making them novel interventional targets and 2) miRNAs enable diagnosis and prognosis of DIKI because of their stable presence in biofluids. In this review, apart from summarizing the literature on miRNAs in DIKI, we report small RNA sequencing results showing miRNA expression profiles at baseline in normal kidney samples from mice and humans. Additionally, we also compared the miRNA expression in biopsies of normal human kidneys to patients with acute tubular necrosis, and found 76 miRNAs significantly downregulated and 47 miRNAs upregulated (FDR adjusted p<0.05, +/−2-fold change). In summary, we highlight the transformative potential of miRNAs in therapeutics and translational medicine with a focus on drug-induced kidney damage. PMID:27126472

  1. Identification of conserved microRNAs in peripheral blood from giant panda: expression of mammary gland-related microRNAs during late pregnancy and early lactation.

    PubMed

    Wang, C D; Long, K; Jin, L; Huang, S; Li, D H; Ma, X P; Wei, M; Gu, Y; Ma, J D; Zhang, H

    2015-11-13

    The giant panda (Ailuropoda melanoleuca) is one of the world's most endangered mammals, and it has evolved several unusual biological and behavioral traits. During puberty, pregnancy, lactation, and involution, the mammary gland undergoes profound morphological and functional changes. A large number of microRNAs (miRNAs) have been identified to be involved in mammary gland development and lactation. In this study, we identified 202 conserved mature miRNAs, corresponding to 147 pre-miRNAs, in giant panda peripheral blood using a small RNA-sequencing approach. In addition, 27 miRNA families and 29 miRNA clusters were identified. We analyzed the arm selection preference of pre-miRNAs and found that: 1) most giant panda pre-miRNAs generated one-strand miRNAs, and the 5p-arm only miRNAs have a higher expression level than 3p-arm only miRNAs; 2) there were more 5p-arm dominant miRNAs than 3p-arm dominant miRNAs; and 3) 5p-arm dominant miRNAs have a larger fold change within miRNA pairs than 3p-arm dominant miRNAs. Expression of 12 lactation-related miRNAs was detected across late pregnancy and early lactation stages by qPCR, and seven miRNAs were identified as clustered in one significant model. Most of these clustered miRNAs exhibited inhibitory roles in proliferation and differentiation of mammary epithelial cells. Functional analysis highlighted important roles of the seven as signed miRNAs in mammary development and metabolic changes, including blood vessel morphogenesis, macromolecule biosynthesis, cell cycle regulation, and protein transport.

  2. A mRNA and cognate microRNAs localize in the nucleolus.

    PubMed

    Reyes-Gutierrez, Pablo; Ritland Politz, Joan C; Pederson, Thoru

    2014-01-01

    We previously discovered that a set of 5 microRNAs are concentrated in the nucleolus of rat myoblasts. We now report that several mRNAs are also localized in the nucleoli of these cells as determined by microarray analysis of RNA from purified nucleoli. Among the most abundant of these nucleolus-localized mRNAs is that encoding insulin-like growth factor 2 (IGF2), a regulator of myoblast proliferation and differentiation. The presence of IGF2 mRNA in nucleoli was confirmed by fluorescence in situ hybridization, and RT-PCR experiments demonstrated that these nucleolar transcripts are spliced, thus arriving from the nucleoplasm. Bioinformatics analysis predicted canonically structured, highly thermodynamically stable interactions between IGF2 mRNA and all 5 of the nucleolus-localized microRNAs. These results raise the possibility that the nucleolus is a staging site for setting up particular mRNA-microRNA interactions prior to export to the cytoplasm.

  3. MicroRNAs Related to Polycystic Ovary Syndrome (PCOS)

    PubMed Central

    Sørensen, Anja Elaine; Wissing, Marie Louise; Salö, Sofia; Englund, Anne Lis Mikkelsen; Dalgaard, Louise Torp

    2014-01-01

    Polycystic ovary syndrome (PCOS) is the most common, though heterogeneous, endocrine aberration in women of reproductive age, with high prevalence and socioeconomic costs. The syndrome is characterized by polycystic ovaries, chronic anovulation and hyperandrogenism, as well as being associated with infertility, insulin resistance, chronic low-grade inflammation and an increased life time risk of type 2 diabetes. MicroRNAs (miRNAs) are small, non-coding RNAs that are able to regulate gene expression at the post-transcriptional level. Altered miRNA levels have been associated with diabetes, insulin resistance, inflammation and various cancers. Studies have shown that circulating miRNAs are present in whole blood, serum, plasma and the follicular fluid of PCOS patients and that they might serve as potential biomarkers and a new approach for the diagnosis of PCOS. In this review, recent work on miRNAs with respect to PCOS will be summarized. Our understanding of miRNAs, particularly in relation to PCOS, is currently at a very early stage, and additional studies will yield important insight into the molecular mechanisms behind this complex and heterogenic syndrome. PMID:25158044

  4. Bacterial Adaptation to Antibiotics through Regulatory RNAs.

    PubMed

    Felden, Brice; Cattoir, Vincent

    2018-05-01

    The extensive use of antibiotics has resulted in a situation where multidrug-resistant pathogens have become a severe menace to human health worldwide. A deeper understanding of the principles used by pathogens to adapt to, respond to, and resist antibiotics would pave the road to the discovery of drugs with novel mechanisms. For bacteria, antibiotics represent clinically relevant stresses that induce protective responses. The recent implication of regulatory RNAs (small RNAs [sRNAs]) in antibiotic response and resistance in several bacterial pathogens suggests that they should be considered innovative drug targets. This minireview discusses sRNA-mediated mechanisms exploited by bacterial pathogens to fight against antibiotics. A critical discussion of the newest findings in the field is provided, with emphasis on the implication of sRNAs in major mechanisms leading to antibiotic resistance, including drug uptake, active drug efflux, drug target modifications, biofilms, cell walls, and lipopolysaccharide (LPS) biosynthesis. Of interest is the lack of knowledge about sRNAs implicated in Gram-positive compared to Gram-negative bacterial resistance. Copyright © 2018 American Society for Microbiology.

  5. miRNAs and ovarian cancer: An overview.

    PubMed

    Deb, Bornali; Uddin, Arif; Chakraborty, Supriyo

    2018-05-01

    Ovarian cancer (OC) is the sixth most common cancer in women globally. However, even with the advances in detection and therapeutics it still represents the most dangerous gynecologic malignancy in women of the industrialized countries. The discovery of micro-RNAs (miRNA), a small noncoding RNA molecule targeting multiple mRNAs and regulation of gene expression by triggering translation repression and/or RNA degradation, has revealed the existence of a new array for regulation of genes involved in cancer. This review summarizes the current knowledge regarding the role of miRNAs expression in OC. It also provides information about potential clinical relevance of circulating miRNAs for OC diagnosis, prognosis, and therapeutics. The identification of functional targets for miRNAs represents a major obstacle in our understanding of microRNA function in OC, but significant progress is being made. The better understanding of the role of microRNA expression in ovarian cancer may provide new array for the detection, diagnosis, and therapy of the OC. © 2017 Wiley Periodicals, Inc.

  6. Genome-wide identification of microRNAs in pomegranate (Punica granatum L.) by high-throughput sequencing

    USDA-ARS?s Scientific Manuscript database

    Background: MicroRNAs (miRNAs), a class of small non-coding endogenous RNAs that regulate gene expression post-transcriptionally, play multiple key roles in plant growth and development and in biotic and abiotic stress response. Knowledge and roles of miRNAs in pomegranate fruit development have not...

  7. Identification of microRNAs in Response to Drought in Common Wild Rice (Oryza rufipogon Griff.) Shoots and Roots.

    PubMed

    Zhang, Jing-Wen; Long, Yan; Xue, Man-de; Xiao, Xing-Guo; Pei, Xin-Wu

    2017-01-01

    Drought is the most important factor that limits rice production in drought-prone environments. Plant microRNAs (miRNAs) are involved in biotic and abiotic stress responses. Common wild rice (Oryza rufipogon Griff.) contains abundant drought-resistant genes, which provide an opportunity to explore these excellent resources as contributors to improve rice resistance, productivity, and quality. In this study, we constructed four small RNA libraries, called CL and CR from PEG6000-free samples and DL and DR from PEG6000-treated samples, where 'R' indicates the root tissue and 'L' indicates the shoot tissue. A total of 200 miRNAs were identified to be differentially expressed under the drought-treated conditions (16% PEG6000 for 24 h), and the changes in the miRNA expression profile of the shoot were distinct from those of the root. At the miRNA level, 77 known miRNAs, which belong to 23 families, including 40 up-regulated and 37 down-regulated in the shoot, and 85 known miRNAs in 46 families, including 65 up-regulated and 20 down-regulated in the root, were identified as differentially expressed. In addition, we predicted 26 new miRNA candidates from the shoot and 43 from the root that were differentially expressed during the drought stress. The quantitative real-time PCR analysis results were consistent with high-throughput sequencing data. Moreover, 88 miRNAs that were differentially-expressed were predicted to match with 197 targets for drought-stress. Our results suggest that the miRNAs of O. rufipogon are responsive to drought stress. The differentially expressed miRNAs that are tissue-specific under drought conditions could play different roles in the regulation of the auxin pathway, the flowering pathway, the drought pathway, and lateral root formation. Thus, the present study provides an account of tissue-specific miRNAs that are involved in the drought adaption of O. rufipogon.

  8. An endogenous small interfering RNA pathway in Drosophila

    PubMed Central

    Czech, Benjamin; Malone, Colin D.; Zhou, Rui; Stark, Alexander; Schlingeheyde, Catherine; Dus, Monica; Perrimon, Norbert; Kellis, Manolis; Wohlschlegel, James A.; Sachidanandam, Ravi; Hannon, Gregory J.; Brennecke, Julius

    2009-01-01

    Drosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of ~22 nucleotides in length, which arise from structured precursors through the action of Drosha–Pasha and Dicer-1–Loquacious complexes1–7. These join Argonaute-1 to regulate gene expression8,9. A second endogenous small RNA class, the Piwi-interacting RNAs, bind Piwi proteins and suppress transposons10,11. Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of single-stranded RNAs12,13. Here we show that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious1,4,5 rather than the canonical Dicer-2 partner, R2D2 (ref. 14). Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles. PMID:18463631

  9. Micropathogen Community Analysis in Hyalomma rufipes via High-Throughput Sequencing of Small RNAs

    PubMed Central

    Luo, Jin; Liu, Min-Xuan; Ren, Qiao-Yun; Chen, Ze; Tian, Zhan-Cheng; Hao, Jia-Wei; Wu, Feng; Liu, Xiao-Cui; Luo, Jian-Xun; Yin, Hong; Wang, Hui; Liu, Guang-Yuan

    2017-01-01

    Ticks are important vectors in the transmission of a broad range of micropathogens to vertebrates, including humans. Because of the role of ticks in disease transmission, identifying and characterizing the micropathogen profiles of tick populations have become increasingly important. The objective of this study was to survey the micropathogens of Hyalomma rufipes ticks. Illumina HiSeq2000 technology was utilized to perform deep sequencing of small RNAs (sRNAs) extracted from field-collected H. rufipes ticks in Gansu Province, China. The resultant sRNA library data revealed that the surveyed tick populations produced reads that were homologous to St. Croix River Virus (SCRV) sequences. We also observed many reads that were homologous to microbial and/or pathogenic isolates, including bacteria, protozoa, and fungi. As part of this analysis, a phylogenetic tree was constructed to display the relationships among the homologous sequences that were identified. The study offered a unique opportunity to gain insight into the micropathogens of H. rufipes ticks. The effective control of arthropod vectors in the future will require knowledge of the micropathogen composition of vectors harboring infectious agents. Understanding the ecological factors that regulate vector propagation in association with the prevalence and persistence of micropathogen lineages is also imperative. These interactions may affect the evolution of micropathogen lineages, especially if the micropathogens rely on the vector or host for dispersal. The sRNA deep-sequencing approach used in this analysis provides an intuitive method to survey micropathogen prevalence in ticks and other vector species. PMID:28861401

  10. siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

    PubMed

    Vickers, Timothy A; Crooke, Stanley T

    2012-07-01

    While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

  11. [The role of microRNAs in molecular pathology of esophageal cancer and their potential usage in clinical oncology].

    PubMed

    Kovaříková, A; Héžová, R; Srovnal, J; Rédová-Lojová, M; Slabý, O

    2014-01-01

    MicroRNAs are an abundant class of noncoding RNAs (approx. 18- 25 nucleotides in length) that suppress translation through binding to their target mRNAs, eventually leading to mRNAs degradation. Sequences of these endogenous RNA molecules are highly conserved, even among unrelated species, indicating their involvement in basic bio-logical processes, such as development, differentiation, proliferation or apoptosis. MiRNAs also participate on regulation of cancer stem cell functioning, immune system and malignant transformation. This review provides a comprehensive overview of miRNAs functions in esophageal cancer, their roles in key pathogenetic pathways and disease development, as well as their potential usage in clinical routine as bio-markers improving dia-gnosis, prognosis and prediction of therapeutic response. Through regulation of signaling pathways important in malignant transformation, miRNAs present also promising therapeutic targets.

  12. 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

  13. Non-coding stem-bulge RNAs are required for cell proliferation and embryonic development in C. elegans

    PubMed Central

    Kowalski, Madzia P.; Baylis, Howard A.; Krude, Torsten

    2015-01-01

    ABSTRACT Stem bulge RNAs (sbRNAs) are a family of small non-coding stem-loop RNAs present in Caenorhabditis elegans and other nematodes, the function of which is unknown. Here, we report the first functional characterisation of nematode sbRNAs. We demonstrate that sbRNAs from a range of nematode species are able to reconstitute the initiation of chromosomal DNA replication in the presence of replication proteins in vitro, and that conserved nucleotide sequence motifs are essential for this function. By functionally inactivating sbRNAs with antisense morpholino oligonucleotides, we show that sbRNAs are required for S phase progression, early embryonic development and the viability of C. elegans in vivo. Thus, we demonstrate a new and essential role for sbRNAs during the early development of C. elegans. sbRNAs show limited nucleotide sequence similarity to vertebrate Y RNAs, which are also essential for the initiation of DNA replication. Our results therefore establish that the essential function of small non-coding stem-loop RNAs during DNA replication extends beyond vertebrates. PMID:25908866

  14. Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli.

    PubMed

    Nakashima, Nobutaka; Tamura, Tomohiro; Good, Liam

    2006-01-01

    Reliable methods for conditional gene silencing in bacteria have been elusive. To improve silencing by expressed antisense RNAs (asRNAs), we systematically altered several design parameters and targeted multiple reporter and essential genes in Escherichia coli. A paired termini (PT) design, where flanking inverted repeats create paired dsRNA termini, proved effective. PTasRNAs targeted against the ackA gene within the acetate kinase-phosphotransacetylase operon (ackA-pta) triggered target mRNA decay and a 78% reduction in AckA activity with high genetic penetrance. PTasRNAs are abundant and stable and function through an RNase III independent mechanism that requires a large stoichiometric excess of asRNA. Conditional ackA silencing reduced carbon flux to acetate and increased heterologous gene expression. The PT design also improved silencing of the essential fabI gene. Full anti-fabI PTasRNA induction prevented growth and partial induction sensitized cells to a FabI inhibitor. PTasRNAs have potential for functional genomics, antimicrobial discovery and metabolic flux control.

  15. Noncoding RNAs and immune checkpoints-clinical implications as cancer therapeutics.

    PubMed

    Smolle, Maria A; Calin, Horatiu N; Pichler, Martin; Calin, George A

    2017-07-01

    A major mechanism of tumor development and progression is silencing of the patient's immune response to cancer-specific antigens. Defects in the so-called cancer immunity cycle may occur at any stage of tumor development. Within the tumor microenvironment, aberrant expression of immune checkpoint molecules with activating or inhibitory effects on T lymphocytes induces immune tolerance and cellular immune escape. Targeting immune checkpoint molecules such as programmed cell death protein 1 (PD-1) and its ligand PD-L1 with specific antibodies has proven to be a major advance in the treatment of several types of cancer. Another way to therapeutically influence the tumor microenvironment is by modulating the levels of microRNAs (miRNAs), small noncoding RNAs that shuttle bidirectionally between malignant and tumor microenvironmental cells. These small RNA transcripts have two features: (a) their expression is quite specific to distinct tumors, and (b) they are involved in early regulation of immune responses. Consequently, miRNAs may be ideal molecules for use in cancer therapy. Many miRNAs are aberrantly expressed in human cancer cells, opening new opportunities for cancer therapy, but the exact functions of these miRNAs and their interactions with immune checkpoint molecules have yet to be investigated. This review summarizes recently reported findings about miRNAs as modulators of immune checkpoint molecules and their potential application as cancer therapeutics in clinical practice. © 2017 Federation of European Biochemical Societies.

  16. Retroposed SNOfall--a mammalian-wide comparison of platypus snoRNAs.

    PubMed

    Schmitz, Jürgen; Zemann, Anja; Churakov, Gennady; Kuhl, Heiner; Grützner, Frank; Reinhardt, Richard; Brosius, Jürgen

    2008-06-01

    Diversification of mammalian species began more than 160 million years ago when the egg-laying monotremes diverged from live bearing mammals. The duck-billed platypus (Ornithorhynchus anatinus) and echidnas are the only potential contemporary witnesses of this period and, thereby, provide a unique insight into mammalian genome evolution. It has become clear that small RNAs are major regulatory agents in eukaryotic cells, and the significant role of non-protein-coding (npc) RNAs in transcription, processing, and translation is now well accepted. Here we show that the platypus genome contains more than 200 small nucleolar (sno) RNAs among hundreds of other diverse npcRNAs. Their comparison among key mammalian groups and other vertebrates enabled us to reconstruct a complete temporal pathway of acquisition and loss of these snoRNAs. In platypus we found cis- and trans-duplication distribution patterns for snoRNAs, which have not been described in any other vertebrates but are known to occur in nematodes. An exciting novelty in platypus is a snoRNA-derived retroposon (termed snoRTE) that facilitates a very effective dispersal of an H/ACA snoRNA via RTE-mediated retroposition. From more than 40,000 detected full-length and truncated genomic copies of this snoRTE, at least 21 are processed into mature snoRNAs. High-copy retroposition via multiple host gene-promoted transcription units is a novel pathway for combining housekeeping function and SINE-like dispersal and reveals a new dimension in the evolution of novel snoRNA function.

  17. Expression profiling of snoRNAs in normal hematopoiesis and AML

    PubMed Central

    Warner, Wayne A.; Spencer, David H.; Trissal, Maria; White, Brian S.; Helton, Nichole; Ley, Timothy J.

    2018-01-01

    Small nucleolar RNAs (snoRNAs) are noncoding RNAs that contribute to ribosome biogenesis and RNA splicing by modifying ribosomal RNA and spliceosome RNAs, respectively. We optimized a next-generation sequencing approach and a custom analysis pipeline to identify and quantify expression of snoRNAs in acute myeloid leukemia (AML) and normal hematopoietic cell populations. We show that snoRNAs are expressed in a lineage- and development-specific fashion during hematopoiesis. The most striking examples involve snoRNAs located in 2 imprinted loci, which are highly expressed in hematopoietic progenitors and downregulated during myeloid differentiation. Although most snoRNAs are expressed at similar levels in AML cells compared with CD34+, a subset of snoRNAs showed consistent differential expression, with the great majority of these being decreased in the AML samples. Analysis of host gene expression, splicing patterns, and whole-genome sequence data for mutational events did not identify transcriptional patterns or genetic alterations that account for these expression differences. These data provide a comprehensive analysis of the snoRNA transcriptome in normal and leukemic cells and should be helpful in the design of studies to define the contribution of snoRNAs to normal and malignant hematopoiesis. PMID:29365324

  18. 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.

  19. Cell Cycle Regulation of Stem Cells by MicroRNAs.

    PubMed

    Mens, Michelle M J; Ghanbari, Mohsen

    2018-06-01

    MicroRNAs (miRNAs) are a class of small non-coding RNA molecules involved in the regulation of gene expression. They are involved in the fine-tuning of fundamental biological processes such as proliferation, differentiation, survival and apoptosis in many cell types. Emerging evidence suggests that miRNAs regulate critical pathways involved in stem cell function. Several miRNAs have been suggested to target transcripts that directly or indirectly coordinate the cell cycle progression of stem cells. Moreover, previous studies have shown that altered expression levels of miRNAs can contribute to pathological conditions, such as cancer, due to the loss of cell cycle regulation. However, the precise mechanism underlying miRNA-mediated regulation of cell cycle in stem cells is still incompletely understood. In this review, we discuss current knowledge of miRNAs regulatory role in cell cycle progression of stem cells. We describe how specific miRNAs may control cell cycle associated molecules and checkpoints in embryonic, somatic and cancer stem cells. We further outline how these miRNAs could be regulated to influence cell cycle progression in stem cells as a potential clinical application.

  20. Soil Nutrient Content Influences the Abundance of Soil Microbes but Not Plant Biomass at the Small-Scale

    PubMed Central

    Koorem, Kadri; Gazol, Antonio; Öpik, Maarja; Moora, Mari; Saks, Ülle; Uibopuu, Annika; Sõber, Virve; Zobel, Martin

    2014-01-01

    Small-scale heterogeneity of abiotic and biotic factors is expected to play a crucial role in species coexistence. It is known that plants are able to concentrate their root biomass into areas with high nutrient content and also acquire nutrients via symbiotic microorganisms such as arbuscular mycorrhizal (AM) fungi. At the same time, little is known about the small-scale distribution of soil nutrients, microbes and plant biomass occurring in the same area. We examined small-scale temporal and spatial variation as well as covariation of soil nutrients, microbial biomass (using soil fatty acid biomarker content) and above- and belowground biomass of herbaceous plants in a natural herb-rich boreonemoral spruce forest. The abundance of AM fungi and bacteria decreased during the plant growing season while soil nutrient content rather increased. The abundance of all microbes studied also varied in space and was affected by soil nutrient content. In particular, the abundance of AM fungi was negatively related to soil phosphorus and positively influenced by soil nitrogen content. Neither shoot nor root biomass of herbaceous plants showed any significant relationship with variation in soil nutrient content or the abundance of soil microbes. Our study suggests that plants can compensate for low soil phosphorus concentration via interactions with soil microbes, most probably due to a more efficient symbiosis with AM fungi. This compensation results in relatively constant plant biomass despite variation in soil phosphorous content and in the abundance of AM fungi. Hence, it is crucial to consider both soil nutrient content and the abundance of soil microbes when exploring the mechanisms driving vegetation patterns. PMID:24637633