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Sample records for 24-nt small rnas

  1. Arabidopsis AGO3 predominantly recruits 24-nt small RNAs to regulate epigenetic silencing.

    PubMed

    Zhang, Zhonghui; Liu, Xiuying; Guo, Xinwei; Wang, Xiu-Jie; Zhang, Xiuren

    2016-01-01

    Argonaute (AGO) proteins recruit 21-24-nucleotide (nt) small RNAs (sRNAs) to constitute RNA-induced silencing complexes (RISCs) to regulate gene expression at transcriptional or posttranscriptional levels(1-3). Arabidopsis encodes nine functional AGO proteins. These proteins are classified into three clusters, AGO1/5/10, AGO2/3/7 and AGO4/6/9, based on their sequence similarity, functional redundancy, as well as species and features of AGO-bound sRNAs(4-7). Although most Arabidopsis AGO proteins have been studied well, AGO3-bound sRNAs and their basic function remain unknown. Here we observed that AGO3 could not complement the signature function of AGO2, the closest genetic paralog of AGO3, in host antiviral defence. We also found, surprisingly, that AGO3 predominantly bound 24-nt sRNAs with 5'-terminal adenine. The spectrum of AGO3-associated sRNAs was different from those bound to AGO2, further indicating their functional divergence. By contrast, approximately 30% of AGO3-bound 24-nt sRNAs overlapped with those bound to AGO4, and over 60% of AGO3-associated 24-nt sRNA-enriched loci were identical to those of AGO4. Moreover, the redundancy of AGO3- and AGO4-bound sRNAs is much more than that of AGO6- and AGO4-recruited sRNAs. In addition, expression of AGO3 driven by the AGO4 promoter partially complemented AGO4 function and rescued a DNA methylation defect in the ago4-1 background. Together, our results indicated that AGO3, similarly to AGO4, is a component in the epigenetic pathway. PMID:27243648

  2. Citrus tristeza virus infection induces the accumulation of viral small RNAs (21-24-nt) mapping preferentially at the 3'-terminal region of the genomic RNA and affects the host small RNA profile.

    PubMed

    Ruiz-Ruiz, Susana; Navarro, Beatriz; Gisel, Andreas; Peña, Leandro; Navarro, Luis; Moreno, Pedro; Di Serio, Francesco; Flores, Ricardo

    2011-04-01

    To get an insight into the host RNA silencing defense induced by Citrus tristeza virus (CTV) and into the counter defensive reaction mediated by its three silencing suppressors (p25, p20 and p23), we have examined by deep sequencing (Solexa-Illumina) the small RNAs (sRNAs) in three virus-host combinations. Our data show that CTV sRNAs: (i) represent more than 50% of the total sRNAs in Mexican lime and sweet orange (where CTV reaches relatively high titers), but only 3.5% in sour orange (where the CTV titer is significantly lower), (ii) are predominantly of 21-22-nt, with a biased distribution of their 5' nucleotide and with those of (+) polarity accumulating in a moderate excess, and (iii) derive from essentially all the CTV genome (ca. 20 kb), as revealed by its complete reconstruction from viral sRNA contigs, but adopt an asymmetric distribution with a prominent hotspot covering approximately the 3'-terminal 2,500 nt. These results suggest that the citrus homologues of Dicer-like (DCL) 4 and 2 most likely mediate the genesis of the 21 and 22 nt CTV sRNAs, respectively, and show that both ribonucleases act not only on the genomic RNA but also on the 3' co-terminal subgenomic RNAs and, particularly, on their double-stranded forms. The plant sRNA profile, very similar and dominated by the 24-nt sRNAs in the three mock-inoculated controls, was minimally affected by CTV infection in sour orange, but exhibited a significant reduction of the 24-nt sRNAs in Mexican lime and sweet orange. We have also identified novel citrus miRNAs and determined how CTV influences their accumulation. PMID:21327514

  3. Dicer-like 3 produces transposable element-associated 24-nt siRNAs that control agricultural traits in rice.

    PubMed

    Wei, Liya; Gu, Lianfeng; Song, Xianwei; Cui, Xiekui; Lu, Zhike; Zhou, Ming; Wang, Lulu; Hu, Fengyi; Zhai, Jixian; Meyers, Blake C; Cao, Xiaofeng

    2014-03-11

    Transposable elements (TEs) and repetitive sequences make up over 35% of the rice (Oryza sativa) genome. The host regulates the activity of different TEs by different epigenetic mechanisms, including DNA methylation, histone H3K9 methylation, and histone H3K4 demethylation. TEs can also affect the expression of host genes. For example, miniature inverted repeat TEs (MITEs), dispersed high copy-number DNA TEs, can influence the expression of nearby genes. In plants, 24-nt small interfering RNAs (siRNAs) are mainly derived from repeats and TEs. However, the extent to which TEs, particularly MITEs associated with 24-nt siRNAs, affect gene expression remains elusive. Here, we show that the rice Dicer-like 3 homolog OsDCL3a is primarily responsible for 24-nt siRNA processing. Impairing OsDCL3a expression by RNA interference caused phenotypes affecting important agricultural traits; these phenotypes include dwarfism, larger flag leaf angle, and fewer secondary branches. We used small RNA deep sequencing to identify 535,054 24-nt siRNA clusters. Of these clusters, ∼82% were OsDCL3a-dependent and showed significant enrichment of MITEs. Reduction of OsDCL3a function reduced the 24-nt siRNAs predominantly from MITEs and elevated expression of nearby genes. OsDCL3a directly targets genes involved in gibberellin and brassinosteroid homeostasis; OsDCL3a deficiency may affect these genes, thus causing the phenotypes of dwarfism and enlarged flag leaf angle. Our work identifies OsDCL3a-dependent 24-nt siRNAs derived from MITEs as broadly functioning regulators for fine-tuning gene expression, which may reflect a conserved epigenetic mechanism in higher plants with genomes rich in dispersed repeats or TEs. PMID:24554078

  4. Mobile small RNAs regulate genome-wide DNA methylation

    PubMed Central

    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.

    2016-01-01

    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. PMID:26787884

  5. Genome-Wide Discovery and Analysis of Phased Small Interfering RNAs in Chinese Sacred Lotus

    PubMed Central

    Zheng, Yun; Wang, Shengpeng; Sunkar, Ramanjulu

    2014-01-01

    Phased small interfering RNA (phasiRNA) generating loci (briefly as PHAS) in plants are a novel class of genes that are normally regulated by microRNAs (miRNAs). Similar to miRNAs, phasiRNAs encoded by PHAS play important regulatory roles by targeting protein coding transcripts in plant species. We performed a genome-wide discovery of PHAS loci in Chinese sacred lotus and identified a total of 106 PHAS loci. Of these, 47 loci generate 21 nucleotide (nt) phasiRNAs and 59 loci generate 24 nt phasiRNAs, respectively. We have also identified a new putative TAS3 and a putative TAS4 loci in the lotus genome. Our results show that some of the nucleotide-binding, leucine-rich repeat (NB-LRR) disease resistance proteins and MYB transcription factors potentially generate phasiRNAs. Furthermore, our results suggest that some large subunit (LSU) rRNAs can derive putative phasiRNAs, which is potentially resulted from crosstalk between small RNA biogenesis pathways that are employed to process rRNAs and PHAS loci, respectively. Some of the identified phasiRNAs have putative trans-targets with less than 4 mismatches, suggesting that the identified PHAS are involved in many different pathways. Finally, the discovery of 24 nt PHAS in lotus suggests that there are 24 nt PHAS in dicots. PMID:25469507

  6. Recent advances in plant-virus interaction with emphasis on small interfering RNAs (siRNAs).

    PubMed

    Sharma, Namisha; Sahu, Pranav Pankaj; Puranik, Swati; Prasad, Manoj

    2013-09-01

    Regulation of several biological functions in plants has now been known to involve diverse RNA silencing pathways. These vital pathways involve various components such as dsRNA, Dicer, RNA-dependent RNA polymerase and Argonaute proteins, which lead to the production of several small RNAs (sRNAs) varying in their sizes. These sRNAs have significant role in the regulation of gene expression at transcriptional and translational levels. Among them, small interfering RNAs (siRNAs; majorly 21, 22 and 24 nt) have been shown to play an important role in plants' resistance against many viruses by inhibiting the viral gene expression. Furthermore, it has also been highlighted that siRNA-mediated methylation of viral DNA confers resistance to various plant DNA viruses. In this review, we have outlined the recent advances made using the siRNA-mediated antiviral strategy, along with methylation-based epigenetic defensive mechanisms as a protective measure against diverse plant viruses. PMID:23086491

  7. Novel microRNAs uncovered by deep sequencing of small RNA transcriptomes in bread wheat (Triticum aestivum L.) and Brachypodium distachyon (L.) Beauv

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brachypodium distachyon is a promising model for studying temperate grasses, most importantly wheat. Small RNA (smRNAs), especially 21 nt and 24 nt smRNAs, play essential roles in plant development and physiology. We performed seep sequencing of smRNA repertoires in Brachypodium and wheat and found ...

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

    PubMed

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

    2006-10-01

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

  9. The Inheritance Pattern of 24 nt siRNA Clusters in Arabidopsis Hybrids Is Influenced by Proximity to Transposable Elements

    PubMed Central

    Li, Ying; Varala, Kranthi; Moose, Stephen P.; Hudson, Matthew E.

    2012-01-01

    Hybrids often display increased size and growth, and thus are widely cultivated in agriculture and horticulture. Recent discoveries demonstrating the important regulatory roles of small RNAs have greatly improved our understanding of many basic biological questions, and could illuminate the molecular basis for the enhanced growth and size of hybrid plants. We profiled small RNAs by deep sequencing to characterize the inheritance patterns of small RNA levels in reciprocal hybrids of two Arabidopsis thaliana accessions, Columbia and Landsberg erecta. We find 24-nt siRNAs predominate among those small RNAs that are differentially expressed between the parents. Following hybridization, the transposable element (TE)-derived siRNAs are often inherited in an additive manner, whereas siRNAs associated with protein-coding genes are often down-regulated in hybrids to the levels observed for the parent with lower relative siRNA levels. Among the protein-coding genes that exhibit this pattern, genes that function in pathogen defense, abiotic stress tolerance, and secondary metabolism are significantly enriched. Small RNA clusters from protein-coding genes where a TE is present within one kilobase show a different predominant inheritance pattern (additive) from those that do not (low-parent dominance). Thus, down-regulation in the form of low-parent dominance is likely the default pattern of inheritance for genic siRNA, and a different inheritance mechanism for TE siRNA is suggested. PMID:23118865

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

    PubMed

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

    2006-01-01

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

  11. Small regulatory RNAs in Archaea

    PubMed Central

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

    2014-01-01

    Small regulatory RNAs (sRNAs) are universally distributed in all three domains of life, Archaea, Bacteria, and Eukaryotes. In bacteria, sRNAs typically function by binding near the translation start site of their target mRNAs and thereby inhibit or activate translation. In eukaryotes, miRNAs and siRNAs typically bind to the 3′-untranslated region (3′-UTR) of their target mRNAs and influence translation efficiency and/or mRNA stability. In archaea, sRNAs have been identified in all species investigated using bioinformatic approaches, RNomics, and RNA-Seq. Their size can vary significantly between less than 50 to more than 500 nucleotides. Differential expression of sRNA genes has been studied using northern blot analysis, microarrays, and RNA-Seq. In addition, biological functions have been unraveled by genetic approaches, i.e., by characterization of designed mutants. As in bacteria, it was revealed that archaeal sRNAs are involved in many biological processes, including metabolic regulation, adaptation to extreme conditions, stress responses, and even in regulation of morphology and cellular behavior. Recently, the first target mRNAs were identified in archaea, including one sRNA that binds to the 5′-region of two mRNAs in Methanosarcina mazei Gö1 and a few sRNAs that bind to 3′-UTRs in Sulfolobus solfataricus, three Pyrobaculum species, and Haloferax volcanii, indicating that archaeal sRNAs appear to be able to target both the 5′-UTR or the 3′-UTRs of their respective target mRNAs. In addition, archaea contain tRNA-derived fragments (tRFs), and one tRF has been identified as a major ribosome-binding sRNA in H. volcanii, which downregulates translation in response to stress. Besides regulatory sRNAs, archaea contain further classes of sRNAs, e.g., CRISPR RNAs (crRNAs) and snoRNAs. PMID:24755959

  12. Creating small transcription activating RNAs.

    PubMed

    Chappell, James; Takahashi, Melissa K; Lucks, Julius B

    2015-03-01

    We expanded the mechanistic capability of small RNAs by creating an entirely synthetic mode of regulation: small transcription activating RNAs (STARs). Using two strategies, we engineered synthetic STAR regulators to disrupt the formation of an intrinsic transcription terminator placed upstream of a gene in Escherichia coli. This resulted in a group of four highly orthogonal STARs that had up to 94-fold activation. By systematically modifying sequence features of this group, we derived design principles for STAR function, which we then used to forward engineer a STAR that targets a terminator found in the Escherichia coli genome. Finally, we showed that STARs could be combined in tandem to create previously unattainable RNA-only transcriptional logic gates. STARs provide a new mechanism of regulation that will expand our ability to use small RNAs to construct synthetic gene networks that precisely control gene expression. PMID:25643173

  13. Genome-wide profiling of Populus small RNAs

    PubMed Central

    2009-01-01

    Background Short RNAs, and in particular microRNAs, are important regulators of gene expression both within defined regulatory pathways and at the epigenetic scale. We investigated the short RNA (sRNA) population (18-24 nt) of the transcriptome of green leaves from the sequenced Populus trichocarpa using a concatenation strategy in combination with 454 sequencing. Results The most abundant size class of sRNAs were 24 nt. Long Terminal Repeats were particularly associated with 24 nt sRNAs. Additionally, some repetitive elements were associated with 22 nt sRNAs. We identified an sRNA hot-spot on chromosome 19, overlapping a region containing both the proposed sex-determining locus and a major cluster of NBS-LRR genes. A number of phased siRNA loci were identified, a subset of which are predicted to target PPR and NBS-LRR disease resistance genes, classes of genes that have been significantly expanded in Populus. Additional loci enriched for sRNA production were identified and characterised. We identified 15 novel predicted microRNAs (miRNAs), including miRNA*sequences, and identified a novel locus that may encode a dual miRNA or a miRNA and short interfering RNAs (siRNAs). Conclusions The short RNA population of P. trichocarpa is at least as complex as that of Arabidopsis thaliana. We provide a first genome-wide view of short RNA production for P. trichocarpa and identify new, non-conserved miRNAs. PMID:20021695

  14. Genome-wide analysis for discovery of new rice miRNA reveals natural antisense miRNA (nat-miRNAs)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Small RNAs (21-24nt) are involved in gene regulation through translation inhibition, mRNA cleavage, or directing chromatin modifications. In rice, currently ~240 miRNAs have been annotated. We sequenced more than four million small RNAs from rice and identified another 24 miRNA genes. Among these, w...

  15. Role of Small RNAs in Trypanosomatid Infections

    PubMed Central

    Linhares-Lacerda, Leandra; Morrot, Alexandre

    2016-01-01

    Trypanosomatid parasites survive and replicate in the host by using mechanisms that aim to establish a successful infection and ensure parasite survival. Evidence points to microRNAs as new players in the host-parasite interplay. MicroRNAs are small non-coding RNAs that control proteins levels via post-transcriptional gene down-regulation, either within the cells where they were produced or in other cells via intercellular transfer. These microRNAs can be modulated in host cells during infection and are among the growing group of small regulatory RNAs, for which many classes have been described, including the transfer RNA-derived small RNAs. Parasites can either manipulate microRNAs to evade host-driven damage and/or transfer small RNAs to host cells. In this mini-review, we present evidence for the involvement of small RNAs, such as microRNAs, in trypanosomatid infections which lack RNA interference. We highlight both microRNA profile alterations in host cells during those infections and the horizontal transfer of small RNAs and proteins from parasites to the host by membrane-derived extracellular vesicles in a cell communication mechanism. PMID:27065454

  16. Detection of small RNAs in Xylella fastidiosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Non-coding small RNAs (sRNAs) are regarded as ubiquitous regulatory elements in bacteria. For Xylella fastidiosa, a plant pathogen causing many economically important crop diseases, research attention to sRNAs has been limited. With the availability of whole genome sequences and increasing bioinfor...

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

    PubMed

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

    2014-08-01

    Viroids are small (246-401 nt) circular and non coding RNAs infecting higher plants. They are targeted by host Dicer-like enzymes (DCLs) that generate small RNAs of 21-24 nt (sRNAs), which are involved in the host RNA silencing pathways. The accumulation in plant tissues of such viroid-derived small RNAs (vd-sRNAs) is a clear sign of an ongoing viroid infection. In this study, next generation sequencing of a sRNAs library and assembling of the sequenced vd-sRNAs were instrumental for the identification of a viroid resembling apple dimple fruit viroid (ADFVd) in a fig accession. After confirming by molecular methods the presence of this viroid in the fig tree, its population was characterized, showing that the ADFVd master sequence from fig diverges from that of the ADFVd reference variant from apple. Moreover, since this viroid accumulates at a low level in fig, a semi-nested RT-PCR assay was developed for detecting it in other fig accessions. ADFVd seems to have a wider host range than thought before and this poses questions about its epidemiology. A further characterization of ADFVd-sRNAs showed similar accumulation of (+) or (-) vd-sRNAs that mapped on the viroid genome generating hotspot profiles. Moreover, similarly to other nuclear-replicating viroids, vd-sRNAs of 21, 22 and 24 nt in size prevailed in the distribution profiles. Altogether, these data support the involvement of double-stranded RNAs and different DCLs, targeting the same restricted viroid regions, in the genesis of ADFVd-sRNAs. PMID:24704673

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

    PubMed Central

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

    2015-01-01

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

  19. A novel class of heat-responsive small RNAs derived from the chloroplast genome of Chinese cabbage (Brassica rapa)

    PubMed Central

    2011-01-01

    Background Non-coding small RNAs play critical roles in various cellular processes in a wide spectrum of eukaryotic organisms. Their responses to abiotic stress have become a popular topic of economic and scientific importance in biological research. Several studies in recent years have reported a small number of non-coding small RNAs that map to chloroplast genomes. However, it remains uncertain whether small RNAs are generated from chloroplast genome and how they respond to environmental stress, such as high temperature. Chinese cabbage is an important vegetable crop, and heat stress usually causes great losses in yields and quality. Under heat stress, the leaves become etiolated due to the disruption and disassembly of chloroplasts. In an attempt to determine the heat-responsive small RNAs in chloroplast genome of Chinese cabbage, we carried out deep sequencing, using heat-treated samples, and analysed the proportion of small RNAs that were matched to chloroplast genome. Results Deep sequencing provided evidence that a novel subset of small RNAs were derived from the chloroplast genome of Chinese cabbage. The chloroplast small RNAs (csRNAs) include those derived from mRNA, rRNA, tRNA and intergenic RNA. The rRNA-derived csRNAs were preferentially located at the 3'-ends of the rRNAs, while the tRNA-derived csRNAs were mainly located at 5'-termini of the tRNAs. After heat treatment, the abundance of csRNAs decreased in seedlings, except those of 24 nt in length. The novel heat-responsive csRNAs and their locations in the chloroplast were verified by Northern blotting. The regulation of some csRNAs to the putative target genes were identified by real-time PCR. Our results reveal that high temperature suppresses the production of some csRNAs, which have potential roles in transcriptional or post-transcriptional regulation. Conclusions In addition to nucleus, the chloroplast is another important organelle that generates a number of small RNAs. Many members of cs

  20. Roles of small RNAs in tumor formation

    PubMed Central

    Di Leva, Gianpiero; Croce, Carlo M.

    2010-01-01

    MicroRNAs (miRNAs) are small noncoding RNAs that act as post-transcriptional repressors of gene expression in organisms ranging from plants to humans. A widespread role for miRNAs in diverse molecular processes driving initiation and progression of various tumor types has recently been described. In this review, we discuss the etiology of aberrant expression of miRNAs in human cancers and their role in tumor metastasis, which may define miRNAs as oncogenes or tumor suppressors. Moreover, we highlight the genomic/epigenetic alterations and transcriptional/post-transcriptional mechanisms associated with misexpression of miRNAs in cancer. A better understanding of miRNA biology may ultimately yield further insight into the molecular mechanisms of tumorigenesis and new therapeutic strategies against cancer. PMID:20493775

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

    PubMed

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

    2016-04-01

    Infection of plant cells by viral pathogens triggers RNA silencing, an innate antiviral defense mechanism. In response to infection, small RNAs (sRNAs) are produced that associate with Argonaute (AGO)-containing silencing complexes which act to inactivate viral genomes by posttranscriptional gene silencing (PTGS). Deep sequencing was used to compare virus-derived small RNAs (vsRNAs) in cassava genotypes NASE 3, TME 204 and 60444 infected with the positive sense single-stranded RNA (+ssRNA) viruses cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), the causal agents of cassava brown streak disease (CBSD). An abundance of 21-24nt vsRNAs was detected and mapped, covering the entire CBSV and UCBSV genomes. The 21nt vsRNAs were most predominant, followed by the 22 nt class with a slight bias toward sense compared to antisense polarity, and a bias for adenine and uracil bases present at the 5'-terminus. Distribution and frequency of vsRNAs differed between cassava genotypes and viral genomes. In susceptible genotypes TME 204 and 60444, CBSV-derived sRNAs were seen in greater abundance than UCBSV-derived sRNAs. NASE 3, known to be resistant to UCBSV, accumulated negligible UCBSV-derived sRNAs but high populations of CBSV-derived sRNAs. Transcript levels of cassava homologues of AGO2, DCL2 and DCL4, which are central to the gene-silencing complex, were found to be differentially regulated in CBSV- and UCBSV-infected plants across genotypes, suggesting these proteins play a role in antiviral defense. Irrespective of genotype or viral pathogen, maximum populations of vsRNAs mapped to the cytoplasmic inclusion, P1 and P3 protein-encoding regions. Our results indicate disparity between CBSV and UCBSV host-virus interaction mechanisms, and provide insight into the role of virus-induced gene silencing as a mechanism of resistance to CBSD. PMID:26811902

  2. 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

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

    PubMed

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

    2015-01-01

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

  4. Production of small RNAs by mammalian Dicer.

    PubMed

    Svobodova, Eliska; Kubikova, Jana; Svoboda, Petr

    2016-06-01

    MicroRNA (miRNA) and RNA interference (RNAi) pathways employ RNase III Dicer for the biogenesis of small RNAs guiding post-transcriptional repression. Requirements for Dicer activity differ in the two pathways. The biogenesis of miRNAs requires a single Dicer cleavage of a short hairpin precursor to produce a small RNA with a precisely defined sequence, while small RNAs in RNAi come from a processive cleavage of a long double-stranded RNA (dsRNA) into a pool of small RNAs with different sequences. While Dicer is generally conserved among eukaryotes, its substrate recognition, cleavage, and biological roles differ. In Metazoa, a single Dicer can function as a universal factor for RNAi and miRNA pathways or as a factor adapted specifically for one of the pathways. In this review, we focus on the structure, function, and evolution of mammalian Dicer. We discuss key structural features of Dicer and other factors defining Dicer substrate repertoire and biological functions in mammals in comparison with invertebrate models. The key for adaptation of Dicer for miRNA or RNAi pathways is the N-terminal helicase, a dynamically evolving Dicer domain. Its functionality differs between mammals and invertebrates: the mammalian Dicer is well adapted to produce miRNAs while its ability to support RNAi is limited. PMID:27048428

  5. Specific argonautes selectively bind small RNAs derived from potato spindle tuber viroid and attenuate viroid accumulation in vivo.

    PubMed

    Minoia, Sofia; Carbonell, Alberto; Di Serio, Francesco; Gisel, Andreas; Carrington, James C; Navarro, Beatriz; Flores, Ricardo

    2014-10-01

    The identification of viroid-derived small RNAs (vd-sRNAs) of 21 to 24 nucleotides (nt) in plants infected by viroids (infectious non-protein-coding RNAs of just 250 to 400 nt) supports their targeting by Dicer-like enzymes, the first host RNA-silencing barrier. However, whether viroids, like RNA viruses, are also targeted by the RNA-induced silencing complex (RISC) remains controversial. At the RISC core is one Argonaute (AGO) protein that, guided by endogenous or viral sRNAs, targets complementary RNAs. To examine whether AGO proteins also load vd-sRNAs, leaves of Nicotiana benthamiana infected by potato spindle tuber viroid (PSTVd) were agroinfiltrated with plasmids expressing epitope-tagged versions of AGO1, AGO2, AGO3, AGO4, AGO5, AGO6, AGO7, AGO9, and AGO10 from Arabidopsis thaliana. Immunoprecipitation analyses of the agroinfiltrated halos revealed that all AGOs except AGO6, AGO7, and AGO10 associated with vd-sRNAs: AGO1, AGO2, and AGO3 preferentially with those of 21 and 22 nt, while AGO4, AGO5, and AGO9 additionally bound those of 24 nt. Deep-sequencing analyses showed that sorting of vd-sRNAs into AGO1, AGO2, AGO4, and AGO5 depended essentially on their 5'-terminal nucleotides, with the profiles of the corresponding AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Furthermore, agroexpression of AGO1, AGO2, AGO4, and AGO5 on PSTVd-infected tissue attenuated the level of the genomic RNAs, suggesting that they, or their precursors, are RISC targeted. In contrast to RNA viruses, PSTVd infection of N. benthamiana did not affect miR168-mediated regulation of the endogenous AGO1, which loaded vd-sRNAs with specificity similar to that of its A. thaliana counterpart. Importance: To contain invaders, particularly RNA viruses, plants have evolved an RNA-silencing mechanism relying on the generation by Dicer-like (DCL) enzymes of virus-derived small RNAs of 21 to 24 nucleotides (nt) that load and guide Argonaute (AGO) proteins to

  6. Small RNAs in the Genus Clostridium

    PubMed Central

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

    2011-01-01

    The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny. PMID:21264064

  7. Characterization of Small RNAs Derived from tRNAs, rRNAs and snoRNAs and Their Response to Heat Stress in Wheat Seedlings

    PubMed Central

    Sun, Qixin; Yao, Yingyin

    2016-01-01

    Small RNAs (sRNAs) derived from non-coding RNAs (ncRNAs), such as tRNAs, rRNAs and snoRNAs, have been identified in various organisms. Several observations have indicated that cleavage of tRNAs and rRNAs is induced by various stresses. To clarify whether sRNAs in wheat derived from tRNAs (stRNAs), rRNAs (srRNAs) and snoRNAs (sdRNAs) are produced specifically in association with heat stress responses, we carried out a bioinformatic analysis of sRNA libraries from wheat seedlings and performed comparisons between control and high-temperature-treated samples to measure the differential abundance of stRNAs, srRNAs and sdRNAs. We found that the production of sRNAs from tRNAs, 5.8S rRNAs, and 28S rRNAs was more specific than that from 5S rRNAs and 18S rRNAs, and more than 95% of the stRNAs were processed asymmetrically from the 3’ or 5’ ends of mature tRNAs. We identified 333 stRNAs and 8,822 srRNAs that were responsive to heat stress. Moreover, the expression of stRNAs derived from tRNA-Val-CAC, tRNA-Thr-UGU, tRNA-Tyr-GUA and tRNA-Ser-UGA was not only up-regulated under heat stress but also induced by osmotic stress, suggesting that the increased cleavage of tRNAs might be a mechanism that developed in wheat seedlings to help them cope with adverse environmental conditions. PMID:26963812

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

    NASA Astrophysics Data System (ADS)

    Chu, Chia-Ying; Rana, Tariq M.

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

  9. High throughput sequencing of small RNAs in Potato Leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. Genome-wide analysis of single non-templated nucleotides in plant endogenous siRNAs and miRNAs

    PubMed Central

    Wang, Feng; Johnson, Nathan R.; Coruh, Ceyda; Axtell, Michael J.

    2016-01-01

    Plant small RNAs are subject to various modifications. Previous reports revealed widespread 3′ modifications (truncations and non-templated tailing) of plant miRNAs when the 2′-O-methyltransferase HEN1 is absent. However, non-templated nucleotides in plant heterochromatic siRNAs have not been deeply studied, especially in wild-type plants. We systematically studied non-templated nucleotide patterns in plant small RNAs by analyzing small RNA sequencing libraries from Arabidopsis, tomato, Medicago, rice, maize and Physcomitrella. Elevated rates of non-templated nucleotides were observed at the 3′ ends of both miRNAs and endogenous siRNAs from wild-type specimens of all species. ‘Off-sized’ small RNAs, such as 25 and 23 nt siRNAs arising from loci dominated by 24 nt siRNAs, often had very high rates of 3′-non-templated nucleotides. The same pattern was observed in all species that we studied. Further analysis of 24 nt siRNA clusters in Arabidopsis revealed distinct patterns of 3′-non-templated nucleotides of 23 nt siRNAs arising from heterochromatic siRNA loci. This pattern of non-templated 3′ nucleotides on 23 nt siRNAs is not affected by loss of known small RNA 3′-end modifying enzymes, and may result from modifications added to longer heterochromatic siRNA precursors. PMID:27207877

  11. Genome-wide analysis of single non-templated nucleotides in plant endogenous siRNAs and miRNAs.

    PubMed

    Wang, Feng; Johnson, Nathan R; Coruh, Ceyda; Axtell, Michael J

    2016-09-01

    Plant small RNAs are subject to various modifications. Previous reports revealed widespread 3' modifications (truncations and non-templated tailing) of plant miRNAs when the 2'-O-methyltransferase HEN1 is absent. However, non-templated nucleotides in plant heterochromatic siRNAs have not been deeply studied, especially in wild-type plants. We systematically studied non-templated nucleotide patterns in plant small RNAs by analyzing small RNA sequencing libraries from Arabidopsis, tomato, Medicago, rice, maize and Physcomitrella Elevated rates of non-templated nucleotides were observed at the 3' ends of both miRNAs and endogenous siRNAs from wild-type specimens of all species. 'Off-sized' small RNAs, such as 25 and 23 nt siRNAs arising from loci dominated by 24 nt siRNAs, often had very high rates of 3'-non-templated nucleotides. The same pattern was observed in all species that we studied. Further analysis of 24 nt siRNA clusters in Arabidopsis revealed distinct patterns of 3'-non-templated nucleotides of 23 nt siRNAs arising from heterochromatic siRNA loci. This pattern of non-templated 3' nucleotides on 23 nt siRNAs is not affected by loss of known small RNA 3'-end modifying enzymes, and may result from modifications added to longer heterochromatic siRNA precursors. PMID:27207877

  12. Deep sequencing of the small RNAs derived from two symptomatic variants of a chloroplastic viroid: implications for their genesis and for pathogenesis.

    PubMed

    Di Serio, Francesco; Gisel, Andreas; Navarro, Beatriz; Delgado, Sonia; Martínez de Alba, Angel-Emilio; Donvito, Giacinto; Flores, Ricardo

    2009-01-01

    Northern-blot hybridization and low-scale sequencing have revealed that plants infected by viroids, non-protein-coding RNA replicons, accumulate 21-24 nt viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs, the hallmarks of RNA silencing. These results strongly support that viroids are elicitors and targets of the RNA silencing machinery of their hosts. Low-scale sequencing, however, retrieves partial datasets and may lead to biased interpretations. To overcome this restraint we have examined by deep sequencing (Solexa-Illumina) and computational approaches the vd-sRNAs accumulating in GF-305 peach seedlings infected by two molecular variants of Peach latent mosaic viroid (PLMVd) inciting peach calico (albinism) and peach mosaic. Our results show in both samples multiple PLMVd-sRNAs, with prevalent 21-nt (+) and (-) RNAs presenting a biased distribution of their 5' nucleotide, and adopting a hotspot profile along the genomic (+) and (-) RNAs. Dicer-like 4 and 2 (DCL4 and DCL2, respectively), which act hierarchically in antiviral defense, likely also mediate the genesis of the 21- and 22-nt PLMVd-sRNAs. More specifically, because PLMVd replicates in plastids wherein RNA silencing has not been reported, DCL4 and DCL2 should dice the PLMVd genomic RNAs during their cytoplasmic movement or the PLMVd-dsRNAs generated by a cytoplasmic RNA-dependent RNA polymerase (RDR), like RDR6, acting in concert with DCL4 processing. Furthermore, given that vd-sRNAs derived from the 12-14-nt insertion containing the pathogenicity determinant of peach calico are underrepresented, it is unlikely that symptoms may result from the accidental targeting of host mRNAs by vd-sRNAs from this determinant guiding the RNA silencing machinery. PMID:19847296

  13. An atlas of soybean small RNAs identifies phased siRNAs from hundreds of coding genes.

    PubMed

    Arikit, Siwaret; Xia, Rui; Kakrana, Atul; Huang, Kun; Zhai, Jixian; Yan, Zhe; Valdés-López, Oswaldo; Prince, Silvas; Musket, Theresa A; Nguyen, Henry T; Stacey, Gary; Meyers, Blake C

    2014-12-01

    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

  14. Ribo-gnome: the big world of small RNAs.

    PubMed

    Zamore, Phillip D; Haley, Benjamin

    2005-09-01

    Small RNA guides--microRNAs, small interfering RNAs, and repeat-associated small interfering RNAs, 21 to 30 nucleotides in length--shape diverse cellular pathways, from chromosome architecture to stem cell maintenance. Fifteen years after the discovery of RNA silencing, we are only just beginning to understand the depth and complexity of how these RNAs regulate gene expression and to consider their role in shaping the evolutionary history of higher eukaryotes. PMID:16141061

  15. Horizontal Transfer of Small RNAs to and from Plants

    PubMed Central

    Han, Lu; Luan, Yu-Shi

    2015-01-01

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

  16. Horizontal Transfer of Small RNAs to and from Plants.

    PubMed

    Han, Lu; Luan, Yu-Shi

    2015-01-01

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

  17. Big Impacts by Small RNAs in Plant Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The identification and study of small RNAs, including microRNAs and trans-acting small interfering RNAs, have added a layer of complexity to the many pathways that regulate plant development. These molecules, which function as negative regulators of gene expression, are now known to have greatly exp...

  18. Small RNAs as Guardians of the Genome

    PubMed Central

    Malone, Colin D.; Hannon, Gregory J.

    2009-01-01

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

  19. Widespread expression of conserved small RNAs in small symbiont genomes

    PubMed Central

    Hansen, Allison K; Degnan, Patrick H

    2014-01-01

    Genome architecture of a microbe markedly changes when it transitions from a free-living lifestyle to an obligate symbiotic association within eukaryotic cells. These symbiont genomes experience numerous rearrangements and massive gene loss, which is expected to radically alter gene regulatory networks compared with those of free-living relatives. As such, it remains unclear whether and how these small symbiont genomes regulate gene expression. Here, using a label-free mass-spec quantification approach we found that differential protein regulation occurs in Buchnera, a model symbiont with a reduced genome, when it transitions between two distinct life stages. However, differential mRNA expression could not be detected between Buchnera life stages, despite the presence of a small number of putative transcriptional regulators. Instead a comparative analysis of small RNA expression profiles among five divergent Buchnera lineages, spanning a variety of Buchnera life stages, reveals 140 novel intergenic and antisense small RNAs and 517 untranslated regions that were significantly expressed, some of which have been conserved for ∼65 million years. In addition, the majority of these small RNAs exhibit both sequence covariation and thermodynamic stability, indicators of a potential structural RNA role. Together, these data suggest that gene regulation at the post-transcriptional level may be important in Buchnera. This is the first study to empirically identify Buchnera small RNAs, and we propose that these novel small RNAs may facilitate post-transcriptional regulation through translational inhibition/activation, and/or transcript stability. Ultimately, post-transcriptional regulation may shape metabolic complementation between Buchnera and its aphid host, thus impacting the animal's ecology and evolution. PMID:25012903

  20. Widespread expression of conserved small RNAs in small symbiont genomes.

    PubMed

    Hansen, Allison K; Degnan, Patrick H

    2014-12-01

    Genome architecture of a microbe markedly changes when it transitions from a free-living lifestyle to an obligate symbiotic association within eukaryotic cells. These symbiont genomes experience numerous rearrangements and massive gene loss, which is expected to radically alter gene regulatory networks compared with those of free-living relatives. As such, it remains unclear whether and how these small symbiont genomes regulate gene expression. Here, using a label-free mass-spec quantification approach we found that differential protein regulation occurs in Buchnera, a model symbiont with a reduced genome, when it transitions between two distinct life stages. However, differential mRNA expression could not be detected between Buchnera life stages, despite the presence of a small number of putative transcriptional regulators. Instead a comparative analysis of small RNA expression profiles among five divergent Buchnera lineages, spanning a variety of Buchnera life stages, reveals 140 novel intergenic and antisense small RNAs and 517 untranslated regions that were significantly expressed, some of which have been conserved for ∼65 million years. In addition, the majority of these small RNAs exhibit both sequence covariation and thermodynamic stability, indicators of a potential structural RNA role. Together, these data suggest that gene regulation at the post-transcriptional level may be important in Buchnera. This is the first study to empirically identify Buchnera small RNAs, and we propose that these novel small RNAs may facilitate post-transcriptional regulation through translational inhibition/activation, and/or transcript stability. Ultimately, post-transcriptional regulation may shape metabolic complementation between Buchnera and its aphid host, thus impacting the animal's ecology and evolution. PMID:25012903

  1. Small RNAs meet their targets: When methylation defends miRNAs from uridylation

    PubMed Central

    Ren, Guodong; Chen, Xuemei; Yu, Bin

    2014-01-01

    Small RNAs are incorporated into Argonaute protein-containing complexes to guide the silencing of target RNAs in both animals and plants. The abundance of endogenous small RNAs is precisely controlled at multiple levels including transcription, processing and Argonaute loading. In addition to these processes, 3′ end modification of small RNAs, the topic of a research area that has rapidly evolved over the last several years, adds another layer of regulation of their abundance, diversity and function. Here, we review our recent understanding of small RNA 3′ end methylation and tailing. PMID:25483033

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

    PubMed

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

    2016-09-01

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

  3. Characterization of two small RNAs of Pseudomonas syringae DC3000

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Small RNAs (sRNAs) are important components of many regulatory pathways and have been shown to have key roles in regulation of factors important for virulence. To date, only a few sRNAs have been described for the bacterial plant pathogen Pseudomonas syringae. One approach to identify novel candid...

  4. The expanding world of small RNAs in plants

    PubMed Central

    Borges, Filipe; Martienssen, Robert A.

    2016-01-01

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

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

    PubMed

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

    2013-01-01

    Apomixis refers to plant asexual reproduction through seeds that give rise to progeny which are genotypically identical to the maternal parent. It has evolved from many different sexual taxa although the underlying genetic factors remain unknown. Previous analyses of the over-representation of transcription factors, in a comparison of microdissected ovules from apomictic and sexual Boechera, showed that many transcription factor mRNAs possessed microRNA (miRNAs) binding sites, thus pointing to miRNAs as potentially important factors that may be involved in the regulatory switch from sexual to apomictic reproduction. A microarray-based approach was used to identify (1) 673 microsatellitelike small RNAs (misRNAs) containing predominantly 2-7 repeats of (GAA)n/(CUU)n, (GCA)n/(CGU)n, (GGA)n/(CCU)n, (GGU)n/(CCA)n and (UGA)n/(ACU)n, and (2) 166 more typical non-repeat small RNAs. In total, 87 small RNAs were found to be located in cDNAs that could fold into stem-loop structures and thus represent miRNA molecules. In addition, 109 Boechera small RNAs including both misRNAs and non-repeat small RNAs, showed significant homology to 407 Arabidopsis thaliana small RNAs including the A. thaliana pollen-specific ath-miR5021. This indicates that only a fraction of the identified small RNAs are unique to Boechera. Ten small RNAs were validated using a Northern blot assay on flower and leaf tissues, eight of which showed flower-specific expression with varying abundance. The potential binding sites of many of the misRNAs and non-repeat small RNAs occur predominantly in exonic regions. This feature coupled with their flower-specific pattern of expression is suggestive of their probable role in post-transcriptional gene regulation. We propose that quantitative variation for misRNA target binding (and hence post-transcriptional gene regulation) could arise via microsatellite length polymorphisms occurring either in misRNA precursors or in their gene targets. PMID:25070713

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

    PubMed Central

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

    2008-01-01

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

  7. Analysis of small RNAs derived from Chinese wheat mosaic virus.

    PubMed

    Yang, Jian; Zheng, Shi-Ling; Zhang, Heng-Mu; Liu, Xiao-Ya; Li, Jing; Li, Jun-Min; Chen, Jian-Ping

    2014-11-01

    The virus-derived small interfering RNAs (vsiRNAs) of Chinese wheat mosaic virus (CWMV), a member of the genus Furovirus, were characterised from wheat plants by deep sequencing. CWMV vsiRNAs of 21-22 nt in length predominated, suggesting that there might be a conserved mechanism of DCL2 and DCL4 involvement in the biogenesis of vsiRNAs, as well as a common RNA silencing pathway in CWMV-infected wheat plants. The 5'-terminal base of vsiRNAs was biased towards A/U, suggesting that CWMV vsiRNAs might be loaded into diverse AGO-containing RISCs to disturb the gene expression of host plants. Possible targets for some of the vsiRNAs were predicted. PMID:24997977

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

    PubMed Central

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

    2015-01-01

    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. PMID:25944477

  9. Sources and Functions of Extracellular Small RNAs in Human Circulation.

    PubMed

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

    2016-07-17

    Various biotypes of endogenous small RNAs (sRNAs) have been detected in human circulation, including microRNAs, transfer RNAs, ribosomal RNA, 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 states and have, therefore, been proposed for use as effective biomarkers. Furthermore, exosome-borne ex-sRNAs have been reported to elicit physiological responses in acceptor cells. Exogenous ex-sRNAs derived from diet (most prominently from plants) and microorganisms have also been reported in human blood. Essential issues that remain to be conclusively addressed concern the (a) presence and sources of exogenous ex-sRNAs in human bodily fluids, (b) detection and measurement of ex-sRNAs in human circulation, (c) selectivity of ex-sRNA export and import, (d) sensitivity and specificity of ex-sRNA delivery to cellular targets, and (e) cell-, tissue-, organ-, and organism-wide impacts of ex-sRNA-mediated cell-to-cell communication. We survey the present state of knowledge of most of these issues in this review. PMID:27215587

  10. Small RNAs regulate plant responses to filamentous pathogens.

    PubMed

    Kuan, Tung; Zhai, Yi; Ma, Wenbo

    2016-08-01

    Small RNAs are central players of RNA silencing in eukaryotes. These short RNA molecules (20-25 nucleotides in length) repress target gene expression based on sequence complementarity. While small RNAs are well-known for their essential function in regulating growth and development, recent research has revealed that they also influence plant immunity. Extensive changes in small RNA accumulation have been observed during infection. This review focuses on specific small RNA changes that are involved in plant responses to filamentous eukaryotic pathogens including fungi and oomycetes. We describe how changes in small RNA accumulation influence plant immunity and summarize the cellular processes affected by these small RNAs. In particular, we discuss secondary small interfering RNAs that directly modulate the expression of defense-related genes. PMID:27208726

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Bai, Baoyan; Laiho, Marikki

    2016-01-01

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

  13. Small RNAs in Xylella fastidiosa and their epidemiological applications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial non-coding small RNAs (sRNAs) have attracted considerable attention due to their roles in regulating numerous cellular processes including survival, adaptation and pathogenesis. Sequence variation in sRNA genes reflect a previously unrecognized source of genomic diversity in bacteria. Xy...

  14. How do base-pairing small RNAs evolve?

    PubMed Central

    Updegrove, Taylor B.; Shabalina, Svetlana A.; Storz, Gisela

    2015-01-01

    The increasing numbers of characterized base-pairing small RNAs (sRNAs) and the identification of these regulators in a broad range of bacteria are allowing comparisons between species and explorations of sRNA evolution. In this review, we describe some examples of trans-encoded base-pairing sRNAs that are species-specific and others that are more broadly distributed. We also describe examples of sRNA orthologs where different features are conserved. These examples provide the background for a discussion of mechanisms of sRNA evolution and selective pressures on the sRNAs and their mRNA target(s). PMID:25934120

  15. Regulatory role of small nucleolar RNAs in human diseases.

    PubMed

    Stepanov, Grigory A; Filippova, Julia A; Komissarov, Andrey B; Kuligina, Elena V; Richter, Vladimir A; Semenov, Dmitry V

    2015-01-01

    Small nucleolar RNAs (snoRNAs) are appreciable players in gene expression regulation in human cells. The canonical function of box C/D and box H/ACA snoRNAs is posttranscriptional modification of ribosomal RNAs (rRNAs), namely, 2'-O-methylation and pseudouridylation, respectively. A series of independent studies demonstrated that snoRNAs, as well as other noncoding RNAs, serve as the source of various short regulatory RNAs. Some snoRNAs and their fragments can also participate in the regulation of alternative splicing and posttranscriptional modification of mRNA. Alterations in snoRNA expression in human cells can affect numerous vital cellular processes. SnoRNA level in human cells, blood serum, and plasma presents a promising target for diagnostics and treatment of human pathologies. Here we discuss the relation between snoRNAs and oncological, neurodegenerative, and viral diseases and also describe changes in snoRNA level in response to artificial stress and some drugs. PMID:26060813

  16. Regulatory Role of Small Nucleolar RNAs in Human Diseases

    PubMed Central

    Stepanov, Grigory A.; Filippova, Julia A.; Komissarov, Andrey B.; Kuligina, Elena V.; Richter, Vladimir A.; Semenov, Dmitry V.

    2015-01-01

    Small nucleolar RNAs (snoRNAs) are appreciable players in gene expression regulation in human cells. The canonical function of box C/D and box H/ACA snoRNAs is posttranscriptional modification of ribosomal RNAs (rRNAs), namely, 2′-O-methylation and pseudouridylation, respectively. A series of independent studies demonstrated that snoRNAs, as well as other noncoding RNAs, serve as the source of various short regulatory RNAs. Some snoRNAs and their fragments can also participate in the regulation of alternative splicing and posttranscriptional modification of mRNA. Alterations in snoRNA expression in human cells can affect numerous vital cellular processes. SnoRNA level in human cells, blood serum, and plasma presents a promising target for diagnostics and treatment of human pathologies. Here we discuss the relation between snoRNAs and oncological, neurodegenerative, and viral diseases and also describe changes in snoRNA level in response to artificial stress and some drugs. PMID:26060813

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-02-01

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

  19. Molecular call and response: the physiology of bacterial small RNAs

    PubMed Central

    Richards, Gregory R.; Vanderpool, Carin K.

    2011-01-01

    The vital role of bacterial small RNAs (sRNAs) in cellular regulation is now well-established. Although many diverse mechanisms by which sRNAs effect changes in gene expression have been thoroughly described, comparatively less is known about their biological roles and effects on cell physiology. Nevertheless, for some sRNAs, insight has been gained into the intricate regulatory interplay that is required to sense external environmental and internal metabolic cues and turn them into physiological outcomes. Here, we review examples of regulation by selected sRNAs, emphasizing signals and regulators required for sRNA expression, sRNA regulatory targets, and the resulting consequences for the cell. We highlight sRNAs involved in regulation of the processes of iron homeostasis (RyhB, PrrF, and FsrA) and carbon metabolism (Spot 42, CyaR, and SgrS). PMID:21843668

  20. Molecular call and response: the physiology of bacterial small RNAs.

    PubMed

    Richards, Gregory R; Vanderpool, Carin K

    2011-10-01

    The vital role of bacterial small RNAs (sRNAs) in cellular regulation is now well-established. Although many diverse mechanisms by which sRNAs bring about changes in gene expression have been thoroughly described, comparatively less is known about their biological roles and effects on cell physiology. Nevertheless, for some sRNAs, insight has been gained into the intricate regulatory interplay that is required to sense external environmental and internal metabolic cues and turn them into physiological outcomes. Here, we review examples of regulation by selected sRNAs, emphasizing signals and regulators required for sRNA expression, sRNA regulatory targets, and the resulting consequences for the cell. We highlight sRNAs involved in regulation of the processes of iron homeostasis (RyhB, PrrF, and FsrA) and carbon metabolism (Spot 42, CyaR, and SgrS). PMID:21843668

  1. Balance and Stealth: The Role of Noncoding RNAs in the Regulation of Virus Gene Expression.

    PubMed

    Cox, Jennifer E; Sullivan, Christopher S

    2014-11-01

    In the past two decades, our knowledge of gene regulation has been greatly expanded by the discovery of microRNAs (miRNAs). miRNAs are small (19-24 nt) noncoding RNAs (ncRNAs) found in metazoans, plants, and some viruses. They have been shown to regulate many cellular processes, including differentiation, maintenance of homeostasis, apoptosis, and the immune response. At present, there are over 300 known viral miRNAs encoded by diverse virus families. One well-characterized function of some viral miRNAs is the regulation of viral transcripts. Host miRNAs can also regulate viral gene expression. We propose that viruses take advantage of both host and viral ncRNA regulation to balance replication and infectious state (for example, latent versus lytic infection). As miRNA regulation can be reversed upon certain cellular stresses, we hypothesize that ncRNAs can serve viruses as barometers for cellular stress. PMID:26958716

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

    PubMed

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

    2016-06-01

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

  3. Probing small non-coding RNAs structures.

    PubMed

    Philippe, Jean-Vincent; Ayadi, Lilia; Branlant, Christiane; Behm-Ansmant, Isabelle

    2015-01-01

    The diverse roles of RNAs depend on their ability to fold so as to form biologically functional structures. Thus, understanding the function of a given RNA molecule often requires experimental analysis of its secondary structure by in vitro RNA probing, which is more accurate than using prediction programs only. This chapter presents in vitro RNA probing protocols that we routinely use, from RNA transcript production and purification to RNA structure determination using enzymatic (RNases T1, T2, and V1) and chemical (DMS, CMCT, kethoxal, and Pb(2+)) probing performed on both unlabeled and end-labeled RNAs. PMID:25791596

  4. Novel Meiotic miRNAs and Indications for a Role of PhasiRNAs in Meiosis

    PubMed Central

    Dukowic-Schulze, Stefanie; Sundararajan, Anitha; Ramaraj, Thiruvarangan; Kianian, Shahryar; Pawlowski, Wojciech P.; Mudge, Joann; Chen, Changbin

    2016-01-01

    Small RNAs (sRNA) add additional layers to the regulation of gene expression, with siRNAs directing gene silencing at the DNA level by RdDM (RNA-directed DNA methylation), and micro RNAs (miRNAs) directing post-transcriptional regulation of specific target genes, mostly by mRNA cleavage. We used manually isolated male meiocytes from maize (Zea mays) to investigate sRNA and DNA methylation landscapes during zygotene, an early stage of meiosis during which steps of meiotic recombination and synapsis of paired homologous chromosomes take place. We discovered two novel miRNAs from meiocytes, zma-MIR11969 and zma-MIR11970, and identified putative target genes. Furthermore, we detected abundant phasiRNAs of 21 and 24 nt length. PhasiRNAs are phased small RNAs which occur in 21 or 24 nt intervals, at a few hundred loci, specifically in male reproductive tissues in grasses. So far, the function of phasiRNAs remained elusive. Data from isolated meiocytes now revealed elevated DNA methylation at phasiRNA loci, especially in the CHH context, suggesting a role for phasiRNAs in cis DNA methylation. In addition, we consider a role of these phasiRNAs in chromatin remodeling/dynamics during meiosis. However, this is not well supported yet and will need more additional data. Here, we only lay out the idea due to other relevant literature and our additional observation of a peculiar GC content pattern at phasiRNA loci. Chromatin remodeling is also indicated by the discovery that histone genes were enriched for sRNA of 22 nt length. Taken together, we gained clues that lead us to hypothesize sRNA-driven DNA methylation and possibly chromatin remodeling during male meiosis in the monocot maize which is in line with and extends previous knowledge. PMID:27313591

  5. Improved Placement of Multi-mapping Small RNAs

    PubMed Central

    Johnson, Nathan R.; Yeoh, Jonathan M.; Coruh, Ceyda; Axtell, Michael J.

    2016-01-01

    High-throughput sequencing of small RNAs (sRNA-seq) is a popular method used to discover and annotate microRNAs (miRNAs), endogenous short interfering RNAs (siRNAs), and Piwi-associated RNAs (piRNAs). One of the key steps in sRNA-seq data analysis is alignment to a reference genome. sRNA-seq libraries often have a high proportion of reads that align to multiple genomic locations, which makes determining their true origins difficult. Commonly used sRNA-seq alignment methods result in either very low precision (choosing an alignment at random), or sensitivity (ignoring multi-mapping reads). Here, we describe and test an sRNA-seq alignment strategy that uses local genomic context to guide decisions on proper placements of multi-mapped sRNA-seq reads. Tests using simulated sRNA-seq data demonstrated that this local-weighting method outperforms other alignment strategies using three different plant genomes. Experimental analyses with real sRNA-seq data also indicate superior performance of local-weighting methods for both plant miRNAs and heterochromatic siRNAs. The local-weighting methods we have developed are implemented as part of the sRNA-seq analysis program ShortStack, which is freely available under a general public license. Improved genome alignments of sRNA-seq data should increase the quality of downstream analyses and genome annotation efforts. PMID:27175019

  6. Improved Placement of Multi-mapping Small RNAs.

    PubMed

    Johnson, Nathan R; Yeoh, Jonathan M; Coruh, Ceyda; Axtell, Michael J

    2016-01-01

    High-throughput sequencing of small RNAs (sRNA-seq) is a popular method used to discover and annotate microRNAs (miRNAs), endogenous short interfering RNAs (siRNAs), and Piwi-associated RNAs (piRNAs). One of the key steps in sRNA-seq data analysis is alignment to a reference genome. sRNA-seq libraries often have a high proportion of reads that align to multiple genomic locations, which makes determining their true origins difficult. Commonly used sRNA-seq alignment methods result in either very low precision (choosing an alignment at random), or sensitivity (ignoring multi-mapping reads). Here, we describe and test an sRNA-seq alignment strategy that uses local genomic context to guide decisions on proper placements of multi-mapped sRNA-seq reads. Tests using simulated sRNA-seq data demonstrated that this local-weighting method outperforms other alignment strategies using three different plant genomes. Experimental analyses with real sRNA-seq data also indicate superior performance of local-weighting methods for both plant miRNAs and heterochromatic siRNAs. The local-weighting methods we have developed are implemented as part of the sRNA-seq analysis program ShortStack, which is freely available under a general public license. Improved genome alignments of sRNA-seq data should increase the quality of downstream analyses and genome annotation efforts. PMID:27175019

  7. Comparative genomics boosts target prediction for bacterial small RNAs.

    PubMed

    Wright, Patrick R; Richter, Andreas S; Papenfort, Kai; Mann, Martin; Vogel, Jörg; Hess, Wolfgang R; Backofen, Rolf; Georg, Jens

    2013-09-10

    Small RNAs (sRNAs) constitute a large and heterogeneous class of bacterial gene expression regulators. Much like eukaryotic microRNAs, these sRNAs typically target multiple mRNAs through short seed pairing, thereby acting as global posttranscriptional regulators. In some bacteria, evidence for hundreds to possibly more than 1,000 different sRNAs has been obtained by transcriptome sequencing. However, the experimental identification of possible targets and, therefore, their confirmation as functional regulators of gene expression has remained laborious. Here, we present a strategy that integrates phylogenetic information to predict sRNA targets at the genomic scale and reconstructs regulatory networks upon functional enrichment and network analysis (CopraRNA, for Comparative Prediction Algorithm for sRNA Targets). Furthermore, CopraRNA precisely predicts the sRNA domains for target recognition and interaction. When applied to several model sRNAs, CopraRNA revealed additional targets and functions for the sRNAs CyaR, FnrS, RybB, RyhB, SgrS, and Spot42. Moreover, the mRNAs gdhA, lrp, marA, nagZ, ptsI, sdhA, and yobF-cspC were suggested as regulatory hubs targeted by up to seven different sRNAs. The verification of many previously undetected targets by CopraRNA, even for extensively investigated sRNAs, demonstrates its advantages and shows that CopraRNA-based analyses can compete with experimental target prediction approaches. A Web interface allows high-confidence target prediction and efficient classification of bacterial sRNAs. PMID:23980183

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

    PubMed

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

    2016-01-01

    Exosomes are small membrane bound vesicles between 30 and 100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. Exosomes play a role in intercellular communication by transferring proteins, lipids, and RNAs to recipient cells.Exosomes from human cells could be used as vectors to provide cells with therapeutic RNAs. Here we describe how exogenous small interfering RNAs may successfully be introduced into various kinds of human exosomes using electroporation and subsequently delivered to recipient cells. Methods used to confirm the presence of siRNA inside exosomes and cells are presented, such as flow cytometry, confocal microscopy, and Northern blot. PMID:26472446

  9. Identification of small Hfq-binding RNAs in Listeria monocytogenes

    PubMed Central

    Christiansen, Janne K.; Nielsen, Jesper S.; Ebersbach, Tine; Valentin-Hansen, Poul; Søgaard-Andersen, Lotte; Kallipolitis, Birgitte H.

    2006-01-01

    The RNA-binding protein Hfq plays important roles in bacterial physiology and is required for the activity of many small regulatory RNAs in prokaryotes. We have previously shown that Hfq contributes to stress tolerance and virulence in the Gram-positive human pathogen Listeria monocytogenes. In the present study, we performed coimmunoprecipitations followed by enzymatic RNA sequencing to identify Hfq-binding RNA molecules in L. monocytogenes. The approach resulted in the discovery of three small RNAs (sRNAs). The sRNAs are conserved between Listeria species, but were not identified in other bacterial species. The initial characterization revealed a number of unique features displayed by each individual sRNA. The first sRNA is encoded from within an annotated gene in the L. monocytogenes EGD-e genome. Analogous to most regulatory sRNAs in Escherichia coli, the stability of this sRNA is highly dependent on the presence of Hfq. The second sRNA appears to be produced by a transcription attenuation mechanism, and the third sRNA is present in five copies at two different locations within the L. monocytogenes EGD-e genome. The cellular levels of the sRNAs are growth phase dependent and vary in response to growth medium. All three sRNAs are expressed when L. monocytogenes multiplies within mammalian cells. This study represents the first attempt to identify sRNAs in L. monocytogenes. PMID:16682563

  10. Computational methods for comparative analysis of plant small RNAs.

    PubMed

    Mahalingam, Gayathri; Meyers, Blake C

    2010-01-01

    Small RNAs play an important role in plant development, stress responses, and epigenetic regulation, primarily through their role in transcriptional and post-transcriptional silencing of specific target genes and loci. Most if not all plants utilize these small RNA signaling networks. We have developed a deep-sequencing based dataset of plant small RNAs, based on the hypothesis that comparisons among the complex pool of small RNAs from diverse plants will identify novel types of conserved, regulated, or species-specific molecules. A database containing upward of hundreds of millions of plant small RNA sequences is being created for comparative analyses. This small RNA database will allow the experimental characterization of the majority of the biologically important small RNAs for a range of plant species. This database can be accessed from our website (http://smallrna.udel.edu/). A variety of web-based tools have been developed for analyses of these data. Here, we focus on these tools, and we describe how the users can implement these tools to analyze and interpret the small RNA data and how the users could use similar approaches for other sets of plant small RNAs from diverse species. PMID:19802596

  11. 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

  12. Small RNAs in angiosperms: sequence characteristics, distribution and generation.

    PubMed

    Chen, Dijun; Meng, Yijun; Ma, Xiaoxia; Mao, Chuanzao; Bai, Youhuang; Cao, Junjie; Gu, Haibin; Wu, Ping; Chen, Ming

    2010-06-01

    High-throughput sequencing (HTS) has opened up a new era for small RNA (sRNA) exploration. Using HTS data for a global survey of sRNAs in 26 angiosperms, elevated GC contents were detected in the monocots, whereas the 5(')-terminal compositions were quite uniform among the angiosperms. Chromosome-wide distribution patterns of sRNAs were investigated by using scrolling-window analysis. We performed de novo natural antisense transcript (NAT) prediction, and found that the overlapping regions of trans-NATs, but not cis-NATs, were hotspots for sRNA generation. One cis-NAT generates phased natural antisense short interfering RNAs (nat-siRNAs) specifically from flowers in Arabidopsis, while one in rice produces phased nat-siRNAs from grains, suggesting their organ-specific regulatory roles. PMID:20378553

  13. Big impacts by small RNAs in plant development.

    PubMed

    Chuck, George; Candela, Héctor; Hake, Sarah

    2009-02-01

    The identification and study of small RNAs, including microRNAs and trans-acting small interfering RNAs, have added a layer of complexity to the many pathways that regulate plant development. These molecules, which function as negative regulators of gene expression, are now known to have greatly expanded roles in a variety of developmental processes affecting all major plant structures, including meristems, leaves, roots, and inflorescences. Mutants with specific developmental phenotypes have also advanced our knowledge of the biogenesis and mode of action of these diverse small RNAs. In addition, previous models on the cell autonomy of microRNAs may have to be revised as more data accumulate supporting their long distance transport. As many of these small RNAs appear to be conserved across different species, knowledge gained from one species is expected to have general application. However, a few surprising differences in small RNA function seem to exist between monocots and dicots regarding meristem initiation and sex determination. Integrating these unique functions into the overall scheme for plant growth will give a more complete picture of how they have evolved as unique developmental systems. PMID:18980858

  14. Conjugation-specific small RNAs in Tetrahymena have predicted properties of scan (scn) RNAs involved in genome rearrangement

    PubMed Central

    Mochizuki, Kazufumi; Gorovsky, Martin A.

    2004-01-01

    We proposed a scan-RNA model for genome rearrangement based on finding small RNAs that hybridized preferentially to micronuclear-specific sequences and on the properties of Twi1p, a PPD protein required for both sequence elimination and small RNA accumulation in Tetrahymena. Here we show that Twi1p interacts with the small RNAs in both the old and the developing macronucleus, and is required for their stability. We show that the specificity of the small RNAs for micronuclear-limited sequences increases during conjugation. These results indicate that the small RNAs observed in conjugating cells have the properties predicted for scan RNAs. PMID:15314029

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

    PubMed Central

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

    2016-01-01

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

  16. Dynamic features of gene expression control by small regulatory RNAs.

    PubMed

    Mitarai, Namiko; Benjamin, Julie-Anna M; Krishna, Sandeep; Semsey, Szabolcs; Csiszovszki, Zsolt; Massé, Eric; Sneppen, Kim

    2009-06-30

    Small regulatory RNAs (sRNAs) in eukaryotes and bacteria play an important role in the regulation of gene expression either by binding to regulatory proteins or directly to target mRNAs. Two of the best-characterized bacterial sRNAs, Spot42 and RyhB, form a complementary pair with the ribosome binding region of their target mRNAs, thereby inhibiting translation or promoting mRNA degradation. To investigate the steady-state and dynamic potential of such sRNAs, we examine the 2 key parameters characterizing sRNA regulation: the capacity to overexpress the sRNA relative to its target mRNA and the speed at which the target mRNA is irreversibly inactivated. We demonstrate different methods to determine these 2 key parameters, for Spot42 and RyhB, which combine biochemical and genetic experiments with computational analysis. We have developed a mathematical model that describes the functional properties of sRNAs with various characteristic parameters. We observed that Spot42 and RyhB function in distinctive parameter regimes, which result in divergent mechanisms. PMID:19541626

  17. Discovery of Ethanol-Responsive Small RNAs in Zymomonas mobilis

    PubMed Central

    Cho, Seung Hee; Lei, Roy; Henninger, Trey D.

    2014-01-01

    Zymomonas mobilis is a bacterium that can produce ethanol by fermentation. Due to its unique metabolism and efficient ethanol production, Z. mobilis has attracted special interest for biofuel energy applications; an important area of study is the regulation of those specific metabolic pathways. Small RNAs (sRNAs) have been studied as molecules that function as transcriptional regulators in response to cellular stresses. While sRNAs have been discovered in various organisms by computational prediction and experimental approaches, their discovery in Z. mobilis has not yet been reported. In this study, we have applied transcriptome analysis and computational predictions to facilitate identification and validation of 15 novel sRNAs in Z. mobilis. We furthermore characterized their expression in the context of high and low levels of intracellular ethanol. Here, we report that 3 of the sRNAs (Zms2, Zms4, and Zms6) are differentially expressed under aerobic and anaerobic conditions, when low and high ethanol productions are observed, respectively. Importantly, when we tested the effect of ethanol stress on the expression of sRNAs in Z. mobilis, Zms2, Zms6, and Zms18 showed differential expression under 5% ethanol stress conditions. These data suggest that in this organism regulatory RNAs can be associated with metabolic functions involved in ethanol stress responses. PMID:24795378

  18. Plant siRNAs from introns mediate DNA methylation of host genes.

    PubMed

    Chen, Dijun; Meng, Yijun; Yuan, Chunhui; Bai, Lin; Huang, Donglin; Lv, Shaolei; Wu, Ping; Chen, Ling-Ling; Chen, Ming

    2011-06-01

    Small RNAs (sRNAs), largely known as microRNAs (miRNAs) and short interfering RNAs (siRNAs), emerged as the critical components of genetic and epigenetic regulation in eukaryotic genomes. In animals, a sizable portion of miRNAs reside within the introns of protein-coding genes, designated as mirtron genes. Recently, high-throughput sequencing (HTS) revealed a huge amount of sRNAs that derived from introns in plants, such as the monocot rice (Oryza sativa). However, the biogenesis and the biological functions of this kind of sRNAs remain elusive. Here, we performed a genome-scale survey of intron-derived sRNAs in rice based on HTS data. Several introns were found to have great potential to form internal hairpin structures, and the short hairpins could generate miRNAs while the larger ones could produce siRNAs. Furthermore, 22 introns, termed "sirtrons," were identified from the rice protein-coding genes. The single-stranded sirtrons produced a diverse set of siRNAs from long hairpin structures. These sirtron-derived siRNAs are dominantly 21 nt, 22 nt, and 24 nt in length, whose production relied on DCL4, DCL2, and DCL3, respectively. We also observed a strong tendency for the sirtron-derived siRNAs to be coexpressed with their host genes. Finally, the 24-nt siRNAs incorporated with Argonaute 4 (AGO4) could direct DNA methylation on their host genes. In this regard, homeostatic self-regulation between intron-derived siRNAs and their host genes was proposed. PMID:21518803

  19. deepBase v2.0: identification, expression, evolution and function of small RNAs, LncRNAs and circular RNAs from deep-sequencing data

    PubMed Central

    Zheng, Ling-Ling; Li, Jun-Hao; Wu, Jie; Sun, Wen-Ju; Liu, Shun; Wang, Ze-Lin; Zhou, Hui; Yang, Jian-Hua; Qu, Liang-Hu

    2016-01-01

    Small non-coding RNAs (e.g. miRNAs) and long non-coding RNAs (e.g. lincRNAs and circRNAs) are emerging as key regulators of various cellular processes. However, only a very small fraction of these enigmatic RNAs have been well functionally characterized. In this study, we describe deepBase v2.0 (http://biocenter.sysu.edu.cn/deepBase/), an updated platform, to decode evolution, expression patterns and functions of diverse ncRNAs across 19 species. deepBase v2.0 has been updated to provide the most comprehensive collection of ncRNA-derived small RNAs generated from 588 sRNA-Seq datasets. Moreover, we developed a pipeline named lncSeeker to identify 176 680 high-confidence lncRNAs from 14 species. Temporal and spatial expression patterns of various ncRNAs were profiled. We identified approximately 24 280 primate-specific, 5193 rodent-specific lncRNAs, and 55 highly conserved lncRNA orthologs between human and zebrafish. We annotated 14 867 human circRNAs, 1260 of which are orthologous to mouse circRNAs. By combining expression profiles and functional genomic annotations, we developed lncFunction web-server to predict the function of lncRNAs based on protein-lncRNA co-expression networks. This study is expected to provide considerable resources to facilitate future experimental studies and to uncover ncRNA functions. PMID:26590255

  20. deepBase v2.0: identification, expression, evolution and function of small RNAs, LncRNAs and circular RNAs from deep-sequencing data.

    PubMed

    Zheng, Ling-Ling; Li, Jun-Hao; Wu, Jie; Sun, Wen-Ju; Liu, Shun; Wang, Ze-Lin; Zhou, Hui; Yang, Jian-Hua; Qu, Liang-Hu

    2016-01-01

    Small non-coding RNAs (e.g. miRNAs) and long non-coding RNAs (e.g. lincRNAs and circRNAs) are emerging as key regulators of various cellular processes. However, only a very small fraction of these enigmatic RNAs have been well functionally characterized. In this study, we describe deepBase v2.0 (http://biocenter.sysu.edu.cn/deepBase/), an updated platform, to decode evolution, expression patterns and functions of diverse ncRNAs across 19 species. deepBase v2.0 has been updated to provide the most comprehensive collection of ncRNA-derived small RNAs generated from 588 sRNA-Seq datasets. Moreover, we developed a pipeline named lncSeeker to identify 176 680 high-confidence lncRNAs from 14 species. Temporal and spatial expression patterns of various ncRNAs were profiled. We identified approximately 24 280 primate-specific, 5193 rodent-specific lncRNAs, and 55 highly conserved lncRNA orthologs between human and zebrafish. We annotated 14 867 human circRNAs, 1260 of which are orthologous to mouse circRNAs. By combining expression profiles and functional genomic annotations, we developed lncFunction web-server to predict the function of lncRNAs based on protein-lncRNA co-expression networks. This study is expected to provide considerable resources to facilitate future experimental studies and to uncover ncRNA functions. PMID:26590255

  1. Small nucleolar RNAs as new biomarkers in chronic lymphocytic leukemia

    PubMed Central

    2013-01-01

    Background Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs are non-coding RNAs involved in the maturation of other RNA molecules. Alterations of sno/scaRNA expression may play a role in cancerogenesis. This study elucidates the patterns of sno/scaRNA expression in 211 chronic lymphocytic leukemia (CLL) patients (Binet stage A) also in comparison with those of different normal B-cell subsets. Methods The patterns of sno/scaRNA expression in highly purified CD19+ B-cells of 211 CLL patients and in 18 normal B-cell samples - 6 from peripheral blood, and 12 from tonsils (4 germinal center, 2 marginal zone, 3 switched memory and 3 naïve B-cells) - were analyzed on the Affymetrix GeneChip® Human Gene 1.0 ST array. Results CLLs display a sno/scaRNAs expression profile similar to normal memory, naïve and marginal-zone B-cells, with the exception of a few down-regulated transcripts (SNORA31, -6, -62, and -71C). Our analyses also suggest some heterogeneity in the pattern of sno/scaRNAs expression which is apparently unrelated to the major biological (ZAP-70 and CD38), molecular (IGHV mutation) and cytogenetic markers. Moreover, we found that SNORA70F was significantly down-regulated in poor prognostic subgroups and this phenomenon was associated with the down-regulation of its host gene COBLL1. Finally, we generated an independent model based on SNORA74A and SNORD116-18 expression, which appears to distinguish two different prognostic CLL groups. Conclusions These data extend the view of sno/scaRNAs deregulation in cancer and may contribute to discover novel biomarkers associated with the disease and potentially useful to predict the clinical outcome of early stage CLL patients. PMID:24004562

  2. Small regulatory RNAs in Streptococcus pneumoniae: discovery and biological functions

    PubMed Central

    Wilton, Joana; Acebo, Paloma; Herranz, Cristina; Gómez, Alicia; Amblar, Mónica

    2015-01-01

    Streptococcus pneumoniae is a prominent human pathogen responsible for many severe diseases and the leading cause of childhood mortality worldwide. The pneumococcus is remarkably adept at colonizing and infecting different niches in the human body, and its adaptation to dynamic host environment is a central aspect of its pathogenesis. In the last decade, increasing findings have evidenced small RNAs (sRNAs) as vital regulators in a number of important processes in bacteria. In S. pneumoniae, a small antisense RNA was first discovered in the pMV158 plasmid as a copy number regulator. More recently, genome-wide screens revealed that the pneumococcal genome also encodes multiple sRNAs, many of which have important roles in virulence while some are implicated in competence control. The knowledge of the sRNA-mediated regulation in pneumococcus remains very limited, and future research is needed for better understanding of functions and mechanisms. Here, we provide a comprehensive summary of the current knowledge on sRNAs from S. pneumoniae, focusing mainly on the trans-encoded sRNAs. PMID:25904932

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

    PubMed

    Bai, Baoyan; Laiho, Marikki

    2016-01-01

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

  4. Efficient degradation and expression prioritization with small RNAs.

    PubMed

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

    2007-09-01

    We build a simple model for feedback systems involving small RNA (sRNA) molecules based on the iron metabolism system in the bacterium E. coli, and compare it with the corresponding system in H. pylori which uses purely transcriptional regulation. This reveals several unique features of sRNA-based regulation that could be exploited by cells. Firstly, we show that sRNA regulation can maintain a smaller turnover of target mRNAs than transcriptional regulation, without sacrificing the speed of response to external shocks. Secondly, we propose that a single sRNA can prioritize the usage of different target mRNAs. This suggests that sRNA regulation would be more common in more complex systems which need to co-regulate many mRNAs efficiently. PMID:17928655

  5. Isolation of Small Noncoding RNAs from Human Serum

    PubMed Central

    Khoury, Samantha; Ajuyah, Pamela; Tran, Nham

    2014-01-01

    The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 µl of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/µl from serum but can also be adapted for other biological tissues. PMID:24998448

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

    PubMed

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

    2013-10-01

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

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

    PubMed

    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

  8. 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

  9. High throughput sequencing of two celery varieties small RNAs identifies microRNAs involved in temperature stress response

    PubMed Central

    2014-01-01

    Background MicroRNAs (miRNAs) are small, non-coding RNAs of 20 to 24 nucleotides that regulate gene expression and responses to biotic and abiotic stress. Till now, no reports have previously been published concerning miRNAs in celery. Results Two small RNAs libraries were constructed from two celery varieties, ‘Jinnan Shiqin’ and ‘Ventura’, and characterized by deep sequencing. A total of 431 (418 known and 13 novel) and 346 (341 known and five novel) miRNAs were identified in celery varieties ‘Jinnan Shiqin’ and ‘Ventura’, respectively. Potential miRNA-target genes were predicted and annotated by screening diverse protein databases, including Gene Ontology, Cluster of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes. Significant differential expression between the two varieties was seen for 221 miRNAs. qRT-PCR was used to analyze the abundance of six miRNAs under cold and heat stress conditions. The results showed that miRNAs may have important functions in controlling temperature stress in celery. Conclusion A large number of miRNAs were identified in celery, and their target genes, functional annotations, and gene expression patterns have been explored. These findings provide the first information on celery miRNAs and enhance understanding of celery miRNA regulatory mechanisms under extreme temperature stress. PMID:24673837

  10. Phloem small RNAs, nutrient stress responses, and systemic mobility

    PubMed Central

    2010-01-01

    Background Nutrient availabilities and needs have to be tightly coordinated between organs to ensure a balance between uptake and consumption for metabolism, growth, and defense reactions. Since plants often have to grow in environments with sub-optimal nutrient availability, a fine tuning is vital. To achieve this, information has to flow cell-to-cell and over long-distance via xylem and phloem. Recently, specific miRNAs emerged as a new type of regulating molecules during stress and nutrient deficiency responses, and miR399 was suggested to be a phloem-mobile long-distance signal involved in the phosphate starvation response. Results We used miRNA microarrays containing all known plant miRNAs and a set of unknown small (s) RNAs earlier cloned from Brassica phloem sap [1], to comprehensively analyze the phloem response to nutrient deficiency by removing sulfate, copper or iron, respectively, from the growth medium. We show that phloem sap contains a specific set of sRNAs that is distinct from leaves and roots, and that the phloem also responds specifically to stress. Upon S and Cu deficiencies phloem sap reacts with an increase of the same miRNAs that were earlier characterized in other tissues, while no clear positive response to -Fe was observed. However, -Fe led to a reduction of Cu- and P-responsive miRNAs. We further demonstrate that under nutrient starvation miR399 and miR395 can be translocated through graft unions from wild type scions to rootstocks of the miRNA processing hen1-1 mutant. In contrast, miR171 was not transported. Translocation of miR395 led to a down-regulation of one of its targets in rootstocks, suggesting that this transport is of functional relevance, and that miR395, in addition to the well characterized miR399, could potentially act as a long-distance information transmitter. Conclusions Phloem sap contains a specific set of sRNAs, of which some specifically accumulate in response to nutrient deprivation. From the observation that mi

  11. Small interfering RNAs targeting the rabies virus nucleoprotein gene.

    PubMed

    Yang, Yu-Jiao; Zhao, Ping-Sen; Zhang, Tao; Wang, Hua-Lei; Liang, Hong-Ru; Zhao, Li-Li; Wu, Hong-Xia; Wang, Tie-Cheng; Yang, Song-Tao; Xia, Xian-Zhu

    2012-10-01

    Rabies virus (RABV) infection continues to be a global threat to human and animal health, yet no curative therapy has been developed. RNA interference (RNAi) therapy, which silences expression of specific target genes, represents a promising approach for treating viral infections in mammalian hosts. We designed six small interfering (si)RNAs (N473, N580, N783, N796, N799 and N1227) that target the conserved region of the RABV challenge virus standard (CVS)-11 strain nucleoprotein (N) gene. Using a plasmid-based transient expression model, we demonstrated that N796, N580 and N799 were capable of significantly inhibiting viral replication in vitro and in vivo. These three siRNAs effectively suppressed RABV expression in infected baby hamster kidney-21 (BHK-21) cells, as evidenced by direct immunofluorescence assay, viral titer measurements, real-time PCR, and Western blotting. In addition, liposome-mediated siRNA expression plasmid delivery to RABV-infected mice significantly increased survival, compared to a non-liposome-mediated delivery method. Collectively, our results showed that the three siRNAs, N796, N580 and N799, targeting the N gene could potently inhibit RABV CVS-11 reproduction. These siRNAs have the potential to be developed into new and effective prophylactic anti-RABV drugs. PMID:22884777

  12. Comparative analyses of endogenous Small RNAs in Sclerotinia sclerotiorum and S. trifoliorum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Endogenous small RNAs (sRNAs) of Sclerotinia sclerotiorum and S. trifoliorum were compared to gain insight into the biology of the two closely related plant pathogens. Random samples of 53 unique sRNAs of S. sclerotiorum and 55 unique sRNAs of S. trifoliorum had, respectively, 221 and 229 target loc...

  13. 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. PMID:26210184

  14. Identification of Small RNAs in Desulfovibrio vulgaris Hildenborough

    SciTech Connect

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

    2010-05-17

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

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

    DOE PAGESBeta

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

    2016-02-17

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

  16. Modular Synthetic Inverters from Zinc Finger Proteins and Small RNAs

    PubMed Central

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

    2016-01-01

    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. Our chosen parts performed insufficiently to create oscillations, but we include future directions for improvement upon our work presented here. PMID:26886888

  17. Deep sequencing of small RNAs in tomato for virus and viroid identification and strain differentiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Small RNAs (sRNA), including microRNAs (miRNA) and small interfering RNAs (siRNA), are produced abundantly in plants and animals and function in regulating gene expression or in defense against virus or viroid infection. Analysis of siRNA profiles upon virus infection in plant may allow for virus i...

  18. 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

    2016-01-01

    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. PMID:27233361

  19. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes.

    PubMed

    Tam, Oliver H; Aravin, Alexei A; Stein, Paula; Girard, Angelique; Murchison, Elizabeth P; Cheloufi, Sihem; Hodges, Emily; Anger, Martin; Sachidanandam, Ravi; Schultz, Richard M; Hannon, Gregory J

    2008-05-22

    Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals. PMID:18404147

  20. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes

    PubMed Central

    Tam, Oliver H.; Aravin, Alexei A.; Stein, Paula; Girard, Angelique; Murchison, Elizabeth P.; Cheloufi, Sihem; Hodges, Emily; Anger, Martin; Sachidanandam, Ravi; Schultz, Richard M.; Hannon, Gregory J.

    2010-01-01

    Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways1. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals. PMID:18404147

  1. Novel small Cajal-body-specific RNAs identified in Drosophila: probing guide RNA function

    PubMed Central

    Deryusheva, Svetlana; Gall, Joseph G.

    2013-01-01

    The spliceosomal small nuclear RNAs (snRNAs) are modified post-transcriptionally by introduction of pseudouridines and 2′-O-methyl modifications, which are mediated by box H/ACA and box C/D guide RNAs, respectively. Because of their concentration in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific (sca) RNAs. In the cell, scaRNAs are associated with the WD-repeat protein WDR79. We used coimmunoprecipitation with WDR79 to recover seven new scaRNAs from Drosophila cell lysates. We demonstrated concentration of these new scaRNAs in the CB by in situ hybridization, and we verified experimentally that they can modify their putative target RNAs. Surprisingly, one of the new scaRNAs targets U6 snRNA, whose modification is generally assumed to occur in the nucleolus, not in the CB. Two other scaRNAs have dual guide functions, one for an snRNA and one for 28S rRNA. Again, the modification of 28S rRNA is assumed to take place in the nucleolus. These findings suggest that canonical scaRNAs may have functions in addition to their established role in modifying U1, U2, U4, and U5 snRNAs. We discuss the likelihood that processing by scaRNAs is not limited to the CB. PMID:24149844

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. Investigating the role of two iron-regulated small RNAs of Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Small RNAs (sRNAs) have emerged as important components of many regulatory pathways and have been shown to have key roles in the regulation of iron homeostasis in a number of bacteria. To date, only a few sRNAs have been described for the bacterial plant pathogen Pseudomonas syringae pathovar tomat...

  4. Plant virus-derived small interfering RNAs originate predominantly from highly structured single-stranded viral RNAs.

    PubMed

    Molnár, Attila; Csorba, Tibor; Lakatos, Lóránt; Várallyay, Eva; Lacomme, Christophe; Burgyán, József

    2005-06-01

    RNA silencing is conserved in a broad range of eukaryotes and includes the phenomena of RNA interference in animals and posttranscriptional gene silencing (PTGS) in plants. In plants, PTGS acts as an antiviral system; a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with positive-strand RNA viruses and provide specificity to this RNA-mediated defense. We present here the results of a survey of virus-specific siRNAs characterized by a sequence analysis of siRNAs from plants infected with Cymbidium ringspot tombusvirus (CymRSV). CymRSV siRNA sequences have a nonrandom distribution along the length of the viral genome, suggesting that there are hot spots for virus-derived siRNA generation. CymRSV siRNAs bound to the CymRSV p19 suppressor protein have the same asymmetry in strand polarity as the sequenced siRNAs and are imperfect double-stranded RNA duplexes. Moreover, an analysis of siRNAs derived from two other nonrelated positive-strand RNA viruses showed that they displayed the same asymmetry as CymRSV siRNAs. Finally, we show that Tobacco mosaic virus (TMV) carrying a short inverted repeat of the phytoene desaturase (PDS) gene triggered more accumulation of PDS siRNAs than the corresponding antisense PDS sequence. Taken together, these results suggest that virus-derived siRNAs originate predominantly by direct DICER cleavage of imperfect duplexes in the most folded regions of the positive strand of the viral RNA. PMID:15919934

  5. Assessing the gene regulatory properties of Argonaute-bound small RNAs of diverse genomic origin

    PubMed Central

    Thomson, Daniel W.; Pillman, Katherine A.; Anderson, Matthew L.; Lawrence, David M.; Toubia, John; Goodall, Gregory J.; Bracken, Cameron P.

    2015-01-01

    High-throughput sequencing reveals an abundance of microRNA-sized fragments derived from larger non-coding RNAs. Roles for these small RNAs in gene silencing are suggested by their co-precipitation with Argonaute, the microRNA effector protein, though the extent to which they suppress gene expression endogenously remains unclear. To address this, we used luciferase reporters to determine the endogenous functionality of small RNAs from a diverse range of sources. We demonstrate small RNAs derived from snoRNAs have the capacity to act in a microRNA-like manner, though we note the vast majority of these are bound to Argonaute at levels below that required for detectable silencing activity. We show Argonaute exhibits a high degree of selectivity for the small RNAs with which it interacts and note that measuring Argonaute-associated levels is a better indicator of function than measuring total expression. Although binding to Argonaute at sufficient levels is necessary for demonstrating microRNA functionality in our reporter assay, this alone is not enough as some small RNAs derived from other non-coding RNAs (tRNAs, rRNAs, Y-RNAs) are associated with Argonaute at very high levels yet do not serve microRNA-like roles. PMID:25452337

  6. Assessing the gene regulatory properties of Argonaute-bound small RNAs of diverse genomic origin.

    PubMed

    Thomson, Daniel W; Pillman, Katherine A; Anderson, Matthew L; Lawrence, David M; Toubia, John; Goodall, Gregory J; Bracken, Cameron P

    2015-01-01

    High-throughput sequencing reveals an abundance of microRNA-sized fragments derived from larger non-coding RNAs. Roles for these small RNAs in gene silencing are suggested by their co-precipitation with Argonaute, the microRNA effector protein, though the extent to which they suppress gene expression endogenously remains unclear. To address this, we used luciferase reporters to determine the endogenous functionality of small RNAs from a diverse range of sources. We demonstrate small RNAs derived from snoRNAs have the capacity to act in a microRNA-like manner, though we note the vast majority of these are bound to Argonaute at levels below that required for detectable silencing activity. We show Argonaute exhibits a high degree of selectivity for the small RNAs with which it interacts and note that measuring Argonaute-associated levels is a better indicator of function than measuring total expression. Although binding to Argonaute at sufficient levels is necessary for demonstrating microRNA functionality in our reporter assay, this alone is not enough as some small RNAs derived from other non-coding RNAs (tRNAs, rRNAs, Y-RNAs) are associated with Argonaute at very high levels yet do not serve microRNA-like roles. PMID:25452337

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

    PubMed

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

    2013-08-01

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

  8. Novel and conserved microRNAs in soybean floral whorls.

    PubMed

    Kulcheski, F R; Molina, L G; da Fonseca, G C; de Morais, G L; de Oliveira, L F V; Margis, R

    2016-01-10

    MicroRNAs (miRNAs) correspond to a class of endogenous small non-coding RNAs (19-24 nt) that regulates the gene expression, through mRNA target cleavage or translation inhibition. In plants, miRNAs have been shown to play pivotal roles in a wide variety of metabolic and biological processes like plant growth, development, and response to biotic and abiotic stress. Soybean is one of the most important crops worldwide, due to the production of oil and its high protein content. The reproductive phase is considered the most important for soybean yield, which is mainly intended to produce the grains. The identification of miRNAs is not yet saturated in soybean, and there are no studies linking them to the different floral organs. In this study, three different mature soybean floral whorls were used in the construction of sRNA libraries. The sequencing of petal, carpel and stamen libraries generated a total of 10,165,661 sequences. Subsequent analyses identified 200 miRNAs sequences, among which, 41 were novel miRNAs, 80 were conserved soybean miRNAs, 31 were new antisense conserved soybean miRNAs and 46 were soybean miRNAs isoforms. We also found a new miRNA conserved in other plant species, and finally one miRNA-sibling of a soybean conserved miRNA. Conserved and novel miRNAs were evaluated by RT-qPCR. We observed a differential expression across the three whorls for six miRNAs. Computational predicted targets for miRNAs analyzed by RT-qPCR were identified and present functions related to reproductive process in plants. In summary, the increased accumulation of specific and novel miRNAs in different whorls indicates that miRNAs are an important part of the regulatory network in soybean flower. PMID:26341053

  9. Small non-coding RNAs and their associated proteins in spermatogenesis.

    PubMed

    Luo, Ling-Feng; Hou, Cong-Cong; Yang, Wan-Xi

    2016-03-10

    The importance of the gene regulation roles of small non-coding RNAs and their protein partners is of increasing focus. In this paper, we reviewed three main small RNA species which appear to affect spermatogenesis. MicroRNAs (miRNAs) are single stand RNAs derived from transcripts containing stem-loops and hairpins which target corresponding mRNAs and affect their stability or translation. Many miRNA species have been found to be related to normal male germ cell development. The biogenesis of piRNAs is still largely unknown but several models have been proposed. Some piRNAs and PIWIs target transposable elements and it is these that may be active in regulating translation or stem cell maintenance. endo-siRNAs may also participate in sperm development. Some possible interactions between different kinds of small RNAs have even been suggested. We also show that male germ granules are seen to have a close relationship with a considerable number of mRNAs and small RNAs. Those special structures may also participate in sperm development. PMID:26692146

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  12. Inhibition of 3' modification of small RNAs in virus-infected plants require spatial and temporal co-expression of small RNAs and viral silencing-suppressor proteins.

    PubMed

    Lózsa, Rita; Csorba, Tibor; Lakatos, Lóránt; Burgyán, József

    2008-07-01

    Plant viruses are inducers and targets of RNA silencing. Viruses counteract with RNA silencing by expressing silencing-suppressor proteins. Many of the identified proteins bind siRNAs, which prevents assembly of silencing effector complexes, and also interfere with their 3' methylation, which protects them against degradation. Here, we investigated the 3' modification of silencing-related small RNAs in Nicotiana benthamiana plants infected with viruses expressing RNA silencing suppressors, the p19 protein of Carnation Italian ringspot virus (CIRV) and HC-Pro of Tobacco etch virus (TEV). We found that CIRV had only a slight effect on viral siRNA 3' modification, but TEV significantly inhibited the 3' modification of si/miRNAs. We also found that p19 and HC-Pro were able to bind both 3' modified and non-modified small RNAs in vivo. The findings suggest that the 3' modification of viral siRNAs occurs in the cytoplasm, though miRNA 3' modification likely takes place in the nucleus as well. Both silencing suppressors inhibited the 3' modification of si/miRNAs when they and small RNAs were transiently co-expressed, suggesting that the inhibition of si/miRNA 3' modification requires spatial and temporal co-expression. Finally, our data revealed that a HEN1-like methyltransferase might account for the small RNA modification at the their 3'-terminal nucleotide in N. benthamiana. PMID:18539609

  13. The expression pattern of small nucleolar and small Cajal body-specific RNAs characterizes distinct molecular subtypes of multiple myeloma

    PubMed Central

    Ronchetti, D; Todoerti, K; Tuana, G; Agnelli, L; Mosca, L; Lionetti, M; Fabris, S; Colapietro, P; Miozzo, M; Ferrarini, M; Tassone, P; Neri, A

    2012-01-01

    Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs involved in the maturation of other RNA molecules and generally located in the introns of host genes. It is now emerging that altered sno/scaRNAs expression may have a pathological role in cancer. This study elucidates the patterns of sno/scaRNAs expression in multiple myeloma (MM) by profiling purified malignant plasma cells from 55 MMs, 8 secondary plasma cell leukemias (sPCLs) and 4 normal controls. Overall, a global sno/scaRNAs downregulation was found in MMs and, even more, in sPCLs compared with normal plasma cells. Whereas SCARNA22 resulted the only sno/scaRNA characterizing the translocation/cyclin D4 (TC4) MM, TC2 group displayed a distinct sno/scaRNA signature overexpressing members of SNORD115 and SNORD116 families located in a region finely regulated by an imprinting center at 15q11, which, however, resulted overall hypomethylated in MMs independently of the SNORD115 and SNORD116 expression levels. Finally, integrative analyses with available gene expression and genome-wide data revealed the occurrence of significant sno/scaRNAs/host genes co-expression and the putative influence of allelic imbalances on specific snoRNAs expression. Our data extend the current view of sno/scaRNAs deregulation in cancer and add novel information to the bio-molecular complexity of plasma cell dyscrasias. PMID:23178508

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

    PubMed Central

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

    2014-01-01

    Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep) sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA) associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs had a unique size distribution consisting of 19–20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA). Sequences from 47 sdRNAs were identified, which mapped to both 5′ and 3′ ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha–mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs. PMID:25203660

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    2012-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Brantl, Sabine; Brückner, Reinhold

    2014-01-01

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

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

    PubMed

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

    2014-05-01

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

  2. Gammaherpesvirus Small Noncoding RNAs Are Bifunctional Elements That Regulate Infection and Contribute to Virulence In Vivo

    PubMed Central

    Diebel, Kevin W.; Oko, Lauren M.; Medina, Eva M.; Niemeyer, Brian F.; Warren, Cody J.; Claypool, David J.; Tibbetts, Scott A.; Cool, Carlyne D.; Clambey, Eric T.

    2015-01-01

    ABSTRACT Many viruses express noncoding RNAs (ncRNAs). The gammaherpesviruses (γHVs), including Epstein-Barr virus, Kaposi’s sarcoma-associated herpesvirus, and murine γHV68, each contain multiple ncRNA genes, including microRNAs (miRNAs). While these ncRNAs can regulate multiple host and viral processes in vitro, the genetic contribution of these RNAs to infection and pathogenesis remains largely unknown. To study the functional contribution of these RNAs to γHV infection, we have used γHV68, a small-animal model of γHV pathogenesis. γHV68 encodes eight small hybrid ncRNAs that contain both tRNA-like elements and functional miRNAs. These genes are transcribed by RNA polymerase III and are referred to as the γHV68 TMERs (tRNA-miRNA-encoded RNAs). To determine the total concerted genetic contribution of these ncRNAs to γHV acute infection and pathogenesis, we generated and characterized a recombinant γHV68 strain devoid of all eight TMERs. TMER-deficient γHV68 has wild-type levels of lytic replication in vitro and normal establishment of latency in B cells early following acute infection in vivo. In contrast, during acute infection of immunodeficient mice, TMER-deficient γHV68 has reduced virulence in a model of viral pneumonia, despite having an enhanced frequency of virus-infected cells. Strikingly, expression of a single viral tRNA-like molecule, in the absence of all other virus-encoded TMERs and miRNAs, reverses both attenuation in virulence and enhanced frequency of infected cells. These data show that γHV ncRNAs play critical roles in acute infection and virulence in immunocompromised hosts and identify these RNAs as a new potential target to modulate γHV-induced infection and pathogenesis. PMID:25691585

  3. Transcriptome of Small Regulatory RNAs in the Development of the Zoonotic Parasite Trichinella spiralis

    PubMed Central

    Tang, Bin; Piao, Xianyu; Hou, Nan; Peng, Shuai; Jiang, Ning; Yin, Jigang; Liu, Mingyuan; Chen, Qijun

    2011-01-01

    Background Trichinella spiralis is a parasite with unique features. It is a multicellular organism but with an intracellular parasitization and development stage. T. spiralis is the helminthic pathogen that causes zoonotic trichinellosis and afflicts more than 10 million people worldwide, whereas the parasite's biology, especially the developmental regulation is largely unknown. In other organisms, small non-coding RNAs, such as microRNAs (miRNA) and small interfering RNAs (siRNA) execute post-transcriptional regulation by translational repression or mRNA degradation, and a large number of miRNAs have been identified in diverse species. In T. spiralis, the profile of small non-coding RNAs and their function remains poorly understood. Methodology and Principal Findings Here, the transcriptional profiles of miRNA and siRNA in three developmental stages of T. spiralis in the rat host were investigated, and compared by high-throughput cDNA sequencing technique (“RNA-seq”). 5,443,641 unique sequence tags were obtained. Of these, 21 represented conserved miRNAs related to 13 previously identified metazoan miRNA families and 213 were novel miRNAs so far unique to T. spiralis. Some of these miRNAs exhibited stage-specific expression. Expression of miRNAs was confirmed in three stages of the life cycle by qRT-PCR and northern blot analysis. In addition, endogenous siRNAs (endo-siRNAs) were found mainly derived from natural antisense transcripts (NAT) and transposable elements (TE) in the parasite. Conclusions and Significance We provide evidence for the presence of miRNAs and endo-siRNAs in T. spiralis. The miRNAs accounted for the major proportion of the small regulatory RNA population of T. spiralis, while fewer endogenous siRNAs were found. The finding of stage-specific expression patterns of the miRNAs in different developmental stages of T. spiralis suggests that miRNAs may play important roles in parasite development. Our data provide a basis for further

  4. FDF-PAGE: a powerful technique revealing previously undetected small RNAs sequestered by complementary transcripts

    PubMed Central

    Harris, C. Jake; Molnar, Attila; Müller, Sebastian Y.; Baulcombe, David C.

    2015-01-01

    Small RNAs, between 18nt and 30nt in length, are a diverse class of non-coding RNAs that mediate a range of cellular processes, from gene regulation to pathogen defense. They guide ribonucleoprotein complexes to their target nucleic acids by Watson–Crick base pairing. We report here that current techniques for small RNA detection and library generation are biased by formation of RNA duplexes. To address this problem, we established FDF-PAGE (fully-denaturing formaldehyde polyacrylamide gel electrophoresis) to prevent annealing of sRNAs to their complement. By applying FDF-PAGE, we provide evidence that both strands of viral small RNA are present in near equimolar ratios, indicating that the predominant precursor is a long double-stranded RNA. Comparing non-denaturing conditions to FDF-PAGE uncovered extensive sequestration of miRNAs in model organisms and allowed us to identify candidate small RNAs under the control of competing endogenous RNAs (ceRNAs). By revealing the full repertoire of small RNAs, we can begin to create a better understanding of small RNA mediated interactions. PMID:26071954

  5. 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. PMID:25389522

  6. 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

  7. Identification of small non-coding RNAs in the planarian Dugesia japonica via deep sequencing.

    PubMed

    Qin, Yun-Fei; Zhao, Jin-Mei; Bao, Zhen-Xia; Zhu, Zhao-Yu; Mai, Jia; Huang, Yi-Bo; Li, Jian-Biao; Chen, Ge; Lu, Ping; Chen, San-Jun; Su, Lin-Lin; Fang, Hui-Min; Lu, Ji-Ke; Zhang, Yi-Zhe; Zhang, Shou-Tao

    2012-05-01

    Freshwater planarian flatworm possesses an extraordinary ability to regenerate lost body parts after amputation; it is perfect organism model in regeneration and stem cell biology. Recently, small RNAs have been an increasing concern and studied in many aspects, including regeneration and stem cell biology, among others. In the current study, the large-scale cloning and sequencing of sRNAs from the intact and regenerative planarian Dugesia japonica are reported. Sequence analysis shows that sRNAs between 18nt and 40nt are mainly microRNAs and piRNAs. In addition, 209 conserved miRNAs and 12 novel miRNAs are identified. Especially, a better screening target method, negative-correlation relationship of miRNAs and mRNA, is adopted to improve target prediction accuracy. Similar to miRNAs, a diverse population of piRNAs and changes in the two samples are also listed. The present study is the first to report on the important role of sRNAs during planarian Dugesia japonica regeneration. PMID:22425900

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

    PubMed Central

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

    2013-01-01

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

  9. 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

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

    PubMed Central

    2013-01-01

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

  11. Identification of microRNA-like small RNAs from fungal parasite Nosema ceranae.

    PubMed

    Huang, Qiang; Evans, Jay D

    2016-01-01

    We previously found transcripts encoding Dicer and Argonaute which are involved in the production of microRNAs, in the honey bee parasite Nosema ceranae. In order to identify microRNAs in N. ceranae, we sequenced small RNAs from midgut tissues of infected honey bees at 24 h intervals for 6 days post infection, covering the complete reproduction cycle for this intracellular parasite. We predicted six microRNA-like small RNAs, all of which were confirmed via RT-qPCR assays. This is the first evidence for microRNA-like small RNAs generated by a microsporidian species, providing new insights into host-parasite interactions involving this widespread taxonomic group. PMID:26678507

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

    PubMed Central

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

    2013-01-01

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

  13. 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

  14. 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. PMID:26914027

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

    PubMed Central

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

    2015-01-01

    Small silencing RNAs, including microRNAs, endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs), have been shown to play important roles in fine-tuning gene expression, defending virus and controlling transposons. Loss of small silencing RNAs or components in their pathways often leads to severe developmental defects, including lethality and sterility. Recently, non-templated addition of nucleotides to the 3′ end, namely tailing, was found to associate with the processing and stability of small silencing RNAs. Next Generation Sequencing has made it possible to detect such modifications at nucleotide resolution in an unprecedented throughput. Unfortunately, detecting such events from millions of short reads confounded by sequencing errors and RNA editing is still a tricky problem. Here, we developed a computational framework, Tailor, driven by an efficient and accurate aligner specifically designed for capturing the tailing events directly from the alignments without extensive post-processing. The performance of Tailor was fully tested and compared favorably with other general-purpose aligners using both simulated and real datasets for tailing analysis. Moreover, to show the broad utility of Tailor, we used Tailor to reanalyze published datasets and revealed novel findings worth further experimental validation. The source code and the executable binaries are freely available at https://github.com/jhhung/Tailor. PMID:26007652

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

    PubMed

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

    2015-09-30

    Small silencing RNAs, including microRNAs, endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs), have been shown to play important roles in fine-tuning gene expression, defending virus and controlling transposons. Loss of small silencing RNAs or components in their pathways often leads to severe developmental defects, including lethality and sterility. Recently, non-templated addition of nucleotides to the 3' end, namely tailing, was found to associate with the processing and stability of small silencing RNAs. Next Generation Sequencing has made it possible to detect such modifications at nucleotide resolution in an unprecedented throughput. Unfortunately, detecting such events from millions of short reads confounded by sequencing errors and RNA editing is still a tricky problem. Here, we developed a computational framework, Tailor, driven by an efficient and accurate aligner specifically designed for capturing the tailing events directly from the alignments without extensive post-processing. The performance of Tailor was fully tested and compared favorably with other general-purpose aligners using both simulated and real datasets for tailing analysis. Moreover, to show the broad utility of Tailor, we used Tailor to reanalyze published datasets and revealed novel findings worth further experimental validation. The source code and the executable binaries are freely available at https://github.com/jhhung/Tailor. PMID:26007652

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

    PubMed

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

    2015-01-01

    Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30 nm) polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture. PMID:26035832

  18. Functional diversification of Dicer-like proteins and small RNAs required for genome sculpting.

    PubMed

    Sandoval, Pamela Y; Swart, Estienne C; Arambasic, Miroslav; Nowacki, Mariusz

    2014-01-27

    In eukaryotes, small RNAs (sRNAs) have key roles in development, gene expression regulation, and genome integrity maintenance. In ciliates, such as Paramecium, sRNAs form the heart of an epigenetic system that has evolved from core eukaryotic gene silencing components to selectively target DNA for deletion. In Paramecium, somatic genome development from the germline genome accurately eliminates the bulk of typically gene-interrupting, noncoding DNA. We have discovered an sRNA class (internal eliminated sequence [IES] sRNAs [iesRNAs]), arising later during Paramecium development, which originates from and precisely delineates germline DNA (IESs) and complements the initial sRNAs ("scan" RNAs [scnRNAs]) in targeting DNA for elimination. We show that whole-genome duplications have facilitated successive differentiations of Paramecium Dicer-like proteins, leading to cooperation between Dcl2 and Dcl3 to produce scnRNAs and to the production of iesRNAs by Dcl5. These innovations highlight the ability of sRNA systems to acquire capabilities, including those in genome development and integrity. PMID:24439910

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

    PubMed

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

    2015-01-01

    MicroRNAs (miRNAs) exhibit diverse and important roles in plant growth, development, and stress responses and regulate gene expression at the post-transcriptional level. Knowledge about the diversity of miRNAs and their roles in leaf development in celery remains unknown. To elucidate the roles of miRNAs in celery leaf development, we identified leaf development-related miRNAs through high-throughput sequencing. Small RNA libraries were constructed using leaves from three stages (10, 20, and 30 cm) of celery cv.'Ventura' and then subjected to high-throughput sequencing and bioinformatics analysis. At Stage 1, Stage 2, and Stage 3 of 'Ventura', a total of 333, 329, and 344 conserved miRNAs (belonging to 35, 35, and 32 families, respectively) were identified. A total of 131 miRNAs were identified as novel in 'Ventura'. Potential miRNA target genes were predicted and annotated using the eggNOG, GO, and KEGG databases to explore gene functions. The abundance of five conserved miRNAs and their corresponding potential target genes were validated. Expression profiles of novel potential miRNAs were also detected. Anatomical characteristics of the leaf blades and petioles at three leaf stages were further analyzed. This study contributes to our understanding on the functions and molecular regulatory mechanisms of miRNAs in celery leaf development. PMID:26057455

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

    PubMed Central

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

    2015-01-01

    Hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer and represents the third leading cause of cancer-related death worldwide. Current available therapeutic approaches are poorly effective, especially for the advanced forms of the disease. In the last year, short double stranded RNA molecules termed small interfering RNAs (siRNAs) and micro interfering RNAs (miRNA), emerged as interesting molecules with potential therapeutic value for HCC. The practical use of these molecules is however limited by the identification of optimal molecular targets and especially by the lack of effective and targeted HCC delivery systems. Here we focus our discussion on the most recent advances in the identification of siRNAs/miRNAs molecular targets and on the development of suitable siRNA/miRNAs delivery systems. PMID:26290628

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

    PubMed Central

    2011-01-01

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

  2. Rapid and selective extraction, isolation, preconcentration, and quantitation of small RNAs from cell lysate using on-chip isotachophoresis.

    PubMed

    Schoch, Reto B; Ronaghi, Mostafa; Santiago, Juan G

    2009-08-01

    We present a technique which enables the separation of small RNAs-such as microRNAs (miRNAs), short interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs)-from >or=66 nucleotide RNAs and other biomolecules contained in a cell lysate. In particular, the method achieves separation of small RNAs from precursor miRNAs (pre-miRNAs) in less than 3 min. We use on-chip isotachophoresis (ITP) for the simultaneous extraction, isolation, preconcentration and quantitation of small RNAs (approximately 22 nucleotides) and employ the high-efficiency sieving matrix Pluronic F-127; a thermo-responsive triblock copolymer which allows convenient microchannel loading at low temperature. We present the isolation of small RNAs from the lysate of 293A human kidney cells, and quantitate the number of short RNA molecules per cell to be 2.9x10(7). We estimate this quantity is an aggregate of roughly 500 types of short RNA molecules per 293A cell. Currently, the minimal cell number for small RNA extraction and detection with our method is approximately 900 (from a 5 microL sample volume), and we believe that small RNA analysis from tens of cells is realizable. Techniques for rapid and sensitive extraction and isolation of small RNAs from cell lysate are much-needed to further uncover their full range and functionality, including RNA interference studies. PMID:19606290

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

    PubMed

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

    2013-04-01

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

  4. Identification of miniature inverted-repeat transposable elements (MITEs) and biogenesis of their siRNAs in the Solanaceae: New functional implications for MITEs

    PubMed Central

    Kuang, Hanhui; Padmanabhan, Chellappan; Li, Feng; Kamei, Ayako; Bhaskar, Pudota B.; Ouyang, Shu; Jiang, Jiming; Buell, C. Robin; Baker, Barbara

    2009-01-01

    Small RNAs regulate the genome by guiding transcriptional and post-transcriptional silencing machinery to specific target sequences, including genes and transposable elements (TEs). Although miniature inverted-repeat transposable elements (MITEs) are closely associated with euchromatic genes, the broader functional impact of these short TE insertions in genes is largely unknown. We identified 22 families of MITEs in the Solanaceae (MiS1–MiS22) and found abundant MiS insertions in Solanaceae genomic DNA and expressed sequence tags (EST). Several Solanaceae MITEs generate genome changes that potentially affect gene function and regulation, most notably, a MiS insertion that provides a functionally indispensable alternative exon in the tobacco mosaic virus N resistance gene. We show that MITEs generate small RNAs that are primarily 24 nt in length, as detected by Northern blot hybridization and by sequencing small RNAs of Solanum demissum, Nicotiana glutinosa, and Nicotiana benthamiana. Additionally, we show that stable RNAi lines silencing DICER-LIKE3 (DCL3) in tobacco and RNA-dependent RNA polymerase 2 (RDR2) in potato cause a reduction in 24-nt MITE siRNAs, suggesting that, as in Arabidopsis, TE-derived siRNA biogenesis is DCL3 and RDR2 dependent. We provide evidence that DICER-LIKE4 (DCL4) may also play a role in MITE siRNA generation in the Solanaceae. PMID:19037014

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

    PubMed Central

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

    2015-01-01

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

  6. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  9. RNA SHAPE Analysis of Small RNAs and Riboswitches

    PubMed Central

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

    2016-01-01

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

  10. Targeting the production of oncogenic microRNAs with multimodal synthetic small molecules.

    PubMed

    Vo, Duc Duy; Staedel, Cathy; Zehnacker, Laura; Benhida, Rachid; Darfeuille, Fabien; Duca, Maria

    2014-03-21

    MicroRNAs (miRNAs) are a recently discovered category of small RNA molecules that regulate gene expression at the post-transcriptional level. Accumulating evidence indicates that miRNAs are aberrantly expressed in a variety of human cancers and revealed to be oncogenic and to play a pivotal role in initiation and progression of these pathologies. It is now clear that the inhibition of oncogenic miRNAs, defined as blocking their biosynthesis or their function, could find an application in the therapy of different types of cancer in which these miRNAs are implicated. Here we report the design, synthesis, and biological evaluation of new small-molecule RNA ligands targeting the production of oncogenic microRNAs. In this work we focused our attention on miR-372 and miR-373 that are implicated in the tumorigenesis of different types of cancer such as gastric cancer. These two oncogenic miRNAs are overexpressed in gastric cancer cells starting from their precursors pre-miR-372 and pre-miR-373, two stem-loop structured RNAs that lead to mature miRNAs after cleavage by the enzyme Dicer. The small molecules described herein consist of the conjugation of two RNA binding motives, i.e., the aminoglycoside neomycin and different natural and artificial nucleobases, in order to obtain RNA ligands with increased affinity and selectivity compared to that of parent compounds. After the synthesis of this new series of RNA ligands, we demonstrated that they are able to inhibit the production of the oncogenic miRNA-372 and -373 by binding their pre-miRNAs and inhibiting the processing by Dicer. Moreover, we proved that some of these compounds bear anti-proliferative activity toward gastric cancer cells and that this activity is likely linked to a decrease in the production of targeted miRNAs. To date, only few examples of small molecules targeting oncogenic miRNAs have been reported, and such inhibitors could be extremely useful for the development of new anticancer therapeutic

  11. 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

  12. A big role for small RNAs in HDL homeostasis

    PubMed Central

    Ouimet, Mireille; Moore, Kathryn J.

    2013-01-01

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

  13. Multiple factors dictate target selection by Hfq-binding small RNAs

    PubMed Central

    Beisel, Chase L; Updegrove, Taylor B; Janson, Ben J; Storz, Gisela

    2012-01-01

    Hfq-binding small RNAs (sRNAs) in bacteria modulate the stability and translational efficiency of target mRNAs through limited base-pairing interactions. While these sRNAs are known to regulate numerous mRNAs as part of stress responses, what distinguishes targets and non-targets among the mRNAs predicted to base pair with Hfq-binding sRNAs is poorly understood. Using the Hfq-binding sRNA Spot 42 of Escherichia coli as a model, we found that predictions using only the three unstructured regions of Spot 42 substantially improved the identification of previously known and novel Spot 42 targets. Furthermore, increasing the extent of base-pairing in single or multiple base-pairing regions improved the strength of regulation, but only for the unstructured regions of Spot 42. We also found that non-targets predicted to base pair with Spot 42 lacked an Hfq-binding site, folded into a secondary structure that occluded the Spot 42 targeting site, or had overlapping Hfq-binding and targeting sites. By modifying these features, we could impart Spot 42 regulation on non-target mRNAs. Our results thus provide valuable insights into the requirements for target selection by sRNAs. PMID:22388518

  14. A Global Identification and Analysis of Small Nucleolar RNAs and Possible Intermediate-Sized Non-Coding RNAs in Oryza sativa

    PubMed Central

    Liu, Ting-Ting; Zhu, Danmeng; Chen, Wei; Deng, Xing Wang

    2013-01-01

    Accumulating evidence suggests that non-coding RNAs (ncRNAs) are both widespread and functionally important in many eukaryotic organisms. In this study, we employed a special size fractionation and cDNA library construction method followed by 454 deep sequencing to systematically profile rice intermediate-size ncRNAs. Our analysis resulted in the identification of 1349 ncRNAs in total, including 754 novel ncRNAs of an unknown functional category. Chromosome distribution of all identified ncRNAs showed no strand bias, and displayed a pattern similar to that observed in protein-coding genes with few chromosome dependencies. More than half of the ncRNAs were centered around the plus-strand of the 5’ and 3’ termini of the coding regions. The majority of the novel ncRNAs were rice specific, while 78% of the small nucleolar RNAs (snoRNAs) were conserved. Tandem duplication drove the expansion of over half of the snoRNA gene families. Furthermore, 90% of the snoRNA candidates were shown to produce small RNAs between 20–30 nt, 80% of which were associated with ARGONAUT proteins generally, and AGO1b in particular. Overall, our findings provide a comprehensive view of an intermediate-size non-coding transcriptome in a monocot species, which will serve as a useful platform for an in-depth analysis of ncRNA functions. PMID:22986792

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

    PubMed

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

    2016-09-01

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

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

    PubMed Central

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

    2016-01-01

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

  17. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  20. Non-coding RNAs: Epigenetic regulators of bone development and homeostasis.

    PubMed

    Hassan, Mohammad Q; Tye, Coralee E; Stein, Gary S; Lian, Jane B

    2015-12-01

    Non-coding RNAs (ncRNAs) have evolved in eukaryotes as epigenetic regulators of gene expression. The most abundant regulatory ncRNAs are the 20-24 nt small microRNAs (miRNAs) and long non-coding RNAs (lncRNAs, <200 nt). Each class of ncRNAs operates through distinct mechanisms, but their pathways to regulating gene expression are interrelated in ways that are just being recognized. While the importance of lncRNAs in epigenetic control of transcription, developmental processes and human traits is emerging, the identity of lncRNAs in skeletal biology is scarcely known. However, since the first profiling studies of miRNA at stages during osteoblast and osteoclast differentiation, over 1100 publications related to bone biology and pathologies can be found, as well as many recent comprehensive reviews summarizing miRNA in skeletal cells. Delineating the activities and targets of specific miRNAs regulating differentiation of osteogenic and resorptive bone cells, coupled with in vivo gain- and loss-of-function studies, discovered unique mechanisms that support bone development and bone homeostasis in adults. We present here "guiding principles" for addressing biological control of bone tissue formation by ncRNAs. This review emphasizes recent advances in understanding regulation of the process of miRNA biogenesis that impact on osteogenic lineage commitment, transcription factors and signaling pathways. Also discussed are the approaches to be pursued for an understanding of the role of lncRNAs in bone and the challenges in addressing their multiple and complex functions. Based on new knowledge of epigenetic control of gene expression to be gained for ncRNA regulation of the skeleton, new directions for translating the miRNAs and lncRNAs into therapeutic targets for skeletal disorders are possible. This article is part of a Special Issue entitled Epigenetics and Bone. PMID:26039869

  1. Revealing editing and SNPs of microRNAs in colon tissues by analyzing high-throughput sequencing profiles of small RNAs

    PubMed Central

    2014-01-01

    Background Editing and mutations in microRNAs (miRNAs) can change the stability of pre-miRNAs and/or complementarities between miRNAs and their targets. Small RNA (sRNA) high-throughput sequencing (HTS) profiles contain miRNAs that are originated from mutated DNAs or are edited during their biogenesis procedures. It is largely unknown whether miRNAs are edited in colon tissues since existing studies mainly focused their attention on the editing of miRNAs in brain tissues. Results Through comprehensive analysis of four high-throughput sequencing profiles of normal and cancerous colon tissues, we identified 548 editing and/or SNPs in miRNAs that are significant in at least one of the sequencing profiles used. Our results show that the most abundant editing events of miRNAs in colon tissues are 3'-A and 3'-U. In addition to four known A-to-I editing sites previously reported in brain tissues, four novel A-to-I editing sites are also identified in colon tissues. Conclusions This suggests that A-to-I editing of miRNAs potentially is a commonly existing mechanism in different tissues to diversify the possible functional roles of miRNAs, but only a small portion of different miRNAs are edited by the A-to-I mechanism at a significant level. Our results suggest that there are other types of editing in miRNAs through unknown mechanisms. Furthermore, several SNPs in miRNAs are also identified. PMID:25521855

  2. Small is beautiful: microRNAs and breast cancer-where are we now?

    PubMed

    Verghese, E T; Hanby, A M; Speirs, V; Hughes, T A

    2008-07-01

    MicroRNAs are a recently discovered class of small regulatory RNAs that influence the stability and translational efficiency of target mRNAs. They have been implicated in an increasing number of biological processes, including neoplasia. Recent studies have shown an involvement for these regulatory molecules in breast cancer. For example, miRNA profiling studies have identified microRNAs that are deregulated in breast cancer. Furthermore, functional studies have uncovered their roles in breast cancer as both tumour suppressor genes (eg miR-335) and oncogenes (eg miR-21). miRNAs deregulated in breast cancer influence the translational regulation of well-established regulatory molecules, such as oestrogen receptor-alpha, which is regulated by miR-206, and novel cancer-related molecules whose functions are not yet fully understood.. Here we present an overview of our current understanding of miRNA in breast cancer. PMID:18446835

  3. MicroRNAs in heart failure: Small molecules with major impact

    PubMed Central

    Kalozoumi, Georgia; Yacoub, Magdi; Sanoudou, Despina

    2014-01-01

    MicroRNAs (miRNAs) have emerged as potent modulators of mammalian gene expression, thereby broadening the spectrum of molecular mechanisms orchestrating human physiological and pathological cellular functions. Growing evidence suggests that these small non-coding RNA molecules are pivotal regulators of cardiovascular development and disease. Importantly, multiple miRNAs have been specifically implicated in the onset and progression of heart failure, thus providing a new platform for battling this multi-faceted disease. This review introduces the basic concepts of miRNA biology, describes representative examples of miRNAs associated with multiple aspects of HF pathogenesis, and explores the prognostic, diagnostic and therapeutic potential of miRNAs in the cardiology clinic. PMID:25419522

  4. Regulation of cytokines by small RNAs during skin inflammation

    PubMed Central

    2010-01-01

    Intercellular signaling by cytokines is a vital feature of the innate immune system. In skin, an inflammatory response is mediated by cytokines and an entwined network of cellular communication between T-cells and epidermal keratinocytes. Dysregulated cytokine production, orchestrated by activated T-cells homing to the skin, is believed to be the main cause of psoriasis, a common inflammatory skin disorder. Cytokines are heavily regulated at the transcriptional level, but emerging evidence suggests that regulatory mechanisms that operate after transcription play a key role in balancing the production of cytokines. Herein, we review the nature of cytokine signaling in psoriasis with particular emphasis on regulation by mRNA destabilizing elements and the potential targeting of cytokine-encoding mRNAs by miRNAs. The proposed linkage between mRNA decay mediated by AU-rich elements and miRNA association is described and discussed as a possible general feature of cytokine regulation in skin. Moreover, we describe the latest attempts to therapeutically target cytokines at the RNA level in psoriasis by exploiting the cellular RNA interference machinery. The applicability of cytokine-encoding mRNAs as future clinical drug targets is evaluated, and advances and obstacles related to topical administration of RNA-based drugs targeting the cytokine circuit in psoriasis are described. PMID:20594301

  5. 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. PMID

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

    PubMed Central

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

    2016-01-01

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

  7. Evidence for widespread exonic small RNAs in the glaucophyte alga Cyanophora paradoxa.

    PubMed

    Gross, Jeferson; Wajid, Sana; Price, Dana C; Zelzion, Ehud; Li, Junyi; Chan, Cheong Xin; Bhattacharya, Debashish

    2013-01-01

    RNAi (RNA interference) relies on the production of small RNAs (sRNAs) from double-stranded RNA and comprises a major pathway in eukaryotes to restrict the propagation of selfish genetic elements. Amplification of the initial RNAi signal by generation of multiple secondary sRNAs from a targeted mRNA is catalyzed by RNA-dependent RNA polymerases (RdRPs). This phenomenon is known as transitivity and is particularly important in plants to limit the spread of viruses. Here we describe, using a genome-wide approach, the distribution of sRNAs in the glaucophyte alga Cyanophora paradoxa. C. paradoxa is a member of the supergroup Plantae (also known as Archaeplastida) that includes red algae, green algae, and plants. The ancient (>1 billion years ago) split of glaucophytes within Plantae suggests that C. paradoxa may be a useful model to learn about the early evolution of RNAi in the supergroup that ultimately gave rise to plants. Using next-generation sequencing and bioinformatic analyses we find that sRNAs in C. paradoxa are preferentially associated with mRNAs, including a large number of transcripts that encode proteins arising from different functional categories. This pattern of exonic sRNAs appears to be a general trend that affects a large fraction of mRNAs in the cell. In several cases we observe that sRNAs have a bias for a specific strand of the mRNA, including many instances of antisense predominance. The genome of C. paradoxa encodes four sequences that are homologous to RdRPs in Arabidopsis thaliana. We discuss the possibility that exonic sRNAs in the glaucophyte may be secondarily derived from mRNAs by the action of RdRPs. If this hypothesis is confirmed, then transitivity may have had an ancient origin in Plantae. PMID:23844054

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

    PubMed Central

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

    2015-01-01

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

  9. Intercellular transfer of small RNAs from astrocytes to lung tumor cells induces resistance to chemotherapy

    PubMed Central

    Menachem, Assaf; Makovski, Victoria; Bodner, Or; Pasmanik-Chor, Metsada; Stein, Reuven; Shomron, Noam; Kloog, Yoel

    2016-01-01

    Brain metastases are resistant to chemotherapy and carry a poor prognosis. Studies have shown that tumor cells are surrounded by activated astrocytes, whose cytoprotective properties they exploit for protection from chemotherapy-induced apoptosis. The mechanism of such astrocytic protection is poorly understood. A non-mutational mechanism of resistance to chemotherapy that is receiving increased attention is the regulation of gene translation mediated by small noncoding RNAs (sRNAs), and particularly microRNAs (miRNAs). With the aim of examining the role of astrocytic sRNAs in promoting resistance of human lung tumor PC14 cells to chemotherapy-induced apoptosis, here we used a miRNA microarray to compare sRNA profiles of human lung tumor cells cultured with and without astrocytes. We found that sRNAs are transferred from astrocytes to PC14 cells in a contact-dependent manner. Transfer was rapid, reaching a plateau after only 6 hours in culture. The sRNA transfer was inhibited by the broad-spectrum gap-junction antagonist carbenoxolone, indicating that transfer occurs via gap junctions. Among the transferred sRNAs were several that are implicated in survival pathways. Enforced expression of these sRNAs in PC14 cells increased their resistance to the chemotherapeutic agent paclitaxel. These novel findings might be of clinical relevance for the treatment of patients with brain metastases. PMID:26871466

  10. Small RNAs--the secret agents in the plant-pathogen interactions.

    PubMed

    Weiberg, Arne; Jin, Hailing

    2015-08-01

    Eukaryotic regulatory small RNAs (sRNAs) that induce RNA interference (RNAi) are involved in a plethora of biological processes, including host immunity and pathogen virulence. In plants, diverse classes of sRNAs contribute to the regulation of host innate immunity. These immune-regulatory sRNAs operate through distinct RNAi pathways that trigger transcriptional or post-transcriptional gene silencing. Similarly, many pathogen-derived sRNAs also regulate pathogen virulence. Remarkably, the influence of regulatory sRNAs is not limited to the individual organism in which they are generated. It can sometimes extend to interacting species from even different kingdoms. There they trigger gene silencing in the interacting organism, a phenomenon called cross-kingdom RNAi. This is exhibited in advanced pathogens and parasites that produce sRNAs to suppress host immunity. Conversely, in host-induced gene silencing (HIGS), diverse plants are engineered to trigger RNAi against pathogens and pests to confer host resistance. Cross-kingdom RNAi opens up a vastly unexplored area of research on mobile sRNAs in the battlefield between hosts and pathogens. PMID:26123395

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

    PubMed

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

    2000-04-01

    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

  12. 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

  13. MicroRNAs As Potential Targets for Abiotic Stress Tolerance in Plants.

    PubMed

    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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    Shortridge, Matthew D.; Varani, Gabriele

    2015-01-01

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

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

    PubMed Central

    Dahlmann, Tim A.; Kück, Ulrich

    2015-01-01

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

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

    PubMed

    Dahlmann, Tim A; Kück, Ulrich

    2015-01-01

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

  18. Comparative genomics of eukaryotic small nucleolar RNAs reveals deep evolutionary ancestry amidst ongoing intragenomic mobility

    PubMed Central

    2012-01-01

    Background Small nucleolar (sno)RNAs are required for posttranscriptional processing and modification of ribosomal, spliceosomal and messenger RNAs. Their presence in both eukaryotes and archaea indicates that snoRNAs are evolutionarily ancient. The location of some snoRNAs within the introns of ribosomal protein genes has been suggested to belie an RNA world origin, with the exons of the earliest protein-coding genes having evolved around snoRNAs after the advent of templated protein synthesis. Alternatively, this intronic location may reflect more recent selection for coexpression of snoRNAs and ribosomal components, ensuring rRNA modification by snoRNAs during ribosome synthesis. To gain insight into the evolutionary origins of this genetic organization, we examined the antiquity of snoRNA families and the stability of their genomic location across 44 eukaryote genomes. Results We report that dozens of snoRNA families are traceable to the Last Eukaryotic Common Ancestor (LECA), but find only weak similarities between the oldest eukaryotic snoRNAs and archaeal snoRNA-like genes. Moreover, many of these LECA snoRNAs are located within the introns of host genes independently traceable to the LECA. Comparative genomic analyses reveal the intronic location of LECA snoRNAs is not ancestral however, suggesting the pattern we observe is the result of ongoing intragenomic mobility. Analysis of human transcriptome data indicates that the primary requirement for hosting intronic snoRNAs is a broad expression profile. Consistent with ongoing mobility across broadly-expressed genes, we report a case of recent migration of a non-LECA snoRNA from the intron of a ubiquitously expressed non-LECA host gene into the introns of two LECA genes during the evolution of primates. Conclusions Our analyses show that snoRNAs were a well-established family of RNAs at the time when eukaryotes began to diversify. While many are intronic, this association is not evolutionarily stable across

  19. Structural and genetic requirements for the biogenesis of tobacco rattle virus-derived small interfering RNAs.

    PubMed

    Donaire, Livia; Barajas, Daniel; Martínez-García, Belén; Martínez-Priego, Llucia; Pagán, Israel; Llave, César

    2008-06-01

    In plants, small RNA-guided processes referred to as RNA silencing control gene expression and serve as an efficient antiviral mechanism. Plant viruses are inducers and targets of RNA silencing as infection involves the production of functional virus-derived small interfering RNAs (siRNAs). Here we investigate the structural and genetic components influencing the formation of Tobacco rattle virus (TRV)-derived siRNAs. TRV siRNAs are mostly 21 nucleotides in length and derive from positive and negative viral RNA strands, although TRV siRNAs of positive polarity are significantly more abundant. This asymmetry appears not to correlate with the presence of highly structured regions of single-stranded viral RNA. The Dicer-like enzyme DCL4, DCL3, or DCL2 targets, alone or in combination, viral templates to promote synthesis of siRNAs of both polarities from all regions of the viral genome. The heterogeneous distribution profile of TRV siRNAs reveals differential contributions throughout the TRV genome to siRNA formation. Indirect evidence suggests that DCL2 is responsible for production of a subset of siRNAs derived from the 3' end region of TRV. TRV siRNA biogenesis and antiviral silencing are strongly dependent on the combined activity of the host-encoded RNA-dependent RNA polymerases RDR1, RDR2, and RDR6, thus providing evidence that perfectly complementary double-stranded RNA serves as a substrate for siRNA production. We conclude that the overall composition of viral siRNAs in TRV-infected plants reflects the combined action of several interconnected pathways involving different DCL and RDR activities. PMID:18353962

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

    PubMed Central

    Peláez, Pablo; Sanchez, Federico

    2013-01-01

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

  1. Identification of phasiRNAs in wild rice (Oryza rufipogon).

    PubMed

    Liu, Yang; Wang, Yu; Zhu, Qian-Hao; Fan, Longjiang

    2013-08-01

    Plant miRNAs can trigger the production of phased, secondary siRNAs from either non-coding or protein-coding genes. In this study, at least 864 and 3,961 loci generating 21-nt and 24-nt phased siRNAs (phasiRNAs),respectively, were identified in three tissues from wild rice. Of these phasiRNA-producing loci, or PHAS genes, biogenesis of phasiRNAs in at least 160 of 21-nt and 254 of 24-nt loci could be triggered by interaction with miRNA(s). Developing seeds had more PHAS genes than leaves and roots. Genetic constrain on miRNA-triggered PHAS genes suggests that phasiRNAs might be one of the driving forces contributed to rice domestication. PMID:23733069

  2. 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

  3. 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

  4. Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs

    PubMed Central

    Wu, Qingfa; Luo, Yingjun; Lu, Rui; Lau, Nelson; Lai, Eric C.; Li, Wan-Xiang; Ding, Shou-Wei

    2010-01-01

    In response to infection, invertebrates process replicating viral RNA genomes into siRNAs of discrete sizes to guide virus clearance by RNA interference. Here, we show that viral siRNAs sequenced from fruit fly, mosquito, and nematode cells were all overlapping in sequence, suggesting a possibility of using siRNAs for viral genome assembly and virus discovery. To test this idea, we examined contigs assembled from published small RNA libraries and discovered five previously undescribed viruses from cultured Drosophila cells and adult mosquitoes, including three with a positive-strand RNA genome and two with a dsRNA genome. Notably, four of the identified viruses exhibited only low sequence similarities to known viruses, such that none could be assigned into an existing virus genus. We also report detection of virus-derived PIWI-interacting RNAs (piRNAs) in Drosophila melanogaster that have not been previously described in any other host species and demonstrate viral genome assembly from viral piRNAs in the absence of viral siRNAs. Thus, this study provides a powerful culture-independent approach for virus discovery in invertebrates by assembling viral genomes directly from host immune response products without prior virus enrichment or amplification. We propose that invertebrate viruses discovered by this approach may include previously undescribed human and vertebrate viral pathogens that are transmitted by arthropod vectors. PMID:20080648

  5. Small and Long Non-Coding RNAs: Novel Targets in Perspective Cancer Therapy

    PubMed Central

    Han Li, Chi; Chen, Yangchao

    2015-01-01

    Non-coding RNA refers to a large group of endogenous RNA molecules that have no protein coding capacity, while having specialized cellular and molecular functions. They possess wide range of functions such as the regulation of gene transcription and translation, post-transcriptional modification, epigenetic landscape establishment, protein scaffolding and cofactors recruitments. They are further divided into small non-coding RNAs with size < 200nt (e.g. miRNA, piRNA) and long non-coding RNAs with size >= 200nt (e.g. lincRNA, NAT). Increasing evidences suggest that both non-coding RNAs groups play important roles in cancer development, progression and pathology. Clinically, non-coding RNAs aberrations show high diagnostic and prognostic values. With improved understanding of the nature and roles of non-coding RNAs, it is believed that we can develop therapeutic treatment against cancer via the modulation of these RNA molecules. Advances in nucleic acid drug technology and computational simulation prompt the development of agents to intervene the malignant effects of non-coding RNAs. In this review, we will discuss the role of non-coding RNAs in cancer, and evaluate the potential of non-coding RNA-based cancer therapies. PMID:27047252

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

    PubMed Central

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

    2016-01-01

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

  7. Analysis of small nucleolar RNAs in sputum for lung cancer diagnosis

    PubMed Central

    Guarnera, Maria A.; Zhan, Min; Fang, HongBin; Stass, Sanford A.; Jiang, Feng

    2016-01-01

    Molecular analysis of sputum presents a noninvasive approach for diagnosis of lung cancer. We have shown that dysregulation of small nucleolar RNAs (snoRNAs) plays a vital role in lung tumorigenesis. We have also identified six snoRNAs whose changes are associated with lung cancer. Here we investigated if analysis of the snoRNAs in sputum could provide a potential tool for diagnosis of lung cancer. Using qRT-PCR, we determined expressions of the six snoRNAs in sputum of a training set of 59 lung cancer patients and 61 cancer-free smokers to develop a biomarker panel, which was validated in a testing set of 67 lung cancer patients and 69 cancer-free smokers for the diagnostic performance. The snoRNAs were robustly measurable in sputum. In the training set, a panel of two snoRNA biomarkers (snoRD66 and snoRD78) was developed, producing 74.58% sensitivity and 83.61% specificity for identifying lung cancer. The snoRNA biomarkers had a significantly higher sensitivity (74.58%) compared with sputum cytology (45.76%) (P < 0.05). The changes of the snoRNAs were not associated with stage and histology of lung cancer (All P >0.05). The performance of the biomarker panel was confirmed in the testing cohort. We report for the first time that sputum snoRNA biomarkers might be useful to improve diagnosis of lung cancer. PMID:26246471

  8. High Throughput Genome-Wide Survey of Small RNAs from the Parasitic Protists Giardia intestinalis and Trichomonas vaginalis

    PubMed Central

    Collins, Lesley J.; Biggs, Patrick J.; Penny, David

    2009-01-01

    RNA interference (RNAi) is a set of mechanisms which regulate gene expression in eukaryotes. Key elements of RNAi are small sense and antisense RNAs from 19 to 26 nt generated from double-stranded RNAs. MicroRNAs (miRNAs) are a major type of RNAi-associated small RNAs and are found in most eukaryotes studied to date. To investigate whether small RNAs associated with RNAi appear to be present in all eukaryotic lineages, and therefore present in the ancestral eukaryote, we studied two deep-branching protozoan parasites, Giardia intestinalis and Trichomonas vaginalis. Little is known about endogenous small RNAs involved in RNAi of these organisms. Using Illumina Solexa sequencing and genome-wide analysis of small RNAs from these distantly related deep-branching eukaryotes, we identified 10 strong miRNA candidates from Giardia and 11 from Trichomonas. We also found evidence of Giardia short-interfering RNAs potentially involved in the expression of variant-specific surface proteins. In addition, eight new small nucleolar RNAs from Trichomonas are identified. Our results indicate that miRNAs are likely to be general in ancestral eukaryotes and therefore are likely to be a universal feature of eukaryotes. PMID:20333187

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

    PubMed Central

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  12. Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways.

    PubMed

    Weiberg, Arne; Wang, Ming; Lin, Feng-Mao; Zhao, Hongwei; Zhang, Zhihong; Kaloshian, Isgouhi; Huang, Hsien-Da; Jin, Hailing

    2013-10-01

    Botrytis cinerea, the causative agent of gray mold disease, is an aggressive fungal pathogen that infects more than 200 plant species. Here, we show that some B. cinerea small RNAs (Bc-sRNAs) can silence Arabidopsis and tomato genes involved in immunity. These Bc-sRNAs hijack the host RNA interference (RNAi) machinery by binding to Arabidopsis Argonaute 1 (AGO1) and selectively silencing host immunity genes. The Arabidopsis ago1 mutant exhibits reduced susceptibility to B. cinerea, and the B. cinerea dcl1 dcl2 double mutant that can no longer produce these Bc-sRNAs displays reduced pathogenicity on Arabidopsis and tomato. Thus, this fungal pathogen transfers "virulent" sRNA effectors into host plant cells to suppress host immunity and achieve infection, which demonstrates a naturally occurring cross-kingdom RNAi as an advanced virulence mechanism. PMID:24092744

  13. 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. PMID:27353039

  14. Small RNA Sequencing Uncovers New miRNAs and moRNAs Differentially Expressed in Normal and Primary Myelofibrosis CD34+ Cells

    PubMed Central

    Saccoman, Claudia; Mannarelli, Carmela; Coppe, Alessandro; Vannucchi, Alessandro M.; Bortoluzzi, Stefania

    2015-01-01

    Myeloproliferative neoplasms (MPN) are chronic myeloid cancers thought to arise at the level of CD34+ hematopoietic stem/progenitor cells. They include essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). All can progress to acute leukemia, but PMF carries the worst prognosis. Increasing evidences indicate that deregulation of microRNAs (miRNAs) might plays an important role in hematologic malignancies, including MPN. To attain deeper knowledge of short RNAs (sRNAs) expression pattern in CD34+ cells and of their possible role in mediating post-transcriptional regulation in PMF, we sequenced with Illumina HiSeq2000 technology CD34+ cells from healthy subjects and PMF patients. We detected the expression of 784 known miRNAs, with a prevalence of miRNA up-regulation in PMF samples, and discovered 34 new miRNAs and 99 new miRNA-offset RNAs (moRNAs), in CD34+ cells. Thirty-seven small RNAs were differentially expressed in PMF patients compared with healthy subjects, according to microRNA sequencing data. Five miRNAs (miR-10b-5p, miR-19b-3p, miR-29a-3p, miR-379-5p, and miR-543) were deregulated also in PMF granulocytes. Moreover, 3’-moR-128-2 resulted consistently downregulated in PMF according to RNA-seq and qRT-PCR data both in CD34+ cells and granulocytes. Target predictions of these validated small RNAs de-regulated in PMF and functional enrichment analyses highlighted many interesting pathways involved in tumor development and progression, such as signaling by FGFR and DAP12 and Oncogene Induced Senescence. As a whole, data obtained in this study deepened the knowledge of miRNAs and moRNAs altered expression in PMF CD34+ cells and allowed to identify and validate a specific small RNA profile that distinguishes PMF granulocytes from those of normal subjects. We thus provided new information regarding the possible role of miRNAs and, specifically, of new moRNAs in this disease. PMID:26468945

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

    PubMed

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

    2013-01-01

    Current technologies that are used for genome-wide microRNA (miRNA) prediction are mainly based on BLAST tool. They often produce a large number of false positives. Here, we describe an effective approach for identifying orthologous pre-miRNAs in several primates based on syntenic information. Some of them have been validated by small RNA high throughput sequencing data. This approach uses the synteny information and experimentally validated miRNAs of human, and incorporates currently available algorithms and tools to identify the pre-miRNAs in five other primates. First, we identified 929 potential pre-miRNAs in the marmoset in which miRNAs have not yet been reported. Then, we predicted the miRNAs in other primates, and we successfully re-identified most of the published miRNAs and found 721, 979, 650 and 639 new potential pre-miRNAs in chimpanzee, gorilla, orangutan and rhesus macaque, respectively. Furthermore, the miRNA transcriptome in the four primates have been re-analyzed and some novel predicted miRNAs have been supported by the small RNA sequencing data. Finally, we analyzed the potential functions of those validated miRNAs and explored the regulatory elements and transcription factors of some validated miRNA genes of interest. The results show that our approach can effectively identify novel miRNAs and some miRNAs that supported by small RNA sequencing data maybe play roles in the nervous system. PMID:24135875

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  18. What parameters to consider and which software tools to use for target selection and molecular design of small interfering RNAs.

    PubMed

    Matveeva, Olga

    2013-01-01

    The design of small gene silencing RNAs with a high probability of being efficient still has some elements of an art, especially when the lowest concentration of small molecules needs to be utilized. The design of highly target-specific small interfering RNAs or short hairpin RNAs is even a greater challenging task. Some logical schemes and software tools that can be used for simplifying both tasks are presented here. In addition, sequence motifs and sequence composition biases of small interfering RNAs that have to be avoided because of specificity concerns are also detailed. PMID:23027043

  19. Prediction of CsrA-regulating small RNAs in bacteria and their experimental verification in Vibrio fischeri

    PubMed Central

    Kulkarni, Prajna R.; Cui, Xiaohui; Williams, Joshua W.; Stevens, Ann M.; Kulkarni, Rahul V.

    2006-01-01

    The role of small RNAs as critical components of global regulatory networks has been highlighted by several recent studies. An important class of such small RNAs is represented by CsrB and CsrC of Escherichia coli, which control the activity of the global regulator CsrA. Given the critical role played by CsrA in several bacterial species, an important problem is the identification of CsrA-regulating small RNAs. In this paper, we develop a computer program (CSRNA_FIND) designed to locate potential CsrA-regulating small RNAs in bacteria. Using CSRNA_FIND to search the genomes of bacteria having homologs of CsrA, we identify all the experimentally known CsrA-regulating small RNAs and also make predictions for several novel small RNAs. We have verified experimentally our predictions for two CsrA-regulating small RNAs in Vibrio fischeri. As more genomes are sequenced, CSRNA_FIND can be used to locate the corresponding small RNAs that regulate CsrA homologs. This work thus opens up several avenues of research in understanding the mode of CsrA regulation through small RNAs in bacteria. PMID:16822857

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  2. Transcriptional Analysis of Deinococcus radiodurans Reveals Novel Small RNAs That Are Differentially Expressed under Ionizing Radiation

    PubMed Central

    Tsai, Chen-Hsun; Liao, Rick; Chou, Brendan

    2014-01-01

    Small noncoding RNAs (sRNAs) are posttranscriptional regulators that have been identified in multiple species and shown to play essential roles in responsive mechanisms to environmental stresses. The natural ability of specific bacteria to resist high levels of radiation has been of high interest to mechanistic studies of DNA repair and biomolecular protection. Deinococcus radiodurans is a model extremophile for radiation studies that can survive doses of ionizing radiation of >12,000 Gy, 3,000 times higher than for most vertebrates. Few studies have investigated posttranscriptional regulatory mechanisms of this organism that could be relevant in its general gene regulatory patterns. In this study, we identified 199 potential sRNA candidates in D. radiodurans by whole-transcriptome deep sequencing analysis and confirmed the expression of 41 sRNAs by Northern blotting and reverse transcriptase PCR (RT-PCR). A total of 8 confirmed sRNAs showed differential expression during recovery after acute ionizing radiation (15 kGy). We have also found and confirmed 7 sRNAs in Deinococcus geothermalis, a closely related radioresistant species. The identification of several novel sRNAs in Deinococcus bacteria raises important questions about the evolution and nature of global gene regulation in radioresistance. PMID:25548054

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

    PubMed Central

    Samir, Mohamed; Pessler, Frank

    2016-01-01

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

  4. 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. PMID:24584845

  5. Transcriptional analysis of Deinococcus radiodurans reveals novel small RNAs that are differentially expressed under ionizing radiation.

    PubMed

    Tsai, Chen-Hsun; Liao, Rick; Chou, Brendan; Contreras, Lydia M

    2015-03-01

    Small noncoding RNAs (sRNAs) are posttranscriptional regulators that have been identified in multiple species and shown to play essential roles in responsive mechanisms to environmental stresses. The natural ability of specific bacteria to resist high levels of radiation has been of high interest to mechanistic studies of DNA repair and biomolecular protection. Deinococcus radiodurans is a model extremophile for radiation studies that can survive doses of ionizing radiation of >12,000 Gy, 3,000 times higher than for most vertebrates. Few studies have investigated posttranscriptional regulatory mechanisms of this organism that could be relevant in its general gene regulatory patterns. In this study, we identified 199 potential sRNA candidates in D. radiodurans by whole-transcriptome deep sequencing analysis and confirmed the expression of 41 sRNAs by Northern blotting and reverse transcriptase PCR (RT-PCR). A total of 8 confirmed sRNAs showed differential expression during recovery after acute ionizing radiation (15 kGy). We have also found and confirmed 7 sRNAs in Deinococcus geothermalis, a closely related radioresistant species. The identification of several novel sRNAs in Deinococcus bacteria raises important questions about the evolution and nature of global gene regulation in radioresistance. PMID:25548054

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

    PubMed Central

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

    2012-01-01

    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. PMID:22266655

  7. Annotation of primate miRNAs by high throughput sequencing of small RNA libraries

    PubMed Central

    2012-01-01

    Background In addition to genome sequencing, accurate functional annotation of genomes is required in order to carry out comparative and evolutionary analyses between species. Among primates, the human genome is the most extensively annotated. Human miRNA gene annotation is based on multiple lines of evidence including evidence for expression as well as prediction of the characteristic hairpin structure. In contrast, most miRNA genes in non-human primates are annotated based on homology without any expression evidence. We have sequenced small-RNA libraries from chimpanzee, gorilla, orangutan and rhesus macaque from multiple individuals and tissues. Using patterns of miRNA expression in conjunction with a model of miRNA biogenesis we used these high-throughput sequencing data to identify novel miRNAs in non-human primates. Results We predicted 47 new miRNAs in chimpanzee, 240 in gorilla, 55 in orangutan and 47 in rhesus macaque. The algorithm we used was able to predict 64% of the previously known miRNAs in chimpanzee, 94% in gorilla, 61% in orangutan and 71% in rhesus macaque. We therefore added evidence for expression in between one and five tissues to miRNAs that were previously annotated based only on homology to human miRNAs. We increased from 60 to 175 the number miRNAs that are located in orthologous regions in humans and the four non-human primate species studied here. Conclusions In this study we provide expression evidence for homology-based annotated miRNAs and predict de novo miRNAs in four non-human primate species. We increased the number of annotated miRNA genes and provided evidence for their expression in four non-human primates. Similar approaches using different individuals and tissues would improve annotation in non-human primates and allow for further comparative studies in the future. PMID:22453055

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

    PubMed Central

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

    2015-01-01

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

  9. New Neurons in Aging Brains: Molecular Control by Small Non-Coding RNAs

    PubMed Central

    Schouten, Marijn; Buijink, M. Renate; Lucassen, Paul J.; Fitzsimons, Carlos P.

    2012-01-01

    Adult neurogenesis generates functional neurons from neural stem cells present in specific brain regions. It is largely confined to two main regions: the subventricular zone of the lateral ventricle, and the subgranular zone of the dentate gyrus (DG), in the hippocampus. With age, the function of the hippocampus and particularly the DG is impaired. For instance, adult neurogenesis is decreased with aging, in both proliferating and differentiation of newborn cells, while in parallel an age-associated decline in cognitive performance is often seen. Surprisingly, the synaptogenic potential of adult-born neurons is only marginally influenced by aging. Therefore, although proliferation, differentiation, and synaptogenesis of adult-born new neurons in the DG are closely related to each other, they are differentially affected by aging. In this review we discuss the crucial roles of a novel class of recently discovered modulators of gene expression, the small non-coding RNAs, in the regulation of adult neurogenesis. Multiple small non-coding RNAs are differentially expressed in the hippocampus. In particular a subgroup of the small non-coding RNAs, the microRNAs, fine-tune the progression of adult neurogenesis. This makes small non-coding RNAs appealing candidates to orchestrate the functional alterations in adult neurogenesis and cognition associated with aging. Finally, we summarize observations that link changes in circulating levels of steroid hormones with alterations in adult neurogenesis, cognitive decline, and vulnerability to psychopathology in advanced age, and discuss a potential interplay between steroid hormone receptors and microRNAs in cognitive decline in aging individuals. PMID:22363255

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. Global analyses of endonucleolytic cleavage in mammals reveal expanded repertoires of cleavage-inducing small RNAs and their targets

    PubMed Central

    Cass, Ashley A.; Bahn, Jae Hoon; Lee, Jae-Hyung; Greer, Christopher; Lin, Xianzhi; Kim, Yong; Hsiao, Yun-Hua Esther; Xiao, Xinshu

    2016-01-01

    In mammals, small RNAs are important players in post-transcriptional gene regulation. While their roles in mRNA destabilization and translational repression are well appreciated, their involvement in endonucleolytic cleavage of target RNAs is poorly understood. Very few microRNAs are known to guide RNA cleavage. Endogenous small interfering RNAs are expected to induce target cleavage, but their target genes remain largely unknown. We report a systematic study of small RNA-mediated endonucleolytic cleavage in mouse through integrative analysis of small RNA and degradome sequencing data without imposing any bias toward known small RNAs. Hundreds of small cleavage-inducing RNAs and their cognate target genes were identified, significantly expanding the repertoire of known small RNA-guided cleavage events. Strikingly, both small RNAs and their target sites demonstrated significant overlap with retrotransposons, providing evidence for the long-standing speculation that retrotransposable elements in mRNAs are leveraged as signals for gene targeting. Furthermore, our analysis showed that the RNA cleavage pathway is also present in human cells but affecting a different repertoire of retrotransposons. These results show that small RNA-guided cleavage is more widespread than previously appreciated. Their impact on retrotransposons in non-coding regions shed light on important aspects of mammalian gene regulation. PMID:26975654

  13. Argonaute-dependent small RNAs derived from single-stranded, non-structured precursors

    PubMed Central

    Chak, Li-Ling; Okamura, Katsutomo

    2014-01-01

    A general feature of Argonaute-dependent small RNAs is their base-paired precursor structures, and precursor duplex structures are often required for confident annotation of miRNA genes. However, this rule has been broken by discoveries of functional small RNA species whose precursors lack a predictable double-stranded (ds-) RNA structure, arguing that duplex structures are not prerequisite for small RNA loading to Argonautes. The biological significance of single-stranded (ss-) RNA loading has been recognized particularly in systems where active small RNA amplification mechanisms are involved, because even a small amount of RNA molecules can trigger the production of abundant RNA species leading to profound biological effects. However, even in the absence of small RNA amplification mechanisms, recent studies have demonstrated that potent gene silencing can be achieved using chemically modified synthetic ssRNAs that are resistant to RNases in mice. Therefore, such ssRNA-mediated gene regulation may have broader roles than previously recognized, and the findings have opened the door for further research to optimize the design of ss-siRNAs toward future pharmaceutical and biomedical applications of gene silencing technologies. In this review, we will summarize studies about endogenous ssRNA species that are bound by Argonaute proteins and how ssRNA precursors are recognized by various small RNA pathways. PMID:24959173

  14. Virus-associated small satellite RNAs and viroids display similarities in their replication strategies.

    PubMed

    Rao, A L N; Kalantidis, Kriton

    2015-05-01

    Since the discovery of non-coding, small, highly structured, satellite RNAs (satRNAs) and viroids as subviral pathogens of plants , have been of great interest to molecular biologists as possible living fossils of pre-cellular evolution in an RNA world. Despite extensive studies performed in the last four decades, there is still mystery surrounding the origin and evolutionary relationship between these subviral pathogens. Recent technical advances revealed some commonly shared replication features between these two subviral pathogens. In this review, we discuss our current perception of replication and evolutionary origin of these petite RNA pathogens. PMID:25731957

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  17. Accumulation of Potato spindle tuber viroid-specific small RNAs is accompanied by specific changes in tomato gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Post-transcriptional gene silencing appears to play a key role in viroid pathogenicity. To better understand the biogenesis of viroid-specific small RNAs and their possible role in disease induction, we have examined the accumulation of these small RNAs in the leaves and stems of potato spindle tub...

  18. Profiling and Characterization of Small RNAs in the Liverwort, Marchantia polymorpha, Belonging to the First Diverged Land Plants.

    PubMed

    Tsuzuki, Masayuki; Nishihama, Ryuichi; Ishizaki, Kimitsune; Kurihara, Yukio; Matsui, Minami; Bowman, John L; Kohchi, Takayuki; Hamada, Takahiro; Watanabe, Yuichiro

    2016-02-01

    MicroRNAs (miRNAs) have important roles in gene regulation during plant development. Previous studies revealed that some miRNAs are highly shared by most land plants. Recently, the liverwort, Marchantia polymorpha, has been studied by molecular genetic approaches, and sequencing of its genome is currently underway. The expression pattern and the detailed functions of miRNAs during Marchantia development are unknown. Here, we profiled the small RNAs expressed in thalli, antheridiophores and archegoniophores of M. polymorpha using high-throughput RNA sequencing. We revealed that a limited number of miRNAs are shared between M. polymorpha and the moss, Physcomitrella patens, and that a number of miRNAs are M. polymorpha specific. Like other land plants, cognate target genes corresponding to conserved miRNAs could be found in the genome database and were experimentally confirmed to guide cleavage of target mRNAs. The results suggested that two genes in the SPL (SQUAMOSA PROMOTER BINDING-LIKE) transcription factor family, which are regulated by miR156 in most land plants, were instead targeted by two distinct miRNAs in M. polymorpha. In order to demonstrate the physiological roles of miRNAs in M. polymorpha, we constructed an miRNA ectopic expression system to establish overexpression transformants for conserved miRNAs, miR166 and miR319. Ectopic expression of these miRNAs induced abnormal development of the thallus and gemma cups, suggesting that balanced expression of miRNA/target mRNAs has a crucial role in developmental regulation in M. polymorpha. Profiling data on miRNA together with the ectopic expression system would provide new information on the liverwort small RNA world and evolutionary divergence/conservation of small RNA function among land plants. PMID:26589267

  19. Translational control and target recognition by Escherichia coli small RNAs in vivo

    PubMed Central

    Urban, Johannes H.; Vogel, Jörg

    2007-01-01

    Small non-coding RNAs (sRNAs) are an emerging class of regulators of bacterial gene expression. Most of the regulatory Escherichia coli sRNAs known to date modulate translation of trans-encoded target mRNAs. We studied the specificity of sRNA target interactions using gene fusions to green fluorescent protein (GFP) as a novel reporter of translational control by bacterial sRNAs in vivo. Target sequences were selected from both monocistronic and polycistronic mRNAs. Upon expression of the cognate sRNA (DsrA, GcvB, MicA, MicC, MicF, RprA, RyhB, SgrS and Spot42), we observed highly specific translation repression/activation of target fusions under various growth conditions. Target regulation was also tested in mutants that lacked Hfq or RNase III, or which expressed a truncated RNase E (rne701). We found that translational regulation by these sRNAs was largely independent of full-length RNase E, e.g. despite the fact that ompA fusion mRNA decay could no longer be promoted by MicA. This is the first study in which multiple well-defined E.coli sRNA target pairs have been studied in a uniform manner in vivo. We expect our GFP fusion approach to be applicable to sRNA targets of other bacteria, and also demonstrate that Vibrio RyhB sRNA represses a Vibrio sodB fusion when co-expressed in E.coli. PMID:17264113

  20. Identification of novel soybean microRNAs involved in abiotic and biotic stresses

    PubMed Central

    2011-01-01

    Background Small RNAs (19-24 nt) are key regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in eukaryotes. Current studies have demonstrated that microRNAs (miRNAs) act in several plant pathways associated with tissue proliferation, differentiation, and development and in response to abiotic and biotic stresses. In order to identify new miRNAs in soybean and to verify those that are possibly water deficit and rust-stress regulated, eight libraries of small RNAs were constructed and submitted to Solexa sequencing. Results The libraries were developed from drought-sensitive and tolerant seedlings and rust-susceptible and resistant soybeans with or without stressors. Sequencing the library and subsequent analyses detected 256 miRNAs. From this total, we identified 24 families of novel miRNAs that had not been reported before, six families of conserved miRNAs that exist in other plants species, and 22 families previously reported in soybean. We also observed the presence of several isomiRNAs during our analyses. To validate novel miRNAs, we performed RT-qPCR across the eight different libraries. Among the 11 miRNAs analyzed, all showed different expression profiles during biotic and abiotic stresses to soybean. The majority of miRNAs were up-regulated during water deficit stress in the sensitive plants. However, for the tolerant genotype, most of the miRNAs were down regulated. The pattern of miRNAs expression was also different for the distinct genotypes submitted to the pathogen stress. Most miRNAs were down regulated during the fungus infection in the susceptible genotype; however, in the resistant genotype, most miRNAs did not vary during rust attack. A prediction of the putative targets was carried out for conserved and novel miRNAs families. Conclusions Validation of our results with quantitative RT-qPCR revealed that Solexa sequencing is a powerful tool for miRNA discovery. The identification of

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

    PubMed Central

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

    2015-01-01

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

  2. A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications.

    PubMed

    Chen, Qiu-Xia; Wang, Wei-Peng; Zeng, Su; Urayama, Shiro; Yu, Ai-Ming

    2015-04-20

    RNA research and therapy relies primarily on synthetic RNAs. We employed recombinant RNA technology toward large-scale production of pre-miRNA agents in bacteria, but found the majority of target RNAs were not or negligibly expressed. We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a). Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP-transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum RNase activities in pancreatic cancer patients. These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications. PMID:25800741

  3. A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications

    PubMed Central

    Chen, Qiu-Xia; Wang, Wei-Peng; Zeng, Su; Urayama, Shiro; Yu, Ai-Ming

    2015-01-01

    RNA research and therapy relies primarily on synthetic RNAs. We employed recombinant RNA technology toward large-scale production of pre-miRNA agents in bacteria, but found the majority of target RNAs were not or negligibly expressed. We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a). Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP-transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum RNase activities in pancreatic cancer patients. These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications. PMID:25800741

  4. Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae

    PubMed Central

    Gowda, Malali; Nunes, Cristiano C.; Sailsbery, Joshua; Xue, Minfeng; Chen, Feng; Nelson, Cassie A.; Brown, Douglas E.; Oh, Yeonyee; Meng, Shaowu; Mitchell, Thomas; Hagedorn, Curt H.; Dean, Ralph A.

    2010-01-01

    Small RNAs are well described in higher eukaryotes such as mammals and plants; however, knowledge in simple eukaryotes such as filamentous fungi is limited. In this study, we discovered and characterized methylguanosine-capped and polyadenylated small RNAs (CPA-sRNAs) by using differential RNA selection, full-length cDNA cloning and 454 transcriptome sequencing of the rice blast fungus Magnaporthe oryzae. This fungus causes blast, a devastating disease on rice, the principle food staple for over half the world’s population. CPA-sRNAs mapped primarily to the transcription initiation and termination sites of protein-coding genes and were positively correlated with gene expression, particularly for highly expressed genes including those encoding ribosomal proteins. Numerous CPA-sRNAs also mapped to rRNAs, tRNAs, snRNAs, transposable elements and intergenic regions. Many other 454 sequence reads could not be mapped to the genome; however, inspection revealed evidence for non-template additions and chimeric sequences. CPA-sRNAs were independently confirmed using a high affinity variant of eIF-4E to capture 5′-methylguanosine-capped RNA followed by 3′-RACE sequencing. These results expand the repertoire of small RNAs in filamentous fungi. PMID:20660015

  5. Regulation of spermatogenesis by small non-coding RNAs: role of the Germ Granule

    PubMed Central

    de Mateo, Sara; Sassone-Corsi, Paolo

    2015-01-01

    The spermatogenic process relays in highly regulated gene expression mechanisms at the transcriptional and post-transcriptional levels to generate the male gamete that is needed for the perpetuation of the species. Small non-coding RNA pathways have been determined to participate in the post-transcriptional regulatory processes of germ cells. The most important sncRNA molecules that are critically involved in spermatogenesis belong to the miRNA and piRNAs pathways as illustrated by animal models where ablation of specific protein components displays male infertility. Several elements of these regulatory pathways have been found in the nuage or germ granule, a non-membranous cytoplasmatic structure that can be seen in spermatocytes and spermatids. This notion suggests that germ granules may act as organizer centers for silencing pathways in the germline. In general, miRNAs regulate spermatogenesis through targeting and down-regulation of specific transcripts to eventually promote sperm development. However, piRNAs are powerful repressors of transposon elements expression in the spermatogenic process. Here we describe the suggested functions that miRNA and piRNAs pathways execute in the regulation of spermatogenesis and include some recent studies in the field. Despite major strides on the detailed molecular mechanisms of sncRNAs in relation to spermatogenesis, there is plenty to discover on this fascinating regulatory program. PMID:24755166

  6. Multiple small RNAs act additively to integrate sensory information and control quorum sensing in Vibrio harveyi

    PubMed Central

    Tu, Kimberly C.; Bassler, Bonnie L.

    2007-01-01

    Quorum sensing is a cell–cell communication mechanism that bacteria use to collectively regulate gene expression and, at a higher level, to coordinate group behavior. In the bioluminescent marine bacterium Vibrio harveyi, sensory information from three independent quorum-sensing systems converges on the shared response regulator LuxO. When LuxO is phosphorylated, it activates the expression of a putative repressor that destabilizes the mRNA encoding the master quorum-sensing transcriptional regulator LuxR. In the closely related species Vibrio cholerae, this repressor was revealed to be the RNA chaperone Hfq together with four small regulatory RNAs (sRNAs) called Qrr1–4 (quorum regulatory RNA). Here, we identify five Qrr sRNAs that control quorum sensing in V. harveyi. Mutational analysis reveals that only four of the five Qrrs are required for destabilization of the luxR mRNA. Surprisingly, unlike in V. cholerae where the sRNAs act redundantly, in V. harveyi, the Qrr sRNAs function additively to control quorum sensing. This latter mechanism produces a gradient of LuxR that, in turn, enables differential regulation of quorum-sensing target genes. Other regulators appear to be involved in control of V. harveyi qrr expression, allowing the integration of additional sensory information into the regulation of quorum-sensing gene expression. PMID:17234887

  7. Nicotine biosynthesis is regulated by two more layers: Small and long non-protein-coding RNAs.

    PubMed

    Xie, Jiahua; Fan, Longjiang

    2016-06-01

    In recent years, many small RNAs and long non-protein-coding RNAs (lncRNAs) have been identified and characterized. They have been proved to play essential regulatory roles in gene expression in both primary and secondary metabolisms. In nature, many plants produce alkaloids. However, there are only few reports on the involvement of non-coding RNAs in alkaloid biosynthesis. Nicotine is major alkaloid in tobacco plants. Its biosynthesis and regulation in tobacco (Nicotiana tabacum) have been well studied; and major structural genes involved in the nicotine biosynthesis and transcriptional regulators related to its biosynthesis have been identified and characterized. In our recent studies, we identified a microRNA (nta-miRX27) and also a lncRNA (nta-eTMX27) as an endogenous target mimicry (eTM) in tobacco targeting the nicotine biosynthesis key gene QPT2 encoding quinolinate phosphoribosyltransferase (QPT) and thereby regulating the nicotine content. Their regulatory pattern leads us to conclude that nicotine biosynthesis is regulated by 2 more layers besides previously known mechanisms. Future study on the relationship between the non-coding RNAs and transcription factors in nicotine biosynthesis was discussed in this article. PMID:27172239

  8. Production of functional small interfering RNAs by an amino-terminal deletion mutant of human Dicer

    PubMed Central

    Kennedy, Edward M.; Whisnant, Adam W.; Kornepati, Anand V. R.; Marshall, Joy B.; Bogerd, Hal P.; Cullen, Bryan R.

    2015-01-01

    Although RNA interference (RNAi) functions as a potent antiviral innate-immune response in plants and invertebrates, mammalian somatic cells appear incapable of mounting an RNAi response and few, if any, small interfering RNAs (siRNAs) can be detected. To examine why siRNA production is inefficient, we have generated double-knockout human cells lacking both Dicer and protein kinase RNA-activated. Using these cells, which tolerate double-stranded RNA expression, we show that a mutant form of human Dicer lacking the amino-terminal helicase domain can process double-stranded RNAs to produce high levels of siRNAs that are readily detectable by Northern blot, are loaded into RNA-induced silencing complexes, and can effectively and specifically inhibit the expression of cognate mRNAs. Remarkably, overexpression of this mutant Dicer, but not wild-type Dicer, also resulted in a partial inhibition of Influenza A virus—but not poliovirus—replication in human cells. PMID:26621737

  9. Bio-functional surfaces for the immunocapture of AGO2-bound microRNAs.

    PubMed

    Vaghi, V; Potrich, C; Lunelli, L; Facci, P; Pasquardini, L; Vanzetti, L; Pederzolli, C

    2016-10-01

    MicroRNAs (miRNAs) are endogenous, small (18-24nt), non-coding RNAs that regulate gene expression. Among miRNAs, those bound to the AGO2 protein are the functionally active fraction which mediates the cell regulatory processes and regulate messages exchanged by cells. Several methods have been developed to purify this fraction of microRNAs, such as immunoprecipitation and immunoprecipitation-derived techniques. However, all these techniques are generally recognized as technically complicated and time consuming. Here, a new bio-functional surface for the specific capture of AGO2-bound microRNAs is proposed. Starting from a silicon oxide surface, a protein A layer was covalently bound via epoxy chemistry to orient specific anti-AGO2 antibodies on the surface. The anti-AGO2 antibodies captured the AGO2 protein present in cell lysate and in human plasma. The AGO2-bound microRNAs were then released by enzymatic digestion and detected via RT-qPCR. Control surfaces were also prepared and tested. Every step in the preparation of the bio-functional surfaces was fully characterized from the chemical, morphological and functional point of view. The resulting bio-functional surface is able to specifically capture the AGO2-bound miRNAs from biologically-relevant samples, such as cell lysate and human plasma. These samples contain different proportions of AGO2-bound microRNAs, as reliably detected with the immunocapture method here proposed. This work opens new perspectives for a simple and faster method to isolate not only AGO2-bound microRNAs, but also the multiprotein complex containing AGO2 and miRNAs. PMID:27449965

  10. Profiling of T helper cell-derived small RNAs reveals unique antisense transcripts and differential association of miRNAs with argonaute proteins 1 and 2

    PubMed Central

    Polikepahad, Sumanth; Corry, David B.

    2013-01-01

    RNA interference mediated through antisense transcripts is a fundamentally important mechanism regulating gene expression that remains incompletely understood. Here, we have used next-generation sequencing to determine from mouse CD4+ T cells the functional implications of antisense transcripts binding to argonaute (AGO) proteins that mediate RNA interference and post-transcriptional gene silencing. This effort identified 90 new microRNAs (miRNAs) and six endogenous hairpin RNA-derived small interfering RNAs (siRNAs) mapping to distinct introns. Unexpectedly, 69 miRNAs were expressed as non-canonical isomiRs as the dominant AGO-binding transcript, with extensive 3′ terminal nucleotide modifications. Furthermore, differential expression analysis between AGO1- and AGO2-bound miRNAs suggested preferential binding of isomiRs ending with 3′ adenine residues to AGO1 and 3′ uridine residues to AGO2. Analysis of the putative targets of all miRNAs suggested a striking preference for regulating transcription and transcription factors with additional evidence of a functional division of labor between AGO proteins in this regard. We further provide evidence that multiple mitochondrial genomic loci serve as the source of endogenous cis-natural antisense transcripts. These findings imply diversity in AGO protein function based on differential miRNA binding and indicate that RNA interference-based gene regulation is more complex than previously recognized. PMID:23185045

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

    PubMed

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Shen, Jian-Wei

    2011-03-01

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

  13. Identification and characterization of noncoding small RNAs in Streptococcus pneumoniae serotype 2 strain D39.

    PubMed

    Tsui, Ho-Ching Tiffany; Mukherjee, Dhriti; Ray, Valerie A; Sham, Lok-To; Feig, Andrew L; Winkler, Malcolm E

    2010-01-01

    We report a search for small RNAs (sRNAs) in the low-GC, gram-positive human pathogen Streptococcus pneumoniae. Based on bioinformatic analyses by Livny et al. (J. Livny, A. Brencic, S. Lory, and M. K. Waldor, Nucleic Acids Res. 34:3484-3493, 2006), we tested 40 candidates by Northern blotting and confirmed the expression of nine new and one previously reported (CcnA) sRNAs in strain D39. CcnA is one of five redundant sRNAs reported by Halfmann et al. (A. Halfmann, M. Kovacs, R. Hakenbeck, and R. Bruckner, Mol. Microbiol. 66:110-126, 2007) that are positively controlled by the CiaR response regulator. We characterized 3 of these 14 sRNAs: Spd-sr17 (144 nucleotides [nt]; decreased in stationary phase), Spd-sr37 (80 nt; strongly expressed in all growth phases), and CcnA (93 nt; induced by competence stimulatory peptide). Spd-sr17 and CcnA likely fold into structures containing single-stranded regions between hairpin structures, whereas Spd-sr37 forms a base-paired structure. Primer extension mapping and ectopic expression in deletion/insertion mutants confirmed the independent expression of the three sRNAs. Microarray analyses indicated that insertion/deletion mutants in spd-sr37 and ccnA exerted strong cis-acting effects on the transcription of adjacent genes, indicating that these sRNA regions are also cotranscribed in operons. Deletion or overexpression of the three sRNAs did not cause changes in growth, certain stress responses, global transcription, or virulence. Constitutive ectopic expression of CcnA reversed some phenotypes of D39 Delta ciaR mutants, but attempts to link CcnA to -E to comC as a target were inconclusive in ciaR(+) strains. These results show that S. pneumoniae, which lacks known RNA chaperones, expresses numerous sRNAs, but three of these sRNAs do not strongly affect common phenotypes or transcription patterns. PMID:19854910

  14. 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. PMID:27184117

  15. Aggregates of Small Nuclear Ribonucleic Acids (snRNAs) in Alzheimer’s Disease’

    PubMed Central

    Hales, Chadwick M.; Dammer, Eric B.; Diner, Ian; Yi, Hong; Seyfried, Nicholas T.; Gearing, Marla; Glass, Jonathan D.; Montine, Thomas J.; Levey, Allan I.; Lah, James J.

    2014-01-01

    We recently discovered that protein components of the ribonucleic acid (RNA) spliceosome form cytoplasmic aggregates in Alzheimer’s disease (AD) brain, resulting in widespread changes in RNA splicing. However, the involvement of small nuclear RNAs (snRNAs), also key components of the spliceosome complex, in the pathology of AD remains unknown. Using immunohistochemical staining of post-mortem human brain and spinal cord, we identified cytoplasmic tangle-shaped aggregates of snRNA in both sporadic and familial AD cases but not in aged controls or other neurodegenerative disorders. Immunofluorescence using antibodies reactive with the 2,2,7-trimethylguanosine cap of snRNAs and transmission electron microscopy demonstrated snRNA localization with tau and paired helical filaments, the main component of neurofibrillary tangles. Quantitative real-time polymerase chain reaction (PCR) showed U1 snRNA accumulation in the insoluble fraction of AD brains whereas other U snRNAs were not enriched. In combination with our previous results, these findings demonstrate that aggregates of U1 snRNA and U1 small nuclear ribonucleoproteins represent a new pathological hallmark of AD. PMID:24571648

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

    PubMed Central

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

    2015-01-01

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

  17. Kinetics of bulge bases in small RNAs and the effect of pressure on it.

    PubMed

    Kumar, Pradeep; Lehmann, Jean; Libchaber, Albert

    2012-01-01

    Due to their self-catalytic properties, small RNAs with bulge bases are hypothesized to be primordial molecules which could form elementary translation systems. Using molecular dynamics simulations, we study the binding propensity of small RNAs by calculating the free energy barrier corresponding to the looped out conformations of bulge bases, which presumably act as the binding sites for ligands in these small RNAs. We find that base flipping kinetics can proceed at atmospheric pressure but with a very small propensity. Furthermore, the free energy barrier associated with base flipping depends on the stacking with neighboring bases. Next, we studied the base flipping kinetics with pressure. We find that the free energy associated with base looping out increases monotonically as the pressure is increased. Furthermore, we calculate the mean first-passage time of conformational looping out of the bulge base using the diffusion of reaction coordinate associated with the base flipping on the underlying free energy surface. We find that the mean first-passage time associated with bulge looping out increases slowly upon increasing pressures P up to 2000 atm but changes dramatically for P>2000 atm. Finally, we discuss our results in the light of the role of hydration shell of water around RNA. Our results are relevant for the RNA world hypothesis. PMID:22916118

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

    PubMed

    Paul, Sujay; Kundu, Anirban; Pal, Amita

    2014-01-01

    MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that play a crucial role in post-transcriptional gene regulation. Several conserved and species-specific miRNAs have been characterized to date, predominantly from the plant species whose genome is well characterized. However, information on the variability of these regulatory RNAs in economically important but genetically less characterized crop species are limited. Vigna mungo is an important grain legume, which is grown primarily for its protein-rich edible seeds. miRNAs from this species have not been identified to date due to lack of genome sequence information. To identify miRNAs from V. mungo, a small RNA library was constructed from young leaves. High-throughput Illumina sequencing technology and bioinformatic analysis of the small RNA reads led to the identification of 66 miRNA loci represented by 45 conserved miRNAs belonging to 19 families and eight non-conserved miRNAs belonging to seven families. Besides, 13 novel miRNA candidates in V. mungo were also identified. Expression patterns of selected conserved, non-conserved, and novel miRNA candidates have been demonstrated in leaf, stem, and root tissues by quantitative polymerase chain reaction, and potential target genes were predicted for most of the conserved miRNAs. This information offers genomic resources for better understanding of miRNA mediated post-transcriptional gene regulation. PMID:24138283

  19. Prediction and characterization of small non-coding RNAs related to secondary metabolites in Saccharopolyspora erythraea.

    PubMed

    Liu, Wei-Bing; Shi, Yang; Yao, Li-Li; Zhou, Ying; Ye, Bang-Ce

    2013-01-01

    Saccharopolyspora erythraea produces a large number of secondary metabolites with biological activities, including erythromycin. Elucidation of the mechanisms through which the production of these secondary metabolites is regulated may help to identify new strategies for improved biosynthesis of erythromycin. In this paper, we describe the systematic prediction and analysis of small non-coding RNAs (sRNAs) in S. erythraea, with the aim to elucidate sRNA-mediated regulation of secondary metabolite biosynthesis. In silico and deep-sequencing technologies were applied to predict sRNAs in S. erythraea. Six hundred and forty-seven potential sRNA loci were identified, of which 382 cis-encoded antisense RNA are complementary to protein-coding regions and 265 predicted transcripts are located in intergenic regions. Six candidate sRNAs (sernc292, sernc293, sernc350, sernc351, sernc361, and sernc389) belong to four gene clusters (tpc3, pke, pks6, and nrps5) that are involved in secondary metabolite biosynthesis. Deep-sequencing data showed that the expression of all sRNAs in the strain HL3168 E3 (E3) was higher than that in NRRL23338 (M), except for sernc292 and sernc361 expression. The relative expression of six sRNAs in strain M and E3 were validated by qRT-PCR at three different time points (24, 48, and 72 h). The results showed that, at each time point, the transcription levels of sernc293, sernc350, sernc351, and sernc389 were higher in E3 than in M, with the largest difference observed at 72 h, whereas no signals for sernc292 and sernc361 were detected. sernc293, sernc350, sernc351, and sernc389 probably regulate iron transport, terpene metabolism, geosmin synthesis, and polyketide biosynthesis, respectively. The major significance of this study is the successful prediction and identification of sRNAs in genomic regions close to the secondary metabolism-related genes in S. erythraea. A better understanding of the sRNA-target interaction would help to elucidate the

  20. Allele dependent silencing of COL1A2 using small interfering RNAs

    PubMed Central

    Lindahl, Katarina; Rubin, Carl-Johan; Kindmark, Andreas; Ljunggren, Östen

    2008-01-01

    Osteogenesis imperfecta (OI) is generally caused by a dominant mutation in Collagen I, encoded by the genes COL1A1 and COL1A2. To date there is no satisfactory therapy for OI, but inactivation of the mutant allele through small interfering RNAs (siRNA) is a promising approach, as siRNAs targeting each allele of a polymorphism could be used for allele-specific silencing irrespective of the location of the actual mutations. In this study we examined the allele dependent effects of several tiled siRNAs targeting a region surrounding an exonic COL1A2 T/C polymorphism (rs1800222) in heterozygous primary human bone cells. Relative abundances of COL1A2 alleles were determined by cDNA sequencing and overall COL1A2 abundance was analyzed by quantitative PCR. One of the siRNAs decreased overall COL1A2 abundance by 71% of which 75% was due to silencing of the targeted T-allele. In conclusion, allele-preferential silencing of Collagen type I genes may be a future therapeutic approach for OI. PMID:19015742

  1. Global Analyses of Small Interfering RNAs from Sour Orange seedlings Infected with Different Citrus tristeza virus Genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA silencing is a sequence-specific regulatory mechanism in development and maintenance of genome integrity and functions in plant antiviral defense mechanisms. Small interfering RNAs (siRNAs) are key mediators of RNA silencing. To study CTV-host interactions and disease expression, profiles of v...

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-03-01

    Until recently, only two small regulatory RNAs encoded by lambdoid bacteriophages were known. These transcripts are derived from paQ and pO promoters. The former one is supposed to act as an antisense RNA for expression of the Q gene, encoding a transcription antitermination protein. The latter transcript, called oop RNA, was initially proposed to have a double role, in establishing expression of the cI gene and in providing a primer for DNA replication. Although the initially proposed mechanisms by which oop RNA could influence the choice between two alternative developmental pathways of the phage and the initiation of phage DNA replication were found not true, the pO promoter has been demonstrated to be important for both regulation of phage development and control of DNA replication. Namely, the pO-derived transcript is an antisense RNA for expression of the cII gene, and pO is a part of a dual promoter system responsible for regulation of initiation of DNA synthesis from the oriλ region. Very recent studies identified a battery of small RNAs encoded by lambdoid bacteriophages existing as prophages in chromosomes of enterohemorrhagic Escherichia coli strains. Some of them have very interesting functions, like anti-small RNAs. PMID:25111672

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

    PubMed Central

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

    2015-01-01

    Small RNA pathways act at the front line of defence against transposable elements across the Eukaryota. In animals, Piwi interacting small RNAs (piRNAs) are a crucial arm of this defence. However, the evolutionary relationships among piRNAs and other small RNA pathways targeting transposable elements are poorly resolved. To address this question we sequenced small RNAs from multiple, diverse nematode species, producing the first phylum-wide analysis of how small RNA pathways evolve. Surprisingly, despite their prominence in Caenorhabditis elegans and closely related nematodes, piRNAs are absent in all other nematode lineages. We found that there are at least two evolutionarily distinct mechanisms that compensate for the absence of piRNAs, both involving RNA-dependent RNA polymerases (RdRPs). Whilst one pathway is unique to nematodes, the second involves Dicer-dependent RNA-directed DNA methylation, hitherto unknown in animals, and bears striking similarity to transposon-control mechanisms in fungi and plants. Our results highlight the rapid, context-dependent evolution of small RNA pathways and suggest piRNAs in animals may have replaced an ancient eukaryotic RNA-dependent RNA polymerase pathway to control transposable elements. PMID:25668728

  5. Small RNA and Degradome Sequencing Reveal Complex Roles of miRNAs and Their Targets in Developing Wheat Grains

    PubMed Central

    Geng, Yuke; Hao, Chenyang; Chen, Xinhong; Zhang, Xueyong

    2015-01-01

    Plant microRNAs (miRNAs) have been shown to play critical roles in plant development. In this study, we employed small RNA combined with degradome sequencing to survey development-related miRNAs and their validated targets during wheat grain development. A total of 186 known miRNAs and 37 novel miRNAs were identified in four small RNA libraries. Moreover, a miRNA-like long hairpin locus was first identified to produce 21~22-nt phased siRNAs that act in trans to cleave target mRNAs. A comparison of the miRNAomes revealed that 55 miRNA families were differentially expressed during the grain development. Predicted and validated targets of these development-related miRNAs are involved in different cellular responses and metabolic processes including cell proliferation, auxin signaling, nutrient metabolism and gene expression. This study provides insight into the complex roles of miRNAs and their targets in regulating wheat grain development. PMID:26426440

  6. Identification of miRNAs and their target genes in developing maize ears by combined small RNA and degradome sequencing

    PubMed Central

    2014-01-01

    Background In plants, microRNAs (miRNAs) are endogenous ~22 nt RNAs that play important regulatory roles in many aspects of plant biology, including metabolism, hormone response, epigenetic control of transposable elements, and stress response. Extensive studies of miRNAs have been performed in model plants such as rice and Arabidopsis thaliana. In maize, most miRNAs and their target genes were analyzed and identified by clearly different treatments, such as response to low nitrate, salt and drought stress. However, little is known about miRNAs involved in maize ear development. The objective of this study is to identify conserved and novel miRNAs and their target genes by combined small RNA and degradome sequencing at four inflorescence developmental stages. Results We used deep-sequencing, miRNA microarray assays and computational methods to identify, profile, and describe conserved and non-conserved miRNAs at four ear developmental stages, which resulted in identification of 22 conserved and 21-maize-specific miRNA families together with their corresponding miRNA*. Comparison of miRNA expression in these developmental stages revealed 18 differentially expressed miRNA families. Finally, a total of 141 genes (251 transcripts) targeted by 102 small RNAs including 98 miRNAs and 4 ta-siRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs. Moreover, the differentially expressed miRNAs-mediated pathways that regulate the development of ears were discussed. Conclusions This study confirmed 22 conserved miRNA families and discovered 26 novel miRNAs in maize. Moreover, we identified 141 target genes of known and new miRNAs and ta-siRNAs. Of these, 72 genes (117 transcripts) targeted by 62 differentially expressed miRNAs may attribute to the development of maize ears. Identification and characterization of these important classes of regulatory genes in maize may improve our understanding of molecular mechanisms controlling ear development

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

    PubMed Central

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

    2016-01-01

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

  8. Target delivery of small interfering RNAs with vitamin E-coupled nanoparticles for treating hepatitis C.

    PubMed

    Duan, Liang; Yan, Yan; Liu, Jingyi; Wang, Bo; Li, Pu; Hu, Qin; Chen, Weixian

    2016-01-01

    RNA interference (RNAi) represents a promising strategy for the treatment of HCV infection. However, the development of an effective system for in vivo delivery of small interfering RNA (siRNA) to target organ remains a formidable challenge. Here, we develop a unique nanoparticle platform (VE-DC) composed of α-tocopherol (vitamin E) and cholesterol-based cationic liposomes (DOTAP-Chol) for systemic delivery of siRNAs to the liver. A HCV-replicable cell line, Huh7.5.1-HCV, and a transient HCV core expressing cell line, Huh7.5.1-Core, were constructed and used to assess the in vitro anti-HCV activity of VE-DC/siRNAs. A transient in vivo HCV model was also constructed by hydrodynamic injection of pCDNA3.1(+)-3FLAG-Core (pCore-3FLAG) plasmid expressing core protein or pGL3-5'UTR-luciferase (pGL3-5'UTR-luc) plasmid expressing luciferase driven by HCV 5'UTR. Nanoscale VE-DC/siRNA was intravenously injected to assess the liver-targeting property as well as antiviral activity. The nanoscale VE-DC effectively exerted an anti-HCV activity in the in vitro cell models. Post-administration of VE-DC/siRNAs also effectively delivered siRNAs to the liver, suppressing core protein production and firefly luciferase activity, without inducing an innate immunity response or off-target and toxicity effects. The VE-DC platform has high potential as a vehicle for delivery of siRNAs to the liver for gene therapy for targeting hepatitis C. PMID:27113197

  9. Target delivery of small interfering RNAs with vitamin E-coupled nanoparticles for treating hepatitis C

    PubMed Central

    Duan, Liang; Yan, Yan; Liu, Jingyi; Wang, Bo; Li, Pu; Hu, Qin; Chen, Weixian

    2016-01-01

    RNA interference (RNAi) represents a promising strategy for the treatment of HCV infection. However, the development of an effective system for in vivo delivery of small interfering RNA (siRNA) to target organ remains a formidable challenge. Here, we develop a unique nanoparticle platform (VE-DC) composed of α-tocopherol (vitamin E) and cholesterol-based cationic liposomes (DOTAP-Chol) for systemic delivery of siRNAs to the liver. A HCV-replicable cell line, Huh7.5.1-HCV, and a transient HCV core expressing cell line, Huh7.5.1-Core, were constructed and used to assess the in vitro anti-HCV activity of VE-DC/siRNAs. A transient in vivo HCV model was also constructed by hydrodynamic injection of pCDNA3.1(+)-3FLAG-Core (pCore-3FLAG) plasmid expressing core protein or pGL3-5′UTR-luciferase (pGL3-5′UTR-luc) plasmid expressing luciferase driven by HCV 5′UTR. Nanoscale VE-DC/siRNA was intravenously injected to assess the liver-targeting property as well as antiviral activity. The nanoscale VE-DC effectively exerted an anti-HCV activity in the in vitro cell models. Post-administration of VE-DC/siRNAs also effectively delivered siRNAs to the liver, suppressing core protein production and firefly luciferase activity, without inducing an innate immunity response or off-target and toxicity effects. The VE-DC platform has high potential as a vehicle for delivery of siRNAs to the liver for gene therapy for targeting hepatitis C. PMID:27113197

  10. Characterization of the Small RNA Transcriptome of the Marine Coccolithophorid, Emiliania huxleyi

    PubMed Central

    Zhang, Xiaoyu; Gamarra, Jaime; Castro, Steven; Carrasco, Estela; Hernandez, Aaron; Mock, Thomas; Hadaegh, Ahmad R.; Read, Betsy A.

    2016-01-01

    Small RNAs (smRNAs) control a variety of cellular processes by silencing target genes at the transcriptional or post-transcription level. While extensively studied in plants, relatively little is known about smRNAs and their targets in marine phytoplankton, such as Emiliania huxleyi (E. huxleyi). Deep sequencing was performed of smRNAs extracted at different time points as E. huxleyi cells transition from logarithmic to stationary phase growth in batch culture. Computational analyses predicted 18 E. huxleyi specific miRNAs. The 18 miRNA candidates and their precursors vary in length (18–24 nt and 71–252 nt, respectively), genome copy number (3–1,459), and the number of genes targeted (2–107). Stem-loop real time reverse transcriptase (RT) PCR was used to validate miRNA expression which varied by nearly three orders of magnitude when growth slows and cells enter stationary phase. Stem-loop RT PCR was also used to examine the expression profiles of miRNA in calcifying and non-calcifying cultures, and a small subset was found to be differentially expressed when nutrients become limiting and calcification is enhanced. In addition to miRNAs, endogenous small RNAs such as ra-siRNAs, ta-siRNAs, nat-siRNAs, and piwiRNAs were predicted along with the machinery for the biogenesis and processing of si-RNAs. This study is the first genome-wide investigation smRNAs pathways in E. huxleyi. Results provide new insights into the importance of smRNAs in regulating aspects of physiological growth and adaptation in marine phytoplankton and further challenge the notion that smRNAs evolved with multicellularity, expanding our perspective of these ancient regulatory pathways. PMID:27101007