Small RNA Populations Research Articles

Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing.

BackgroundIn recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequence long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins.ResultsAnalysis of these data sets reveals that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently cataloged. We further find that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs reveals that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein.ConclusionsWe conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon.

MicroRNA expression profiles in avian haemopoietic cells

MicroRNAs (miRNAs) are small, abundant, non-coding RNAs that modulate gene expression by interfering with translation or stability of mRNA transcripts in a sequence-specific manner. A total of 734 precursor and 996 mature miRNAs have so far been identified in the chicken genome. A number of these miRNAs are expressed in a cell type-specific manner, and understanding their function requires detailed examination of their expression in different cell types. We carried out deep sequencing of small RNA populations isolated from stimulated or transformed avian haemopoietic cell lines to determine the changes in the expression profiles of these important regulatory molecules during these biological events. There were significant changes in the expression of a number of miRNAs, including miR-155, in chicken B cells stimulated with CD40 ligand. Similarly, avian leukosis virus (ALV)-transformed DT40 cells also showed changes in miRNA expression in relation to the naïve cells. Embryonic stem cell line BP25 demonstrated a distinct cluster of upregulated miRNAs, many of which were shown previously to be involved in embryonic stem cell development. Finally, chicken macrophage cell line HD11 showed changes in miRNA profiles, some of which are thought to be related to the transformation by v-myc transduced by the virus. This work represents the first publication of a catalog of microRNA expression in a range of important avian cells and provides insights into the potential roles of miRNAs in the hematopoietic lineages of cells in a model non-mammalian species.

Advanced engineering of lipid metabolism in Nicotiana benthamiana using a draft genome and the V2 viral silencing-suppressor protein.

The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18∶1 to elongation reactions beginning with 18∶1 as substrate. These V2-based leaf assays produced ∼50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays.

Asymmetric purine-pyrimidine distribution in cellular small RNA population of papaya

BackgroundThe small RNAs (sRNA) are a regulatory class of RNA mainly represented by the 21 and 24-nucleotide size classes. The cellular sRNAs are processed by RNase III family enzyme dicer (Dicer like in plant) from a self-complementary hairpin loop or other type of RNA duplexes. The papaya genome has been sequenced, but its microRNAs and other regulatory RNAs are yet to be analyzed.ResultsWe analyzed the genomic features of the papaya sRNA population from three sRNA deep sequencing libraries made from leaves, flowers, and leaves infected with Papaya Ringspot Virus (PRSV). We also used the deep sequencing data to annotate the micro RNA (miRNA) in papaya. We identified 60 miRNAs, 24 of which were conserved in other species, and 36 of which were novel miRNAs specific to papaya. In contrast to the Chargaff’s purine-pyrimidine equilibrium, cellular sRNA was significantly biased towards a purine rich population. Of the two purine bases, higher frequency of adenine was present in 23nt or longer sRNAs, while 22nt or shorter sRNAs were over represented by guanine bases. However, this bias was not observed in the annotated miRNAs in plants. The 21nt species were expressed from fewer loci but expressed at higher levels relative to the 24nt species. The highly expressed 21nt species were clustered in a few isolated locations of the genome. The PRSV infected leaves showed higher accumulation of 21 and 22nt sRNA compared to uninfected leaves. We observed higher accumulation of miRNA* of seven annotated miRNAs in virus-infected tissue, indicating the potential function of miRNA* under stressed conditions.ConclusionsWe have identified 60 miRNAs in papaya. Our study revealed the asymmetric purine-pyrimidine distribution in cellular sRNA population. The 21nt species of sRNAs have higher expression levels than 24nt sRNA. The miRNA* of some miRNAs shows higher accumulation in PRSV infected tissues, suggesting that these strands are not totally functionally redundant. The findings open a new avenue for further investigation of the sRNA silencing pathway in plants.

Regulatory Impact of RNA Secondary Structure across the Arabidopsis Transcriptome

The secondary structure of an RNA molecule plays an integral role in its maturation, regulation, and function. However, the global influence of this feature on plant gene expression is still largely unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach in conjunction with transcriptome-wide sequencing of rRNA-depleted (RNA sequencing), small RNA, and ribosome-bound RNA populations to investigate the impact of RNA secondary structure on gene expression regulation in Arabidopsis thaliana. From this analysis, we find that highly unpaired and paired RNAs are strongly correlated with euchromatic and heterochromatic epigenetic histone modifications, respectively, providing evidence that secondary structure is necessary for these RNA-mediated posttranscriptional regulatory pathways. Additionally, we uncover key structural patterns across protein-coding transcripts that indicate RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in this model plant. We further reveal that RNA folding is significantly anticorrelated with overall transcript abundance, which is often due to the increased propensity of highly structured mRNAs to be degraded and/or processed into small RNAs. Finally, we find that secondary structure affects mRNA translation, suggesting that this feature regulates plant gene expression at multiple levels. These findings provide a global assessment of RNA folding and its significant regulatory effects in a plant transcriptome.

SmRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni

BackgroundMicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data.ResultsTo identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways.ConclusionsThis study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to understand their roles in key stevia traits.

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

BackgroundSmall 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.ResultsUsing 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. Detailed analysis of the size classes associated with ribosomal RNAs and transposable element families showed greater diversity of smRNAs in the 22- and 24-nt size classes.ConclusionsThe flux of endogenous smRNAs within multiple stages and tissues of seed development was contrasted with vegetative tissues of soybean, one of the dominant sources of protein and oil in world markets. The smRNAs varied in size class, complexity of origins, and possible targets. Sequencing revealed tissue-preferential expression for certain smRNAs and expression differences among closely related miRNA family members.

Small RNAs of Sequoia sempervirens during rejuvenation and phase change

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

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

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

Dynamic expression of small RNA populations in larch (Larix leptolepis)

Small RNAs (sRNAs) are emerging as essential regulators of biological processes. However, several studies have reported that gymnosperms do not express appreciable amounts of 24-nt sRNAs, and conifers in particular may have a unique sRNA-silencing signature. Here, we compared the sRNA transcriptomes of Japanese larch somatic embryos (SE) and seedlings. SE sRNAs exhibited a length bias toward 24nt, while seedlings showed a bias toward a 21-nt length. We also confirmed that larch is capable of producing 24-nt sRNAs based on a polyacrylamide gel analysis. The sRNA expression patterns varied according to developmental stage, which might be associated with Dicer-like 3 and RNA-dependent RNA polymerase2 (RDR2) levels. Our data suggest that many MIR loci that produce canonical microRNAs (miRNAs, 20-22nt) and long sRNAs (23-26nt) have dual functions; the latter were preferentially produced in SE compared to seedlings. However, the ratio of miRNAs to total sRNAs in seedlings was higher than in SE, and most miRNAs were upregulated in seedlings. Trans-acting small interfering RNAs (ta-siRNAs) generated from TAS3 triggered by miR390 were identified, and levels of the three detected ta-siRNAs peaked in mature embryos, which was not consistent with the lowest RDR6 level. These findings indicate that larch, and possibly other gymnosperms, shares a common sRNA pathway with other land plants, and that the sRNA distribution pattern varies according to developmental stage, which may be attributable to the expression of sRNA pathway genes.

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

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

Generation of PVY Coat Protein siRNAs in Transgenic Potatoes Resistant to PVY

Global climate change has the potential to quickly alter the distribution and profile of crop pests and pathogens. Ready tools for mobilization of resistance into elite crops will be part of an integrated strategy to maintain agricultural productivity. Engineered virus resistance through expression of coat protein is well established. The mechanisms of virus suppression by small RNAs (sRNAs) have only recently been elucidated. Understanding the role of sRNAs in gene regulation and development is rapidly evolving. High throughput sequencing (HTS) can generate large data sets of sRNA sequences which can be analyzed to recognize the abundant sRNAs that result from transcript degradation and to identify the regulatory micro RNAs (miRNA) involved in gene regulation and small inhibitory RNAs (siRNA) that confer virus resistance. Transgenic Solanum tuberosum that are glass house or field grown and that express the potato virus Y coat protein (PVY-CP) inverted hairpin RNA (ihRNA) from the 409s promoter with GBSS6 intron as a spacer exhibited resistance to PVY. Southern blots of genomic DNA indicated one or two copies of the transgene and analysis of HTS data of the small RNA population indicated high levels of siRNA production from the transgenic hairpin construct. Up to 47 of the top 200 most frequent sRNA reads were attributable to the PVY-CP transgenic construct in Ranger Russet line #5. This combination of transgene and transcription strength is functional for constructing efficient resistance cassettes for crop protection. Up to 95 of the top 200 sRNA sequences were 21 nucleotides in length with as many as 19 sequences corresponding to known miRNA transcripts. Additional endogenous sRNAs with homology to resistance gene analogs may be indicative of additional or complimentary resistance mechanisms produced in concert with the PVY siRNAs arising from PVY-CP RNA transcription.

P141 Viral genome sequencing and small RNA detection by next generation sequencing

Introduction Next generation sequencing technologies are increasingly used in many domains. Their application to virology opens the door to various investigations such as virus sequencing and genotyping; analysis at quasispecies level; identification of causative agents of undiagnosed diseases; and finally study of host responses induced by viral infections. In this study, we used influenza- and rhinoviruses as models to validate some of these applications. Methods First we used influenza A virus infected cells to compare complete genome coverage after purification and ultra-deep sequencing of small RNAs (20–30 nt) or total fragmented RNAs (150–250 nt). Second, we sequenced small RNA population in rhinovirus (HRB-B14 and HRV-A39) infected HeLa cells and analyzed human micro RNA (miRNA) changes and the distribution of sequence reads on the viral genome. Results As expected the number of viral reads and genome coverage were much higher by sequencing total RNA, despite a lower ratio of viral versus host sequences. In contrast, small RNA purification from rhinovirus infected cells allowed us to point out to miRNA expression changes upon viral infection as well as over-expressed small viral RNAs (svRNAs). Conclusion svRNAs match the rhinovirus 5′UTR cloverleaf region, an important structural element implicated in the regulation of replication. The generation and the functional role of these svRNAs is currently under investigation.

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

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

Identification and Characterization of Argonaute Protein, Ago2 and Its Associated Small RNAs in Schistosoma japonicum

BackgroundThe complex life cycle of the genus Schistosoma drives the parasites to employ subtle developmentally dependent gene regulatory machineries. Small non-coding RNAs (sncRNAs) are essential gene regulatory factors that, through their impact on mRNA and genome stability, control stage-specific gene expression. Abundant sncRNAs have been identified in this genus. However, their functionally associated partners, Argonaute family proteins, which are the key components of the RNA-induced silencing complex (RISC), have not yet been fully explored.Methodology/Principal FindingsTwo monoclonal antibodies (mAbs) specific to Schistosoma japonicum Argonaute protein Ago2 (SjAgo2), but not SjAgo1 and SjAgo3, were generated. Soluble adult worm antigen preparation (SWAP) was subjected to immunoprecipitation with the mAbs and the captured SjAgo2 protein was subsequently confirmed by Western blot and mass spectrometry (MS) analysis. The small RNA population associated with native SjAgo2 in adult parasites was extracted from the immunoprecipitated complex and subjected to library construction. High-through-put sequencing of these libraries yielded a total of ≈50 million high-quality reads. Classification of these small RNAs showed that endogenous siRNAs (endo-siRNAs) generated from transposable elements (TEs), especially from the subclasses of LINE and LTR, were prominent. Further bioinformatics analysis revealed that siRNAs derived from ten types of well-defined retrotransposons were dramatically enriched in the SjAgo2-specific libraries compared to small RNA libraries constructed with total small RNAs from separated adult worms. These results suggest that a key function of SjAgo2 is to maintain genome stability through suppressing the activities of retrotransposons.Conclusions/SignificanceIn this study, we identified and characterized one of the three S. japonicum Argonautes, SjAgo2, and its associated small RNAs were found to be predominantly derived from particular classes of retrotransposons. Thus, a major function of SjAgo2 appears to associate with the maintenance of genome stability via suppression of retroelements. The data advance our understanding of the gene regulatory mechanisms in the blood fluke.

Shutdown is a component of the Drosophila piRNA biogenesis machinery

In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors, which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping-pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown (shu), a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germline and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins. Shu contains domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones, though the relevance of these domains to piRNA biogenesis is unknown.

RNA polymerase V-dependent small RNAs in Arabidopsis originate from small, intergenic loci including most SINE repeats

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM.

A Baculovirus-Encoded MicroRNA (miRNA) Suppresses Its Host miRNA Biogenesis by Regulating the Exportin-5 Cofactor Ran

MicroRNAs have emerged as key players in the regulation of various biological processes in eukaryotes, including host-pathogen interactions. Recent studies suggest that viruses encode miRNAs to manipulate their host gene expression to ensure their effective proliferation, whereas the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we demonstrate that an insect virus, Bombyx mori nucleopolyhedrosis virus (BmNPV), modulates the small-RNA-mediated defense of its host, B. mori, by encoding an miRNA (bmnpv-miR-1) that downregulates the expression of the host GTP-binding nuclear protein Ran, an essential component of the exportin-5-mediated nucleocytoplasmic transport machinery mainly involved in small-RNA transport from the nucleus to the cytoplasm. We demonstrate the sequence-dependent interaction of bmnpv-miR-1 with Ran mRNA using cell culture and in vivo assays, including RNA interference (RNAi) of Ran. Our results clearly show that bmnpv-miR-1 represses Ran, leading to reduction in the host small-RNA population, and consequently, the BmNPV load increases in the infected larvae. Blocking of bmnpv-miR-1 resulted in higher expression levels of Ran and a decrease in BmNPV proliferation. In contrast, blockage of host miRNA, bmo-miR-8, which targets the immediate-early gene of the virus and whose production was repressed upon bmnpv-miR-1 and Ran dsRNA administration, resulted in a significant increase in the virus load in the infected B. mori larvae. The present study provides an insight into one of the evasion strategies used by the virus to counter the host defense for its effective proliferation and has relevance to the development of insect virus control strategies.

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

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

A Global Microarray Expression Profile of Grapevine miRNAs Isolated from Cell Suspensions Pre-Treated with Apoptosis Activators

A Global Microarray Expression Profile of Grapevine miRNAs Isolated from Cell Suspensions Pre-Treated with Apoptosis Activators In an attempt to identify novel and apoptosis/pathogen-regulated microRNAs (miRNAs) and small interfering RNAs, we performed a robust microarray screening of small RNA population from Vitis vinifera L. cv. Limberger cell suspension exposed to apoptosis activators (e.g. methyl jasmonate) or elicitors (botrycin and cinerein) derived from necrotrophic fungus Botrytis cinerea Pers. et Fries. Using a microarray expression profiling approach, we identified 22 miRNAs. We found that a majority of these miRNAs were predicted to target stress/defense-related genes of plants. Of the 22 V. vinifera miRNAs, 11 have sequence conservation in Arabidopsis thaliana but exhibited species-specific developmental and/or stress/defense-related expression patterns. Ten of the miRNAs are highly conserved in other plant species, suggesting that even conserved miRNAs may have different regulatory roles in various species. Our results show that these grapevine miRNAs can be also induced by various apoptosis inducers. Fifty-one potential targets were predicted to the newly identified miRNAs based on sequence complementarity. In addition to miRNAs, we identified 102 other novel endogenous small RNAs in Vitis, indicating that a large number of miRNAs and other small regulatory RNAs are encoded by the Vitis vinifera genome.