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

RNA silencing is a mechanism of genetic regulation that is mediated by short noncoding RNAs, or small RNAs (sRNAs). Regulatory interactions are established based on nucleotide sequence complementarity between the sRNAs and their targets. The development of new high-throughput sequencing technologies has accelerated the discovery of sRNAs in a variety of plants and animals. The use of these and other high-throughput technologies, such as microarrays, to measure RNA and protein concentrations of gene products potentially regulated by sRNAs has also been important for their functional characterisation. mRNAs targeted by sRNAs can produce new sRNAs or the protein encoded by the target mRNA can regulate other mRNAs. In either case the targeting sRNAs are parts of complex RNA networks therefore identifying and characterising sRNAs contribute to better understanding of RNA networks. In this chapter we will review RNA silencing, the different types of sRNAs that mediate it and the computational methods that have been developed to use high-throughput technologies in the study of sRNAs and their targets.

microRNAs are non-coding RNAs that regulate gene expression. A significant proportion of microRNAs is perfectly conserved across the vertebrate clade, including miR-140, which is specifically expressed in cartilage. Although it has been computationally predicted that a large majority of microRNA targets are conserved, experimental evidence for this hypothesis remains scarce. In this work we use mRNA expression profiles obtained after manipulation of miR-140 activity levels in human and chicken primary chondrocytes to explore the extent of miR-140 target conservation. Our data suggest that miR-140 has a large number of targets conserved between human and chicken and we validate one of these, BMP2. However, we also found a significant number of non-conserved targets in the two species. In addition, we found that a commercially available scrambled siRNA, which is regularly used as a negative control, regulate the accumulation of many genes.

Genomic RNA of cymbidium ringspot tombusvirus (CyRSV) contains five large open reading frames (ORFs). The two 5′ proximal ORFs encode proteins of 33 and 92 kDa and the three 3′ proximal ORFs encode proteins of 41,22, and 19 kDa. In addition, a small reading frame encoding a protein of 4 kDa was recently observed by computer analysis of the 3′ nontranslated region of CyRSV and other tombusvirus RNAs (ORF 6). The function of putative gene products was investigated with the use of several mutants. Mutants in ORFs 1 and 2 were not viable indicating that both 33- and 92-kDa proteins are required for replication. Deletion of a large portion of the coat protein gene, encoding a 41-kDa protein, did not prevent replication of viral RNA and cell-to-cell movement, but interfered severely with long-distance translocation. No virus particles or viral RNA could be detected in inoculated or upper leaves of plants inoculated with transcripts obtained from mutants not expressing the 22-kDa protein. However, replication and encapsidation occurred normally in inoculated protoplasts indicating that this gene product is a transport protein. Conversely, no role could be assigned to putative gene products of ORF 5 (19-kDa protein) and QRF 6. It was also shown that factors other than gene expression can influence the replication of RNA mutants, probably due to instability of RNA molecules.

Defective interfering (DI) RNA of cymbidium ringspot tombusvirus was cloned downstream from the bacteriophage T7 RNA polymerase promoter. In vitro synthesized RNA was biologically active when coinoculated with parental genomic RNA onto Nicotiana benthamiana plants and prevented the occurrence of apical necrosis. N. benthamiana plants were transformed with the DI RNA sequences in both the positive and negative orientations relative to the cauliflower mosaic virus 35S promoter. Integration of DI RNA sequences in the plant genome was verified using PCR amplification of DNA extracts and Northern blot analysis of RNA extracts. DI RNA-related transcripts were detected in uninfected transgenic plants, but inoculation with the parental virus induced replication of the DI RNA only in transgenic plants expressing DI RNA in the positive orientation. Transgenic plants in which DI RNA accumulated were protected from apical necrosis and death.

s / Osteoarthritis and Cartilage 20 (2012) S10–S53 S42 the synovium expressed complement activating proteins, and found a strong upregulation of COMP, lumican, osteomodulin, biglycan, decorin and fibromodulin. In addition, biglycan expression was strongly enhanced (2.5-fold) in human CHECK-samples from patients with joint damage. Conclusions: All in all, these data suggest an active role for the synovium in OA pathology, and identifies pathways that are likely to be involved. One of the strongest associations was of the complement-pathway with cartilage damage. In addition, TGFb-, BMPand wnt-signaling in the synovium, may contribute to further joint damage. The enhanced expression of cartilage damaging MMP-1, MMP-3 and MMP-13 again suggests an active role of the synovium in OA pathology. Future studies will focus on association of gene expression patterns with progression of damage of CHECKpatients. 66 IDENTIFICATION AND CHARACTERISATION OF MICRORNAS INVOLVED IN CHONDROCYTE DIFFERENTIATION AND OSTEOARTHRITIS T.E. Swingler , N. Crowe , K. Sorefan , L. Le , G. Wheeler , V. Carmont , H.R. Elliott , M.J. Barter , M. Abu-Elmagd , S.T. Donell , R.P. Boot-Handford , M.K. Hajihosseini , A. Munsterberg , T. Dalmay , D.A. Young , I.M. Clark . Univ. of East Anglia, Norwich, United Kingdom; Univ. of Newcastle, Norwich, United Kingdom; Univ. of Manchester, Norwich, United Kingdom Purpose: The majority of bones develop through the process of endochondral ossification where a cartilaginous template is calcified and remodelled into bone. During this process chondrocytes undergo proliferation and differentiation. Many of the signalling pathways and transcription factors which control this developmental programme have been established. MicroRNAs are 20-24 nucleotide non-coding RNA molecules that post-transcriptionally regulate gene expression. There is strong evidence that they can influence both chondrogenesis and the initiation and progression of osteoarthritis (OA), however the exact mechanisms are still undetermined. In this study we aimed to profile expression of miRNAs in a cell model of chondrocyte differentiation and use this to prioritise miRNAs for functional analyses in OA. Using deep sequencing we also aimed to identify novel miRNAs expressed in human articular cartilage. Methods: The ATDC5 murine embryonic carcinoma cell line was induced to differentiate through chondrogenesis in vitro. An Exiqon miRNA microarray was used to profile the expression of all known murine miRNAs across this cell model. Expression of regulated miRNAs was verified in the mouse and chick embryo by in situ hybridisation. The. 3’ UTRs of potential target genes were subcloned downstream of a luciferase gene for experimental. Novel miRNAs expressed in human articular cartilage were identified using deep sequencing with Illumina's GAIIX system and validated in primary chondrocyte culture in vitro. Results: The expression of a number of microRNAs was regulated across chondrogenesis. This includes 39 microRNAs co-expressed with microRNA-140, known to be involved in cartilage homeostasis and osteoarthritis. Of these, microRNA-455 resides within an intron of COL27A1 which encodes a cartilage collagen. Comparing human osteoarthritic cartilage with femoral neck fracture controls, both microRNA140-5p and microRNA-455-3p show increased expression in osteoarthritic cartilage. In situ hybridisation shows microRNA-455-3p expression in the developing limbs of chicks and mice and in human osteoarthritic cartilage. The expression of microRNA-455-3p is regulated by TGFb ligands and the microRNA regulates TGFb signalling. ACVR2B, SMAD2 and CHRDL1 are direct targets of miR-455-3p and may mediate its functional impact on TGFb signalling. Deep sequencing of the small RNA pool from chondrocytes extracted from primary human osteoarthritic cartilage identified 16 potential novel miRNAs. These have been validated in cultured chondrocytes using qRT-PCR, Northern blot and Dicer knockdown. Conclusions:MicroRNA-455 is expressed during chondrogenesis and in adult articular cartilage where it can regulate TGFb signalling, suppressing the Smad2/3 pathway. Diminished signalling through this pathway in ageing and osteoarthritic chondrocytes is known to contribute to cartilage destruction. We propose that the increase in microRNA-455 in osteoarthritis exacerbates this process and contributes to disease pathology. Novel miRNAs expressed in cartilage may regulate cartilage homeostasis and contribute to disease. 67 THE ANALYSIS OF THE GENOME-WIDE DNA METHYLATION PROFILE OF HUMAN ARTICULAR CHONDROCYTES REVEALS DIFFERENT FORMS OF OA. J. Fernandez-Tajes , M. Fernandez-Moreno , M. Tamayo-Novas , A. Mosquera-Rey , N. Oreiro-Villar , C. Fernandez-Lopez , J.L. Fernandez-Garcia , I. Rego-Perez , F.J. Blanco . 1 INIBIC-CHUAC. Rheumatology Div.. Genomic Group., A Coruna, Spain; 2 INIBIC-CHUAC. Genetic Dept.., A Coruna, Spain Purpose: To identify and analyze the genome-wide DNA methylation profiles of human articular chondrocytes from a population-based casecontrol study of OA. Methods: DNAmethylation profiling was performed using the Infinium HumanMethylation27 beadchip (Illumina Inc.), which allows interrogation of 27,578 highly informative CpG loci. Previously, cartilage isolated DNA from 23 OA patients and 19 healthy controls was bisulfitemodified, using the EZ DNA methylation kit (Zymo Research) and hybridized according to the manufacturer’s instructions. DNA methylation b-values were normalized using GenomeStudio v3.0 (Illumina Inc.). Appropriate bioinformatics analyses were carried out using both R bioconductor software packages and Babelomics suite v 4.2 (babelomics.bioinfo.cipf.es). Results: A first approach based on an unsupervised clustering method for the most variable CpG loci (n1⁄4508) showed three distinct groups of samples, called cluster 1 (5 OA patiens), cluster 2 (6 OA patients) and cluster 3 (12 OA patients and 17 healthy controls). Specifically, cluster 2 formed a particularly tight cluster with a characteristic DNA methylation profile (Figure 1). The analyses of the biological relevance of the differentially methylated genes in cluster 2 compared with noncluster 2 by means of a gene set enrichment approach, showed that the biological processes significantly altered were those related to the superoxide metabolic process, morphogenesis/angiogenesis and regulation of cell proliferation, all of them hypermethylated in cluster 2; on the contrary, those mechanisms related to both IL-8 biosynthetic process and apoptosis appeared significantly hypomethylated in cluster 2. Conclusions: The genome-wide methylation analysis shows a clearly distinct epigenetic profile for OA. The DNA methylation profile could be one of the reasons of the existence of different forms of OA and could also be related to both the prevalence and progression of this

Plant Dicer-like (DCL) and Argonaute (AGO) are the key enzymes involved in anti-virus post-transcriptional gene silencing (AV-PTGS). Here we show that AV-PTGS exhibited nucleotide preference by calculating a relative AV-PTGS efficiency on processing viral RNA substrates. In comparison with genome sequences of dicot-infecting Turnip mosaic virus (TuMV) and monocot-infecting Cocksfoot streak virus (CSV), viral-derived small interfering RNAs (vsiRNAs) displayed positive correlations between AV-PTGS efficiency and G+C content (GC%). Further investigations on nucleotide contents revealed that the vsiRNA populations had G-biases. This finding was further supported by our analyses of previously reported vsiRNA populations in diverse plant-virus associations, and AGO associated Arabidopsis endogenous siRNA populations, indicating that plant AGOs operated with G-preference. We further propose a hypothesis that AV-PTGS imposes selection pressure(s) on the evolution of plant viruses. This hypothesis was supported when potyvirus genomes were analysed for evidence of GC elimination, suggesting that plant virus evolution to have low GC% genomes would have a unique function, which is to reduce the host AV-PTGS attack during infections.

Gene expression is regulated at several levels in plants, and one of the most recently discovered regulatory layers involve short RNAs. Short RNAs are produced through several pathways and target either mRNAs or genomic DNA. Different classes of short RNAs have slightly different sizes and detection of their accumulation is an important step in validating and studying non-coding short RNAs. Northern blotting is routinely used to detect short RNAs because it gives information about both the amount and size of the analysed short RNAs. Choice of the right RNA extraction protocol is crucial when short RNAs are being studied, because several routinely used commercial RNA extraction kits do not yield any short RNAs. This chapter describes optimised RNA extraction methods, which give good yields of short RNAs, and separation, transfer and hybridisation protocols to study the accumulation of short RNAs.

Virus-derived short interfering RNAs (vsiRNAs) isolated from grapevine V. vinifera Pinot Noir clone ENTAV 115 were analyzed by high-throughput sequencing using the Illumina Solexa platform. We identified and characterized vsiRNAs derived from grapevine field plants naturally infected with different viruses belonging to the genera Foveavirus, Maculavirus, Marafivirus and Nepovirus. These vsiRNAs were mainly of 21 and 22 nucleotides (nt) in size and were discontinuously distributed throughout Grapevine rupestris stem-pitting associated virus (GRSPaV) and Grapevine fleck virus (GFkV) genomic RNAs. Among the studied viruses, GRSPaV and GFkV vsiRNAs had a 5′ terminal nucleotide bias, which differed from that described for experimental viral infections in Arabidopsis thaliana. VsiRNAs were found to originate from both genomic and antigenomic GRSPaV RNA strands, whereas with the grapevine tymoviruses GFkV and Grapevine Red Globe associated virus (GRGV), the large majority derived from the antigenomic viral strand, a feature never observed in other plant–virus interactions.

mRNA profiling is routinely used to identify microRNA targets, however, this high-throughput technology is not suitable for identifying targets regulated only at protein level. Here, we have developed and validated a novel methodology based on computational analysis of promoter sequences combined with mRNA microarray experiments to reveal transcription factors that are direct microRNA targets at the protein level. Using this approach we identified Smad3, a key transcription factor in the TGFβ signaling pathway, as a direct miR-140 target. We showed that miR-140 suppressed the TGFβ pathway through repression of Smad3 and that TGFβ suppressed the accumulation of miR-140 forming a double negative feedback loop. Our findings establish a valid strategy for the discovery of microRNA targets regulated only at protein level, and we propose that additional targets could be identified by re-analysis of existing microarray datasets.

In plants, microRNAs (miRNAs) comprise one of three classes of small RNAs regulating gene expression at the post-transcriptional level. Many plant miRNAs are conserved, and play a role in development, abiotic stress responses or pathogen responses. However, some miRNAs have only been found in certain species. Here, we use deep-sequencing, computational and molecular methods to identify, profile, and describe conserved and non-conserved miRNAs in four grapevine (Vitis vinifera) tissues. A total of 24 conserved miRNA families were identified in all four tissues, and 26 known but non-conserved miRNAs were also found. In addition to known miRNAs, we also found 21 new grapevine-specific miRNAs together with their star strands. We have also shown that almost all of them originated from single genes. Furthermore, 21 other plausible miRNA candidates have been described. We have found that many known and new miRNAs showed tissue-specific expression. Finally, 112 target mRNAs of known and 44 target mRNAs of new grapevine-specific miRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs.

MicroRNAs (miRNAs) are a new class of negative regulators that repress gene expression by pairing with their target messenger RNAs (mRNAs). There are hundreds of miRNAs coded in the human genome and thousands of target mRNAs participating in a wide variety of physiological processes such as development and cell identity. It is therefore not surprising that several recent reports involved deregulated miRNAs in the complex mechanism of human carcinogenesis, and proposed them as new key regulators to correct the unbalanced expression of oncogenes and tumour suppressor genes exhibited in cancer cells. This review summarises most of the recent patents related to the use of miRNA signatures in cancer diagnosis and prognosis, the detection and profiling of miRNAs from tumour samples and the identification of oncogenes and tumour suppressor genes targeted by miRNAs, as well as new cancer therapies based on miRNA modulators.

Plant microRNAs have been implicated in various abiotic stress responses. We identified several conserved microRNAs that showed differential expression in Medicago truncatula plants subjected to water deficit: miR169 is down-regulated only in the roots and miR398a/b and miR408 are strongly up-regulated in both shoots and roots. Down-regulation of miR169 in the roots did not correlate with accumulation of its target MtHAP2-1 transcripts, suggesting that its regulation may not occur at the mRNA level or may depend on other regulatory mechanisms, which do not involve this miRNA, in water-deficit conditions. The up-regulation of miR398a/b and miR408 and the clear down-regulation of their respective target genes, which encode the copper proteins COX5b (subunit 5b of mitochondrial cytochrome c oxidase) and plantacyanin, highlight the involvement of these miRNAs in response to water deprivation in M. truncatula. Also, miR398 up-regulation is inversely correlated with the down-regulation of copper superoxide dismutase, CSD1, during water deficit. The regulation of genes encoding copper proteins by miR398a/b and miR408 suggests a link between copper homeostasis and M. truncatula adaptation to progressive water deficit.

revealed a marked reduction in radial thickness starting at E13.5, and defective postnatal cortical layering. Whereas the former was due to neuronal apoptosis starting at E12.5, which was the earliest detectable phenotype, the latter reflected dramatic impairment of neuronal differentiation. Remarkably, the primary target cells of Dicer ablation, the neuroepithelial cells, and the neurogenic progenitors derived from them, were unaffected by miRNA depletion with regard to cell cycle progression, cell division, differentiation and viability during the early stage of neurogenesis, and only underwent apoptosis starting at E14.5. Our results support the emerging concept that progenitors are less dependent on miRNAs than their differentiated progeny. In order to identify miRNAs targets, we extracted total RNA from E13.5 cortices of Dicer-ablated and of control littermates, and mRNAs were subjected to microarray analysis. Our results show that upon miRNA depletion the number of upregulated mRNAs is higher than that of downregulated ones, suggesting that identified mRNAs might represent primary miRNAs targets rather than mRNAs indirectly affected by miRNAs depletion.

Background High-throughput sequencing technology is capable to identify novel short RNAs in plant species. We used Solexa sequencing to find new microRNAs in one of the model legume species, barrel medic (Medicago truncatula). Results 3,948,871 reads were obtained from two separate short RNA libraries generated from total RNA extracted from M. truncatula leaves, representing 1,563,959 distinct sequences. 2,168,937 reads were mapped to the available M. truncatula genome corresponding to 619,175 distinct sequences. 174,504 reads representing 25 conserved miRNA families showed perfect matches to known miRNAs. We also identified 26 novel miRNA candidates that were potentially generated from 32 loci. Nine of these loci produced eight distinct sequences, for which the miRNA* sequences were also sequenced. These sequences were not described in other plant species and accumulation of these eight novel miRNAs was confirmed by Northern blot analysis. Potential target genes were predicted for most conserved and novel miRNAs. Conclusion Deep sequencing of short RNAs from M. truncatula leaves identified eight new miRNAs indicating that specific miRNAs exist in legume species.

During their infection in plants, viruses can form double stranded (ds) RNA structures. These dsRNAs can be recognized by plants as "aberrant" signals and short interfering RNA (siRNA) molecules of 19-25 nt will be produced with sequences derived from the viral source. Knowledge about antiviral siRNA profiles including siRNA size, distribution, polarity, etc. provides valuable insights to plant-virus interactions. In this chapter, we describe a simple method for cloning siRNA from virus-infected plants. This protocol includes isolation of small RNAs, their ligation to a pair of 5' and 3' adapters, RT-PCR/PCR amplification, and subsequent concatamerization before pGEM-T cloning and sequencing. Concatamers containing as many as 15 small RNA inserts can be produced. This protocol has successfully been applied to leaf materials of monocots and dicots infected with poty-, carmo-, and sobemo-viruses.

The Nobel Prize in Medicine and Physiology was awarded to the RNA interference (RNAi) field in 2006 because of the huge therapeutic potential this technique harbours. However, the RNAi technology is based on a natural mechanism that utilizes short noncoding RNA molecules (microRNAs) to regulate gene expression. This paper reviews our current knowledge about microRNAs focusing on their involvement in cancer and their potential as diagnostics and therapeutics.

Dicot Dicer-like (DCL) enzymes operate preferably on GC rich regions when producing small interfering (si)RNA and micro (mi)RNA. This GC bias, however, is not generic in monocot miRNA productions. From wild Dactylis glomerata naturally infected by Cocksfoot streak potyvirus (CSV), CSV-siRNAs had a greater GC% than the virus genome, indicating that GC rich regions were also preferred by the grass DCLs. This supports the notion that GC preference is an ancient feature for plant DCLs, and suggests that monocot miRNA genes might have evolved to a high GC% resulting in GC bias being not detectable during mature miRNA production.

During their infection in plants, viruses can form double stranded (ds) RNA structures. These dsRNAs can be recognized by plants as ‘aberrant’ signals and short interfering RNA (siRNA) molecules of 19 - 25 nt will be produced with sequences derived from the viral source. Knowledge about antiviral siRNA profiles including siRNA size, distribution, polarity, etc., provides valuable insights to plant-virus interactions. In this chapter we describe a simple method for cloning siRNA from virus-infected plants. This protocol includes isolation of small RNAs, their ligation to a pair of 5’ and 3’ adapters, RT-PCR/PCR amplification, and subsequent concatamerization before pGEM-T cloning and sequencing. Concatamers containing as many as 15 small RNA inserts can be produced. This protocol has successfully been applied to leaf materials of monocots and dicots infected with poty-, carmo-, and sobemo-viruses.

RNA silencing has evolved as a defense against molecular parasites in plants, such as viruses. It is also useful in regulating gene expression for growth and development. These pathways overlap and operate through the production of small RNAs. Small RNA molecules serve as a guide for complexes to find homologous sequences where they can exert their effect. The biogenesis of small RNAs is diverse and they operate in a variety of phenomena. In the model plant Arabidopsis thaliana, the diversification of small RNAs and gene silencing pathways may be explained by the existence of multiple paralogs of RNA-silencing-associated proteins such as ten AGOs, six RDRs, and four DCLs. Many of these proteins are involved in virus defense. However, viruses also develop strategies to counteract this defense system. As a counterdefense they express silencing suppressor proteins, which efficiently inhibit RNA silencing by interacting with various steps of the different silencing pathways. Insights into the molecular bases of the induction and the suppression of RNA silencing have dramatically improved our knowledge about the intimate interaction of plants and viruses during infection. Furthermore, it has also provided valuable tools to unravel the diversity, regulation, and evolution of RNA-silencing pathways.

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

MicroRNAs (miRNAs) are recently discovered short regulatory RNA molecules representing a new layer in posttranscriptional gene expression regulation. Although more than 450 human miRNAs have been identified, only a very few of them have been characterized in detail. The precise understanding of miRNA-mediated processes requires the reliable spatial and temporal analyses of miRNA accumulation at tissue/cell level. However, the detection of miRNAs by in situ hybridization (ISH) is technically challenging because of the small size of target sequences. It was shown recently that locked nucleic acid nucleotide-containing probes can anneal to short nucleic acids with high specificity. This enabled several research groups to analyze the expression patterns of miRNAs in both plant and animal tissues. This review focuses on the results of recent publications on the detection of miRNAs by ISH.

Small interfering (si)RNAs isolated from Brassica juncea leaves infected by Turnip mosaic virus (TuMV) were characterized by cloning and sequencing. The TuMV siRNA population was dominated by 21 and 22-nt long species originated mainly from the same siRNA hotspots, indicating operational similarity between the plant Dicer-like (DCL) enzymes. Robust GC bias was observed for TuMV siRNAs versus the virus genome, indicating that DCL was more likely to target GC-rich regions. Furthermore, dicot micro-(mi)RNAs displayed higher GC% than their DCL1 substrate RNAs, implicating that the GC bias may be ancient, therefore may be important for the RNAi technology.

Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation process conserved in fungi, plants and animals. The trigger of the mechanism is double-stranded RNA derived from transgenic or endogenous loci and formed by intra- or inter-molecular interactions of single-stranded RNAs or the action of RNA-dependent RNA polymerases (RDRs). Double-stranded RNA from various sources is processed by one of the four Dicer-like (DCL) proteins in Arabidopsis, and the resulting short RNAs enter into at least four different pathways, one of which involves the production of trans-acting short interfering RNAs (tasiRNAs). We report here a novel gene (SDE5) that is required for transgene silencing and the production of tasiRNAs. Mutation in SDE5 also results in hyper-susceptibility to cucumber mosaic virus but not turnip mosaic virus. However, like RDR6, SDE5 is not involved in inverted repeat-induced transgene silencing or the biogenesis of microRNAs and 24 nt siRNAs produced by DCL3. Based on these results, we propose that SDE5 acts together with RDR6 in generating double-stranded RNA from specific single-stranded RNAs. As the sequence of SDE5 has sequence features shared by TAP, a human mRNA export factor, we propose that its role could be in the transport of RNA molecules that are converted into a double-stranded form by RDR6.

To date, the majority of plant small RNAs (sRNA) have been identified in rice, poplar and Arabidopsis. To identify novel tomato sRNAs potentially involved in tomato specific processes such as fruit development and/or ripening, we cloned 4,018 sRNAs from tomato fruit tissue at the mature green stage. From this pool of sRNAs, we detected tomato homologues of nine known miRNAs, including miR482; a poplar miRNA not conserved in Arabidopsis or rice. We identified three novel putative miRNAs with flanking sequence that could be folded into a stem-loop precursor structure and which accumulated as 19-24nt RNA. One of these putative miRNAs (Put-miRNA3) exhibited significantly higher expression in fruit compared with leaf tissues, indicating a specific role in fruit development processes. We also identified nine sRNAs that accumulated as 19-24nt RNA species in tomato but genome sequence was not available for these loci. None of the nine sRNAs or three putative miRNAs possessed a homologue in Arabidopsis that had a precursor with a predicted stem-loop structure or that accumulated as a sRNA species, suggesting that the 12 sRNAs we have identified in tomato may have a species specific role in this model fruit species.

It has become clear that particular microRNAs (miRNAs) function either as tumour suppressors or oncogenes, whose loss or overexpression, respectively, has diagnostic and prognostic significance. In several cases, miRNAs have been shown to affect target genes that are involved in the control of cell proliferation and apoptosis. However, malignant tumours display additional traits beyond the acquisition of enhanced growth potential and decreased cell death. Malignant disease is associated with altered tumour-host interactions leading to sustained angiogenesis and the ability to invade and metastasize. It is possible that miRNAs may act as master regulators of these aspects of tumour biology. Bioinformatic analysis of putative miRNA binding sites has indicated several novel potential gene targets of cancer-associated miRNAs that function in aspects of cell adhesion, neovascularization and tissue invasion. Among others, we speculate that miRNAs may find new roles in the regulation of E-cadherin, integrin alphavbeta3, hypoxia-inducible factor-1alpha, syndecan-1, lysyl oxidase, adamalysin metalloproteinase-17, tissue inhibitors of metalloproteinase-3, c-Met and CXCR-4 that underpin the tissue architectural changes associated with malignancy.

The microRNAs (miRNAs) are recently discovered short, noncoding RNAs, that regulate gene expression in metazoans. We have cloned short RNAs from chicken embryos and identified five new chicken miRNA genes. Genome analysis identified 17 new chicken miRNA genes based on sequence homology to previously characterized mouse miRNAs. Developmental Northern blots of chick embryos showed increased accumulation of most miRNAs analyzed from 1.5 days to 5 days except, the stem cell-specific mir-302, which was expressed at high levels at early stages and then declined. In situ analysis of mature miRNAs revealed the restricted expression of mir-124 in the central nervous system and of mir-206 in developing somites, in particular the developing myotome. In addition, we investigated how miR-206 expression is controlled during somite development using bead implants. These experiments demonstrate that fibroblast growth factor (FGF) -mediated signaling negatively regulates the initiation of mir-206 gene expression. This may be mediated through the effects of FGF on somite differentiation. These data provide the first demonstration that developmental signaling pathways affect miRNA expression. Thus far, miRNAs have not been studied extensively in chicken embryos, and our results show that this system can complement other model organisms to investigate the regulation of many other miRNAs.

MicroRNAs (miRNA) are short RNA molecules regulating the expression of specific mRNAs. We investigated the expression pattern and potential targets of mouse miR-140 and found that miR-140 is specifically expressed in cartilage tissues of mouse embryos during both long and flat bone development. MiR-140 expression was detected in the limbs of E11.5 embryos in the primorida of future bones both in the fore and hindlimb and across autopod, zeugopod and stylopod. All digits of E14.5 fore- and hindlimbs showed accumulation of miR-140, except the first digit of the hindlimb. MiR-140 expression was also detected in the cartilagenous base of E17.5 skulls and in the sternum, the proximal rib heads and the developing vertebral column of E15.5 embryos. A potential target of miR-140, histone deacetylase 4, was validated experimentally and the possible role of miR-140 in long bone development is discussed.

MicroRNAs (miRNAs) are small regulatory molecules suppressing mRNA activity in metazoans. Here we describe two new miRNAs cloned from brain tissue of mouse embryos. These miRNAs are expressed mainly during embryogenesis and specifically in the central nervous system. We also established the expression patterns of three recently identified miRNAs that were found in our short RNA library. All of them were expressed in the brain and spinal chord but while miR-410 and miR-431 were central nervous system specific, miR-500 was also expressed in limb buds. In addition, the expression of miR-500 in limb buds showed very strong asymmetry in favour of the left hand side.

Post-transcriptional gene silencing (PTGS) provides protection in plants against virus infection and can suppress expression of transgenes. Arabidopsis plants carrying mutations at the SDE3 locus are defective in PTGS mediated by a green fluorescent protein transgene. However, PTGS mediated by tobacco rattle virus (TRV) was not affected by sde3. From these results we conclude that SDE3, like the previously described RNA polymerase encoded by SDE1, acts at a stage in the mechanism that is circumvented when PTGS is mediated by TRV. The product of SDE3 is similar to RNA helicase-like proteins including GB110 in mouse and other proteins in Drosophila and humans. These proteins are similar to, but clearly distinct from Upf1p and SMG-2, which are required for nonsense-mediated mRNA decay in yeast and Caenorhabditis elegans and, in the case of SMG-2, for PTGS.

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

Posttranscriptional gene silencing is a defense mechanism in plants that is similar to quelling in fungi and RNA interference in animals. Here, we describe four genetic loci that are required for posttranscriptional gene silencing in Arabidopsis. One of these, SDE1, is a plant homolog of QDE-1 in Neurospora crassa that encodes an RNA-dependent RNA polymerase. The sde1 mutation was specific for posttranscriptional gene silencing induced by transgenes rather than by viruses. We propose that the role of SDE1 is to synthesize a double-stranded RNA initiator of posttranscriptional gene silencing. According to this idea, when a virus induces posttranscriptional gene silencing, the virus-encoded RNA polymerase would produce the double-stranded RNA and SDE1 would be redundant.

Summary: Recent developments in high-throughput sequencing technologies have generated considerable demand for tools to analyse large datasets of small RNA sequences. Here, we describe a suite of web-based tools for processing plant small RNA datasets. Our tools can be used to identify micro RNAs and their targets, compare expression levels in sRNA loci, and find putative trans-acting siRNA loci. Availability: The tools are freely available for use at http://srna-tools.cmp.uea.ac.uk Contact: vincent.moulton@cmp.uea.ac.uk

Mutational analysis of defective interfering (DI) RNAs of Cymbidium ringspot virus (CymRSV) was used to study the mechanism of DI RNA evolution. It was shown that a highly base-paired structure in the 3' region of the longer DI RNA directed the formation of smaller DI RNA molecules. Mutations which increased the stability of the computer-predicted, highly structured 3' region of the longest DI RNA of CymRSV significantly enhanced the generation and accumulation of the smaller derivatives. Sequence analysis of smaller progeny molecules revealed that the highly base-paired region was deleted from the precursor DI RNA. Moreover, sites of recombination were found in other regions of the DI RNA progenies due to transposition of the highly base-paired structure. It is likely that the deletion event was structure- and not sequence-specific, and operated when a foreign sequence containing a 37-nt-long base-paired stem was inserted at the appropriate position of DI RNA.

The smallest defective interfering RNA (DI-2) of cymbidium ringspot tombusvirus (CyRSV) was used to identify the cis-acting sequences necessary for its replication by making a series of deletions throughout the 404 nt long molecule and testing the biological activity of mutants. Deletion or substitution of the conserved sequence blocks (A, B and C) always yielded inactive molecules. The deletion of only a few nucleotides could be tolerated beyond the natural deletion sites in blocks A and B. However, either half of block C1 (34 nt) and the first 25 nt of C2 (102 nt) could be deleted without loss of infectivity. It was also demonstrated that either one of the two halves of block C1 was specifically required for replication. We suggest that the last 77 nt of the viral genome and either half of block C1 represent the complementary strand promoter sequence recognized by the viral replicase.

The present study identified Arabidopsis miR394 and its target, an F-box (SKP1-Cullin/CDC53-F-box) gene At1g27340 (here referred to as LEAF CURLING RESPONSIVENESS, LCR), for regulation of leaf curling-related morphology. The loss-of-function lcr mutants exhibit pleiotropic defects with semi-dwarfism, altered leaf shape and a shorter stem. Overexpression of an miR394-resistant version of LCR under the 35S promoter (35S:m5LCR) and target mimicry MIM394 resulted in a curled-down leaf defect. Conversely, transgenic plants overexpressing 35S:MIR394a/b display a curled-up leaf phenotype. Detailed analyses show that there is a certain level of LCR that is optimal for leaf morphology, but lower or higher levels lead to abnormal leaf development, indicating that expression of miR394 in the leaf lamina is necessary for proper leaf morphology. Because the phytohormone auxin plays a crucial role in leaf morphogenesis and patterning, the DR5-GUS reporter gene was used to monitor the auxin response. We show that DR5 expression patterns in lcr and 35S::m5LCR plants were significantly different from those in the wild type. Also, overexpression of LCR in 35S::m5LCR plants drastically decreased the expression of the auxin-responsive genes IAA3, AXR3 and IAMT1, whereas increased expression of the genes was found in 35S::MIR394a plants. These results indicate that miR394 and its target LCR are involved in the regulation of leaf development.

Small RNAs (sRNAs) are a class of short (20-25 nt) non-coding RNAs that play important regulatory roles in gene expression. An essential first step in understanding their function is to confidently identify sRNA targets. In plants, several classes of sRNAs such as microRNAs (miRNAs) and trans-acting small interfering RNAs have been shown to bind with near-perfect complementarity to their messenger RNA (mRNA) targets, generally leading to cleavage of the mRNA. Recently, a high-throughput technique known as Parallel Analysis of RNA Ends (PARE) has made it possible to sequence mRNA cleavage products on a large-scale. Computational methods now exist to use these data to find targets of conserved and newly identified miRNAs. Due to speed limitations such methods rely on the user knowing which sRNA sequences are likely to target a transcript. By limiting the search to a tiny subset of sRNAs it is likely that many other sRNA/mRNA interactions will be missed. Here, we describe a new software tool called PAREsnip that allows users to search for potential targets of all sRNAs obtained from high-throughput sequencing experiments. By searching for targets of a complete 'sRNAome' we can facilitate large-scale identification of sRNA targets, allowing us to discover regulatory interaction networks.

Defective interfering (DI) RNA of cymbidium ringspot tombusvirus (CyRSV) was tested as a potential RNA vector. The coat protein-encoding gene (CP) of the same virus or of the unrelated tomato aspermy cucumovirus (TAV) was inserted in a biologically active clone of CyRSV DI-3 RNA. Both homologous and heterologous CP genes were inserted into DI-3 cDNA clone in two different positions. The CyRSV CP was expressed only in the leaves which were inoculated with DI-3CPWt plus CP-less mutant helper virus (ΔABgII). Chimaeric DI RNA carrying a heterologous CP gene (DI-3HCPtav) was able to replicate and express the inserted TAV CP in the presence of a wild-type (wt) helper genome. Both constructs, which were stable and active for gene expression, carried the inserted CP genes in the same position, between the A and B blocks of DI-3 RNA. Other constructs in which the CP were cloned between the B and C blocks of DI-3 RNA were not able to direct the translation of the encoded CP. The expression level of CP derived from recombinant DI RNA was lower relative to expression of CP from wt virus infection.

The multimeric forms of cymbidium ringspot tombusvirus (CyRSV) satellite (sat) RNA were analysed. Attempts to amplify the putative junction region of oligomers using the polymerase chain reaction (PCR) failed, indicating the absence of such structures. SatRNA-related species having the size double than the unit length were shown to be double-stranded monomers and not single-stranded dimers. Similarly, satRNA species of a size four times the unit length were shown to be constituted by aggregates of double-stranded monomers. The absence of single-stranded CyRSV satRNA oligomers indicates that the formation of multimers is not a step in the replication of this RNA molecule.

Hybrid cDNA clones were constructed by fusing the coat protein-encoding gene and/or the 3'-terminal region (including the 22- and 19-kDa protein-encoding genes) derived from a clone of artichoke mottled crinkle tombusvirus to the 5'-terminal region of a full-length clone of cymbidium ringspot tombusvirus. In vitro transcripts from recombinant clones were infectious when inoculated into Nicotiana clevelandii plants. Inoculated plants showed symptoms different from those induced by parent viruses. In particular, systemic invasion depended very much, although not exclusively, on the type of protein that coated progeny viral RNA, suggesting a role of the capsid protein in the long-distance movement of tombusvirus infections.

Progeny RNA of CyRSV RNA transcripts ending with -GGG, instead of -CCC as in wild-type RNA, was analyzed and shown to revert to wild-type. A new full-length cDNA clone of CyRSV was prepared, bearing the correct 3′ terminus, from which transcripts could be synthesized having very high infectivity. Mutants were prepared which lacked the three terminal Cs; transcripts were infectious and most cDNA clones recovered from progeny RNA had regained the wild-type sequence. Transcripts ending in -GGCCA n were also infectious, and the 3′ sequence of most cDNA clones recovered after inoculation was the same as the inoculum sequence. Deletion of G at position -4 completely abolished infectivity. The occurrence of a repair mechanism at the 3′ end of CyRSV similar to telomerase activity on chromosomes is suggested.

A sequence of 9703 nucleotides (nt) is reported for the genomic RNA of potato virus Y (Hungarian isolate, PVY-H), which causes necrotic rings around the buds on the tubers and mottling of leaves. The sequence contains one large open reading frame of 3061 amino acids (aa), a noncoding region of 189 nt at the 5' end and a 330-nt 3' nontranslated region. The nt sequence and the predicted aa sequence of the polyprotein of PVY-H were analysed pairwise with the only available complete sequence of PVY strain N (PVYn) and with the partial sequences of different PVY strains, as well as with other potyviruses and potyvirus-related plant viruses. The overall relationship between PVY-H and PVYn shows a nt sequence identity of 88.5% and an aa sequence identity of 94.2%. The lowest degree of homology was detected at the 5' terminus of the genome, including the 5' noncoding region (70.3%) and the 275-aa P1 protein (78%). A fivefold sequence repeat block of 5'-UUUCA was found in the 5' noncoding region of PVY-H, which seems to be characteristic of PVY strains.

The coat protein (CP) gene from potato virus Y (Hungarian isolate, PVY-H) was engineered into Agrobacterium tumefaciens binary vector for expression in different tobacco lines. Three different Nicotiana tabacum breeding lines were transformed and the integration of the CP gene was confirmed by PCR technique using genomic DNA preparations. The transcription and expression of the integrated CP gene was detected by Northern and Western blots. Pathogen-derived resistance was demonstrated by inoculation of the R1 progeny of the transformed lines with purified PVY-H. The efficiency of protection varied between different transgenic plants ranging from almost complete to no protection. Five CP expressing tobacco lines were resistant to challenge infection with PVY-H as indicated by attenuation or absence of symptom development associated with reduction or lack of detectable virus accumulation. Data from Western blots showed that there is no correlation between the level of the expressed CP and the extent of protection. This suggests that the mechanism of the observed resistance is independent of the level of CP accumulation in the transgenic tobacco plants.

BackgroundViroids are circular, highly structured, non-protein-coding RNAs that, usurping cellular enzymes and escaping host defense mechanisms, are able to replicate and move through infected plants. Similarly to viruses, viroid infections are associated with the accumulation of viroid-derived 21-24 nt small RNAs (vd-sRNAs) with the typical features of the small interfering RNAs characteristic of RNA silencing, a sequence-specific mechanism involved in defense against invading nucleic acids and in regulation of gene expression in most eukaryotic organisms.Methodology/principal findingsTo gain further insights on the genesis and possible role of vd-sRNAs in plant-viroid interaction, sRNAs isolated from Vitis vinifera infected by Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1) were sequenced by the high-throughput platform Solexa-Illumina, and the vd-sRNAs were analyzed. The large majority of HSVd- and GYSVd1-sRNAs derived from a few specific regions (hotspots) of the genomic (+) and (-) viroid RNAs, with a prevalence of those from the (-) strands of both viroids. When grouped according to their sizes, vd-sRNAs always assumed a distribution with prominent 21-, 22- and 24-nt peaks, which, interestingly, mapped at the same hotspots.Conclusions/significanceThese findings show that different Dicer-like enzymes (DCLs) target viroid RNAs, preferentially accessing to the same viroid domains. Interestingly, our results also suggest that viroid RNAs may interact with host enzymes involved in the RNA-directed DNA methylation pathway, indicating more complex scenarios than previously thought for both vd-sRNAs genesis and possible interference with host gene expression.