This EB has been in office for a relatively short time. Its election towards the end of last year was somewhat unusual, in the sense that only its Secretary remained on the Board – most of its members are thus new. This has therefore been a period of change, and a learning process for us all. At the same time, the global bioinformatics landscape is also changing. We therefore felt that the 2010 AGM would provide an ideal opportunity to examine those changes, to consider EMBnet’s position within this evolving landscape and to explore its aspirations and motivations, moving forward.

Cancer is a multi-stage process often driven by progressive accumulation of genomic rearrangements that can result in cells acquiring cancer properties such as tumor invasive and metastatic behavior. Many genes associated with cancer are the result of complex somatically and inherited chromosomal rearrangements, resulting in aberrant transcripts or defects in transcription [1-5]. The classical approach for the identification of genome rearrangements such as G-banded cytogenetics, spectral karyotyping and FISH, are poor in sensitivity, while copy number array can identify just imbalanced breakpoints and do not describe the resulted genome structure produced by the events, which may cause the breakpoints. The aim of this project is to obtain, by the paired-end mapping (PEM) approach applied to the massive parallel sequencing, an high resolution virtual karyotype of the genome of a breast-cancer-patient of which we obtained previously the transcriptomic portrait [6].The introduction of massively parallel high throughput sequencing (HTS) techniques have created a broad range of new and exciting research applications by increasing the output sequencing data dramatically. In recent years, the continuous technical improvements of next-generation sequencing technology have made RNA sequencing (RNA-seq) particularly effective for the detection of gene fusions, which are involved in several diseases. Gene fusions are found in many cancer types, and they have proved to be prognostic biomarkers in several studies [7-9]. In addition, gene fusions have often a direct functional impact on the molecular processes in the cell [10].Several analysis steps are needed to process the data provided by the sequencer and to use them for robust gene fusion detection.We propose a workflow to analyze NGS paired-end sequences in order to identify possible candidates to be the results of a fusion between different genes, looking for fusion events occurring on the same chromosome (intra-chromosomal rearrangement).The basic idea is to map the reads onto the reference genome and to study the insert size length distribution of the paired-end, looking at its peak and select all the mapping pairs having an insert size value quite far from the observed peak. In this way we are sure to select paired-end sequences mapping on different regions of the genome far from each other connecting different genes.

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 50 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 30-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 30 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

In plants, which are particularly sensitive to changes of environmental conditions, modulation of DNA methylation is a crucial mechanism of regulation of gene expression in response to abiotic and biotic stresses. Monitoring plant's immune system in response to bacterial pathogen infection demonstrated that also dynamic DNA methylation changes, and not only gene imprinting, have regulatory effect in plant pathogen defense. Critical elements for epigenetic modifications of plant genomes are non-coding smallRNA e same RNA family is also a hallmark of plant reaction to virus infection. Interestingly, sRNA have a central role in both plant genome methylation and resistance upon virus infection, however, the interaction between sRNA expression and DNA methylation regulating the immune system in response to virus infection has not been investigated so far.To correlate dynamic DNA methylation and differential sRNA expression in response to virus infection, we have performed genome-wide methylation and sRNA expression profiling on Arabidopsis leaves systemically infected with either the DNA-genome virus Caulifower mosaic virus or the RNA virus Cucumber mosaic virus. We developed a software package to analyze the sRNA expression and the DNA methylation profile and deploy a genome wide comparison of control and infected samples to search regions significantly different either in the methylation profile or in the sRNA expression, or in both. In the regions where we observe significant correlation of methylation (mainly CHH methylation) and sRNA expression modifications, we found that both hypo- and hypermethylation correlated with downregulation of 21/24nt sRNAs. These regions mostly comprised transposons and few of them contained promoter or coding sequences of genes involved, according to gene ontology, in DNA-binding and DNA-dependent regulation of transcription and response to abiotic or biotic stimulus. This confirms virus-induced infection regulation of sRNA and DNA methylation. We are presently still in the process of data analysis and more details about correlation of virus-induced modification of sRNA and DNA methylation levelswill be reported.

To integrate heterogeneous and large omics data constitutes not only a conceptual challenge but a practical hurdle in the daily analysis of omics data. With the rise of novel omics technologies and through large-scale consortia projects, biological systems are being further investigated at an unprecedented scale generating heterogeneous and often large data sets. These data-sets encourage researchers to develop novel data integration methodologies. In this introduction we review the definition and characterize current efforts on data integration in the life sciences. We have used a web-survey to assess current research projects on data-integration to tap into the views, needs and challenges as currently perceived by parts of the research community.

Cytosine methylation is a stable and heritable modification of the DNA that imparts epigenetic control throughout the genome, including regulation of coding and noncoding elements. In plants, previous studies have contributed to shape the epigenetic landscape in developmental processes, whereas the potential for these pathways to be dynamically regulated during non-developmental processes, such as stress responses, has not been investigated thoroughly at date. Recently, it has been demonstrated that DNA methylation is involved in controlling the Arabidopsis thaliana defence response against bacterial pathogens, and several lines of evidence suggest that plant immune response to viral infections imply an involvement of DNA methylation. It has been shown that plant viruses have the capability to modify the methylation profile of the host genome, although with scarce loci-specific methylation. However a high-resolution quantitative analysis of DNA methylation changes, a technology now available for genome-wide investigations (also known as BS-Seq) is still missing in the case of plant-virus interactions. With the aim of investigating how epigenetic mechanisms contribute to the onset and progression of diseases, we used BS-Seq to analyse methylation processes on the host genome induced by a RNA virus (Cucumber mosaic virus, CMV) and a DNA virus (Cauliflower mosaic virus, CaMV). First analysis of the BS-seq data demonstrates that infection of each virus modifies the methylation landscape of all 5 chromosomes. Our data strongly indicate that the mechanism induced by RNA- and DNA-virus infection must be different. Interestingly, the RNA-virus infection mainly induces de-methylation whereas CaMV infection triggers a general hyper-methylation. Investigations on the genomic localization of these modifications result in a similar list of regions affected by the different viruses. On the other side, analysis of the differentially methylated sites according to annotated genes reveals significantly more differentially methylated genes in CMV-than in CaMV-infected plants. Furthermore, gene feature analysis shows prevalent promoter regions de-methylation upon infection of both viruses. However, we find many hypermethylated coding regions in CMV infected plants versus hardly differentially methylated coding regions in DNA-virus infected Arabidopsis.Finally, perhaps the most intriguing concluding remark of this preliminary analysis is that despite we find differentially methylated regions within gene features, such as transcripts, transposon and promoter, the large majority of methylation modifications are located in intergenic (not yet annotated) regions and might cover and regulate still unknown non-coding RNA products.

The establishment and maintenance of DNA methylation are relatively well understood whereas little is known about their dynamics and biological relevance in innate immunity [1-2]. In plants, modulation of DNA methylation might be an effective mechanism to regulate gene expression in response to abiotic and biotic stresses. Recent evidences from large-scale epigenomic approaches indicate that dynamic DNA methylation changes are not limited to gene imprinting but can regulate the plant's immune system in response to pathogens.In plants, virus infections trigger the expression of non-coding small RNAs (smRNAs) by also influencing the epigenetic status of the host genome; however, the involvement of DNA methylation in regulation of plant immune system in response to virus infection has not been so far investigated. In this context, we are carrying out a study aiming to elucidate the impact of DNA and RNA virus infections on genomic DNA methylation in plants, and their correlation with also the expression of smallRNA, by integrating the analysis of multiple "omics" datasets obtained by using next-generation sequencing technologies.In this paper we present the results of the analysis on the methylation modifications induced by the viruses infection on the whole genome and on coding and non-coding gene regions.

The world that gave rise to EMBnet 22 years ago has changed; EMBnet has changed too. Initially focusing on shared European community needs, the organisation now embraces partners from around the world, whose aims, aspirations and communities are very different. Over time, its centre of gravity has shifted: no longer needed as a mechanism for distributing the EMBL databases, EMBnet has moved on. In the past, its activities have been funded by membership fees and a series of European grants; now, however, against a background of emerging (government-funded) European infrastructural initiatives, EMBnet’s funding mechanisms need to be reviewed, in order to continue to sustain its core activities.EMBnet is at a cross-roads. Before taking its next steps, it is appropriate to consider how the global bioinformatics landscape is evolving, and how EMBnet needs to adapt. This paper outlines a number of practical steps that could be taken, tempered by today’s funding climate. Its principal recommendations are that EMBnet should:i) review and properly define the roles, aims and goals of its Executive Board (EB) and Project Committees (PCs), and consider establishing additional PCs or Special Interest Groups (SIGs), with well-defined roles, aims and goals;ii) review how EMBnet and its collection of PCs/SIGs might achieve its goals, with or without further funding;iii) identify and exploit its Unique Selling Point (USP);iv) review and better understand who its communities are, what their needs are, and how to be more responsive to those needs;v) review, streamline and clarify its current membership scheme;vi) review how and why it might interact with other networks and organisations;vii) establish internal infrastructures that would allow it to make strategic ties to other bioinformatics networks and organisations;viii) establish internal infrastructures that would allow it to make more strategic responses to global funding opportunities;ix) review the evolving role and internal structure of EMBnet.Journal, and consider more tactical publishing strategies; and, in light of these considerations,x) review and revamp its current name, brand and Website.This paper is an open invitation for every member of the constituency to help with this critical evaluation of EMBnet’s unique attributes and strengths; to consider how to build on these to create a competent, valuable and focused organisation that complements existing and emerging bioinformatics institutes, networks, associations and societies worldwide; ultimately, to maintain EMBnet’s relevance in 2010 and beyond.

Sequencing has seen major breakthroughs in recent years and has paved the way for developing novel life-science applications for the life sciences. Consequently, the life sciences are facing a rapidly increasing demand for data handling capacity. The workshop "ICT needs and challenges for Big Data in the Life Sciences" invited experts from different areas of the life sciences, bioinformatics and systems biology across Europe to discuss the topic. The aim was to identify and describe current and future ICT needs from sample analysis to model generation.

The CNR Institute for Biomedical Technologies (ITB) in Bari (IT), with support from the Italian Flagship project InterOmics, organised a Tutorial-Day as a satellite event of the BiP-Day 2013 workshop (see related article in the present volume). The tutorial was organised in three 3-hour events, covering metagenomics, phylogenetics and data analysis of non-coding RNA. The event took place on 6 December 2013 at the Department of Physics Michelangelo Merlin of the University of Bari and INFN.

The goal of the present paper is to establish and validate the link between cancer diagnosis and therapy by microRNAs detection. The induction in vitro of some specific microRNAs after treatment with MDR ligands has been outlined. Starting from the results obtained by in vitro induction of MDCK and MDCK-MDR1 cells treated by a MDR1 ligand, a new scenario in the early diagnosis and chemotherapy could be disclosed. To corroborate this perspective a short overview on pancreatic cancer diagnosis and chemotherapeutic treatment has been reported. © 2014 by the authors.

High-throughput technologies (HT), such as microarray and especially Next-Generation Sequencing (NGS) technologies, have provided tremendous potential for profiling protein-coding and non- protein coding RNAs (ncRNAs). Recent reports of the ENCODE project underline that while 80% of the human genome is transcribed, only 2% is protein coding, suggesting that the vast majority of the genome is transcribed as non-protein-coding RNA.We present the development of a web-based bioinformatics platform, nc-aReNA, for the mapping, classification and annotation of human and mouse ncRNAs from HT-NGS data. The platform is based on a data-warehouse approach and workflow environment that includes data quality control, genome and nc-RNAome sequence alignment, differential expression profiling analysis and statistics of classified data.MethodsThe nc-aReNA architecture is based on a modular analysis pipeline, flanked by a data-warehouse, for the classification and annotation of small-RNAseqdata. The pipeline takes in input the sequenced reads in FASTQ format. After the initial steps of adaptor removal and quality check, the input reads are mapped to an in-house non-redundant ncRNA reference database (http://ncRNAdb.ba.itb.cnr.it) which collects and integrates ncRNA gene lists, from MGI (Mouse Genome Informatics) and HGNC (Human Genome Nomenclature Committee), with sequences and biotype annotations from VEGA (Vertebrate Genome Annotation), ENSEMBL, RefSeq, RFam (for tRNA sequence) and miRBase (for miRNA). NGS reads mapped in this step are classified by using Sequence Ontology (SO) (Eilbeck K. et al., 2005). Unmapped reads are aligned to the reference genome and tagged to the corresponding genomic locus.Integrated statistics are used for RPM (Reads Per Million), fold changes and False Discovery Rate (FDR) corrected p-values calculation and differential expression analysis of all (or user-chosen) ncRNA classes, by comparing two or more experimental conditions or time-courses data.An additional module, called "miRNA identification", provides the analysis of all unmapped miRNA-like reads by mean of the miRDeep2 software.All the analysis results and annotation are stored in a data-warehouse implemented with Infobright (http://www.infobright.org). A user-friendly web-based Graphical User Interface (GUI), developed by using the JAVA platform, guides the user in the submission process and displays results in tables and graphs.ResultsThe main features of the nc-aReNA are:- identification and classification of reads in known functional ncRNA categories in SO;- identification and filtering of reads mapping to ribosomal RNAs and mtDNA transcripts;- RPMs calculation for each known ncRNA;- the export of user-selected classesof ncRNA for further specific investigation;- quantification of ncRNAs expression and differential expression analysis for all identified ncRNAclasses;- graphical visualization of sample expression profiles;- additional annot

MOTIVATION:The recent availability of next generation sequencing (NGS) technologies, has provided the scientific community with an unprecedented opportunity for large-scale analysis of genome in a large number of organisms. One of the most challenging task for bioinformaticians is to develop tools that provide biologists with an easy access to curated and non-redundant collections of sequence data.Non-coding RNAs, for a long time believed to be not-functional, are emerging as the most large and important family of gene regulators.METHODS:NonCode aReNA DataBase is a comprehensive and non-redundant source of manually curated and automatically annotated ncRNA transcripts collected from major public resources.The database is built through a set of ETL (Extraction Transformation Loading) automated processes which extracts and collects data from VEGA, ENSEMBL, RefSeq, miRBase, GtRNAdb and piRNABank. The automatic process guarantees also recurring updates.The identification of redundant sequences is made by analyzing both cross-link references and sequence similarity. Furthermore non-coding RNA sequences have been classified in diverse biotypes and associated to Sequence Ontology terms.NonCode aReNA DataBase is originally developed as a component of a bigger project, represented by a datawarehouse and an analysis workflow, for the functional annotation of ncRNAs from NGS data.RESULTS:NonCode aReNA Database is currently available as a web-resource at http://ncrnadb.ba.itb.cnr.it/. The database can be queried by using multi-criteria and ontological search, through an easy-to-use web interface. Query results can be exported as non-redundant collections of ncRNA transcripts.Currently NonCode aReNA DataBase contains 134,908 human ncRNAs classified in 24 biotypes, and next updates will include transcripts of Mus musculus and Arabidopsis thaliana

The establishment and maintenance of DNA methylation are relatively well understood whereas little is known about their dynamics and biological relevance in innate immunity. In plants, modulation of DNA methylation might be an effective mechanism to regulate gene expression in response to abiotic and biotic stresses. Recent evidence through large-scale epigenomicapproaches indicate that dynamic DNA methylation changes are not limited to gene imprinting but can regulate the plant's immune system in response to pathogens. In plants, virus infections trigger expression and regulation of non-coding smallRNAs, and genomic regions are epigenetically modified through the action of the same molecules; however, the involvement of DNA methylation in regulation of plant immune system in response to virus infection was not investigated before. We have examined for the first time the impact of virus infections on genomic DNA methylation and the correlation with smallRNA regulation and gene expression by integrating together analysis of multiple "omics" datasets based on next-generation sequencing platforms. To investigate the possibility that DNA methylation dynamically responds to virus infection, we performed whole-genome bisulfite sequencing on Arabidopsis leaves systemically infected with either the DNA genome virus Cauliflower mosaic virus (CaMV-Arabidopsis) or the RNA virus Cucumber mosaic virus (CMV-Arabidopsis). Single-base resolution methylome analysis revealed more than 3.7million methyl-cytosines (mCs) for the control plant. Interestingly in CMV Arabidopsis we found 300.000 more mCs (hypermethylated) and in CaMV-Arabidopsis 700.000 mCs less (hypomethylated). Focusing on differentially methylated regions (DMR, 250nt in length) we observed a balanced distribution of hyper- and hypomethylation in CG and CHH context in CMV-Arabidopsis (total DMRs 2700) but in CaMV-Arabidopsis we have predominantly hypomethylated DMRs in CHH context (total DMRs 5600). Gene features including coding, non-coding and promoter sequences were assigned to unique gene identifiers according to the TAIR nomenclature. Among differentially methylated gene features, promoter regions were the vast majority, accounting, in specific mCs contexts, for up to 80% of the total. The whole gene ID dataset was subjected to gene functional enrichment analysis by using the DAVID package tool. Interestingly, definite functional categories such as "plant defense" and "auxin signalling pathway" resulted significantly enriched. The correlation between the DNA methylation status and the transcriptional modulation of those genes is under investigation. A comparison between methylation profiles induced by either CaMV or CMV infections revealed conspicuous qualitative and quantitative differences. Taken together our results indicate that RNA- and DNA-genome virus infection induce different regulation of DNA methylation and, at least in part, different immune response in Arabidopsis.

More than 20 years after its inception as an organisation for disseminating data, knowledge and services to support molecular biology/biotechnology research across Europe, EMBnet has arrived at a cross-roads. Since 1988, its membership has expanded outside Europe, and the nature of many of the needs it was created to support has changed. Against this background, major (government-supported) initiatives have begun to emerge to try to satisfy the modern European data infrastructure need. The ‘EMBnet moving forward’ workshop was an opportunity to review the current context and how EMBnet should embrace the future, to the benefit of its global membership. The following pages summarise the workshop discussions and conclusions.

The 2011 AGM workshop took place at the Instituto Gulbenkian de Ciência (IGC) in Oeiras, Portugal, from 23-25 May (Figure 1). The goal of the workshop was to build on the demonstrable progress made during the previous year, in particular by helping to deliver on some of the plans outlined during the 2010 AGM. It was also an opportunity to build on our commitment to take EMBnet forward by embracing new partners and new activities. The following pages summarise the workshop content, discussions and conclusions.

The detection of viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs (siRNAs, 21 to 24 nucleotides [nt]) in plants infected by nuclear-replicating members of the family Pospiviroidae (type species, Potato spindle tuber viroid [PSTVd]) indicates that they are inducers and targets of the RNA-silencing machinery of their hosts. RNA-dependent RNA polymerase 6 (RDR6) catalyzes an amplification circuit producing the double-stranded precursors of secondary siRNAs. Recently, the role of RDR6 in restricting systemic spread of certain RNA viruses and precluding their invasion of the apical growing tip has been documented using RDR6-silenced Nicotiana benthamiana (NbRDR6i) plants. Here we show that RDR6 is also engaged in regulating PSTVd levels: accumulation of PSTVd genomic RNA was increased in NbRDR6i plants with respect to the wild-type controls (Nbwt) early in infection, whereas this difference decreased or disappeared in later infection stages. Moreover, in situ hybridization revealed that RDR6 is involved in restricting PSTVd access in floral and vegetative meristems, thus providing firm genetic evidence for an antiviroid RNA silencing mechanism. RNA gel blot hybridization and deep sequencing showed in wt and RDR6i backgrounds that PSTVd sRNAs (i) accumulate to levels paralleling their genomic RNA, (ii) display similar patterns with prevailing 22- or 21-nt plus-strand species, and (iii) adopt strand-specific hot spot profiles along the genomic RNA. Therefore, the surveillance mechanism restraining entry of some RNA viruses into meristems likely also controls PSTVd access in N. benthamiana. Unexpectedly, deep sequencing also disclosed in NbRDR6i plants a profile of RDR6-derived siRNA dominated by 21-nt plus-strand species mapping within a narrow window of the hairpin RNA stem expressed transgenically for silencing RDR6, indicating that minus-strand siRNAs silencing the NbRDR6 mRNA represent a minor fraction of the total siRNA population.

How viroids, tiny non-protein-coding RNAs (~250-400 nt), incite disease is unclear. One hypothesis is that viroid-derived small RNAs (vd-sRNAs; 21-24 nt) resulting from the host defensive response, via RNA silencing, may target for cleavage cell mRNAs and trigger a signal cascade, eventually leading to symptoms. Peach latent mosaic viroid (PLMVd), a chloroplast-replicating viroid, is particularly appropriate to tackle this question because it induces an albinism (peach calico, PC) strictly associated with variants containing a specific 12-14-nt hairpin insertion. By dissecting albino and green leaf sectors of Prunus persica (peach) seedlings inoculated with PLMVd natural and artificial variants, and cloning their progeny, we have established that the hairpin insertion sequence is involved in PC. Furthermore, using deep sequencing, semi-quantitative RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends (RACE), we have determined that two PLMVd-sRNAs containing the PC-associated insertion (PC-sRNA8a and PC-sRNA8b) target for cleavage the mRNA encoding the chloroplastic heat-shock protein 90 (cHSP90), thus implicating RNA silencing in the modulation of host gene expression by a viroid. Chloroplast malformations previously reported in PC-expressing tissues are consistent with the downregulation of cHSP90, which participates in chloroplast biogenesis and plastid-to-nucleus signal transduction in Arabidopsis. Besides PC-sRNA8a and PC-sRNA8b, both deriving from the less-abundant PLMVd (-) strand, we have identified other PLMVd-sRNAs potentially targeting peach mRNAs. These results also suggest that sRNAs derived from other PLMVd regions may downregulate additional peach genes, ultimately resulting in other symptoms or in a more favorable host environment for viroid infection.

Biodiversity research concerns with data coming from many different domains (e.g., Biology, Geography, Evolutionary Studies, Genomics, Taxonomy, Environmental Sciences, etc.) which need to be integrated for leading to valuable Biodiversity knowledge. Collecting and integrating data from so many heterogeneous resources is not a trivial task. Data are extremely scattered, heterogeneous in format and purpose, and protected in repositories of several research institutes. Driven by the widely diffused trend of the web of sharing information through aggregation of people with the same interests (social networks), and by the new type of database architecture defined as dynamic distributed federated database, we are proposing a new paradigm of data integration in the Biodiversity domain. Here we present a new approach for the development of a Knowledge Base aiming to the collection, integration and analysis of biodiversity data implemented as a product of the MBLab project.

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.

The recent availability of high throughput tech- nologies, like next generation sequencing (NGS) platforms, has providedthescientific community with an unprecedented opportunity for large- scale analysis of genome in a large number of organisms.However,among others, one of the most challenging task for bioinformaticians is to developtools that providebiologists withaneasy access to curated and non-redundant collec- tions of sequence data.Non-coding RNAs, for a long time believed tobe not-functional, are emerging as themost large and important family of gene regulators. NonCode aReNA Database is a comprehensive and non-redundant source ofmanually curated and automatically annotated ncRNA transcripts. Originally developed as a component of a big- ger project, composed by a datawarehouse for the functional annotation of ncRNAs fromNGS data, NonCode aReNA DB is currently availableas a web-resource at http://ncrnadb.ba.itb.cnr. it/. Sequences have been classified in diverse biotypes and associated to SequenceOntology terms. The database can be queried by using multi-criteria and ontological search, through an easy-to-use web interface, and data exported as non-redundant collections of transcripts an- notated in VEGA, ENSEMBL, RefSeq, miRBase, GtRNAdb and piRNABank. The database is up- dated through an automatic pipeline and last updatewasonJanuary 2015. PresentlyNonCode aReNA DB contains 134,908 human ncRNAs clas- sified in 24 biotypes, and next update will include transcripts ofMusmusculus and Arabidopsis thal- iana.AcknowledgementsThis work was supported by the Italian MIUR Flagship Project "Epigen".

Despite being composed by a single-stranded, circular, non-protein-coding RNA of just 246-401 nucleotides (nt), viroids can incite in their host plants symptoms similar to those caused by DNA and RNA viruses, which have genomes at least 20-fold bigger and encode proteins. On the other hand, certain non-protein-coding plant satellite RNAs display structural similarities with viroids but for replication and transmission they need to parasitize specific helper viruses (modifying concomitantly the symptoms they induce). While phenotypic alterations accompanying infection by viruses may partly result from expressing the proteins they code for, how the non-protein-coding viroids (and satellite RNAs) cause disease remains a conundrum. Initial ideas on viroid pathogenesis focused on a direct interaction of the genomic RNA with host proteins resulting in their malfunction. With the advent of RNA silencing, it was alternatively proposed that symptoms could be produced by viroid-derived small RNAs (vd-sRNAs) -generated by the host defensive machinery- targeting specific host mRNA or DNA sequences for post-transcriptional or transcriptional gene silencing, respectively, a hypothesis that could also explain pathogenesis of non-protein-coding satellite RNAs. Evidence sustaining this view has been circumstantial, but recent data provide support for it in two cases: i) the yellow symptoms associated with a specific satellite RNA result from a 22-nt small RNA (derived from the 24-nt fragment of the satellite genome harboring the pathogenic determinant), which is complementary to a segment of the mRNA of the chlorophyll biosynthetic gene CHLI and targets it for cleavage by the RNA silencing machinery, and ii) two 21-nt vd-sRNAS containing the pathogenic determinant of the albino phenotype induced by a chloroplast-replicating viroid target for cleavage the mRNA coding for the chloroplastic heat-shock protein 90 via RNA silencing too. This evidence, which is compelling for the satellite RNA, does not exclude alternative mechanisms.