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A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5'-Phosphate-Dependent Exonuclease to study the 5' end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5' monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5' end of short RNAs or after replacing any potential 5' ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase.

Background: Viroids 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 Findings: To 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/significance: These 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.

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.

In plants there are several classes of 21-24-nt short RNAs that regulate gene expression. The most conserved class is the microRNAs (miRNAs), although some miRNAs are found only in specific species. We used high-throughput pyrosequencing to identify conserved and nonconserved miRNAs and other short RNAs in tomato fruit and leaf. Several conserved miRNAs showed tissue-specific expression, which, combined with target gene validation results, suggests that miRNAs may play a role in fleshy fruit development. We also identified four new nonconserved miRNAs. One of the validated targets of a novel miRNA is a member of the CTR family involved in fruit ripening. However, 62 predicted targets showing near perfect complementarity to potential new miRNAs did not validate experimentally. This suggests that target prediction of plant short RNAs could have a high false-positive rate and must therefore be validated experimentally. We also found short RNAs from a Solanaceae-specific foldback transposon, which showed a miRNA/miRNA*-like distribution, suggesting that this element may function as a miRNA gene progenitor. The other Solanaceae-specific class of short RNA was derived from an endogenous pararetrovirus sequence inserted into the tomato chromosomes. This study opens a new avenue in the field of fleshy fruit biology by raising the possibility that fruit development and ripening may be under miRNA regulation.

Unlabelled: 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.

Background: Experimental identification of microRNA (miRNA) targets is a difficult and time consuming process. As a consequence several computational prediction methods have been devised in order to predict targets for follow up experimental validation. Current computational target prediction methods use only the miRNA sequence as input. With an increasing number of experimentally validated targets becoming available, utilising this additional information in the search for further targets may help to improve the specificity of computational methods for target site prediction., Results: We introduce a generic target prediction method, the Stacking Binding Matrix (SBM) that uses both information about the miRNA as well as experimentally validated target sequences in the search for candidate target sequences. We demonstrate the utility of our method by applying it to both animal and plant data sets and compare it with miRanda, a commonly used target prediction method., Conclusion: We show that SBM can be applied to target prediction in both plants and animals and performs well in terms of sensitivity and specificity. Open source code implementing the SBM method, together with documentation and examples are freely available for download from the address in the Availability and Requirements section.

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.

Pfam is a database of protein families that currently contains 7973 entries (release 18.0). A recent development in Pfam has enabled the grouping of related families into clans. Pfam clans are described in detail, together with the new associated web pages. Improvements to the range of Pfam web tools and the first set of Pfam web services that allow programmatic access to the database and associated tools are also presented. Pfam is available on the web in the UK (http://www.sanger.ac.uk/Software/Pfam/), the USA (http://pfam.wustl.edu/), France (http://pfam.jouy.inra.fr/) and Sweden (http://pfam.cgb.ki.se/).

Rfam is a comprehensive collection of non-coding RNA (ncRNA) families, represented by multiple sequence alignments and profile stochastic context-free grammars. Rfam aims to facilitate the identification and classification of new members of known sequence families, and distributes annotation of ncRNAs in over 200 complete genome sequences. The data provide the first glimpses of conservation of multiple ncRNA families across a wide taxonomic range. A small number of large families are essential in all three kingdoms of life, with large numbers of smaller families specific to certain taxa. Recent improvements in the database are discussed, together with challenges for the future. Rfam is available on the Web at http://www.sanger.ac.uk/Software/Rfam/ and http://rfam.wustl.edu/.

A three-dimensional block model of the cochlear nucleus of the cat was constructed from histologic sections. Boundaries of various subdivisions, based on cytoarchitectonic criteria, were included in the model. Usage of the block model in correlating physiological and anatomical data is illustrated by localizing characteristic waveforms of gross evoked responses and characteristic frequencies of single units.

The past few years have witnessed a growing literature on the behavioural, cognitive and performance effects, both beneficial and hazardous, of caffeine ingestion. A brief overview of these studies is provided and methodological difficulties discussed. Disagreement over such factors as the quantity of caffeine required to produce deleterious effects, difficulties obtaining valid measures of caffeine intake from self-reports and differing interpretations of tolerance levels and absorption rates between studies, often prevent the derivation of meaningful results. The need for a rigorous, scientific appraisal of the effects of caffeine on psychological functioning is emphasised. This requires agreement about criteria referred to above, a more valid data base, and in some cases, the adoption of alternative methodology.