Natalie Wilson

RNA silencing: Probing the silence

Reporting in The Plant Cell, Olivier Voinnet and colleagues show that viral silencing suppressors can be used to help unravel the complexity of RNA silencing. In plants, RNA silencing is involved in the anti-viral adaptive immune response through posttranscriptional gene silencing (PTGS; known as RNA interference (RNAi) in animals). PTGS involves endonucleolytic cleavage of RNAs guided by sequence-specific small interfering RNAs (siRNAs). RNA silencing is also linked to development, in which sequence-specific microRNAs (miRNAs) either promote endonucleolytic cleavage of RNAs or inhibit the translation of target RNAs. The similarities between the siRNA and miRNA pathways led the authors to investigate RNA silencing using the suppressor proteins that viruses use to counteract PTGS. Although viral silencing suppressors have been studied before, their mode and point of action in the PTGS pathway have remained unknown. The authors devised an experimental system in Arabidopsis thaliana to allow the comparative side-by-side analysis of five distinct suppressors. They expressed the suppressors transgenically in the same Arabidopsis ecotype, in which PTGS of the endogenous chalcone synthase (CHS) transcript was activated. All five suppressors inhibited PTGS of the CHS transcript, but only three altered miRNA accumulation or miRNA-guided functions. This indicates that, as in animals, the siRNA and miRNA pathways overlap only partially. The three suppressors that altered the miRNA pathway are diverse, so the authors propose that their induction of similar developmental abnormalities might be secondary to the inhibition of the siRNA pathway. The authors also found that other Arabidopsis small silencing RNAs were resistant to all five silencing suppressors, which indicates that these RNAs have distinct biosynthetic and functional pathways. Furthermore, miRNAs were shown to fall into 2 size classes (as per siRNAs), and the authors suggest that 21-nucleotide miRNAs are incorporated into the RNA-induced silencing complex (RISC), whereas 24-nucleotide miRNAs might direct transcriptional silencing events. Using this system, the authors showed that not all suppressors are equal — one partially reduces dsRNA processing by Dicer and prevents mRNA degradation, perhaps by inhibiting RISC activity, others might act downstream of Dicer, as they have no effect on dsRNA processing, and another functions by sequestering both siRNAs and miRNAs. Because one suppressor was also found to suppress RNAi in a human cell line, it is hoped that these results will ultimately help to decipher the mechanisms that underlie RNAi in animals, as well as in plants.

Technology: A robot scientist

Peer review — the big secret? Three quarters of the British public have no idea what peer review is, according to a new poll that was commissioned by the Science Media Centre and Nature. The poll, conducted by the MORI Social Research Institute, involved interviewing more than a 1,000 adults aged 15 and over. The results were startling or unsurprising, depending on your point of view — only a quarter of those interviewed described peer review as “society’s scrutiny of other scientists’ work, generally” (BBC Radio 4, Today programme). Intriguingly, however, the survey also showed that the public supports rigorous scrutiny of scientific results before publication, and if peer review did not exist already they would want to create it. “The vast majority (71%) of the public favour either the kind of scrutiny provided by peer review or more stringent controls in which experiments are repeated independently before being published” (The Guardian). Fiona Fox, director of the Science Media Centre, encouraged the scientists to “get out there and share their big secret” of peer review. These findings are of course timely — they were published only a few days after the “IVF specialist Dr Panos Zavos announced to the press that he had cloned a baby” having “refused to submit his experiment to peer review” (The Guardian). So, the poll’s results seem to say that it is not only the scientists who are frustrated with this kind of science reporting, but that the public is weary as well. There is a constructive outcome to this survey — the Science Media Centre has published a new guide for scientists “in an effort to help them better communicate their work” (The Guardian). Magdalena Skipper IN THE NEWS

Development: A new twist to bone formation

Human Ageing Genomics Resources • http://genomics. senescence.info/index.html If you are working on or are interested in human ageing, you should know about the Human Ageing Genomics Resources (HAGR): a web site that brings together databases and computational tools to help understand the biology of ageing. The resource started in 2002 as a collaborative project at the University of Namur, Belgium. A searchable and browsable database of genes that are related to human ageing, GenAge, forms the core of the resource. Each gene page contains nomenclature, cytogenetic and protein information. Among other useful features are links to relevant publications and a list of orthologues with links to NCBI Entrez. There are also further external links to OMIM, Swiss-Prot and GeneCard, to name but a few. Another database, AnAge, caters for those who work on ageing in other species. It has information on ageing in Drosophila melanogaster, Caenorhabditis elegans, red and purple sea urchin, and 2,469 chordate species! But databases are not all the HAGR has to offer: there is also the Ageing Research Computational Tool — ARCT. This Perl-based toolkit allows you to generate phylogenetic profiles locally or through NCBI’s BLAST, to data-mine multiple sequences to find regulatory or functionally important regions, to display protein–protein interactions and phylogenetic trees, and to access other data-analysis programs such as ClustalW and Gibbs. The project team is busy making continuous improvements to HAGR. For example, the inclusion of gene-expression information is on the cards. The team plan to include genes that are differently expressed between young and old tissues, focusing mainly on microarray data. Magdalena Skipper WEB WATCH

Shining a new light on genetic variation

microemulsions are prepared for PCR. The water phase contains all of the reagents that are needed for PCR plus the oligonucleotide-bound beads and template DNA. Third, PCR cycling of the microemulsions is carried out. Each microemulsion contains an average of less than one bead and less than one template, but if the template and bead are present in the same microemulsion then amplification occurs. Fourth, the microemulsions are ‘broken’ by the addition of non-ionic detergent and the beads are captured with a magnet. Fifth, fluoresceinconjugated or biotin-conjugated oligonucleotides, which can detect sequence variations among the templates, are hybridized to the templates, and, again, the beads are magnetically captured. The beads are then incubated with fluorescently labelled antibodies that label the bound hybridization probes — after laser excitation the beads that contain a PCR product appear as red or green. Sixth, flow cytometry, using standard two-colour analysis, is used to count the two different populations of DNA molecules. As well as being sensitive, reliable and accessible, BEAMing has various other benefits: by analysing more beads its sensitivity can be increased; the fraction of variant sequences can be quantified; variant sequences can be purified after flow cytometry and analysed further; and it also has the potential to be automated. Although the authors chose to focus on the application of BEAMing to sequence variation, they say that “it could also be used to quantify epigenetic alterations such as methylation”. The list of potential applications goes on. And so it seems that this technique really does promise to shine a new light on genetic variation. Natalie Wilson