Research
Higher eukaryotes have developed a mechanism of sequence-specific RNA degradation known as ‘RNA silencing', an idiom combining the terms ‘posttranscriptional gene silencing (PTGS)' and ‘RNA interference (RNAi)'. Despite common features of RNA silencing, there are differences between the animal and plant kingdoms and also amongst species (for review see e.g . [1-6]. The central path of the RNA degradation pathway is the generation of short interfering RNA (siRNA) from a double-stranded RNA by a double-strand-specific RNase, called Dicer. The siRNAs are incorporated into the RNA-induced silencing complex (RISC) and after strand separation, the remaining single-stranded RNA strand guides the sequence-specific cleavage of a target RNA.

The mechanism of RNA silencing in plants is more complex than in most animals. Different size classes of siRNAs ranging from 21 to 24 nt can be found [7] , as well as different forms of Dicer [8]. Further, plant RNA silencing is non-cell-autonomous. It can spread from an initially silenced cell to a neighbouring cell and silencing can spread over a long distance to different parts of the plant [9-11].

Another class of small RNA molecules named microRNAs (miRNAs) were identified as negative modulators of gene expression. These short (21-24nt) RNA molecules are processed from longer precursors transcribed from endogenous sequences. Although they have been found both in plants and animals- but not in fungi- there is mounting evidence that the mode of action of miRNAs in the two kingdoms is somewhat different. Unlike animal miRNAs, plant miRNAs are usually encoded in intergenic regions and down-regulate the expression of the gene mainly by guiding an Argonaute protein complex in slicing a highly complementary mRNA-target molecule. Plants also express trans-acting siRNAs (tasiRNAs), small RNAs that share many characteristics with siRNAs but require the activity of specific miRNAs for their biogenesis.

Recently in humans it has been shown that miRNAs encompass novel regulatory subunits present within cancer associated genomic regions (CAGRs). Genomic studies have shown that various regions of the human genome are often associated with a cancerous phenotype. These regions (denoted CAGRs) are often hotspots for genetic perturbations such as deletions. In most of these cases the actual regulators present in CAGRs, which govern the molecular mechanisms leading to a tumourigenic phenotype, are scarcely known. Frequently, known tumour suppressors or oncogenes are absent from these regions, strengthening the hypothesis that the primary regulators in specific CAGRs take the form of non-coding microRNA molecules.

Other small RNA species such as endogenous-siRNAs (endo-siRNAs) in plants, repeat-associated-siRNAs (rasiRNAs) in S. pombe and piRNAs in mammals and flies, are involved in phenomena related to RNA-guided DNA and/or chromatin methylation phenomena.

Further information on RNA silencing phenomena can be found on the literature cited above but also in open source sites such as the ones given below.

http://en.wikipedia.org/wiki/RNA_interference

http://en.wikipedia.org/wiki/Small_interfering_RNA

Research Interests
•  Sense-Post Transcriptional Gene Silencing (S-PTGS): The mechanism by which the expression of a gene in a plant cell leads to the activation of RNA silencing against its own sequences.
•  Stability of Transgene expression
•  Endogenous and exogenous factors affecting S-PTGS
•  The systemic signal of PTGS
•  The cross-talk between RNA silencing and specific pathogens (viruses, viroids and bacteria).
•  Virus resistance
•  The cross-talk between chloroplasts and the RNA silencing mechanism
•  Elucidating the molecular and regulatory mechanisms underlying novel human miRNA genes present in CAGRs.

Major contributions
Our group was one of the first to report a number of important features of RNA silencing such as the susceptibility of viroids to the silencing mechanism [12], the conservation of the RNA silencing mechanism between plants and nematods [13], the effects of temperature on the generation of siRNAs in plants [14] , and the effects of light on S-PTGS [15]. The group has also made important contributions in differentiating between short-range [16] ) and long-range silencing and in characterising some of the rules defining the systemic movement of the silencing signal [17]. The group was also the first to show the regulation of c-myc by the miR let-7 in mammalian cells [16].

Moreover our laboratory recently collaborated with the Computational Biology lab at IMBB which lead to the publication of a combined computational as well as experimental approach for predicting and verifying novel miRNA gene candidates within CAGRs [18].

Bibliography
1. Vance, V. and H. Vaucheret, RNA silencing in plants--defense and counterdefense. Science, 2001. 292 (5525): p. 2277-80.
2. Voinnet, O., RNA silencing: small RNAs as ubiquitous regulators of gene expression. Curr Opin Plant Biol, 2002. 5 (5): p. 444-51.
3. Sontheimer, E.J., Assembly and function of RNA silencing complexes. Nat Rev Mol Cell Biol, 2005. 14 : p. 14.
4. Baulcombe, D., RNA silencing in plants. Nature, 2004. 431 (7006): p. 356-63.
5. Meister, G. and T. Tuschl, Mechanisms of gene silencing by double-stranded RNA. Nature, 2004. 431 (7006): p. 343-9.
6. Hutvagner, G., et al., Sequence-specific inhibition of small RNA function. PLoS Biol, 2004. 2 (4): p. E98.
7. Hamilton, A., et al., Two classes of short interfering RNA in RNA silencing. Embo J, 2002. 21 (17): p. 4671-9.
8. Schauer, S.E., et al., DICER-LIKE1: blind men and elephants in Arabidopsis development. Trends Plant Sci, 2002. 7 (11): p. 487-91.
9. Winston, W.M., C. Molodowitch, and C.P. Hunter, Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science, 2002. 295 (5564): p. 2456-9.
10. Palauqui, J.C., et al., Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. Embo J, 1997. 16 (15): p. 4738-45.
11. Voinnet, O. and D.C. Baulcombe, Systemic signalling in gene silencing. Nature, 1997. 389 (6651): p. 553.
12. Papaefthimiou, I., et al., Replicating potato spindle tuber viroid RNA is accompanied by short RNA fragments that are characteristic of post-transcriptional gene silencing. Nucleic Acids Res, 2001. 29 (11): p. 2395-2400.
13. Boutla, A., et al., Induction of RNA interference in Caenorhabditis elegans by RNAs derived from plants exhibiting post-transcriptional gene silencing. Nucleic Acids Res, 2002. 30 (7): p. 1688-94.
14. Kalantidis, K., et al., The occurrence of CMV-specific short Rnas in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus. Mol Plant Microbe Interact, 2002. 15 (8): p. 826-33.
15. Kotakis, C., et al., Light intensity affects RNA silencing of a transgene in Nicotiana benthamiana plants. BMC Plant Biol. 10 : p. 220.
16. Koscianska, E., et al., Prediction and preliminary validation of oncogene regulation by miRNAs. BMC Mol Biol, 2007. 8 : p. 79.
17. Tournier, B., M. Tabler, and K. Kalantidis, Phloem flow strongly influences the systemic spread of silencing in GFP Nicotiana benthamiana plants. Plant J, 2006. 47 (3): p. 383-94.
18. Oulas, A., et al., Prediction of novel microRNA genes in cancer-associated genomic regions--a combined computational and experimental approach. Nucleic Acids Res, 2009. 37 (10): p. 3276-87.

Current Research
The groups' main focus is on RNA silencing in plants. However, RNA silencing phenomena in other model organisms interest us as well. We have been working on the plant homologue of ERI1, a 3' exonuclease shown to be involved in the regulation of RNA silencing in animals. In parallel, we have been also studying the Drosophila ERI1-homologue known as Snipper whose function in vivo is still not know.

Recently we have shown that HL increases spontaneous silencing of a Transgene in Nicotiana and we are also currently studying the mechanism through which this effect is mediated.

Further, we are interested on how are viroids, which are subviral RNA pathogens not known to encode for any proteins, affected by the hosts RNA silencing machinery.

The group is also pursuing research in order to elucidate the molecular mechanisms that are distorted as a result of the deletion of recently identified miRNAs within CAGRs and furthermore provide a link to tumourigenic processes.

Support
During the last five years the laboratory has been successful in a number of EU and National grant applications. As a result It has been involved in two EU financed activities: The team is coordinating the EU STREP Project (FOSRAK) “Functional analysis of small regulatory RNAs across kingdoms” (www.fosrak.org).In addition the team has been a partner site within a Marie Curie training program of the EU the Early Stage Training site FAMED, “Functional analysis of miRNA during early development” (MEST-CT-2004-007295). In addition the laboratory is currently coordinating a national “Cooperation” grant on Criniviruses and is a partner in a second “Cooperation” grant, both financed through the “General Secretariat for Research and Technology”. In addition, the team is involved in a collaborative project with the group of Pascale Romby at CNRS in Strassburg (Bilatelar collaborative project Greece-France). Finally the group is a host for two “Heraklitos” PhD scholarships.