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    Research

    Background

    RNA silencing

    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].

    A 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.

    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 in 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

    Viroids
    Viroids are infectious, naked circular RNAs sized from 246 to 401nt, capable of infecting a wide range of hosts, causing important economic loses [1]. They are divided into two families, Pospiviroidae and Avsunviroidae [12–14]. Potato spindle tuber viroid (PSTVd), a type species of the Pospiviroidae family, has a rod-like secondary structure with five distinct domains, and replicates in the nucleus through an asymmetric rolling circle mechanism using RNA polymerase II (RNAPII) [12, 15–17]. It is known to infect crop plants of the Solanaceae family such as tomato and potato, as well as some ornamental plants of the Scrophulariaceae and Asteracae family, but does not infect the plant model Arabidopsis thaliana systemically.

    Since viroids do not encode any protein they rely on plant available resources and / or mechanisms for their infectivity. One of the mechanisms they have been proposed to exploit is RNA interference (RNAi), especially because of their particular double stranded RNA structures (dsRNA) [15,16].Further information on viroids can be found in the literature cited above but also in open source sites such as the ones given below.

    https://en.wikipedia.org/wiki/Viroid
     

    Research Interests

    • RNA Silencing Pathways: RNA-mediated Regulation and Defence mechanisms
    • Viroids
    • Stability of Transgene expression
    • The systemic signal of PTGS
    • Plant/RNA-Virus interactions
    • Virus resistance, Viral suppressors of RNA silencing
    • Diatoms (Marine unicellular photosynthesising organisms): RNA silencing and terpenoids
     

    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 [18], the conservation of the RNA silencing mechanism between plants and nematods [19], the effects of temperature on the generation of siRNAs in plants [20] , and the effects of light on S-PTGS [21].

    The group has also made important contributions in differentiating between short-range [22] ) and long-range silencing and in characterising some of the rules defining the systemic movement of the silencing signal [23]. Our group in collaboration with Dr. Hatzigeorgiou's lab, then at UPen, was one of the first to characterise miRNA promoters in plants [24].

    The group was also the first to show the regulation of c-myc by the miR let-7 in mammalian cells [25]. Moreover our laboratory in a collaboration 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 [26].

    Recently, we showed that the plant homologue of Enhancer of RNAi, ERL1, functions as a 3' exonuclease in the processing of rRNAs in the chloroplasts, and not in RNA silencing [27].

    Finally, we have shown that DCL4, the plant Dicer a key player in antiviral silencing pathways has a contrasting effect on viroid pathogenicity [28]. In addition we have shown that in in contrast, it is the combined activity of DCL2 and DCL3 that defend the plant from Pospoviroids [29].
     

    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. Flores R, Di Serio F, Navarro B, Duran-Vila N, Owens R. Viroids and viroid diseases of plants. Wiley-Blackwell. 2011; 307–341.
    13. Gago-Zachert S. Viroids, infectious long non-coding RNAs with autonomous replication. Virus Res.; 2015;15.
    14. Katsarou K, Rao ALN, Tsagris M, Kalantidis K. Infectious long non-coding RNAs. Biochimie. 2015;117: 37–47.
    15. Tabler M, Tsagris M. Viroids: petite RNA pathogens with distinguished talents. Trends Plant Sci. 2004;9: 339–348.
    16. Tsagris EM, et al. Viroids. Cell Microbiol. 2008;10: 2168–2179.
    17. Rao ALN, Kalantidis K. Virus-associated small satellite RNAs and viroids display similarities in their replication strategies. Virology. 2015;479–480: 627–636.
    18. 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.
    19. 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.
    20. 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.
    21. Kotakis, C., et al., Light intensity affects RNA silencing of a transgene in Nicotiana benthamiana plants. BMC Plant Biol. 10 : p. 220.
    23. Tournier, B, et al. Phloem flow strongly influences the systemic spread of silencing in GFP Nicotiana benthamiana plants. Plant J, 2006. 47 (3): p. 383-94.
    24. Megraw M, et al. MicroRNA Promoter Element Discovery in Arabidopsis. RNA, 2006, 12(9): 1612.
    25. Koscianska, E., et al., Prediction and preliminary validation of oncogene regulation by miRNAs. BMC Mol Biol, 2007. 8 : p. 79.
    26. 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.
    27. Mermigka G, Verret F., and Kalantidis K.*. 2016. RNA silencing movement in plants. Invited expert review, JIPB, 2016 Apr;58(4):328-42.
    28. Dadami E. et al. . 2013. RNA silencing pathways may have a positive effect on Potato spindle tuber viroid infectivity in Nicotiana benthamiana. Mol. Plant 6:232-234.
    29. Katsarou K, et al. Combined Activity of DCL2 and DCL3 is crucial in The Defense Against Potato Spindle Tuber Viroid.. PLoS Path., 2016, In Press.

     

    Current Research

    The groups' main focus is on viroid- virus/ plant interactions and RNA silencing pathways in plants. However, we are interested also in RNA silencing phenomena in diatoms and coccolithophores, marine unicellular photosynthetic organisms with a wide genetic reservoir. In the plant RNA silencing field we are currently trying to understand the phenomenon of spontaneous silencing of transgenes, i.e. the ability of plants to silence specifically transgene expression, a process that often leads to unstable transgenic phenotype.

    In relation to viroids, we are studying how these tiny RNA pathogens not encoding for any protein can escape the plants defence mechanisms and especially the hosts RNA silencing machinery. In addition, we are aiming in elucidating how specific host factors, such as Virp1, are used by the viroid to achieve infection.

    The group is also interested in RNA viruses that infect plants and especially how these pathogens manage to overcome the plant resistance mechanisms. In this direction we have isolated virally encoded suppressors of RNA silencing and are working in elucidating their function.
     

    Support

    During the last five years the laboratory has been successful in a number National grant applications within the ESPA framework securing more than 600K research money for the lab. In three of these projects we functioned as coordinators.

    The laboratory is currently member in two Horizon 2020 RISE projects and in two National Research Infrastructure projects.