Developmental defects by antisense-mediated inactivation of micro-RNAs 2 and 13 in Drosophila and the identification of putative target genes

Micro-RNAs are a class of small non-coding regulatory RNAs that impair translation by imperfect base pairing to mRNAs. For analysis of their cellular function we injected different miRNA-specific DNA antisense oligonucleotides in Drosophila embryos. In four cases we observed severe interference with normal development, one had a moderate impact and six oligonucleotides did not cause detectable phenotypes. We further used the miR-13a DNA antisense oligonucleotide as a PCR primer on a cDNA library template. In this experimental way we identified nine Drosophila genes, which are characterised by 3' untranslated region motifs that allow imperfect duplex formation with miR-13 or related miRNAs. These genes, which include Sos and Myd88, represent putative targets for miRNA regulation. Mutagenesis of the target motif of two genes followed by transfection in Drosophila Schneider 2 (S2) cells and subsequent reporter gene analysis confirmed the hypothesis that the binding potential of miR-13 is inversely correlated with gene expression.     

Variations of the 3' protruding ends in synthetic short interfering RNA (siRNA) tested by microinjection in Drosophila embryos

Short interfering RNAs (siRNAs) are the processing product originating from long double-stranded RNAs (dsRNAs) that are cleaved by the RNase III-like ribonuclease Dicer. As siRNAs mediate cleavage of specific single-stranded target RNAs, they are essential intermediates of RNA interference (RNAi). When applied in synthetic form, siRNAs likewise can induce the silencing process in the absence of long dsRNAs. Here, we tested variations of a conventional synthetic siRNA that had been used successfully to silence the Drosophila notch gene. The variants had two 3 ' -terminal deoxynucleotides in their protruding single-stranded ends. In one case, the deoxynulceotides would match to the notch mRNA, whereas the other variant had nonmatching deoxy-T residues, representing a widely used siRNA design. siRNAs with different combinations of sense and antisense strands were injected into Drosophila embryos at two different concentrations. We found that the all-ribonucleotide siRNA gave the best inhibition of notch expression. The combination of two modified strands with 3 ' -terminal deoxynucleotides was effective, but if combined with a sense or antisense ribostrand, the efficacy dropped. The siRNAs with nonmatching 3 ' -terminal TT residues showed a reduced silencing potential, which became evident at low concentration. An siRNA with a nonmatching 3 ' -terminal ribonucleotide in the antisense strand retained most of its silencing potential in accordance with the hypothesis that primer extension for generation of ssRNA from single-stranded mRNA does not operate in Drosophila.     

Prediction of novel microRNA genes in cancer-associated genomic regions—a combined computational and experimental approach

The majority of existing computational tools rely on sequence homology and/or structural similarity to identify novel microRNA (miRNA) genes. Recently supervised algorithms are utilized to address this problem, taking into account sequence, structure and comparative genomics information. In most of these studies miRNA gene predictions are rarely supported by experimental evidence and prediction accuracy remains uncertain. In this work we present a new computational tool (SSCprofiler) utilizing a probabilistic method based on Profile Hidden Markov Models to predict novel miRNA precursors. Via the simultaneous integration of biological features such as sequence, structure and conservation, SSCprofiler achieves a performance accuracy of 88.95% sensitivity and 84.16% specificity on a large set of human miRNA genes. The trained classifier is used to identify novel miRNA gene candidates located within cancer-associated genomic regions and rank the resulting predictions using expression information from a full genome tiling array. Finally, four of the top scoring predictions are verified experimentally using northern blot analysis. Our work combines both analytical and experimental techniques to show that SSCprofiler is a highly accurate tool which can be used to identify novel miRNA gene candidates in the human genome. SSCprofiler is freely available as a web service at http://www.imbb.forth.gr/SSCprofiler.html.     

Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing

In this study we applied RNA silencing to engineer potato plants that are resistant to potato virus Y (PVY). We expressed double-stranded (ds) RNA derived from the 3 terminal part of the coat protein gene of PVY, which is highly conserved in sequence amongst different PVY isolates, in transgenic potatoes of the commercial variety Spunta. Transgenic plants were analyzed for generation of transgene-derived short interfering RNAs (siRNAs) prior to virus inoculation. Twelve of fifteen transgenic lines produced siRNAs and were highly resistant to three strains of PVY, each belonging to three different subtypes of the virus (PVY^N, PVY^O and PVY^NTN). Infection of transgenic plants with Potato virus X (PVX) simultaneously or prior to the challenge with PVY did not interfere with PVY-resistance.Anastasia Missiou: M.A. and K.K. have contributed equally to this workKriton Kalantidis: M.A. and K.K. have contributed equally to this work