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.

Short interfering RNAs specific for potato spindle tuber viroid are found in the cytoplasm but not in the nucleus.

Short interfering (si) and micro (mi) RNAs influence gene expression at post-transcriptional level. In plants, different classes of DICER-LIKE (DCL) enzymes are responsible for the generation of these small regulatory RNAs from different precursors. To characterize the cellular site of their generation and accumulation, we purified nuclei from tomato plants infected with potato spindle tuber viroid (PSTVd) RNA, which is known to replicate in the nucleus via double-stranded (ds) RNA intermediates. We could detect PSTVd-specific siRNAs in the cytoplasmic fraction, but not in the nuclear fraction. To correlate the localization of the PSTVd-specific siRNAs with that of similarly sized small RNAs, we studied the compartmentalization of a naturally occurring miRNA. We could detect the precursor of miR167 in the nucleus, but the mature miRNA was found only in the cytoplasmic fraction. We discuss the consequences of this finding for the model of viroid replication and heterochromatin formation.

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.

Induction of RNA interference in Caenorhabditis elegans by RNAs derived from plants exhibiting post-transcriptional gene silencing.

The term 'gene silencing' refers to transcriptional and post-transcriptional control of gene expression. Related processes are found across kingdoms in plants and animals. We intended to test whether particular RNA constituents of a silenced plant can induce silencing in an animal. We generated Nicotiana benthamiana lines that expressed green fluorescent protein (GFP) from a transgene. Plants in which GFP expression was spontaneously silenced showed siRNAs characteristic of post-transcriptional gene silencing (PTGS). RNA extracts prepared from silenced plants were injected into a GFP-expressing strain of Caenorhabditis elegans, where they induced RNA interference (RNAi). Extracts from non-silenced plants were inactive. This directly demonstrates a relationship and a mechanistic link between PTGS and RNAi. Controls confirmed that the silencing agent was an RNA. Size fractionation on denaturing gels revealed that an RNA of approximately 85 nt was most active in inducing silencing in the worm. Northern blot analysis of the region in question did not detect a prominent GFP-specific RNA of sense or antisense polarity, indicating that the RNA species which induced silencing was present only in low concentration or did not hybridize due to formation of an intramolecular double strand. In view of its high activity, it is possible that this agent is responsible for the systemic spread of silencing in plants and it might represent the aberrant RNA, a previously postulated inducer of silencing.