We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.

EMBO Press is an editorially independent publishing platform for the development of EMBO scientific publications.

Virus infections induce the expression of ARGONAUTE1 (AGO1) mRNA and in parallel enhance the accumulation of miR168 (regulator of AGO1 mRNA). Here, we show that in virus‐infected plants the enhanced expression of AGO1 mRNA is not accompanied by increased AGO1 protein accumulation. We also show that the induction of AGO1 mRNA level is a part of the host defence reaction, whereas the induction of miR168, which overlaps spatially with virus‐occupied sectors, is mediated mainly by the Tombusvirus p19 RNA‐silencing suppressor. The absence of p19 results in the elimination of miR168 induction and accompanied with the enhanced accumulation of AGO1 protein. In transient expression study, p19 mediates the induction of miR168 and the down‐regulation of endogenous AGO1 level. P19 is not able to efficiently bind miR168 in virus‐infected plants, indicating that this activity is uncoupled from the small RNA‐binding capacity of p19. Our results imply that plant viruses can inhibit the translational capacity of AGO1 mRNA by modulating the endogenous miR168 level to alleviate the anti‐viral function of AGO1 protein.

▪ Abstract Although many subviral RNAs reduce or intensify disease symptoms caused by the helper virus, only recently have clues concerning the mechanism of disease modulation been revealed. New models for DI RNA-mediated reduction in helper virus levels and symptom attenuation include DI RNA enhancement of posttranscriptional gene silencing (PTGS), which is an antiviral defense mechanism in plants. Symptom enhancement by the satRNA of Cucumber mosaic virus is caused by minus-strand induction of the programmed cell death pathway. In contrast, symptom enhancement by satC of Turnip crinkle virus is due to satC interference with virion formation, leading to increased levels of free coat protein, which is the viral suppressor of PTGS. Mutualism between satRNA and helper virus can be seen for the satRNA of Groundnut rosette virus, which contributes to the virus by allowing virion assembly. These novel findings are leading to re-evaluation of the relationships between subviral RNAs, helper viruses, and hosts.