Chemical enhancers of posttranscriptional gene silencing in Arabidopsis.

RNAi mediated by small-interfering RNAs (siRNAs) operates via transcriptional (TGS) and posttranscriptional gene silencing (PTGS). In Arabidopsis thaliana, TGS relies on DICER-LIKE-3 (DCL3)-dependent 24-nt siRNAs loaded into AGO4-clade ARGONAUTE effector proteins. PTGS operates via DCL4-dependent 21-nt siRNAs loaded into AGO1-clade proteins. We set up and validated a medium-throughput, semi-automatized procedure enabling chemical screening, in a 96-well in vitro format, of Arabidopsis transgenic seedlings expressing an inverted-repeat construct from the phloem companion cells. The ensuing quantitative PTGS phenotype was exploited to identify molecules, which, upon topical application, either inhibit or enhance siRNA biogenesis/activities. The vast majority of identified modifiers were enhancers, among which Sortin1, Isoxazolone, and [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) provided the most robust and consistent results, including upon their application onto soil-grown plants in which their effect was nonautonomous and long lasting. The three molecules increased the RNAi potency of the inverted-repeat construct, in large part by enhancing 21-nt siRNA accumulation and loading into AGO1, and concomitantly reducing AGO4 and DCL3 levels in planta. A similar, albeit not identical effect, was observed on 22-nt siRNAs produced from a naturally occurring inverted-repeat locus, demonstrating that the molecules also enhance endogenous PTGS. In standardized assays conducted in seedling extracts, the three enhancers selectively increased DCL4-mediated processing of in vitro-synthesized double-stranded RNAs, indicating the targeting of a hitherto unknown PTGS component probably independent of the DCL4-cofactor DOUBLE-STRANDED RNA-BINDING 4 (DRB4). This study establishes the proof-of-concept that RNAi efficacy can be modulated by chemicals in a whole organism. Their potential applications and the associated future research are discussed.

Autophagy selectively regulates miRNA homeostasis.

MicroRNAs (miRNAs) form a class of ~21 nucleotide (nt) RNAs that post-transcriptionally repress partially complementary messenger RNAs. miRNA-mediated silencing is critical for control of many key biological processes such as tumorigenesis, neuronal synaptic plasticity and defense against bacteria and viruses. Thus, unsurprisingly, miRNA biogenesis, abundance and action are under refined feedback control that is only beginning to be experimentally uncovered. We recently discovered that DICER1 and EIF2C/AGO are targeted for degradation by autophagy as miRNA-free entities by the selective autophagy receptor CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2/nuclear dot protein, 52 kDa). Strikingly, autophagy establishes a checkpoint for continued loading of miRNA, and this checkpoint is required for maintenance of miRNA abundance and proper miRNA activity. This newfound role for autophagy in miRNA biology suggests that human diseases exhibiting misregulated autophagy may be interdependent with defects in miRNA-mediated regulation of gene networks.

Selective autophagy degrades DICER and AGO2 and regulates miRNA activity.

MicroRNAs (miRNAs) form a class of short RNAs (∼ 21 nucleotides) that post-transcriptionally regulate partially complementary messenger RNAs. Each miRNA may target tens to hundreds of transcripts to control key biological processes. Although the biochemical reactions underpinning miRNA biogenesis and activity are relatively well defined and the importance of their homeostasis is increasingly evident, the processes underlying regulation of the miRNA pathway in vivo are still largely elusive. Autophagy, a degradative process in which cytoplasmic material is targeted into double-membrane vacuoles, is recognized to critically contribute to cellular homeostasis. Here, we show that the miRNA-processing enzyme, DICER (also known as DICER1), and the main miRNA effector, AGO2 (also known as eukaryotic translation initiation factor 2C, 2 (EIF2C2)), are targeted for degradation as miRNA-free entities by the selective autophagy receptor NDP52 (also known as calcium binding and coiled-coil domain 2 (CALCOCO2)). Autophagy establishes a checkpoint required for continued loading of miRNA into AGO2; accordingly, NDP52 and autophagy are required for homeostasis and activity of the tested miRNAs. Autophagy also engages post-transcriptional regulation of the DICER mRNA, underscoring the importance of fine-tuned regulation of the miRNA pathway. These findings have implications for human diseases linked to misregulated autophagy, DICER- and miRNA-levels, including cancer.

RNA silencing bridging the gaps in wheat extracts.

In plants, RNA silencing plays important roles in antiviral defence, genome integrity and development. This process involves nucleotide sequence-specific interactions that are mediated by small RNA molecules of 21-25 nucleotides. Although the core biochemical reactions of RNA silencing have been well characterized in animals, such information was crucially missing in plants. Recent work now addresses this question and reveals an overall similarity between the plant and animal RNA-silencing pathways, as well as some intriguing plant-specific aspects.