RNAi-induced off-target effects in Drosophila melanogaster: frequencies and solutions.

Genes can be silenced with short-interfering RNA molecules (siRNA). siRNAs are widely used to identify gene functions and have high potential for therapeutic treatments. It is critical that the siRNA specifically targets the expression of the gene of interest but has no off-target effects on other genes. Although siRNAs were initially considered to be exclusively active on mature mRNAs in the cytoplasm, additional studies have shown that siRNAs are present in the nucleus as well, suggesting that pre-mRNA sequences containing introns and other untranslated regions can also be targeted. In this study, we investigated the extent to which off-targets may occur in Drosophila melanogaster by looking at mature mRNA sequences and pre-mature RNA sequences separately. First, an in silico approach revealed that, based on sequence similarity, numerous off-targets are predicted to occur in RNAi experiments. Second, existing microarray data were used to investigate a possible effect of the predicted off-targets based on analysis of in vitro data. We found that the occurrence of off-targets in both mature and pre-mature RNA sequences in RNAi experiments can be extensive and significant. Possibilities are discussed how to minimize off-target effects.

RNAi experiments in D. melanogaster: solutions to the overlooked problem of off-targets shared by independent dsRNAs.

BACKGROUND: RNAi technology is widely used to downregulate specific gene products. Investigating the phenotype induced by downregulation of gene products provides essential information about the function of the specific gene of interest. When RNAi is applied in Drosophila melanogaster or Caenorhabditis elegans, often large dsRNAs are used. One of the drawbacks of RNAi technology is that unwanted gene products with sequence similarity to the gene of interest can be down regulated too. To verify the outcome of an RNAi experiment and to avoid these unwanted off-target effects, an additional non-overlapping dsRNA can be used to down-regulate the same gene. However it has never been tested whether this approach is sufficient to reduce the risk of off-targets. METHODOLOGY: We created a novel tool to analyse the occurrence of off-target effects in Drosophila and we analyzed 99 randomly chosen genes. PRINCIPAL FINDINGS: Here we show that nearly all genes contain non-overlapping internal sequences that do show overlap in a common off-target gene. CONCLUSION: Based on our in silico findings, off-target effects should not be ignored and our presented on-line tool enables the identification of two RNA interference constructs, free of overlapping off-targets, from any gene of interest.

Pantethine rescues a Drosophila model for pantothenate kinase-associated neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN), a progressive neurodegenerative disorder, is associated with impairment of pantothenate kinase function. Pantothenate kinase is the first enzyme required for de novo synthesis of CoA, an essential metabolic cofactor. The pathophysiology of PKAN is not understood, and there is no cure to halt or reverse the symptoms of this devastating disease. Recently, we and others presented a PKAN Drosophila model, and we demonstrated that impaired function of pantothenate kinase induces a neurodegenerative phenotype and a reduced lifespan. We have explored this Drosophila model further and have demonstrated that impairment of pantothenate kinase is associated with decreased levels of CoA, mitochondrial dysfunction, and increased protein oxidation. Furthermore, we searched for compounds that can rescue pertinent phenotypes of the Drosophila PKAN model and identified pantethine. Pantethine feeding restores CoA levels, improves mitochondrial function, rescues brain degeneration, enhances locomotor abilities, and increases lifespan. We show evidence for the presence of a de novo CoA biosynthesis pathway in which pantethine is used as a precursor compound. Importantly, this pathway is effective in the presence of disrupted pantothenate kinase function. Our data suggest that pantethine may serve as a starting point to develop a possible treatment for PKAN.

A high throughput experimental approach to identify miRNA targets in human cells.

The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation-gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines. Ten to thirty percent of the gene transcripts from the genome were enriched in the Ago2-IP fraction of untreated cells, representing the HL miRNA-targetome. In silico analysis indicated that approximately 40% of these gene transcripts represent targets of the abundantly co-expressed miRNAs. To identify targets of miR-17/20/93/106, RIP-Chip with anti-miR-17/20/93/106 treated cells was performed and 1189 gene transcripts were identified. These genes were analyzed for miR-17/20/93/106 target sites in the 5'-UTRs, coding regions and 3'-UTRs. Fifty-one percent of them had miR-17/20/93/106 target sites in the 3'-UTR while 19% of them were predicted miR-17/20/93/106 targets by TargetScan. Luciferase reporter assay confirmed targeting of miR-17/20/93/106 to the 3'-UTRs of 8 out of 10 genes. In conclusion, we report a method which can establish the miRNA-targetome in untreated human cells and identify miRNA specific targets in a high throughput manner. This approach is applicable to identify miRNA targets in any human tissue sample or purified cell population in an unbiased and physiologically relevant manner.