Silencing of antiapoptotic survivin gene by multiple approaches of RNA interference technology

Silencing of mammalian gene expression by RNA interference (RNAi) technology can be achieved using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, the relative effectiveness of these two approaches is not known. It is also not clear whether gene-specific shRNA transcribed from an RNA polymerase II (Pol II)-directed promoter in a fusion form can disrupt the targeted gene expression. Here, we report that using both luciferase and antiapoptotic survivin genes as targets, both siRNA and shRNA approaches significantly silenced the targeted gene expression in cancer cells. We further demonstrated that shRNAs transcribed from an RNA Pol II-mediated promoter in a green fluorescent protein (GFP) fusion form at the 3'-untranslated region silenced luciferase and survivin expression as well, suggesting that the extra RNA sequence outside of the shRNA hairpin does not disrupt shRNA function. We also showed that silencing of survivin expression selectively induces apoptosis in transfected cells. Together, we have validated multiple approaches of RNAi technology using both survivin and luciferase genes as targets and demonstrated for the first time that GFP-shRNAs transcribed from an RNA Pol II-mediated promoter could mediate gene silencing, which may lead to new directions for the application of RNAi technology.     

Killing of Trypanosoma brucei by concanavalin A: structural basis of resistance in glycosylation mutants

Concanavalin A (Con A) kills procyclic (insect) forms of Trypanosoma brucei by binding to N-glycans on EP-procyclin, a major surface glycosyl phosphatidylinositol (GPI)-anchored protein which is rich in Glu-Pro repeats. We have previously isolated and studied two procyclic mutants (ConA 1-1 and ConA 4-1) that are more resistant than wild-type (WT) to Con A killing. Although both mutants express the same altered oligosaccharides compared to WT cells, ConA 4-1 is considerably more resistant to lectin killing than is ConA 1-1. Thus, we looked for other alterations to account for the differences in sensitivity. Using mass spectrometry, together with chemical and enzymatic treatments, we found that both mutants express types of EP-procyclin that are either poorly expressed or not found at all in WT cells. ConA 1-1 expresses mainly EP1-3, a novel procyclin that contains 18 EP repeats, is partially N-glycosylated, and bears hybrid-type glycans. On the other hand, ConA 4-1 cells express almost exclusively EP2-3, a novel non-glycosylated procyclin isoform with 23 EP repeats and no site for glycosylation. The predominance of EP2-3 in ConA 4-1 cells explains their high resistance to ConA killing. Thus, switching the procyclin repertoire, a process that could be relevant to parasite development in the insect vector, modulates the sensitivity of trypanosomes to cytotoxic lectins.     

RNA interference in Trypanosoma brucei: a high-throughput engine for functional genomics in trypanosomatids?

RNA interference (RNAi) is the technique of choice for down-regulating the gene function of suitable genes in African trypanosomes. A recent report by Subramanian and co-workers describes a high-throughput method for gene function discovery using RNAi in Trypanosoma brucei. The phenotype of most of the Open Reading Frames from chromosome 1 of T. brucei was analysed using a battery test of standard protocols. The authors propose that this technique could be used to mine the full genome of T. brucei and to reveal the core proteomic map of the other two major trypanosomatids, Trypanosoma cruzi and Leishmania major, despite the lack of a homologous mechanism of genetic silencing.     

pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.     

Adipocyte differentiation induced using nonspecific siRNA controls in cultured human mesenchymal stem cells

RNA interference (RNAi) is gene silencing induced by double-stranded RNA of 21-23 nucleotides in length, termed small interfering RNA, or siRNA. RNAi-based techniques have been widely applied to elucidate gene function, identify drug targets, and used in trials as a promising adjunct to silence disease-causing genes. However, emerging evidence suggests unexpected changes in expression of untargeted genes as a consequence of an off-target effect by RNAi in mammalian cells. To date, our understanding of such effects on stem cells is limited. We transfected human fetal femur-derived mesenchymal stem cells using commercially available nonspecific siRNA controls and examined adipocyte differentiation in the cells using morphology, histochemistry, and quantitative real-time PCR to examine the expression of key genes for adipogenic or osteogenic differentiation. We report here the induction of adipocyte differentiation in human mesenchymal stem cells using nonspecific siRNAs raising concerns as to the specificity of RNAi in stem cells and, critically, a need to understand and delineate the rules governing the specificity of RNAi.