Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research最新文献

Many dominantly inherited disorders are caused by missense amino acid substitutions resulting from a single nucleotide exchange in the encoding gene. For these disorders, where proteins expressed from the mutant alleles are often pathogenic and present throughout life, gene silencing, through intervention at the mRNA level, holds promise as a therapeutic approach. We have used mutations that underlie the slow channel congenital myasthenic syndrome (SCCMS) as a model system to study allele-specific gene silencing of RNA transcripts by DNAzymes. We tested the ability of DNAzymes to give allele-specific cleavage for i) mutations that create cleavage sites, and ii) mutations located close to a DNAzyme cleavage site that create a potential mismatch in the binding arms. For both we demonstrate selective cleavage of mutant transcripts under simulated physiological conditions. For DNAzymes with binding arm mismatches the degree of selectivity for mutant over wild type may be enhanced by optimising the mismatch position as well as the binding arm length. The optimal sites for mismatches are 1.1 and 1.2 in arm I, and 16.2 in arm II. Asymmetric binding arm DNAzymes with a shorter arm I are more discriminative. Our results show it should be possible to apply DNAzyme-mediated cleavage of mutant alleles even when the mutant does not itself create a putative cleavage site. This therapeutic approach may be well suited to dominantly inherited disorders such as SCCMS, where loss of some wild type transcripts is unlikely to have pathogenic consequences.

Here we report a versatile mammalian expression vector called pRIGHT11 for production of small interfering RNA (siRNA) in cells. This vector uses opposing eukaryotic RNA polymerase III promoters U6 and H1 to drive the expression of short siRNA. We have demonstrated the effectiveness of pRIGHT11-generated siRNA in sequence-specific inhibition of transfected reporter genes and endogenous genes. Furthermore, this retrovirus-based vector can carry a random library of siRNA, and thus can be applied to rapid screening of novel genes involved in specific cellular responses.

Slow channel congenital myasthenic syndrome (SCCMS) is a dominant disorder caused by missense mutations in muscle acetylcholine receptors (AChR). Expression from mutant alleles causes prolonged AChR ion-channel activations. This 'gain of function' results in excitotoxic damage due to excess entry of calcium ions that manifests as an endplate myopathy. The biology of SCCMS provides a model system to investigate the potential of catalytic nucleic acids for therapy in dominantly inherited disorders involving single missense mutations. Hammerhead ribozymes can catalytically cleave RNA transcripts in a sequence-specific manner. We designed hammerhead ribozymes to target transcripts from four SCCMS mutations, alphaT254I, alphaS226F, alphaS269I and epsilonL221F. Ribozymes were incubated with cRNA transcripts encoding wild type and mutant AChR subunits. The ribozymes efficiently cleaved the mutant allele cRNA transcripts but left the wild type cRNA intact. Cleavage efficiency was optimised for alphaS226F. We were able to demonstrate robust catalytic activity under simulated physiological conditions and at high Ca(2+) concentrations, which is likely to be accumulated at the endplate region of the SCCMS patient muscles. These results demonstrate the potential for gene therapy applications of ribozymes to specifically down-regulate expression of mutant alleles in dominantly inherited disorders.

microRNAs (miRNAs) are small RNAs that regulate translation and hence control a variety of cellular processes in metazoans. The quantitation and identification of miRNAs has been hampered by their small size and low abundance. Here we describe two robust PCR-based assays of miRNA expression based on the original cloning strategy. The non-quantitative PCR method allows detection and identification of miRNAs and we utilise this method in the discovery of a new miRNA (miR-532) in retinoic acid differentiated P19 cells. The second and quantitative method (QM-RT-PCR) is simple and accurate, and uses commonly available technology. Of particular interest is the specificity of this PCR-based technology compared to hybridisation-based methods including arrays and northern blotting. Here we have shown that a single base pair mismatch in the priming sequence results in a two order of magnitude reduction in the amplification of let-7f. These streamlined methods will complement previously described methods and will facilitate analysis of miRNA expression in rare cell populations where the amount of RNA is limited.

Application of siRNA in high-throughput fashion is still in its early phase although the principle has been established for three years. In this review, we outline the different vector-based siRNA delivery platforms as well as resources that are becoming available for high-throughput applications, and some initial outcomes of vector siRNA high-throughput screening efforts using vector encoded siRNA. It is expected that further improvement of the siRNA technology and availability of the siRNA resources will help to materialize the potential of siRNA for functional genomics and drug target validation.

RNA interference (RNAi) is mediated by a multicomponent RNA-induced silencing complex (RISC). Here we examine the phosphorylation state of three Drosophila RISC-associated proteins, VIG, R2D2 and a truncated form of Argonaute2 devoid of the nonconserved N-terminal glutamine-rich domain. We show that of the three studied proteins, only VIG is phosphorylated in cultured Drosophila cells. We also demonstrate that the phosphorylation state of VIG remains unchanged after cell transfection with exogenous dsRNA. A sequence similarity search revealed that VIG shares significant similarity with the human phosphoprotein Ki-1/57, a known in vivo substrate for protein kinase C (PKC). In vitro kinase assays followed by tryptic phosphopeptide mapping showed that PKC could efficiently phosphorylate VIG on multiple sites, suggesting PKC as a candidate kinase for VIG phosphorylation in vivo. Taken together, our results identify the RISC component VIG as a novel kinase substrate in cultured Drosophila cells and suggest a possible involvement of PKC in its phosphorylation.