ABSTRACT
Double-stranded (ds) RNA causes the specific degradation of homologous RNAs in a process called "RNA interference (RNAi)"[1-4]; this process is called "posttranscriptional gene silencing (PTGS)" in plants [5-7]. Both classes of gene silencing have been reviewed extensively [8-13]. The duplex RNA becomes processed by Dicer [14] or another RNase III-like enzyme to short dsRNA fragments of about 21-23 nucleotides (nt) [15], which are incorporated in the RNA-induced silencing complex (RISC)[16] that directs target-specific RNA degradation [17, 18]. Here, we show that different synthetic dsRNA cassettes, consisting of two 5'-phosphorylated RNA strands of 22 nt each, can initiate RNAi in Drosophila embryos. The cassettes were active at similar quantities required to initiate RNAi by conventional dsRNA. Their sequence specificity was confirmed using synthetic dsRNA cassettes for two different genes, Notch and hedgehog; each time, only the relevant embryonic phenotype was observed. Introduction of point mutations had only a moderate effect on the silencing potential, indicating that the silencing machinery does not require perfect sequence identity. 5'-phosphorylated synthetic RNA was more active than its hydroxylated form. Substitution of either RNA strand by DNA strongly reduced activity. Synthetic cassettes of siRNA will provide a new tool to induce mutant phenotypes of genes with unknown function.
Subject(s)
5' Untranslated Regions , Drosophila Proteins , Gene Silencing , RNA, Double-Stranded , RNA, Untranslated , Animals , DNA , Drosophila/embryology , Drosophila/genetics , Hedgehog Proteins , Insect Proteins/genetics , Membrane Proteins/genetics , Mutagenesis , Nucleic Acid Hybridization , Phosphorylation , RNA, Double-Stranded/chemical synthesis , RNA, Small Interfering , Receptors, NotchABSTRACT
Mac1p is a Saccharomyces cerevisiae DNA binding transcription factor that activates genes involved in copper uptake. A copper-induced N-C-terminal intramolecular interaction and copper-independent homodimerization affect its function. Here, we present a functional analysis of Mac1p deletion derivatives that attributes new roles to the second cysteine-rich (REPII) domain of the protein. This domain exhibits the copper-responsive potent transactivation function when assayed independently and, in the context of the entire protein, modulates the efficiency of Mac1p binding to DNA. The efficiency of binding to both copper-response promoter elements can determine the in vivo functionality of Mac1p independent of homodimerization.
