RNAi in cultured Drosophila cells.

Abstract

Double-stranded RNA (dsRNA)-mediated interference, or RNAi, has emerged as an effective technique to phenocopy the loss of function of a given gene product. With this tool researchers can study the functions of individual molecules in living cells and elucidate the mechanisms that regulate cell division. For example, many molecules that are important for regulating mitosis and for controlling the assembly of the mitotic spindle are mutated in different cancer cell types (for a review, see ref. 1). Functional analysis in vivo of molecules that play a role in mitosis is best implemented by a genetic analysis. For this, genetically malleable organisms such as Drosophila, Caenorhabditis elegans, yeast, and other micro-organisms have been extremely useful. Whereas genetic analysis usually requires a long-term effort, RNAi provides a rapid method for the reverse genetic analysis of gene product function and can be exploited to great advantage. In the era of sequenced genomes, this technique provides a valuable tool for functional genomics. Here, a detailed procedure for RNAi in Drosophila cells in culture is presented. RNA interference was first described using C. elegans (2), although the phenomenon has been described in plants as posttranscriptional gene silencing (PTGS) (3) and as “quelling” in Neurospora (4). Furthermore, RNAi has been demonstrated on a number of organisms (2-9). For Drosophila, RNAi has been accomplished by injection of dsRNA into early syncytial cleavage stage embryos (6). Subsequently, heritable RNAi has been achieved in C. elegans and Drosophila using transgenic dsRNA “hairpin”-generating constructs (10-13). An important advance came when RNAi was demonstrated with cultured Drosophila cells (7). More recently, RNAi has been applied successfully to vertebrate cells in culture using short interfering RNAs (siRNAs) (14,15). For this, the first step in the cellular response mechanism to dsRNA (see below) has to be bypassed, because full-length dsRNAs produce nonspecific effects in vertebrate cells (16-19). RNAi-mediated interference occurs by a posttranscriptional mechanism that targets mRNA homologous to the dsRNA that is introduced for destruction (for reviews, see refs. 20-22). Using Drosophila embryo and S2 cell extracts, the mechanisms for RNAi are being elucidated (16,23). In these extracts, dsRNA is cleaved into 21- to 25-bp siRNAs with 5′ phosphates, 3′ hydroxyl groups, and contain two to three nucleotide 3′ overhangs. dsRNA cleavage is mediated by Dicer, an ATP-dependent RNaseIII family RNase (24). siRNAs assemble into an approx 360-kDa complex called RNA-induced silencing complex (RISC) in Drosophila extracts (25). The siRNAs then unwind in an ATP-dependent manner (23,25). The single-stranded siRNAs in the RISC complex provide the homologous targeting to mRNA (23,26), enabling degradation by the RNase associated with RISC. Furthermore, Argonaute proteins are components of RISC (27) with homologs in plants, fungi, and C. elegans that are required for RNAi in those organisms (28-30). The degraded target mRNA appears to then be cycled into new siRNAs that repeat the process in an RNA polymerase-dependent cycle of mRNA degradation and siRNA production (31). The complete mechanism for RNAi has not been elucidated. A teleological explanation for the existence of a mechanism to destroy mRNAs in response to homologous dsRNA has been proposed (32). It has been suggested that RNAi evolved as a mechanism to combat invading dsRNA viruses or to inhibit the activity of retrotransposons. Moreover, there is at least one gene in Drosophila, Stellate, that is regulated by dsRNA-mediated gene silencing in the testis (33). This chapter describes the application of RNAi to cultured Drosophila cells, with a particular emphasis on the imaging of the cytoskeleton and chromosomes in affected cells. Materials and methods are provided to enable the researcher to implement the design and production of dsRNA from polymerase chain reaction (PCR) templates, the culture of Drosophila cells and their treatment by RNAi, the analysis of target protein depletion by Western blotting, and the fixation and treatment of cells for microscopic imaging. The depletion of centrosomin (Cnn) a centrosomal protein that is required for mitotic centrosome assembly and function (34-37) from S2 cells is presented for example, but the technique is widely applicable to different targets and cell lines (7,17,38). Importantly, Drosophila cells in culture readily take up exogenous dsRNA, and there is no need to use carriers or transfection methods like those required with mammalian cell culture (7). Thus, RNAi holds great promise for the analysis of protein function in living cells.