The discovery that dsRNA could induce gene silencing in animals and plants has raised the possibility that RNAi might be a nearly universal mechanism of gene silencing. An outline of the processes involoved in RNAi and several components have been identified in C.elegans
and plants, but details of the mechanism and many of the necessary participants in RNAi in mammalian cells remain unclear. In this study, to clarify the relationship between the localization and the target specificity of RNAi in mammalian cells, we constructed two kinds of dsRNA expression plasmid, namely, tRNA-dsRNA and U6-dsRNA expression plasmids. We demonstrated that tRNA-dsRNA which was localized in the cytoplasm was efficiently processed by the RNase III complex. An initial step in RNAi is the cleavage by Dicer, which is localized in the cytoplasm (20
), of long dsRNAs. Although short dsRNAs are cleaved less effectively by Dicer in vitro
), our tRNA-dsRNA was processed with significant efficiency in mammalian cells (Fig. ). In addition, U6-dsRNAs that had a microRNA-based loop motif were transported to the cytoplasm and were processed. Although transcripts from the U6 promoter are generally localized in the nucleus, the microRNA-loop motif promotes the transport of dsRNAs to the cytoplasm. Thus, cytoplasmic localization of dsRNAs is important for processing by Dicer.
The degradation step of mRNA is a very interesting aspect of RNAi-mediated gene silencing. Although two groups proposed that an RNA-directed RNA polymerase (RdRP) chain reaction with siRNA amplifies the interference caused by a small amount of ‘trigger’ dsRNA in C.elegans
), the mechanism is unclear in mammalian cells because an RdRP homolog does not exist in mammalian cells and siRNAs function as guides, not primers, in the Drosophila
and human RNAi pathways (58
). In this study, we demonstrated that a cytoplasmic fraction from cells which expressed tRNA-dsRNA had mRNA degradation activity (Fig. A). In addition, synthetic siRNAs mixed with the cytoplasmic fraction also had mRNA degradation activity (Fig. B; 36
). Moreover, it was recently reported that the level of HIV RRE-containing mRNA in the nucleus was not affected by siRNAs (59
). Thus, it is likely that a siRNA-associated silencing complex (SASC) including Dicer or -Slicer- is localized in the cytoplasm of mammalian cells.
RNAi has been shown to be a powerful tool for studies of gene function in C.elegans
and plants. However, in mammalian cells, a long dsRNA causes the non-specific reduction in expression of many genes. Thus, it was believed initially that RNAi could not be used for gene inactivation in mammalian cells. However, Tuschle’s group demonstrated that siRNA could specifically suppress the expression of a target gene specifically (24
). Exploitation of RNAi in mammalian cells requires evasion of non-specific reduction of mRNAs. In addition, since the putative SASC is located in the cytoplasm, it is important that dsRNA transcripts be localized in the cytoplasm. If properly designed, tRNA-dsRNAs (with a short hairpin structure) can be transported to the cytoplasm and can escape the non-specific reduction of mRNAs. Indeed, we found that PKR was not activated in cells that expressed tRNA-dsRNA (data not shown).
Although our U6-dsRNA with a general loop motif that consisted of five nucleotides was not transported to the cytoplasm and we could detect only marginal RNAi by our U6-dsRNA (Figs and ), transcripts of U6-dsRNAs (Loop 2) that had a microRNA-based loop motif were transported to the cytoplasm and were processed by Dicer (Fig. ). In addition, they induced RNAi-mediated gene silencing (Fig. ).
When alternative conditions were used, other U6-dsRNAs prepared by several independent groups (length of dsRNA and the size and the sequence of the hairpin-loop in these constructs were different from those of our U6-dsRNA) could support RNAi in mammalian cells (25
). In one case, a microRNA motif was used as the loop motif of dsRNA and the efficacy could have been enhanced by a microRNA pathway (32
). Thus, the length and the nucleotide sequence of the loop in hairpin types of dsRNAs are important for construction of effective hairpin types of dsRNAs. Now we are examining effects of both different length and structure of the loop on the localization and RNAi-mediated gene silencing by hairpin types of dsRNAs in detail.
Taken together, our results indicate that RNAi in mammalian cells occurs in the cytoplasm and is very specific in mammalian cells. Our tRNA-dsRNAs should be powerful tools for studies of the mechanism of RNAi and the functions of specific genes in mammalian cells, and they might also be useful as therapeutic agents.