The minimal active human RISC is composed of three proteins, Dicer, TRBP and Ago2 [5, 15]. Within this complex, Dicer and Ago2 have RNase activities, whereas TRBP is important in loading the appropriate dsRNA into the complex [13, 14]. Other proteins have been shown to associate with the RISC, however their roles remain to be fully determined . We further defined the requirement for TRBP in RNAi using murine cells that are completely deficient for TRBP (tarbp2-/-). Using an shRNA and an miRNA targeting EGFP, these cells displayed low or no RNAi activity as compared to murine cells expressing TRBP (tarbp2+/- and MEF) (Fig. 4), confirming that TRBP is an essential protein for RNAi activity in these cells. The different approaches used to assess RNAi activity yielded slightly different intensities of EGFP knock-down, which can be attributed to the different sensitivities of each technique. Because no human cell has been shown to have a complete TRBP deficit, we evaluated the RNAi function in astrocytes, which have a weak expression of TRBP. This correlated with a low and moderate activity for the sh- and miRNAs, respectively, compared to HeLa cells (Fig. 6). In this case, the difference in RNAi activity between U251MG and HeLa cells follows the same trend in fluorescence, FACS and western blotting. The ability of U251MG cells to knock-down GFP expression using miRNA-based silencing is likely explained by the residual TRBP2 expression in this cell type. Our results demonstrate that in both murine and human cells, TRBP is required for efficient RNAi activity.
The interaction between Dicer and TRBP has been shown to be direct in vitro and in vivo with no requirement for ssRNA, dsRNA or another protein . Although the site of interaction in TRBP was determined to be in the C-terminal end, no distinct functional domain had been defined for Dicer . Two-hybrid assays further defined these domains as aa 267–431 (called D1) in Dicer and aa 298–366 in TRBP2 (called C4) (Fig. 1). D1 is located between the ATPase and the helicase domains in Dicer. It was shown previously that ATP is not required for Dicer function in RNAi [35, 36], suggesting that either this domain is not functional in the human protein or that it plays a role in other processes. Interestingly, although a complete knock-down of Dicer is lethal, cells defective for only the ATPase/helicase domain are viable with only a defect in miRNA biogenesis [9, 37]. Here we show that the functional importance of the ATPase/helicase domain is in supporting the interaction with TRBP. These combined results suggest that the ATPase/helicase domain is not functional in the human protein, and that the sole function of this region may be to recruit TRBP through the D1 domain. Interestingly, when searched in BLASTP, D1 has no homology with any other protein in the databases, showing its unique properties.
Further demonstration that the C4 domain is solely responsible for TRBP binding to Dicer is shown by the IP assays in the presence of complete cell extracts from tarbp2-/- cells (Fig. 2). In this assay, neither TRBP1ΔC4, nor TRBP2ΔC4, immunoprecipitated with Dicer. Together with previous results that showed a direct interaction between TRBP and Dicer in the absence of RNA or other proteins, these results confirm that no bridge between Dicer and TRBP occurs via dsRNA or another protein. TRBPsΔC4 and Dicer both have functional dsRBDs that are able to bind dsRNA. The absence of interaction between the TRBP mutants and Dicer suggests that they do not bind the same RNA at the same time.
Dicer and TRBP are cytoplasmic proteins but their colocalization has not been studied. Here, we show that endogenous Dicer can be observed in both the nucleus and the cytoplasm, while TRBP is distributed predominantly in the cytoplasm (Fig. 1C). Colocalization of the two proteins was found to occur predominantly in the perinuclear space. Overexpression of TRBPs did not affect their cellular distribution as immunofluorescence of Myc-tagged constructs revealed similar staining to that of the endogenous protein (Fig. 1C compared to 3A). As neither Myc-TRBP1ΔC4, nor Myc-TRBP2ΔC4 colocalized with Dicer in the perinuclear space, we conclude that binding of TRBP to Dicer is a major determinant of TRBP cellular distribution and our results suggest that RNAi takes place in the perinuclear space. In contrast to Myc-TRBP1ΔC4, Myc-TRBP2ΔC4 was partially associated to the cell membrane. Because TRBP2 has only 21 more aa in its N-terminus than TRBP1, these aa might be responsible for the membrane association, which is only revealed when its interaction with Dicer is abolished. A closer analysis of the 21 aa sequence and the protein's posttranslational modifications will be necessary to elucidate this function.
This slight difference between TRBP1 and TRBP2 also correlates with the rescue of the RNAi function mediated by shRNAs against EGFP (Figs. 5A and 7A). In these assays, TRBP1 completely rescued the shRNA activity while TRBP2 rescued it partially. In contrast, we could not rescue shRNA activity using the ΔC4 mutants (Figs. 5B and 7C). This demonstrates that the C4 domain, which mediates TRBP interaction with Dicer, is also required for RNAi activity to occur. These results are compatible with a role of Dicer in the recognition and cleavage of precursor miRNA and for TRBP in loading small siRNAs into the RISC [5, 13, 14]. This study provides new insights into the interaction between TRBP and Dicer in the RISC. These results open the way for further studies into differences between the specific roles of TRBP1 and TRBP2 in the RNAi pathway.