6614 Nucleic Acids Research, 2010, Vol. 38, No. 19 Figure 2. The N-terminus of Drosha is critical for nuclear localization. (A) Schematic illustration of domain deletion constructs of Drosha tagged with GFP at the N-terminus. (B) Cellular localization of different Drosha deletion mutants. Top panel: nuclear localization of GFP–Drosha with C-terminal 524 amino acid deletion. Upper middle panel: cytoplasmic localization of GFP–Drosha C-terminus. Lower middle panel: nuclear local- ization of GFP–Drosha N-terminus. Bottom panel: nuclear localization of GFP–Drosha with N-terminal 217 amino acid deletion. the N-terminus using our various constructs. The purest experiment, after all, would be to use a Drosha cell and introduce our various constructs as well as miRNA expression cassettes to examine their downstream effect on miRNA processing. To our knowledge, no Drosha human cell line exists. We therefore relied upon ectopic expression of both our various Drosha constructs as well as miRNA expression cassettes. We ﬁrst conﬁrmed that all our Drosha constructs produced similar levels of protein upon cellular transfection, thereby removing the possibil- ity that differential protein expression of our constructs was the ultimate effector of miRNA processing activity (Figure 6A). As previously reported, HEK293T cells harbor relatively low levels of mature miRNA-143 (27). Interestingly, these cells also express relatively low levels of endogenous Drosha when compared to other cell lines such as HCT116, NIH3T3, etc. Accordingly, we trans- fected cells with the GFP vector alone (empty vector), GFP–Drosha WT or the various GFP–Drosha mutated constructs along with an expression plasmid encoding miRNA-143. FACS was used to obtain GFP expressing cells. Real-time PCR was performed to measure