Nucleic Acids Research, 2010, Vol. 38, No. 19 6615 Figure 3. The domain 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 N-terminal 269 amino acid deletion. Middle panel: nuclear localization of GFP–Drosha with NLS2 deletion. Bottom panel: nuclear localization of GFP–Drosha with N-terminal 269 amino acid and NLS2 deletion. miRNA-143 and northern blot was performed to identify pre- and mature miRNA-143 species. Our data revealed that overexpression of GFP–Drosha WT, S300A, S302A, S300E, S302D or S300E/S302D led to the production of pre- and mature miRNA-143 (Figure 6B and D). However, cells expressing GFP–DroshaS300/302A or GFP–Drosha C-terminus (aa 391–1374) did not retain the ability to produce mature or pre-miRNA-143. These results were further validated by miRNA sensor assays. For example, co-transfection of the sensor along with GFP–Drosha led to a statistically signiﬁcant reduction in Renilla activity compared to empty vector. GFP– DroshaS300A, S302A, S300E, S302D or S300E/S302D behaved similarly to GFP–Drosha. In contrast, cellular introduction of GFP–DroshaS300/302A had no signiﬁ- cant effect on sensor activity compared to empty vector (Figure 6C). We then repeated this experiment but used sensors for miRNA-26b and 125a. Again, similar results were encountered with the stereotypical loss of miRNA activity in transfections involving only GFP–DroshaS300/302A or GFP–Drosha C-terminus (aa 391–1374; Supplementary Figure S3). Lastly, we further reasoned that these nonfunctional Drosha mutants should impede the processing of pri-miRNA and thereby lead to an accumulation of this species. Indeed, real-time PCR quantiﬁcation of pri-miRNA-143 revealed a 2-fold increase in experiments involving the cytoplasmic localized Drosha variants [GFP– DroshaS300/302A or GFP–Drosha C-terminus (aa 391–1374)] compared to bonaﬁde nuclear localized variants (Supplementary Figure S4). DISCUSSION A myriad of proteins exist in a single cell and their correct subcellular localization is critical for protein function and cellular health. The RNaseIII enzyme Drosha is a nuclear protein and correct localization is essential to its function in processing pri-miRNA species in the canonical path- way of miRNA biogenesis. The nuclear transporting