Figure 1. Drosha localizes to the nucleus. (A) Endogenous Drosha protein was detected in the nuclei of HEK293T, Huh-7 and HeLa cells by western blot analysis. Cells were harvested when 100% conﬂu- ent. Cytoplasmic and nuclear fractionation was performed. IkBa and Lamin A served as cytoplasmic and nuclear markers, respectively (C, cytoplasmic; N, nuclear). (B) Overexpression of a construct encoding Drosha tagged with GFP on the N-terminus localizes to the nucleus. HEK293T cells were transfected with GFP backbone vector alone or GFP-tagged Drosha plasmid, respectively. Hoechst 33342 was used to stain the nuclei 30 min before ﬂuorescent imaging was per- formed. Top panel: GFP backbone vector expression showing diffuse localization. Bottom panel: GFP–Drosha expression showing nuclear localization. To pinpoint its location, we constructed a series of GFP– Drosha deletion constructs as illustrated in Figure 2A. A construct in which the C-terminal 524 amino acids was deleted (Drosha C524) localized exclusively to the nucleus (Figure 2B, top panel). In contrast, when we deleted the entire N-terminus (aa 1–390), Drosha localized exclusively to the cytoplasm (Figure 2B, upper middle panel) providing a general location for its NLS. We then focused on better deﬁning the molecular anatomy of the N-terminus which localizes to the nucleus exclusively (Figure 2B, down middle panel). We deleted N-terminal 217 amino acids and a corresponding reporter construct GFP–Drosha N217 still localized to the nucleus exclu- sively (Figure 2B, bottom panel). All these results were conﬁrmed in other cell lines including Huh-7 and HeLa cells (Supplementary Figure S1). A domain between aa 270 and aa 390 of Drosha harbors a NLS In silico screening of the Drosha protein sequence predicted two potential NLSs: NLS1 (243-RHRSLDRRER-252) and NLS2 (276-RHRSYERSRERERERHRHR-295). We constructed GFP–Drosha reporter constructs with either NLS1 or NLS2 or both being deleted (Figure 3A). GFP– Drosha270–1374 with NLS1 deleted localized to the nucleus (Figure 3B, top panel) indicating that this predicted NLS1 site is not essential for correct Nucleic Acids Research, 2010, Vol. 38, No. 19 6613 cellular localization. We next focused on the NLS2 site. Surprisingly, we encountered similar results in that the NLS2 deleted variant also localized to the nucleus (Figure 3B, middle panel). Lastly, a reporter construct GFP–Drosha270–1374 NLS2 in which both NLS1 and NLS2 were deleted also localized to the nucleus (Figure 3B, bottom panel) clearly suggesting that a nuclear localization mechanism distinct from the canonic- al NLS in the domain between aa 270 and aa 390 of Drosha is operational. Identiﬁcation of phosphorylation sites by mass spectrometry analysis Protein phosphorylation plays an important role in nuclear localization (28,29). For example, phosphoryl- ation of extracellular signal-regulated kinase (ERK)-2 at Ser244 and Ser246 induced its nuclear translocation. Additionally, phosphorylation of b-catenin at Ser191 and Ser605 controls its nuclear localization. We next asked if Drosha is constitutively phosphorylated and whether phosphorylation status impacts nuclear localiza- tion. The cell lysate from HEK293T cells transfected with GFP–Drosha for 48 h without any treatment was immunoprecipitated with anti-GFP mouse monoclonal antibody and Protein G Plus-Agarose beads. The precipi- tate was resolved by SDS–PAGE and stained with Coomassie Brilliant Blue R-250 Dye. The gel showed a clear band corresponding to the calculated size of GFP– Drosha (Figure 4A). This band was excised and subjected to LC-MS/MS mass spectrometry. The results conﬁrmed that the excised band was indeed puriﬁed GFP–Drosha with two phosphorylation sites at Serine300 and Serine302 being identiﬁed (Figure 4B). Phosphorylation at Serine300 or Serine302 locates Drosha to the nucleus Based upon the MS results, we made a series of GFP– Drosha constructs with different point mutations involving the putative phosphorylation sites as indicated. Single S!A mutation at S300 or S302 did not change the nuclear localization of GFP–Drosha (Figure 5A and B, respectively). Double mutations at S300 and S302, however, totally prevented nuclear localization producing a cellular staining pattern similar to GFP without NLS (Figure 5C). These results indicated that S300 and S302 were constitutively phosphorylated and that phosphoryl- ation at either site was sufﬁcient to locate Drosha to the nucleus. This was further supported by mutating Serine300 to glutamic acid (E), and Serine302 to aspartic acid (D). Indeed, all of these phosphorylation mimics (GFP–DroshaS300E, GFP–DroshaS302D and GFP– DroshaS300E/S302D) located Drosha in the nuclei of HEK293T cells (Figure 5D–F). Again, these results were reproducible in other cell lines including HeLa cells (Supplementary Figures S1 and S2). Nuclear localization of Drosha is critical for its functionality in miRNA processing The fact that Drosha is indispensible for cellular health posed a challenge for assigning a critical in vivo function to