6612 Nucleic Acids Research, 2010, Vol. 38, No. 19 was performed with a Licor Odyssey Infrared Imaging System (26). LC-MS/MS mass spectrometry analysis of protein with post-translational modiﬁcations HEK293T cells were transfected with 20 mg cDNA of GFP–Drosha. Two milligrams of protein lysate was incubated with 5 mg anti-GFP monoclonal antibody at 4 C for 1 h. Protein G Plus-Agarose beads (80 ml) were added into the lysate. The lysate-antibody-bead mixture was rotated at 4 C overnight. The immunoprecipitated beads were subsequently washed three times with RIPA buffer, resolved by 10% SDS–PAGE and visualized using Coomassie blue staining. The GFP–Drosha protein band was excised and subjected to mass spectrometric analysis performed by the Taplin Biological Mass Spectrometry Facility (Boston, MA, USA; 26). Confocal ﬂuorescent imaging HEK293T, Huh-7 or HeLa cells were transfected with constructs expressing GFP or GFP-tagged wild-type or mutated Drosha, respectively. Twenty-four hours post- transfection, the cells were trypsinized and reseeded at 1:10 dilution. The cells were incubated for another 24 h. Hoechst 33342 (ﬁnal concentration 1 mg/ml) was added to cell culture 30 min before confocal ﬂuorescent imaging which was performed with a LeicaTCS SP2 AOBS confocal laser microscope. Cell sorting and real-time PCR HEK293T cells were transfected with constructs express- ing GFP alone (as EV control) or GFP-tagged wild-type or mutated Drosha, respectively. Twenty-four hours post-transfection, the cells were trypsinized and resus- pended in a sort buffer (1 Ca/Mg++-free phosphate buffered saline, 1 mM EDTA, 25 mM HEPES pH 7.0, 1% heat-inactivated fetal bovine serum, 10 m/ml DNase I). GFP-positive cells were sorted out with FACSVantage SE (Becton Dickinson) to respective collec- tion tubes. The collected cells were used for RNA extrac- tion. Total RNA was extracted by TRIZOL (Invitrogen) and 1 mg of total RNA was used for cDNA synthesis using MMLV reverse transcriptase (New England Biolabs). TaqMan microRNA assay (Applied Biosystems) that include RT primers and TaqMan probes was used to quantify the levels of mature miRNA and 18S RNA was used for normalization. All PCR reactions were run in triplicate. Luciferase assays We used the psi-CHECK2 system (Promega) to create sensor assays for quantifying mature miRNA-143 function by placing the anti-sense sequence of miRNA-143 in the 30UTR of the gene encoding Renilla luciferase. In the presence of mature miRNA-143, the luciferase activity of Renilla decreases through the classic- al RNAi pathway. HEK 293T cells were co-transfected with psiCHECK2, pcDNA3miR-143 (a RNAP II promoter driven miRNA-143 expression vector) and Drosha expression constructs or empty vector. Fireﬂy and Renilla luciferase activities were quantiﬁed using the Dual-Luciferase Reporter Assay System (Promega) and Renilla luciferase activity was normalized to ﬁreﬂy luciferase activity. For each experiment, a control employ- ing an empty vector was used and corrected luciferase values were averaged, arbitrarily set to a value of ‘1’ and served as a reference for comparison of fold-differences in experimental values. Northern blot A miRNA-143 expression plasmid and Drosha wt or mutant constructs were transfected into HEK293T cells using lipofectamine reagent. Forty-eight hour post- transfection, total RNA was prepared with Trizol reagent. Twenty micrograms of total RNA was used for northern blotting analysis following conventional proto- cols but using a biotin-labeled probe and BrightStar BioDectectTM Nonisotopic Detection Kit (Ambion). Statistical/Bioinformatics analysis The Drosha protein sequence was mined for potential phosphorylation sites using the PredictProtein program (http://www.predictprotein.org). Mixed linear models (proc mixed, SAS version 9.2, SAS Institute, Cary, NC, USA) were used for statistical analysis as described previ- ously (27). RESULTS GFP-tagged Drosha locates in the nucleus We ﬁrst quantiﬁed Drosha expression in a number of human cell lines including HEK293T, Huh-7 and HeLa cells by cytoplasmic and nuclear fractionation followed by western blot analysis. As expected, our data (Figure 1A, top panel) revealed that Drosha exclusively localized to the nuclei of the tested cells. Cytoplasmic marker IkBa and nuclear marker Lamin A were used as controls to conﬁrm the stringency of our nuclear isolation methods (Figure 1A, middle panel and bottom panel, respectively). We then subcloned Drosha into pEGFP-C1 vector to generate the reporter construct GFP-Drosha. Importantly, sequences encoding GFP do not harbor a nuclear localization signal (NLS) or a nuclear export signal (NES). As expected, introduction of the parental GFP construct into HEK293T cells led to diffuse GFP expression (Figure 1B, top panel). In contrast, introduc- tion of GFP–Drosha was associated with exclusive GFP expression in the nuclei of HEK293T cells (Figure 1B, bottom panel). We repeated these experiments in other human cell lines such as HeLa (Supplementary Figure S1) and Huh-7 and obtained exactly similar results. These experiments suggested that sequences encoding Drosha harbored a putative NLS that was not affected by the addition of sequence encoding GFP. The N-terminus of Drosha harbors a NLS As GFP–Drosha was exclusively expressed in the nucleus, we reasoned that there must be a strong NLS in Drosha.