protein–protein interaction (22). In an elegant experiment, deletion of Drosha N-terminal 390 amino acids had no effect on its binding with DGCR8 and its ability to process pri-miRNA in vitro (21). While these results sug- gested that the N-terminal region is dispensable vis-a-vis the protein’s catalytic activity, they also raised an interest- ing question: what role, if any, does N-terminal sequence play in Drosha function in vivo? Here, we examine the subcellular localization of wild-type Drosha and N-terminally deleted Drosha. We ﬁnd that wild-type Drosha locates to the nucleus, as expected, but Drosha with N-terminal 390 amino acids deleted is located exclusively in the cytoplasm. These results were in agreement with previous work which found that the N-terminus of Drosha is not dispensable but rather essential for its nuclear localization and ultimate miRNA processing function (24). We further narrowed down the domain for nuclear localization to the N-terminal region between aa 270 and aa 390. We show by microscopy and functional assays that phosphor- ylation at Serine300 or Serine302 is absolutely required for the nuclear localization and subsequent miRNA process- ing activities of Drosha. MATERIALS AND METHODS Plasmids and primers pcDNA4/TO/cmycDrosha was originally created by Thomas Tuschl and deposited at Addgene (Addgene plasmid 10828; 25). Wild-type and mutated Drosha were subcloned into pEGFP-C1 vector (GenBank Accession #:U55763) using restriction endonucleases HindIII and KpnI (New England Biolabs cat.# R0104L and cat.#R0142L, respectively). Point mutants were generated with site-directed mutagenesis techniques as previously described (26). The primers used for PCR were ordered from Integrated DNA Technologies. The primer se- quences are shown in Supplementary Table S1. psiCHECK2-miRNA-143 plasmid was created by insert- ing antisense target sequences of miRNA-143 ds-oligos (sense:CCGCTCGAGGAGCTATAGTGCTTCATCTC AGCGGCCGCAAAAGGAAAA and antisense: TTTTC CTTTTGCGGCCGCTGAGATGAAGCACTATAGCT CCTCGAGCGG) into psiCHECK2 vector (Promega) using NotI and XhoI (New England Biolabs) restriction endonuclease sites. All the cDNA constructs were veriﬁed by DNA sequencing at Yale KECK Foundation Biotechnology Resource Laboratory. Antibodies and reagents Anti-RNase III Drosha (H-300) rabbit polyclonal antibody (sc-33778), Anti-IkB-a (C-21) rabbit polyclonal antibody (sc-371), anti-Lamin A (H-102) rabbit poly- clonal antibody (sc-20680), anti-GFP (B-2) mouse mono- clonal antibody (sc-9996) and Protein G Plus-Agarose (sc-2002) were purchased from Santa Cruz Biotechnology. Pfu DNA Polymerase (cat.#600136) and dNTP mix (cat.#200415-51) were purchased from Stratagene. Lipofectamine Reagent (cat.#18324-020) and Lipofectamine 2000 Reagent (cat.#11668-019) were Nucleic Acids Research, 2010, Vol. 38, No. 19 6611 purchased from Invitrogen. Alexa Fluor 680 goat anti- rabbit IgG (H&L) (cat.#A21076) and Hoechst 33342 (cat#H3570) were purchased from Invitrogen Molecular Probes. IRDye800-conjugated Afﬁnity Puriﬁed Anti- Mouse IgG (H&L) (cat.#610-132-121) was purchased from Rockland Immunochemicals. Tris-Glycine-SDS Running Buffer (cat.#BP-150) and Transfer Buffer (cat.#BP-190) were purchased from Boston Bioproducts. LB Agar Kanamycin-50 Plates (cat.#L0643) and EDTA (cat.#E7889) were purchased from Sigma. The 10 Tris- Buffered Saline (cat.#170-6435) and 10% Tween20 solution (cat.#161-0781) were purchased from Bio-Rad Laboratories. Complete Mini, EDTA-free Protease Inhibitor Cocktail Tablets (cat.#11 836 170 001) was produced by Roche Diagnostics GmbH. QIAprep Spin Miniprep Kit (cat.#27106), QIAquick PCR Puriﬁcation Kit (cat.#28106) and MinElute Gel Extraction Kit (cat.#28604) were purchased from QIAGEN. Coomassie Brilliant Blue R-250 Dye (cat.#20278) was purchased from Thermo Scientiﬁc. Cell culture and transfection HEK293T and Huh-7 cells were cultured in Dulbecco’s modiﬁed Eagle’s medium (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum and 2 mM L-glutam- ine. HeLa cells were grown in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and non-essential amino acids. Cells were trypsinized and reseeded in culture plates 1 day before transfection. HEK293T transfection was per- formed with Lipofectamine when cell conﬂuency was 60%. Huh-7 and HeLa cells were transfected with Lipofectamine 2000 when cell conﬂuency was 80%. Cytoplasmic and nuclear fractionation Cytoplasmic and nuclear fractionation was performed using EZ Nuclei Isolation Kit (Sigma, cat.#NUC-101) ac- cording to the manufacturer’s instructions. Brieﬂy, cells were harvested and washed once with cold PBS. The cells were then suspended in EZ Nuclei Isolation buffer and rotated at 4 C for 5 min. After centrifugation at 4 C for 5 min, supernatant was collected containing the cyto- plasmic fraction. Cell lysis and centrifugation were repeated three times. The ﬁnal pellets were collected as the nuclear fraction and lysed in modiﬁed RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1% Triton X-100). Western blotting Cell lysates (100 mg protein each) were separated by 10% SDS–PAGE electrophoresis and electroblotted to nitrocel- lulose membrane (Bio-Rad, cat.#162-0115). Blotted mem- branes were probed with their respective primary antibodies rotating at 4 C overnight. The membranes were washed three times in TBST buffer and probed with secondary antibody (Alexa Fluor 680 goat anti- rabbit IgG or IRDye800-conjugated Afﬁnity Puriﬁed Anti-Mouse IgG, respectively) at room temperature for 1 h. Membranes were then washed three times in TBST buffer and direct infrared ﬂuorescence detection