6616 Nucleic Acids Research, 2010, Vol. 38, No. 19 Figure 4. Identiﬁcation of phosphorylation sites by mass spectrometry. (A) Immunoprecipitation of GFP–Drosha with anti-GFP monoclonal antibody. HEK293T cells were transfected with a GFP–Drosha construct (or empty vector as a control) using Lipofectamine. Whole-cell lysate was prepared with modiﬁed RIPA buffer 48 h post-transfection and resultant immunoprecipitants were submitted for MS analysis (lane 1, GFP– Drosha; lane 2, a transfection with an irrelevant construct as a negative control). (B) Identiﬁcation of phosphorylation sites by mass spectrometry. The Coomassie stained GFP–Drosha protein band was excised and mass spectrometry analysis was performed. The asterisk shows the phosphorylated serine. mechanisms involving Drosha are unknown. Given the central role of miRNA in many basic cellular processes, any factor that adversely affects the nuclear location of Drosha is likely to disturb miRNA biogenesis and even- tually inﬂuence the normal function of the cell. Most nuclear proteins use a NLS to target themselves to the nucleus. Typical NLS consist of an amino acid sequence with positively charged lysines or arginines (30). In silico screening predicted two potential NLSs (NLS1 and NLS2) on Drosha. To determine if NLS1 and NLS2 were functional for Drosha, we generated a series of wild-type and mutant Drosha constructs with GFP tagged to the N-termini to monitor the protein localization. GFP is a widely used reporter protein that does not change the function and subcellular location when added as a protein tag (31,32). Furthermore, GFP does not harbor a NLS or a nuclear export signal (NES), thereby accounting for its diffuse expression upon cellular introduction. GFP–Drosha and GFP–Drosha1-390 localized to the nucleus. However, GFP–Drosha391–1374 exclusively localized to the cyto- plasm. These results raised the possibility that the C-terminus of Drosha might contain a NES which perhaps was inhibited by a strong NLS in the full-length context. Deletion of the N-terminus may have exposed this NES or abolished its inhibition. In silico analysis revealed the presence of two putative NLS sites in the N-terminus of Drosha. GFP–Drosha270–1374 with deletion of NLS1 located in the nucleus. GFP–Drosha270–1374 NLS2 with deletion of both NLS1 and NLS2 still located in the nucleus indicating that both NLS1 and NLS2 were dispensable for the nuclear localization of Drosha. This prompted us to hypothesize that Drosha might use a nuclear localiza- tion mechanism other than the canonical NLS to locate to the nucleus. In reality, the canonical NLS motif is absent in many proteins that localize to the nucleus. Proteins without canonical NLS may contain Serine–Proline– Serine (SPS) motif. Phosphorylation at either serine can facilitate binding with nuclear transporter (28). This prompted us to identify putative phosphorylation sites in the N-terminus of Drosha. A series of reporter constructs revealed that phosphorylation at Serine 300 or Serine 302 is critical for Drosha nuclear localization. A phosphorylated SPS motif acting as a NLS was initially found in signal proteins such as ERKs, Smad3 and MEK1 (28). Our results suggest that Drosha may use a similar pathway for correct nuclear localization. We note that phosphorylation at either or both serine sites could change the conformation of Drosha and thereby alter its afﬁnity for putative binding partners. Further investiga- tion is clearly warranted to identify proteins that interact with Drosha on the SPS motif as this motif appears highly conserved among mammals (Supplementary Figure S5). Reversible phosphorylation of proteins is an important regulatory mechanism in which kinases (phosphorylation)