6618 Nucleic Acids Research, 2010, Vol. 38, No. 19 Figure 6. Nuclear localization of Drosha is critical for its functionality in miRNA processing. (A) Protein expression levels of wt Drosha and mutants. GFP-tagged Drosha wt or mutant constructs were transfected into HEK293T cells using lipofectamine reagent. Forty-eight hours post-transfection, protein lysates were prepared from the transfected cells and protein levels were measured by western blot analysis. GAPDH was used as a loading control. All constructs produced equivalent levels of Drosha protein. (B) Quantiﬁcation of mature miRNA-143 levels/function by real-time PCR (B), miRNA sensor assays (C) and northern blot (D). GFP vector alone (empty vector, EV), GFP–Drosha wide type (WT) or different mutant constructs as indicated were transfected into HEK 293T cells along with a miRNA-143 expression vector. Twenty-four hours post-transfection, GFP-positive cells were cell sorted for RNA extraction. Endogenous mature miRNA-143 level was quantiﬁed by using a speciﬁc miRNA-143 Taqman real-time PCR kit. All experiments were performed in triplicate (compared to EV, *P < 0.05). (C) miRNA sensor assays revealed that compared to EV control conditions, cells that had been transfected with Drosha constructs that localized to the cytoplasm (GFP–DroshaC’ and GFP–DroshaS300/302A) were associated with impaired miRNA function. All experiments were performed in triplicate (compared to EV, *P < 0.05). (D). Effects of overexpressed Drosha wt or mutants on miRNA-143 biogenesis by northern blotting analysis. Cells transfected with cytoplasmic Drosha localized constructs did not retain the ability to process pri-miRNA-143 with the absence of pre- and mature species in GFP–DroshaC’ and GFP–DroshaS300/302A treated cells. deregulation that is seen in diverse disease states including cancer. and Jin Song Gao for technical support in performing the miRNA sensor assays. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank Ross Tomaino for help in mass spectrometry analysis, Ginny Hovanesian for assistance in ﬂuorescent imaging, Sara Spangenberger for assistance in cell sorting FUNDING National Institutes of Health (P20RR025179 and R01AI058697); Lifespan/Brown/Tufts CFAR (P30AI042853); National Institutes of Health (number T32DA013911 to X.T.). Funding for open access charge: National Institutes of Health. Conﬂict of interest statement. None declared.