6610–6619 Nucleic Acids Research, 2010, Vol. 38, No. 19 doi:10.1093/nar/gkq547 Published online 16 June 2010 Phosphorylation of the RNase III enzyme Drosha at Serine300 or Serine302 is required for its nuclear localization Xiaoli Tang, Yingjie Zhang, Lynne Tucker and Bharat Ramratnam* Laboratory of Retrovirology, Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical School of Brown University, Providence, RI 02903, USA Received January 29, 2010; Revised May 27, 2010; Accepted May 28, 2010 ABSTRACT The RNaseIII enzyme Drosha plays a pivotal role in microRNA (miRNA) biogenesis by cleaving primary miRNA transcripts to generate precursor miRNA in the nucleus. The RNA binding and enzymatic domains of Drosha have been characterized and are on its C-terminus. Its N-terminus harbors a nuclear localization signal. Using a series of truncated Drosha constructs, we narrowed down the segment responsible for nuclear translocation to a domain between aa 270 and aa 390. We further identified two phosphorylation sites at Serine300 (S300) and Serine302 (S302) by mass spectrometric analysis. Double mutations of S!A at S300 and S302 completely disrupted nuclear localization. Single mutation of S!A at S300 or S302, however, had no effect on nuclear localization indicating that phosphorylation at either site is suf- ficient to locate Drosha to the nucleus. Furthermore, mimicking phosphorylation status by mutating S!E at S300 and/or S!D at S302 restored nuclear local- ization. Our findings add a further layer of complex- ity to the molecular anatomy of Drosha as it relates to miRNA biogenesis. INTRODUCTION MicroRNAs (miRNAs) are a class of endogenous non-protein-coding small RNAs of 22 nt in length that impact gene expression by sequence-speciﬁc interaction with homologous mRNA (1). Presently, it is thought that miRNAs most commonly repress gene expression by base-pairing with the 30-untranslated region (UTR) of their target mRNAs. However, recent work has also identiﬁed miRNA that target coding sequences of genes (2). Additionally, miRNA may also target promoter regions and thereby act as pro-transcriptional elements, as in the case of miRNA-373 and the gene encoding E-cadherin (3). One speciﬁc miRNA may inhibit many target genes and one speciﬁc gene may be regulated by more than one miRNA. For example, both miRNA-125 a and b, the genes of which are located on different chromosomes, target the p53 protein as both miRNAs harbor similar seed sequences that share similar- ity to the p53 30UTR (4,5). MiRNAs play increasingly recognized roles in several basic processes including cell signal transduction, tumorigenesis, tumor invasion and metastasis, stem cell renewal, immune function, apoptosis and reaction to stress (6–11). The vast majority of miRNA genes are thought to be under the control of RNAP II with others being recently identiﬁed as substrates of RNAP III (12,13). Irrespectively, miRNA genes are ini- tially transcribed to yield a primary, long transcript that undergoes successive processing in both the nucleus and cytoplasm. Nuclear processing is mediated by the RNase III enzyme Drosha–DGCR8 (DiGeorge Syndrome Critical Region Gene 8) microprocessor complex to generate precursor miRNAs (pre-miRNAs) of 70 nt in length. Pre-miRNAs are subsequently transported to the cytoplasm by export 5-Ran-GTP where they are cleaved by the RNase III enzyme Dicer to generate mature miRNAs (14–18). Investigation into the molecular mechanisms of miRNA biogenesis at the transcriptional and translational levels has been intensively pursued (19–22). Drosha plays a central role in miRNA biogenesis and recent work suggests that its expression level directly inﬂuences clinical outcomes in malignant disease thus underlying the importance of better understanding mechanisms that impact Drosha expression and function (23). Along these lines, Drosha has been found to interact with other host proteins including DGCR8. Drosha and DGCR8 regulate each other post-transcriptionally. The Drosha–DGCR8 complex destabilizes DGCR8 mRNA by cleaving its hairpin structure. DGCR8, in turn, stabilizes Drosha via *To whom correspondence should be addressed. Tel: +1 401 444 5219; Fax: +1 401 444 2939; Email: bramratnam@lifespan.org The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.