Phosphorylation of the RNase III enzyme Drosha at Serine300 or Serine302 is required for its nuclear localization.
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 sufficient to locate Drosha to the nucleus. Furthermore, mimicking phosphorylation status by mutating S→E at S300 and/or S→D at S302 restored nuclear localization. Our findings add a further layer of complexity to the molecular anatomy of Drosha as it relates to miRNA biogenesis.
Tang
Xiaoli
X
Laboratory of Retrovirology, Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical School of Brown University, Providence, RI 02903, USA.
Zhang
Yingjie
Y
Tucker
Lynne
L
Ramratnam
Bharat
B
D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D035683:MicroRNAs / Q000378:metabolism; D019913:Nuclear Localization Signals; D010766:Phosphorylation; D012323:RNA Processing, Post-Transcriptional; D043244:Ribonuclease III / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion; D012694:Serine / Q000378:metabolism*
D035683:MicroRNAs; D019913:Nuclear Localization Signals; D012694:Serine; C517918:DROSHA protein, human; D043244:Ribonuclease III