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Phosphorylation of the RNase III enzyme Drosha at Serine300 or Serine302 is required for its nuclear localization.

Tang X, Zhang Y, Tucker L, Ramratnam B - Nucleic Acids Res. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Retrovirology, Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical School of Brown University, Providence, RI 02903, USA.

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 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.

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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.
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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.

Mentions: Protein phosphorylation plays an important role in nuclear localization (28,29). For example, phosphorylation of extracellular signal-regulated kinase (ERK)-2 at Ser244 and Ser246 induced its nuclear translocation. Additionally, phosphorylation of β-catenin at Ser191 and Ser605 controls its nuclear localization. We next asked if Drosha is constitutively phosphorylated and whether phosphorylation status impacts nuclear localization. The cell lysate from HEK293T cells transfected with GFP–Drosha for 48 h without any treatment was immunoprecipitated with anti-GFP mouse monoclonal antibody and Protein G Plus-Agarose beads. The precipitate was resolved by SDS–PAGE and stained with Coomassie Brilliant Blue R-250 Dye. The gel showed a clear band corresponding to the calculated size of GFP–Drosha (Figure 4A). This band was excised and subjected to LC-MS/MS mass spectrometry. The results confirmed that the excised band was indeed purified GFP–Drosha with two phosphorylation sites at Serine300 and Serine302 being identified (Figure 4B).Figure 4.


Phosphorylation of the RNase III enzyme Drosha at Serine300 or Serine302 is required for its nuclear localization.

Tang X, Zhang Y, Tucker L, Ramratnam B - Nucleic Acids Res. (2010)

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.
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Related In: Results  -  Collection

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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.
Mentions: Protein phosphorylation plays an important role in nuclear localization (28,29). For example, phosphorylation of extracellular signal-regulated kinase (ERK)-2 at Ser244 and Ser246 induced its nuclear translocation. Additionally, phosphorylation of β-catenin at Ser191 and Ser605 controls its nuclear localization. We next asked if Drosha is constitutively phosphorylated and whether phosphorylation status impacts nuclear localization. The cell lysate from HEK293T cells transfected with GFP–Drosha for 48 h without any treatment was immunoprecipitated with anti-GFP mouse monoclonal antibody and Protein G Plus-Agarose beads. The precipitate was resolved by SDS–PAGE and stained with Coomassie Brilliant Blue R-250 Dye. The gel showed a clear band corresponding to the calculated size of GFP–Drosha (Figure 4A). This band was excised and subjected to LC-MS/MS mass spectrometry. The results confirmed that the excised band was indeed purified GFP–Drosha with two phosphorylation sites at Serine300 and Serine302 being identified (Figure 4B).Figure 4.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Retrovirology, Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical School of Brown University, Providence, RI 02903, USA.

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 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.

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