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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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The domain for nuclear localization. (A) Schematic illustration of domain deletion constructs of Drosha tagged with GFP at the N-terminus. (B) Cellular localization of different Drosha deletion mutants. Top panel: nuclear localization of GFP–Drosha with N-terminal 269 amino acid deletion. Middle panel: nuclear localization of GFP–Drosha with NLS2 deletion. Bottom panel: nuclear localization of GFP–Drosha with N-terminal 269 amino acid and NLS2 deletion.
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Figure 3: The domain for nuclear localization. (A) Schematic illustration of domain deletion constructs of Drosha tagged with GFP at the N-terminus. (B) Cellular localization of different Drosha deletion mutants. Top panel: nuclear localization of GFP–Drosha with N-terminal 269 amino acid deletion. Middle panel: nuclear localization of GFP–Drosha with NLS2 deletion. Bottom panel: nuclear localization of GFP–Drosha with N-terminal 269 amino acid and NLS2 deletion.

Mentions: In silico screening of the Drosha protein sequence predicted two potential NLSs: NLS1 (243-RHRSLDRRER-252) and NLS2 (276-RHRSYERSRERERERHRHR-295). We constructed GFP–Drosha reporter constructs with either NLS1 or NLS2 or both being deleted (Figure 3A). GFP–Drosha270–1374 with NLS1 deleted localized to the nucleus (Figure 3B, top panel) indicating that this predicted NLS1 site is not essential for correct cellular localization. We next focused on the NLS2 site. Surprisingly, we encountered similar results in that the NLS2 deleted variant also localized to the nucleus (Figure 3B, middle panel). Lastly, a reporter construct GFP–Drosha270–1374ΔNLS2 in which both NLS1 and NLS2 were deleted also localized to the nucleus (Figure 3B, bottom panel) clearly suggesting that a nuclear localization mechanism distinct from the canonical NLS in the domain between aa 270 and aa 390 of Drosha is operational.Figure 3.


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)

The domain for nuclear localization. (A) Schematic illustration of domain deletion constructs of Drosha tagged with GFP at the N-terminus. (B) Cellular localization of different Drosha deletion mutants. Top panel: nuclear localization of GFP–Drosha with N-terminal 269 amino acid deletion. Middle panel: nuclear localization of GFP–Drosha with NLS2 deletion. Bottom panel: nuclear localization of GFP–Drosha with N-terminal 269 amino acid and NLS2 deletion.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: The domain for nuclear localization. (A) Schematic illustration of domain deletion constructs of Drosha tagged with GFP at the N-terminus. (B) Cellular localization of different Drosha deletion mutants. Top panel: nuclear localization of GFP–Drosha with N-terminal 269 amino acid deletion. Middle panel: nuclear localization of GFP–Drosha with NLS2 deletion. Bottom panel: nuclear localization of GFP–Drosha with N-terminal 269 amino acid and NLS2 deletion.
Mentions: In silico screening of the Drosha protein sequence predicted two potential NLSs: NLS1 (243-RHRSLDRRER-252) and NLS2 (276-RHRSYERSRERERERHRHR-295). We constructed GFP–Drosha reporter constructs with either NLS1 or NLS2 or both being deleted (Figure 3A). GFP–Drosha270–1374 with NLS1 deleted localized to the nucleus (Figure 3B, top panel) indicating that this predicted NLS1 site is not essential for correct cellular localization. We next focused on the NLS2 site. Surprisingly, we encountered similar results in that the NLS2 deleted variant also localized to the nucleus (Figure 3B, middle panel). Lastly, a reporter construct GFP–Drosha270–1374ΔNLS2 in which both NLS1 and NLS2 were deleted also localized to the nucleus (Figure 3B, bottom panel) clearly suggesting that a nuclear localization mechanism distinct from the canonical NLS in the domain between aa 270 and aa 390 of Drosha is operational.Figure 3.

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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