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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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Drosha localizes to the nucleus. (A) Endogenous Drosha protein was detected in the nuclei of HEK293T, Huh-7 and HeLa cells by western blot analysis. Cells were harvested when 100% confluent. Cytoplasmic and nuclear fractionation was performed. IkBa and Lamin A served as cytoplasmic and nuclear markers, respectively (C, cytoplasmic; N, nuclear). (B) Overexpression of a construct encoding Drosha tagged with GFP on the N-terminus localizes to the nucleus. HEK293T cells were transfected with GFP backbone vector alone or GFP-tagged Drosha plasmid, respectively. Hoechst 33342 was used to stain the nuclei 30 min before fluorescent imaging was performed. Top panel: GFP backbone vector expression showing diffuse localization. Bottom panel: GFP–Drosha expression showing nuclear localization.
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Figure 1: Drosha localizes to the nucleus. (A) Endogenous Drosha protein was detected in the nuclei of HEK293T, Huh-7 and HeLa cells by western blot analysis. Cells were harvested when 100% confluent. Cytoplasmic and nuclear fractionation was performed. IkBa and Lamin A served as cytoplasmic and nuclear markers, respectively (C, cytoplasmic; N, nuclear). (B) Overexpression of a construct encoding Drosha tagged with GFP on the N-terminus localizes to the nucleus. HEK293T cells were transfected with GFP backbone vector alone or GFP-tagged Drosha plasmid, respectively. Hoechst 33342 was used to stain the nuclei 30 min before fluorescent imaging was performed. Top panel: GFP backbone vector expression showing diffuse localization. Bottom panel: GFP–Drosha expression showing nuclear localization.

Mentions: We first quantified Drosha expression in a number of human cell lines including HEK293T, Huh-7 and HeLa cells by cytoplasmic and nuclear fractionation followed by western blot analysis. As expected, our data (Figure 1A, top panel) revealed that Drosha exclusively localized to the nuclei of the tested cells. Cytoplasmic marker IkBa and nuclear marker Lamin A were used as controls to confirm the stringency of our nuclear isolation methods (Figure 1A, middle panel and bottom panel, respectively). We then subcloned Drosha into pEGFP-C1 vector to generate the reporter construct GFP-Drosha. Importantly, sequences encoding GFP do not harbor a nuclear localization signal (NLS) or a nuclear export signal (NES). As expected, introduction of the parental GFP construct into HEK293T cells led to diffuse GFP expression (Figure 1B, top panel). In contrast, introduction of GFP–Drosha was associated with exclusive GFP expression in the nuclei of HEK293T cells (Figure 1B, bottom panel). We repeated these experiments in other human cell lines such as HeLa (Supplementary Figure S1) and Huh-7 and obtained exactly similar results. These experiments suggested that sequences encoding Drosha harbored a putative NLS that was not affected by the addition of sequence encoding GFP.Figure 1.


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)

Drosha localizes to the nucleus. (A) Endogenous Drosha protein was detected in the nuclei of HEK293T, Huh-7 and HeLa cells by western blot analysis. Cells were harvested when 100% confluent. Cytoplasmic and nuclear fractionation was performed. IkBa and Lamin A served as cytoplasmic and nuclear markers, respectively (C, cytoplasmic; N, nuclear). (B) Overexpression of a construct encoding Drosha tagged with GFP on the N-terminus localizes to the nucleus. HEK293T cells were transfected with GFP backbone vector alone or GFP-tagged Drosha plasmid, respectively. Hoechst 33342 was used to stain the nuclei 30 min before fluorescent imaging was performed. Top panel: GFP backbone vector expression showing diffuse localization. Bottom panel: GFP–Drosha expression showing nuclear localization.
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Figure 1: Drosha localizes to the nucleus. (A) Endogenous Drosha protein was detected in the nuclei of HEK293T, Huh-7 and HeLa cells by western blot analysis. Cells were harvested when 100% confluent. Cytoplasmic and nuclear fractionation was performed. IkBa and Lamin A served as cytoplasmic and nuclear markers, respectively (C, cytoplasmic; N, nuclear). (B) Overexpression of a construct encoding Drosha tagged with GFP on the N-terminus localizes to the nucleus. HEK293T cells were transfected with GFP backbone vector alone or GFP-tagged Drosha plasmid, respectively. Hoechst 33342 was used to stain the nuclei 30 min before fluorescent imaging was performed. Top panel: GFP backbone vector expression showing diffuse localization. Bottom panel: GFP–Drosha expression showing nuclear localization.
Mentions: We first quantified Drosha expression in a number of human cell lines including HEK293T, Huh-7 and HeLa cells by cytoplasmic and nuclear fractionation followed by western blot analysis. As expected, our data (Figure 1A, top panel) revealed that Drosha exclusively localized to the nuclei of the tested cells. Cytoplasmic marker IkBa and nuclear marker Lamin A were used as controls to confirm the stringency of our nuclear isolation methods (Figure 1A, middle panel and bottom panel, respectively). We then subcloned Drosha into pEGFP-C1 vector to generate the reporter construct GFP-Drosha. Importantly, sequences encoding GFP do not harbor a nuclear localization signal (NLS) or a nuclear export signal (NES). As expected, introduction of the parental GFP construct into HEK293T cells led to diffuse GFP expression (Figure 1B, top panel). In contrast, introduction of GFP–Drosha was associated with exclusive GFP expression in the nuclei of HEK293T cells (Figure 1B, bottom panel). We repeated these experiments in other human cell lines such as HeLa (Supplementary Figure S1) and Huh-7 and obtained exactly similar results. These experiments suggested that sequences encoding Drosha harbored a putative NLS that was not affected by the addition of sequence encoding GFP.Figure 1.

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.

Show MeSH