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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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Nuclear localization of Drosha is critical for its functionality in miRNA processing. (A) Protein expression levels of wt Drosha and mutants. GFP-tagged Drosha wt or mutant constructs were transfected into HEK293T cells using lipofectamine reagent. Forty-eight hours post-transfection, protein lysates were prepared from the transfected cells and protein levels were measured by western blot analysis. GAPDH was used as a loading control. All constructs produced equivalent levels of Drosha protein. (B) Quantification of mature miRNA-143 levels/function by real-time PCR (B), miRNA sensor assays (C) and northern blot (D). GFP vector alone (empty vector, EV), GFP–Drosha wide type (WT) or different mutant constructs as indicated were transfected into HEK 293T cells along with a miRNA-143 expression vector. Twenty-four hours post-transfection, GFP-positive cells were cell sorted for RNA extraction. Endogenous mature miRNA-143 level was quantified by using a specific miRNA-143 Taqman real-time PCR kit. All experiments were performed in triplicate (compared to EV, *P < 0.05). (C) miRNA sensor assays revealed that compared to EV control conditions, cells that had been transfected with Drosha constructs that localized to the cytoplasm (GFP–DroshaC’ and GFP–DroshaS300/302A) were associated with impaired miRNA function. All experiments were performed in triplicate (compared to EV, *P < 0.05). (D). Effects of overexpressed Drosha wt or mutants on miRNA-143 biogenesis by northern blotting analysis. Cells transfected with cytoplasmic Drosha localized constructs did not retain the ability to process pri-miRNA-143 with the absence of pre- and mature species in GFP–DroshaC’ and GFP–DroshaS300/302A treated cells.
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Figure 6: Nuclear localization of Drosha is critical for its functionality in miRNA processing. (A) Protein expression levels of wt Drosha and mutants. GFP-tagged Drosha wt or mutant constructs were transfected into HEK293T cells using lipofectamine reagent. Forty-eight hours post-transfection, protein lysates were prepared from the transfected cells and protein levels were measured by western blot analysis. GAPDH was used as a loading control. All constructs produced equivalent levels of Drosha protein. (B) Quantification of mature miRNA-143 levels/function by real-time PCR (B), miRNA sensor assays (C) and northern blot (D). GFP vector alone (empty vector, EV), GFP–Drosha wide type (WT) or different mutant constructs as indicated were transfected into HEK 293T cells along with a miRNA-143 expression vector. Twenty-four hours post-transfection, GFP-positive cells were cell sorted for RNA extraction. Endogenous mature miRNA-143 level was quantified by using a specific miRNA-143 Taqman real-time PCR kit. All experiments were performed in triplicate (compared to EV, *P < 0.05). (C) miRNA sensor assays revealed that compared to EV control conditions, cells that had been transfected with Drosha constructs that localized to the cytoplasm (GFP–DroshaC’ and GFP–DroshaS300/302A) were associated with impaired miRNA function. All experiments were performed in triplicate (compared to EV, *P < 0.05). (D). Effects of overexpressed Drosha wt or mutants on miRNA-143 biogenesis by northern blotting analysis. Cells transfected with cytoplasmic Drosha localized constructs did not retain the ability to process pri-miRNA-143 with the absence of pre- and mature species in GFP–DroshaC’ and GFP–DroshaS300/302A treated cells.

Mentions: The fact that Drosha is indispensible for cellular health posed a challenge for assigning a critical in vivo function to the N-terminus using our various constructs. The purest experiment, after all, would be to use a Drosha cell and introduce our various constructs as well as miRNA expression cassettes to examine their downstream effect on miRNA processing. To our knowledge, no Drosha human cell line exists. We therefore relied upon ectopic expression of both our various Drosha constructs as well as miRNA expression cassettes. We first confirmed that all our Drosha constructs produced similar levels of protein upon cellular transfection, thereby removing the possibility that differential protein expression of our constructs was the ultimate effector of miRNA processing activity (Figure 6A). As previously reported, HEK293T cells harbor relatively low levels of mature miRNA-143 (27). Interestingly, these cells also express relatively low levels of endogenous Drosha when compared to other cell lines such as HCT116, NIH3T3, etc. Accordingly, we transfected cells with the GFP vector alone (empty vector), GFP–Drosha WT or the various GFP–Drosha mutated constructs along with an expression plasmid encoding miRNA-143. FACS was used to obtain GFP expressing cells. Real-time PCR was performed to measure miRNA-143 and northern blot was performed to identify pre- and mature miRNA-143 species. Our data revealed that overexpression of GFP–Drosha WT, S300A, S302A, S300E, S302D or S300E/S302D led to the production of pre- and mature miRNA-143 (Figure 6B and D). However, cells expressing GFP–DroshaS300/302A or GFP–Drosha C-terminus (aa 391–1374) did not retain the ability to produce mature or pre-miRNA-143. These results were further validated by miRNA sensor assays. For example, co-transfection of the sensor along with GFP–Drosha led to a statistically significant reduction in Renilla activity compared to empty vector. GFP–DroshaS300A, S302A, S300E, S302D or S300E/S302D behaved similarly to GFP–Drosha. In contrast, cellular introduction of GFP–DroshaS300/302A had no significant effect on sensor activity compared to empty vector (Figure 6C). We then repeated this experiment but used sensors for miRNA-26b and 125a. Again, similar results were encountered with the stereotypical loss of miRNA activity in transfections involving only GFP–DroshaS300/302A or GFP–Drosha C-terminus (aa 391–1374; Supplementary Figure S3). Lastly, we further reasoned that these nonfunctional Drosha mutants should impede the processing of pri-miRNA and thereby lead to an accumulation of this species. Indeed, real-time PCR quantification of pri-miRNA-143 revealed a 2-fold increase in experiments involving the cytoplasmic localized Drosha variants [GFP–DroshaS300/302A or GFP–Drosha C-terminus (aa 391–1374)] compared to bonafide nuclear localized variants (Supplementary Figure S4).Figure 6.


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)

Nuclear localization of Drosha is critical for its functionality in miRNA processing. (A) Protein expression levels of wt Drosha and mutants. GFP-tagged Drosha wt or mutant constructs were transfected into HEK293T cells using lipofectamine reagent. Forty-eight hours post-transfection, protein lysates were prepared from the transfected cells and protein levels were measured by western blot analysis. GAPDH was used as a loading control. All constructs produced equivalent levels of Drosha protein. (B) Quantification of mature miRNA-143 levels/function by real-time PCR (B), miRNA sensor assays (C) and northern blot (D). GFP vector alone (empty vector, EV), GFP–Drosha wide type (WT) or different mutant constructs as indicated were transfected into HEK 293T cells along with a miRNA-143 expression vector. Twenty-four hours post-transfection, GFP-positive cells were cell sorted for RNA extraction. Endogenous mature miRNA-143 level was quantified by using a specific miRNA-143 Taqman real-time PCR kit. All experiments were performed in triplicate (compared to EV, *P < 0.05). (C) miRNA sensor assays revealed that compared to EV control conditions, cells that had been transfected with Drosha constructs that localized to the cytoplasm (GFP–DroshaC’ and GFP–DroshaS300/302A) were associated with impaired miRNA function. All experiments were performed in triplicate (compared to EV, *P < 0.05). (D). Effects of overexpressed Drosha wt or mutants on miRNA-143 biogenesis by northern blotting analysis. Cells transfected with cytoplasmic Drosha localized constructs did not retain the ability to process pri-miRNA-143 with the absence of pre- and mature species in GFP–DroshaC’ and GFP–DroshaS300/302A treated cells.
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Figure 6: Nuclear localization of Drosha is critical for its functionality in miRNA processing. (A) Protein expression levels of wt Drosha and mutants. GFP-tagged Drosha wt or mutant constructs were transfected into HEK293T cells using lipofectamine reagent. Forty-eight hours post-transfection, protein lysates were prepared from the transfected cells and protein levels were measured by western blot analysis. GAPDH was used as a loading control. All constructs produced equivalent levels of Drosha protein. (B) Quantification of mature miRNA-143 levels/function by real-time PCR (B), miRNA sensor assays (C) and northern blot (D). GFP vector alone (empty vector, EV), GFP–Drosha wide type (WT) or different mutant constructs as indicated were transfected into HEK 293T cells along with a miRNA-143 expression vector. Twenty-four hours post-transfection, GFP-positive cells were cell sorted for RNA extraction. Endogenous mature miRNA-143 level was quantified by using a specific miRNA-143 Taqman real-time PCR kit. All experiments were performed in triplicate (compared to EV, *P < 0.05). (C) miRNA sensor assays revealed that compared to EV control conditions, cells that had been transfected with Drosha constructs that localized to the cytoplasm (GFP–DroshaC’ and GFP–DroshaS300/302A) were associated with impaired miRNA function. All experiments were performed in triplicate (compared to EV, *P < 0.05). (D). Effects of overexpressed Drosha wt or mutants on miRNA-143 biogenesis by northern blotting analysis. Cells transfected with cytoplasmic Drosha localized constructs did not retain the ability to process pri-miRNA-143 with the absence of pre- and mature species in GFP–DroshaC’ and GFP–DroshaS300/302A treated cells.
Mentions: The fact that Drosha is indispensible for cellular health posed a challenge for assigning a critical in vivo function to the N-terminus using our various constructs. The purest experiment, after all, would be to use a Drosha cell and introduce our various constructs as well as miRNA expression cassettes to examine their downstream effect on miRNA processing. To our knowledge, no Drosha human cell line exists. We therefore relied upon ectopic expression of both our various Drosha constructs as well as miRNA expression cassettes. We first confirmed that all our Drosha constructs produced similar levels of protein upon cellular transfection, thereby removing the possibility that differential protein expression of our constructs was the ultimate effector of miRNA processing activity (Figure 6A). As previously reported, HEK293T cells harbor relatively low levels of mature miRNA-143 (27). Interestingly, these cells also express relatively low levels of endogenous Drosha when compared to other cell lines such as HCT116, NIH3T3, etc. Accordingly, we transfected cells with the GFP vector alone (empty vector), GFP–Drosha WT or the various GFP–Drosha mutated constructs along with an expression plasmid encoding miRNA-143. FACS was used to obtain GFP expressing cells. Real-time PCR was performed to measure miRNA-143 and northern blot was performed to identify pre- and mature miRNA-143 species. Our data revealed that overexpression of GFP–Drosha WT, S300A, S302A, S300E, S302D or S300E/S302D led to the production of pre- and mature miRNA-143 (Figure 6B and D). However, cells expressing GFP–DroshaS300/302A or GFP–Drosha C-terminus (aa 391–1374) did not retain the ability to produce mature or pre-miRNA-143. These results were further validated by miRNA sensor assays. For example, co-transfection of the sensor along with GFP–Drosha led to a statistically significant reduction in Renilla activity compared to empty vector. GFP–DroshaS300A, S302A, S300E, S302D or S300E/S302D behaved similarly to GFP–Drosha. In contrast, cellular introduction of GFP–DroshaS300/302A had no significant effect on sensor activity compared to empty vector (Figure 6C). We then repeated this experiment but used sensors for miRNA-26b and 125a. Again, similar results were encountered with the stereotypical loss of miRNA activity in transfections involving only GFP–DroshaS300/302A or GFP–Drosha C-terminus (aa 391–1374; Supplementary Figure S3). Lastly, we further reasoned that these nonfunctional Drosha mutants should impede the processing of pri-miRNA and thereby lead to an accumulation of this species. Indeed, real-time PCR quantification of pri-miRNA-143 revealed a 2-fold increase in experiments involving the cytoplasmic localized Drosha variants [GFP–DroshaS300/302A or GFP–Drosha C-terminus (aa 391–1374)] compared to bonafide nuclear localized variants (Supplementary Figure S4).Figure 6.

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
Related in: MedlinePlus