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Regulation of human Dicer by the resident ER membrane protein CLIMP-63.

Pépin G, Perron MP, Provost P - Nucleic Acids Res. (2012)

Bottom Line: CLIMP-63 is required for stabilizing Dicer protein and for optimal regulation of a reporter gene coupled to the 3' untranslated region of HMGA2 mRNA in human cells.Interacting with a portion of the luminal domain of CLIMP-63 and within minutes of its synthesis, our results suggest that Dicer transits through the ER, is glycosylated and can be secreted by cultured human cells with CLIMP-63.Our findings define CLIMP-63 as a novel protein interactor and regulator of Dicer function, involved in maintaining Dicer protein levels in human cells.

View Article: PubMed Central - PubMed

Affiliation: CHUQ Research Center/CHUL, 2705 Blvd Laurier, QC, G1V 4G2, Canada.

ABSTRACT
The ribonuclease Dicer plays a central role in the microRNA pathway by catalyzing the formation of microRNAs, which are known to regulate messenger RNA (mRNA) translation. In order to improve our understanding of the molecular context in which Dicer functions and how it is regulated in human cells, we sought to expand its protein interaction network by employing a yeast two-hybrid screening strategy. This approach led to the identification and characterization of cytoskeleton-linking endoplasmic reticulum (ER) membrane protein of 63 kDa (CLIMP-63) as a novel Dicer-interacting protein. CLIMP-63 interacts with Dicer to form a high molecular weight complex, which is electrostatic in nature, is not mediated by RNA and is catalytically active in pre-microRNA processing. CLIMP-63 is required for stabilizing Dicer protein and for optimal regulation of a reporter gene coupled to the 3' untranslated region of HMGA2 mRNA in human cells. Interacting with a portion of the luminal domain of CLIMP-63 and within minutes of its synthesis, our results suggest that Dicer transits through the ER, is glycosylated and can be secreted by cultured human cells with CLIMP-63. Our findings define CLIMP-63 as a novel protein interactor and regulator of Dicer function, involved in maintaining Dicer protein levels in human cells.

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The Dicer•CLIMP-63 complex is electrostatic in nature and is not mediated by RNA. (A–C) CLIMP-63–HA IP, prepared from HEK 293 cells transiently expressing Flag–Dicer and/or CLIMP-63–HA proteins, were incubated in the presence of increasing concentrations of NaCl (A), in the absence or presence of RNases A/T1/V1 (B), or without or with phosphatase inhibitors, CIAP, CaCl2 or EGTA (C). The presence of Dicer and CLIMP-63 in the immune complexes was monitored by western blot.
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gks903-F2: The Dicer•CLIMP-63 complex is electrostatic in nature and is not mediated by RNA. (A–C) CLIMP-63–HA IP, prepared from HEK 293 cells transiently expressing Flag–Dicer and/or CLIMP-63–HA proteins, were incubated in the presence of increasing concentrations of NaCl (A), in the absence or presence of RNases A/T1/V1 (B), or without or with phosphatase inhibitors, CIAP, CaCl2 or EGTA (C). The presence of Dicer and CLIMP-63 in the immune complexes was monitored by western blot.

Mentions: To characterize further this interaction, we exposed Flag–Dicer•CLIMP-63–HA protein complexes, derived from transiently transfected HEK 293 cells, to various conditions. As shown in Figure 2A, the association between Dicer and CLIMP-63 was gradually weakened by increasing the concentration of salt (NaCl), suggesting that the formation of the Dicer•CLIMP-63 protein complex is electrostatic, rather than hydrophobic, in nature. Because Dicer can bind to RNA and that RNA can assemble proteins that do not interact with each other, we exposed CLIMP-63–HA immune complexes to RNases A/T1/V1. This treatment had no effect, indicating that the Dicer–CLIMP-63 protein interaction is unlikely to be mediated by RNA (Figure 2B). The phosphorylation status of both Dicer (our unpublished data) and CLIMP-63 (28) proteins did not seem to influence their ability to interact together, as the presence of phosphatase inhibitors, which preserved protein phosphorylation, or calf intestinal alkaline phosphatase (CIAP), which induced protein dephosphorylation, did not modify the interaction (Figure 2C, lanes 1–7). Dicer•CLIMP-63 complex formation was also not influenced by the presence of CaCl2 or EGTA, which is a chelator of divalent cations (Figure 2C, lanes 8 and 9). These latter findings are particularly relevant, because the ER is an organelle that stocks high concentrations of calcium (29). Taken together, our results suggest that the Dicer•CLIMP-63 complex is electrostatic in nature, is independent of the phosphorylation status of the proteins and of Ca2+ and is not mediated by RNA.Figure 2.


Regulation of human Dicer by the resident ER membrane protein CLIMP-63.

Pépin G, Perron MP, Provost P - Nucleic Acids Res. (2012)

The Dicer•CLIMP-63 complex is electrostatic in nature and is not mediated by RNA. (A–C) CLIMP-63–HA IP, prepared from HEK 293 cells transiently expressing Flag–Dicer and/or CLIMP-63–HA proteins, were incubated in the presence of increasing concentrations of NaCl (A), in the absence or presence of RNases A/T1/V1 (B), or without or with phosphatase inhibitors, CIAP, CaCl2 or EGTA (C). The presence of Dicer and CLIMP-63 in the immune complexes was monitored by western blot.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526294&req=5

gks903-F2: The Dicer•CLIMP-63 complex is electrostatic in nature and is not mediated by RNA. (A–C) CLIMP-63–HA IP, prepared from HEK 293 cells transiently expressing Flag–Dicer and/or CLIMP-63–HA proteins, were incubated in the presence of increasing concentrations of NaCl (A), in the absence or presence of RNases A/T1/V1 (B), or without or with phosphatase inhibitors, CIAP, CaCl2 or EGTA (C). The presence of Dicer and CLIMP-63 in the immune complexes was monitored by western blot.
Mentions: To characterize further this interaction, we exposed Flag–Dicer•CLIMP-63–HA protein complexes, derived from transiently transfected HEK 293 cells, to various conditions. As shown in Figure 2A, the association between Dicer and CLIMP-63 was gradually weakened by increasing the concentration of salt (NaCl), suggesting that the formation of the Dicer•CLIMP-63 protein complex is electrostatic, rather than hydrophobic, in nature. Because Dicer can bind to RNA and that RNA can assemble proteins that do not interact with each other, we exposed CLIMP-63–HA immune complexes to RNases A/T1/V1. This treatment had no effect, indicating that the Dicer–CLIMP-63 protein interaction is unlikely to be mediated by RNA (Figure 2B). The phosphorylation status of both Dicer (our unpublished data) and CLIMP-63 (28) proteins did not seem to influence their ability to interact together, as the presence of phosphatase inhibitors, which preserved protein phosphorylation, or calf intestinal alkaline phosphatase (CIAP), which induced protein dephosphorylation, did not modify the interaction (Figure 2C, lanes 1–7). Dicer•CLIMP-63 complex formation was also not influenced by the presence of CaCl2 or EGTA, which is a chelator of divalent cations (Figure 2C, lanes 8 and 9). These latter findings are particularly relevant, because the ER is an organelle that stocks high concentrations of calcium (29). Taken together, our results suggest that the Dicer•CLIMP-63 complex is electrostatic in nature, is independent of the phosphorylation status of the proteins and of Ca2+ and is not mediated by RNA.Figure 2.

Bottom Line: CLIMP-63 is required for stabilizing Dicer protein and for optimal regulation of a reporter gene coupled to the 3' untranslated region of HMGA2 mRNA in human cells.Interacting with a portion of the luminal domain of CLIMP-63 and within minutes of its synthesis, our results suggest that Dicer transits through the ER, is glycosylated and can be secreted by cultured human cells with CLIMP-63.Our findings define CLIMP-63 as a novel protein interactor and regulator of Dicer function, involved in maintaining Dicer protein levels in human cells.

View Article: PubMed Central - PubMed

Affiliation: CHUQ Research Center/CHUL, 2705 Blvd Laurier, QC, G1V 4G2, Canada.

ABSTRACT
The ribonuclease Dicer plays a central role in the microRNA pathway by catalyzing the formation of microRNAs, which are known to regulate messenger RNA (mRNA) translation. In order to improve our understanding of the molecular context in which Dicer functions and how it is regulated in human cells, we sought to expand its protein interaction network by employing a yeast two-hybrid screening strategy. This approach led to the identification and characterization of cytoskeleton-linking endoplasmic reticulum (ER) membrane protein of 63 kDa (CLIMP-63) as a novel Dicer-interacting protein. CLIMP-63 interacts with Dicer to form a high molecular weight complex, which is electrostatic in nature, is not mediated by RNA and is catalytically active in pre-microRNA processing. CLIMP-63 is required for stabilizing Dicer protein and for optimal regulation of a reporter gene coupled to the 3' untranslated region of HMGA2 mRNA in human cells. Interacting with a portion of the luminal domain of CLIMP-63 and within minutes of its synthesis, our results suggest that Dicer transits through the ER, is glycosylated and can be secreted by cultured human cells with CLIMP-63. Our findings define CLIMP-63 as a novel protein interactor and regulator of Dicer function, involved in maintaining Dicer protein levels in human cells.

Show MeSH
Related in: MedlinePlus