Limits...
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.

Show MeSH
CLIMP-63 co-localizes with Dicer proteins in cultured human cells. HeLa cells transiently transfected with pcDNA-Dicer and pcDNA-CLIMP-63–HA vectors were fixed using formaldehyde (4%) and permeabilized using Triton X-100 (1%). (A) Cells were visualized by differential interference contrast (DIC). (B) Nuclei were stained using 1,5-bis (2-(di-methylamino)ethylamino)-4,8-dihydroxyanthracène-9,10-dione (DRAQ5) (in blue). (C, D) CLIMP-63–HA protein was labeled with a polyclonal anti-HA antibody and a secondary anti-rabbit-IgG coupled to AlexaFluor 546 fluorophore (in red) (C), whereas Dicer protein was labeled using monoclonal anti-Dicer antibody and a secondary murine anti-IgG coupled to AlexaFluor 488 fluorophore (in green) (D). (E) The merged image of panels B, C and D. Panel F represents an enlarged area of the white frame shown in (E). Proteins were visualized using a confocal microscope (Quorum spinning Disc Wave Fx, Quorum Technologies) and a 63X lens.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks903-F7: CLIMP-63 co-localizes with Dicer proteins in cultured human cells. HeLa cells transiently transfected with pcDNA-Dicer and pcDNA-CLIMP-63–HA vectors were fixed using formaldehyde (4%) and permeabilized using Triton X-100 (1%). (A) Cells were visualized by differential interference contrast (DIC). (B) Nuclei were stained using 1,5-bis (2-(di-methylamino)ethylamino)-4,8-dihydroxyanthracène-9,10-dione (DRAQ5) (in blue). (C, D) CLIMP-63–HA protein was labeled with a polyclonal anti-HA antibody and a secondary anti-rabbit-IgG coupled to AlexaFluor 546 fluorophore (in red) (C), whereas Dicer protein was labeled using monoclonal anti-Dicer antibody and a secondary murine anti-IgG coupled to AlexaFluor 488 fluorophore (in green) (D). (E) The merged image of panels B, C and D. Panel F represents an enlarged area of the white frame shown in (E). Proteins were visualized using a confocal microscope (Quorum spinning Disc Wave Fx, Quorum Technologies) and a 63X lens.

Mentions: Interacting with the resident ER protein CLIMP-63, we confirmed the ER co-localization of Dicer and CLIMP-63–HA proteins in HeLa cells by confocal immunofluorescence microscopy (Figure 7), suggesting that a pool of Dicer proteins is located in the same subcellular compartment than CLIMP-63.Figure 7.


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

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

CLIMP-63 co-localizes with Dicer proteins in cultured human cells. HeLa cells transiently transfected with pcDNA-Dicer and pcDNA-CLIMP-63–HA vectors were fixed using formaldehyde (4%) and permeabilized using Triton X-100 (1%). (A) Cells were visualized by differential interference contrast (DIC). (B) Nuclei were stained using 1,5-bis (2-(di-methylamino)ethylamino)-4,8-dihydroxyanthracène-9,10-dione (DRAQ5) (in blue). (C, D) CLIMP-63–HA protein was labeled with a polyclonal anti-HA antibody and a secondary anti-rabbit-IgG coupled to AlexaFluor 546 fluorophore (in red) (C), whereas Dicer protein was labeled using monoclonal anti-Dicer antibody and a secondary murine anti-IgG coupled to AlexaFluor 488 fluorophore (in green) (D). (E) The merged image of panels B, C and D. Panel F represents an enlarged area of the white frame shown in (E). Proteins were visualized using a confocal microscope (Quorum spinning Disc Wave Fx, Quorum Technologies) and a 63X lens.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks903-F7: CLIMP-63 co-localizes with Dicer proteins in cultured human cells. HeLa cells transiently transfected with pcDNA-Dicer and pcDNA-CLIMP-63–HA vectors were fixed using formaldehyde (4%) and permeabilized using Triton X-100 (1%). (A) Cells were visualized by differential interference contrast (DIC). (B) Nuclei were stained using 1,5-bis (2-(di-methylamino)ethylamino)-4,8-dihydroxyanthracène-9,10-dione (DRAQ5) (in blue). (C, D) CLIMP-63–HA protein was labeled with a polyclonal anti-HA antibody and a secondary anti-rabbit-IgG coupled to AlexaFluor 546 fluorophore (in red) (C), whereas Dicer protein was labeled using monoclonal anti-Dicer antibody and a secondary murine anti-IgG coupled to AlexaFluor 488 fluorophore (in green) (D). (E) The merged image of panels B, C and D. Panel F represents an enlarged area of the white frame shown in (E). Proteins were visualized using a confocal microscope (Quorum spinning Disc Wave Fx, Quorum Technologies) and a 63X lens.
Mentions: Interacting with the resident ER protein CLIMP-63, we confirmed the ER co-localization of Dicer and CLIMP-63–HA proteins in HeLa cells by confocal immunofluorescence microscopy (Figure 7), suggesting that a pool of Dicer proteins is located in the same subcellular compartment than CLIMP-63.Figure 7.

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