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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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Dicer interacts with a luminal portion of CLIMP-63 via its N-terminal protein interaction domain. (A) Identification of various Dicer-interacting clones encoding for CLIMP-63 in a yeast two-hybrid screen of a human lung cDNA library using human Dicer as bait. (B) Delineation of the domain of Dicer involved in mediating the Dicer–CLIMP-63 interaction. Yeast transformants were tested for activation of the adenine (Ade), histidine (His) and LacZ reporter genes.
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gks903-F6: Dicer interacts with a luminal portion of CLIMP-63 via its N-terminal protein interaction domain. (A) Identification of various Dicer-interacting clones encoding for CLIMP-63 in a yeast two-hybrid screen of a human lung cDNA library using human Dicer as bait. (B) Delineation of the domain of Dicer involved in mediating the Dicer–CLIMP-63 interaction. Yeast transformants were tested for activation of the adenine (Ade), histidine (His) and LacZ reporter genes.

Mentions: The yeast two-hybrid CLIMP-63 cDNA clones were found to interact with Dicer-encoded luminal fragments of the protein (Figure 6A). One of the yeast two-hybrid CLIMP-63 cDNA clones was of particular interest, as it contained an in-frame premature stop codon (TAG) in its coding sequence, resulting in a truncated form of the CLIMP-63 protein comprising amino acids 192–258 (Figure 6A). Identified serendipitously, this deletion mutant is the shortest form of CLIMP-63 capable of interacting with Dicer.Figure 6.


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

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

Dicer interacts with a luminal portion of CLIMP-63 via its N-terminal protein interaction domain. (A) Identification of various Dicer-interacting clones encoding for CLIMP-63 in a yeast two-hybrid screen of a human lung cDNA library using human Dicer as bait. (B) Delineation of the domain of Dicer involved in mediating the Dicer–CLIMP-63 interaction. Yeast transformants were tested for activation of the adenine (Ade), histidine (His) and LacZ reporter genes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks903-F6: Dicer interacts with a luminal portion of CLIMP-63 via its N-terminal protein interaction domain. (A) Identification of various Dicer-interacting clones encoding for CLIMP-63 in a yeast two-hybrid screen of a human lung cDNA library using human Dicer as bait. (B) Delineation of the domain of Dicer involved in mediating the Dicer–CLIMP-63 interaction. Yeast transformants were tested for activation of the adenine (Ade), histidine (His) and LacZ reporter genes.
Mentions: The yeast two-hybrid CLIMP-63 cDNA clones were found to interact with Dicer-encoded luminal fragments of the protein (Figure 6A). One of the yeast two-hybrid CLIMP-63 cDNA clones was of particular interest, as it contained an in-frame premature stop codon (TAG) in its coding sequence, resulting in a truncated form of the CLIMP-63 protein comprising amino acids 192–258 (Figure 6A). Identified serendipitously, this deletion mutant is the shortest form of CLIMP-63 capable of interacting with Dicer.Figure 6.

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