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

• Published in: Nucleic acids research Vol. 40; no. 22; pp. 11603 - 11617
• Main Authors: Pépin, Geneviève, Perron, Marjorie P, Provost, Patrick
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-12-2012
• Subjects: DEAD-box RNA Helicases - biosynthesis; DEAD-box RNA Helicases - chemistry; DEAD-box RNA Helicases - metabolism; Dicer protein; Endoplasmic reticulum; Endoplasmic Reticulum - enzymology; Enzyme Stability; Glycosylation; HEK293 Cells; HeLa Cells; Humans; Membrane proteins; Membrane Proteins - metabolism; MicroRNAs - metabolism; miRNA; mRNA; Nucleic Acid Enzymes; Protein interaction; Protein Interaction Domains and Motifs; Reporter gene; ribonuclease A; ribonuclease G; Ribonuclease III - biosynthesis; Ribonuclease III - chemistry; Ribonuclease III - metabolism; RNA Interference; RNA Precursors - metabolism; RNA Processing, Post-Transcriptional; Translation
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gks903

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.