RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated~21±23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produced an apparently mature let-7 RNA. Mg 2+ was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer´dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer-related processes.

We have recently identified coactosin-like protein (CLP) in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. In this report, we demonstrate a direct interaction between 5LO and CLP. 5LO associated with CLP, which was expressed as a glutathione S-transferase fusion protein, in a dose-dependent manner. Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP proteins transiently expressed in human embryonic kidney 293 cells revealed the presence of CLP in 5LO immunoprecipitates. In reciprocal experiments, 5LO was detected in CLP immunoprecipitates. Non-denaturing polyacrylamide gel electrophoresis and cross-linking experiments showed that 5LO binds CLP in a 1:1 molar stoichiometry in a Ca 2؉ -independent manner. Site-directed mutagenesis suggested an important role for lysine 131 of CLP in mediating 5LO binding. In view of the ability of CLP to bind 5LO and filamentous actin (F-actin), we determined whether CLP could physically link 5LO to actin filaments. However, no F-actin-CLP⅐5LO ternary complex was observed. In contrast, 5LO appeared to compete with F-actin for the binding of CLP. Moreover, 5LO was found to interfere with actin polymerization. Our results indicate that the 5LO-CLP and CLP-F-actin interactions are mutually exclusive and suggest a modulatory role for 5LO in actin dynamics. 5-Lipoxygenase (5LO)1 is of central importance in cellular leukotriene (LT) synthesis. This enzyme converts arachidonic acid released from the membranes by the cytosolic phospholipase A 2 into 5(S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and subsequently into the epoxide intermediate LTA 4 (1). LTA 4 is further metabolized into LTB 4 by the LTA 4 hydrolase or into LTC 4 through the action of the LTC 4 synthase. LTC 4 is then sequentially degraded into LTD 4 and LTE 4 .Whereas LTB 4 exerts potent stimulatory effects on various leukocyte functions, including chemotaxis, adhesion, degranulation, and aggregation, the cysteinyl-LTs (LTC 4 , LTD 4 , and LTE 4 ) are known to contract airway smooth muscle, increase vascular permeability, and promote mucus secretion (2). 5LO and LTs are, therefore, key components involved in inflammatory disorders, including arthritis, asthma, and allergic reactions.Recently, novel modulatory mechanisms determining cellular 5LO activity were identified. 5LO is phosphorylated by p38 mitogen-activated protein kinase-activated protein (MAPKAP) kinases prepared from stimulated myeloid cells (3). In addition, Mg 2ϩ increases 5LO activity in vitro (4). Furthermore, a stimulatory Ca 2ϩ binding site has been localized in the N-terminal domain of 5LO, that may function as a C2 domain in the calcium regulation of 5LO catalytic activity (5). C2 domains have also been shown to mediate protein-protein interactions (6).Additional lines of evidence indicate that cellular 5LO activity and distribution is regulated by interaction with other proteins. For example, the subcellular distribution of 5LO differs among cell types and changes in response to various stimuli. In particular...

Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction.

SummaryDicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) in the vast majority of eukaryotes. In human, Dicer has been shown to interact with cellular proteins via its N-terminal domain. Here, we demonstrate the ability of Dicer C-terminus to interact with 5-lipoxygenase (5LO), an enzyme involved in the biosynthesis of inflammatory mediators, in vitro and in cultured human cells. Yeast two-hybrid and GST binding assays delineated the smallest 5-lipoxygenase binding domain (5LObd) of Dicer to its C-terminal 140 amino acids comprising the double-stranded RNA (dsRNA) binding domain (dsRBD). The Dicer 5LObd-5LO association was disrupted upon Ala substitution of Trp residues 13, 75 and 102 in 5LO, suggesting that the Dicer 5LObd may recognize 5LO via its N-terminal C2-like domain. Whereas a catalytically active 5LObd-containing Dicer fragment was found to enhance 5LO enzymatic activity in vitro, human 5LO modified the miRNA precursor processing activity of Dicer. In addition to revealing the dual RNA and protein binding properties of Dicer C-terminus, our results may provide a link between miRNA-mediated regulation of gene expression and inflammation.

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