Human Dicer C-terminus functions as a 5-lipoxygenase binding domain - PubMed (original) (raw)

Human Dicer C-terminus functions as a 5-lipoxygenase binding domain

Vildan Dincbas-Renqvist et al. Biochim Biophys Acta. 2009 Feb.

Abstract

Dicer 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. Providing a link between miRNA-mediated regulation of gene expression and inflammation, our results suggest that the formation of miRNAs may be regulated by 5LO in leukocytes and cancer cells expressing this lipoxygenase.

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Figures

Figure 1

5LO interacts with Dicer C-terminal (C-term) domain in the yeast two-hybrid system. (A–B) Yeast strain PJ69-4A was cotransformed with plasmids expressing the Gal4 BD-5LO and Gal4 AD-human Dicer deletion mutants. (A) Transformants were tested for the Ade, His and LacZ reporter genes. +++, denotes yeast growth visible at 2 days; −, denotes absence of yeast growth at 7 days. Color of the colonies was noted at 7 days. RIII, RNase III motifs; dsRBD, dsRNA binding domain. (B) Expression of the Gal4 AD-human Dicer deletion mutant (upper panel) and the Gal4 BD-5LO (lower panel) proteins was confirmed by immunoblot (IB) analysis.

Figure 2

Dicer C-terminal domain colocalizes and interacts with 5LO in transiently transfected human cells. (A) Confocal immunofluorescence microscopy of HeLa cells expressing epitope-tagged 5LO and Dicer C-terminal (C-term) proteins. The merged image demonstrates enrichment and colocalization of 5LO and Dicer C-term proteins at the perinuclear region of HeLa cells. Scale bar = 20 μm. (B) Immunoprecipitation experiments using HEK 293 cells expressing epitope-tagged 5LO and Dicer C-term proteins. Proteins present in the cell lysates (5% input) (lanes 1 to 3) and immunoprecipitates (lanes 4 to 6) were detected by immunoblot analysis (IB) using anti-Flag (upper panels) and anti-5LO (lower panels) antibodies.

Figure 3

Delineation of the smallest 5LO-interacting domain of Dicer to its C-terminal 140 amino acids comprising the dsRBD in the yeast two-hybrid system. (A–C) Yeast strain PJ69-4A was cotransformed with plasmids expressing the Gal4 BD-5LO and Gal4 AD-Dicer deletion mutants. (A) Transformants were tested for the Ade, His and LacZ reporter genes. +++, denotes yeast growth visible at 2 days; −, denotes absence of yeast growth at 7 days. Color of the colonies was noted at 7 days. (B) Expression of the Gal4 AD-Dicer deletion mutant (upper panel) and the Gal4 BD-5LO (lower panel) proteins was confirmed by immunoblot (IB) analysis. (C) Transformants were tested for the His reporter gene in the presence of increasing concentrations of 3-AT. Growth of the colonies was scored when visible at 2 (+++), 4 (++) or 7 (+) days. −, denotes absence of yeast growth at 7 days.

Figure 4

Dicer 5LObd interacts directly with 5LO in vitro. (A–C) GST binding assays (lanes 2 to 6) using GST-Dicer 5LObd (A and C), or GST (B) protein, coupled to GSH-Sepharose 4B beads and incubated in the presence of increasing amounts of recombinant 5LO proteins (A and B). (C) Beads bearing the GST-Dicer 5LObd fusion protein was incubated with 5LO protein in the absence (lane 2) or presence of dsRNA (0.1–25.0 μg) (lanes 3 to 6). Bound 5LO was visualized by immunoblot (IB) analysis using an anti-5LO antibody, and compared with a reference of 5LO (0.02 μg) (lane 1 of each panel).

Figure 5

5LO interacts with Dicer 5LObd via its N-terminal non-catalytic domain. (A) GST binding assays were performed, as described in the legend of Figure panels 4A and 4B, using 0.5 μg 5LO (lanes 5 and 6) or 5LO W13/75/102A (lanes 2 and 3) recombinant protein. (A, left panel) Bound 5LO protein was analyzed by immunoblotting (IB), and compared with a reference (Ref.) of 5LO (0.05 μg) (lane 1). (A, right panel) 5LO and 5LO W13/75/102A mutant proteins (0.15 μg each), analyzed on the same gel by IB analysis, exhibited similar immunoreactivities (lanes 7 and 8). (B) Immunoprecipitation experiments using HEK 293 cells expressing epitope-tagged Dicer C-term and 5LO or 5LO W13/75/102A mutant proteins. Proteins present in the cell lysates (5% input) (lanes 1 to 4) and immunoprecipitates (lanes 5 to 8) were detected by IB using anti-Flag (upper panels) and anti-5LO (lower panels) antibodies.

Figure 6

The smallest 5LObd-containing fragment of Dicer exerts stimulatory effects on 5LO activity. (A–C) 5LO enzyme activity assays. Recombinant human 5LO was incubated in the absence or presence of human Dicer 1650-1912 deletion mutant at a 2:1 or 10:1 ratio, or PC, prior to addition to the 5LO activity assays. After incubation for 10 minutes at room temperature, the reactions were stopped and the 5-HPETE and 5-HETE (A), leukotriene (B) formation and total 5LO products (C) were measured by HPLC by using 17-OH-22:4 (A) or prostaglandin B2 (B) as the internal standard.

Figure 7

5LO modifies the pre-miRNA processing activity of human Dicer. (A–C) Dicer RNase activity assays. Recombinant human Dicer full-length or 1650-1912 mutant protein was incubated in the absence or presence of human 5LO prior to addition of a 32P-labeled microRNA precursor substrate (pre-let-7a-3). (A) Recombinant human Dicer full-length (66 ng) or increasing amounts of the 1650-1912 mutant (lanes 4–8: 5, 20, 100, 500 or 2500 ng and lanes 11–13: 2.5 μg) proteins were incubated in the absence (lanes 1–9) or presence (lanes 10–13) of proteinase K for 10 min at 37°C, prior to addition of a 32P-labeled pre-miRNA substrate (pre-let-7a-3). BSA (1 μg) is annotated as (B) in the figure. (B–C) Recombinant human Dicer full-length (33 ng) or 1650-1912 mutant (100 ng) protein was incubated in the absence (5LO buffer only, denoted as −) or presence of 5LO (panel B, 200 ng) for 30 min at room temperature (B), or at 4°C (C), prior to addition of 32P-labeled pre-let-7a-3. After a 1-h incubation at 37°C, the samples were analyzed by denaturing PAGE and autoradiography. A 10-nt RNA size marker was used.

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Human Dicer C-terminus functions as a 5-lipoxygenase binding domain - PubMed (original) (raw)