Characterization of the interactions between mammalian PAZ PIWI domain proteins and Dicer - PubMed (original) (raw)

Characterization of the interactions between mammalian PAZ PIWI domain proteins and Dicer

Nasser Tahbaz et al. EMBO Rep. 2004 Feb.

Abstract

PAZ PIWI domain (PPD) proteins, together with the RNA cleavage products of Dicer, form ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs). RISCs mediate gene silencing through targeted messenger RNA cleavage and translational suppression. The PAZ domains of PPD and Dicer proteins were originally thought to mediate binding between PPD proteins and Dicer, although no evidence exists to support this theory. Here we show that PAZ domains are not required for PPD protein-Dicer interactions. Rather, a subregion of the PIWI domain in PPD proteins, the PIWI-box, binds directly to the Dicer RNase III domain. Stable binding between PPD proteins and Dicer was dependent on the activity of Hsp90. Unexpectedly, binding of PPD proteins to Dicer inhibits the RNase activity of this enzyme in vitro. Lastly, we show that PPD proteins and Dicer are present in soluble and membrane-associated fractions, indicating that interactions between these two types of proteins may occur in multiple compartments.

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Figures

Figure 1

Figure 1

PIWI domains interact with Dicer. (A) Schematic representation of AGO2 and Hiwi GST-fusion proteins used for binding studies. PIWI domains, PIWI-box (Pb) and PAZ domains are indicated. (B) GST and GST–PPD fusion proteins were co-expressed with CFP–Dicer in HEK293T cells. Proteins were purified on glutathione–sepharose beads, resolved by SDS–PAGE, and visualized by immunoblotting with antibodies to GST and GFP.

Figure 2

Figure 2

The Dicer RNase III domain is required for binding to AGO2. (A) Schematic representation of Dicer and GST-fusion proteins used for binding studies. The three regions of Dicer are indicated: ATPase/helicase, PAZ and RNase III domains. The RNase III domain is further subdivided to indicate the presence of two RNase III motifs A and B and a dsRNA-binding domain (DSRM; D). (B) Plasmids encoding GST or GST fused to domains of Dicer were transfected into a cell line overexpressing AGO2. Following GST pulldowns, proteins were visualized by silver staining (top panel) and immunoblotting (bottom panels). Arrowheads point to the silverstained GST-fusion proteins, whereas AGO2 is indicated by asterisks.

Figure 3

Figure 3

PPD–Dicer interactions are direct. (A) Protein A–sepharose beads coated with affinity-purified Abs against Dicer or the control protein GAR1 were incubated with mixtures of purified Dicer and GST–AGO2 (upper panel), GST–Hiwi (middle panel) or GST alone (lower panel). Bead-associated (B) and unbound (S) material was subjected to SDS–PAGE and immunoblotting with anti-GST Abs. (B) Different combinations of pGBKT7 (AGO2 or Hiwi subdomains) and pGADT7 (Dicer subdomains) constructs were transformed into AH109 strain and tested for interaction by growth on selective media. Four serial dilutions were spotted for each combination. Scoring ranged from − to ++++ depending upon how well the strains grew on media lacking leucine, tryptophan, adenine and histidine. Graded examples of some PPD–Dicer interactions are shown. A complete listing of all the tested interactions is shown in supplementary table 2 online.

Figure 4

Figure 4

PPD proteins inhibit the RNase activity of Dicer in vitro. (A) Effect of Hiwi and AGO2 on Dicer-mediated cleavage of dsRNA. Increasing amounts (indicated in B) of GST–Hiwi, GST–AGO2 or GST alone were added to the cleavage reactions containing 130-bp 32P-labelled dsRNA substrate. Reaction products were analysed by 8 M urea PAGE. The positions of 14-, 21- and 27-nt size markers are indicated (the 21-nt marker was only visible upon longer exposure). (B) PhosphorImager quantitation of the cleavage reactions from a typical experiment.

Figure 5

Figure 5

GD inhibits the binding of PPD proteins to Dicer. GST–AGO2 (A) or GST–Hiwi (B) were transiently co-expressed with CFP–Dicer in HEK293T cells. Cells were incubated with (+) or without (−) 5 μM GD for 2 h before lysis. Proteins were recovered on glutathione–sepharose beads and visualized by immunoblotting. CFP–Dicer was detected using a polyclonal anti-GFP Ab, whereas GST–AGO2 and GST–Hiwi were detected using anti-GST Abs.

Figure 6

Figure 6

Dicer and PPD proteins are present in soluble and membrane-associated pools. Homogenates of HEK293T cells transiently expressing GST–Hiwi were subjected to membrane flotation assays on discontinuous sucrose gradients. Fractions were collected from the tops (T) of gradients, TCA precipitated, resolved by SDS–PAGE and subjected to immunoblotting with Abs specific for AGO2, GST, Dicer, calnexin and Hsp90. Membrane-associated material is found at the top of the gradient, whereas soluble proteins remain in the bottom (B) three fractions.

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