Autoinhibition of human dicer by its internal helicase domain - PubMed (original) (raw)

Autoinhibition of human dicer by its internal helicase domain

Enbo Ma et al. J Mol Biol. 2008.

Abstract

Dicer, a member of the ribonuclease III family of enzymes, processes double-stranded RNA substrates into approximately 21- to 27-nt products that trigger sequence-directed gene silencing by RNA interference. Although the mechanism of RNA recognition and length-specific cleavage by Dicer has been established, the way in which dicing activity is regulated is unclear. Here, we show that the N-terminal domain of human Dicer, which is homologous to DExD/H-box helicases, substantially attenuates the rate of substrate cleavage. Deletion or mutation of this domain activates human Dicer in both single- and multiple-turnover assays. The catalytic efficiency (k(cat)/K(m)) of the deletion construct is increased by 65-fold over that exhibited by the intact enzyme. Kinetic analysis shows that this activation is almost entirely due to an enhancement in k(cat). Modest stimulation of catalysis by the full-length Dicer enzyme was observed in the presence of the TAR-RNA binding protein, which physically interacts with the DExD/H-box domain. These results suggest that the DExD/H-box domain likely disrupts the functionality of the Dicer active site until a structural rearrangement occurs, perhaps upon assembly with its molecular partners.

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Figures

Figure 1

Figure 1. Domain structure and expression of human Dicer (hDcr)

A, Domain structure of hDcr variants; B, 10% SDS-PAGE analysis of recombinant hDcr proteins; C, Single time-point dsRNase activity assays (60 min., 37□, 60 nM protein, 2 nM 32P-labeled 37ab RNA); M, protein size marker.

Figure 2

Figure 2. Single-turnover activity of hDcr proteins

A, Substrates and dicing reactions. Left panels, perfect-duplex dsRNA substrate, 37ab; right panels, hairpin pre-miRNA substrate, pre-hlet-7; asterisks (*) indicate 5′-end labeled with 32P. hDcr generates two products from RNA 37ab, 22-nt (P1) or 15-nt (P2), and one product (P) from pre-hlet-7. Relative rates of P1 and P2 production were the same for all hDcrs tested and were combined to give the total cleavage product for 37ab. B, Single-turnover reaction of hDcrs (60 nM) with 2 nM (3000 c.p.m.) duplex RNA 37ab (left panel) or pre-hlet-7 (right panel); values are the average from two independent experiments. Data were fit to the equation S_=(a-b)exp(−_k_obsd_t)+b, where S is the fraction of dsRNA cleaved at each time point, a is the fraction of dsRNA at the beginning of the reaction, b is the fraction of dsRNA at the reaction plateau (t-->∞), and _k_obsd is the observed rate constant; _k_obsd values (fmol/min) for the 37ab substrate were as follows: hDcr (wildtype), 0.32; hWalker, 0.8; Δhelicase (Δhel), 2; ΔDUF, 0.0034; ΔRBD, 0.2; 2DD, 0.46; _k_obsd values (fmol/min) for the pre-hlet-7 substrate were as follows: hDcr (wildtype), 3; hWalker, 2; Δhelicase, 2.2; ΔDUF, 2.6; ΔRBD, 0.74; 2DD, 0.74; C, summary of initial reaction rates of hDcr variants calculated for 20% substrate cleavage; S, RNA substrate; FL, wild-type hDicer; hW, hWalker mutant; dhel, Δhelicase mutant; dDUF, ΔDUF mutant; dRBD, ΔRBD mutant.

Figure 3

Figure 3. Duplex RNA/hDcr dissociation constants

Equilibrium filter binding assays for hDcr proteins complexed with 37ab RNA (A) or pre-hlet-7 RNA (B). Values are averages from two independent assays. C, summary of dissociation constants (Kd, nM).

Figure 4

Figure 4. Steady-state kinetic analysis of wild-type and Δhelicase hDcr constructs

Plots of initial velocity versus substrate concentration: A, hDcr; and B, Δhelicase. C, summary of kinetic values.

Figure 5

Figure 5. Dicer-TRBP interaction and kinetics

A, size exclusion chromatography of the hDcr-TRBP complex formed by incubation of hDcr (molecular weight 219 kDa; 2.5 nmol) and excess TRBP (molecular weight 39 kDa; 9 nmol) in 20 μl dicing buffer for 60 min. on ice; chromatogram peak C, hDcr-TRBP complex; chromatogram peak T, TRBP; chromatogram peak hD, hDcr. SDS-PAGE gel analysis of fractions are shown below each chromatogram. B, multiple-turnover assay for 37ab cleavage using 100 nM dsRNA and 5 nM hDcr or chromatographically pure hDcr-TRBP; the data shown are representative of two independent experiments.

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