Discriminatory RNP remodeling by the DEAD-box protein DED1

Abstract

DExH/D proteins catalyze NTP-driven rearrangements of RNA and RNA-protein complexes during most aspects of RNA metabolism. Although the vast majority of DExH/D proteins displays virtually no sequence-specificity when remodeling RNA complexes in vitro, the enzymes clearly distinguish between a large number of RNA and RNP complexes in a physiological context. It is unknown how this discrimination between potential substrates is achieved. Here we show one possible way by which a non-sequence specific DExH/D protein can discriminately remodel similar RNA complexes. We have measured in vitro the disassembly of model RNPs by two distinct DExH/D proteins, DED1 and NPH-II. Both enzymes displace the U1 snRNP from a tightly bound RNA in an active, ATP-dependent fashion. However, DED1 cannot actively displace the protein U1A from its binding site, whereas NPH-II can. The dissociation rate of U1A dictates the rate by which DED1 remodels RNA complexes with U1A bound. We further show that DED1 disassembles RNA complexes with slightly altered U1A binding sites at different rates, but only when U1A is bound to the RNA. These findings suggest that the "inability" to actively displace other proteins from RNA can provide non-sequence specific DExH/D proteins with the capacity to disassemble similar RNA complexes in a discriminatory fashion. In addition, our study illuminates possible mechanisms for protein displacement by DExH/D proteins.

FIGURE 1.

U1 snRNP model system. (A) U1 snRNP bound to RNA containing a 5′ splice site. Lines in the U1 snRNP symbolize the U1 snRNA, gray shapes are U1 snRNP specific proteins on their approximate binding sites (Stark et al. 2001). Substrate RNA is depicted by the curved line; the sequence surrounding the 5′ splice site and the complementary part in the U1 snRNA are indicated. The asterisk shows the radiolabel at the 5′ end of the substrate RNA. (B) Measurement of U1 snRNP dissociation and displacement. U1 snRNP is immunoprecipiated by an antibody against the U1A protein. Thus, radiolabeled substrate RNA bound to U1 snRNP precipitates as well. Radiolabeled substrate RNA released from the U1 snRNP (through spontaneous dissociation or by displacement) is found in the supernatant. (C) Representative time course for spontaneous dissociation of the U1 snRNP from the RNA. Reactions were preformed as described under Materials and Methods and data points were fitted to the integrated rate law for a heterogeneous reaction with two first order components. A fraction of ∼0.05 of the U1 snRNP dissociated faster than experimental accessible, the majority of U1 snRNP (∼95%) dissociated with a rate constant of k _{D [U1 snRNP]} = (1.2 ± 0.1) × 10⁻³ min⁻¹.

FIGURE 2.

Displacement of the U1 snRNP by NPH-II. (A) Representative time course for U1 snRNP displacement by NPH-II in the presence (filled circles) and in the absence (open circles) of ATP. Data were fitted to the integrated rate law for a first order reaction. With ATP, the displacement rate constant is k _displ > 6 min⁻¹ (limit is given since the reaction amplitude has already reached >90% of its final value at the first timepoint). (B) Representative PAGE of an U1 snRNP displacement reaction performed in the presence of a control duplex to test the integrity of the NPH-II helicase activity. The duplex of a control RNA at 0.5 nM is unwound, reaction time is given underneath panel C. No significant degradation of the U1 snRNP substrate RNA (mRNA) is detected. The U1 snRNP substrate RNA is labeled with more radioactivity than the control duplex to minimize the influence of the radiolabeled control RNA on the measurement of U1 snRNP displacement. (C) Representative PAGE of U1 snRNP substrate RNA (mRNA) during the reaction shown in panel B, but at lower detection intensity to illustrate the virtually unchanged level of RNA throughout the reaction. (D) Amount of radioactivity in U1 snRNP substrate RNA normalized to the amount of radioactivity in the control duplex (panel B). The constant value indicates no significant degradation of either RNA during the displacement reaction.

FIGURE 3.

Displacement of the U1 snRNP by DED1. Representative time course for U1 snRNP displacement by DED1 in the presence (filled circles) and in the absence (open circles) of ATP. Data were fitted to the integrated rate law for a first order reaction. With ATP, the displacement rate constant was k _displ > 6.4 min⁻¹.

FIGURE 4.

U1A-based RNP. (A) RNP design. Sequence of the RNA strands. U1A binds to the single-stranded loops. (B) Equilibrium binding of U1A to the RNA. Data points represent the average of at least three independent measurements. Error bars represent one standard deviation. Data were fit to the Hill-equation (K _D = 5.1 ± 0.5 nM, n = 1.4 ± 0.1). (C) Spontaneous dissociation of U1A from the RNA. The representative time course was fit to the sum of two exponentials (dissociation rate constants were, for the first phase: k ^I _d = 0.24 ± 0.1 min⁻¹, and for the second phase: k ^II _d = (1.8 ± 0.2) × 10⁻³ min⁻¹).

FIGURE 5.

NPH-II but not DED1 actively displaces U1A from the RNA. (A) Displacement of U1A by NPH-II (representative PAGE). Protein displacement is indicated by the ability of the DExH/D protein to separate the RNA strands with U1A bound (lane 3, Jankowsky et al. 2001). Mobilities of the RNA complex and the single-stranded RNA are indicated by cartoons on the left. Reactions were allowed to proceed for 5 min. Lanes are as follows: (1) NPH-II only; (2) NPH-II and ATP; (3) NPH-II and ATP with U1A; (4) boiled control. (B) Inability of DED1 to actively displace U1A from the RNA (representative PAGE). Lanes are as follows: (1) DED1 only; (2) DED1 and ATP; (3) DED1 and ATP with U1A; (4) boiled control.

FIGURE 6.

Time course of DED1-catalyzed unwinding of the RNA complex with and without U1A bound. RNA strand separation by DED1 with U1A bound (open circles) and in the absence of U1A (filled circles). Data points represent the average of least two independent measurements, error bars indicate one standard deviation. The resulting time course for the reaction without U1A was fit against the integrated rate law for a homogenous first-order process, yielding an unwinding rate constant of k _r ^[RNA] = 0.98 ± 0.02 min⁻¹. The time course for the reaction with U1A was fit to the sum of two exponentials, yielding unwinding rate constants for the first phase of k ^I _r ^[RNP] = 0.10 ± 0.01 min⁻¹, and for the second phase of k ^II _r ^[RNP] = (8.0 ± 1.7) × 10⁻⁴ min⁻¹.

FIGURE 7.

Altered U1A-based RNP. (A) RNP design. Three nucleotides were deleted from the upper RNA strand (indicated by triangles) of the RNA complex with the authentic U1A binding site (Fig. 4A). (B) Equilibrium binding of U1A to the altered RNA complex. Data points represent the average of three independent measurements. Data were fit to the Hill-equation (K _D = 13.5 ± 0.7 nM, n = 1.9 ± 0.2). (C) Spontaneous dissociation of U1A from the RNA. The representative time course was fit to the sum of two exponentials. Dissociation rate constants were, for the first phase: k ^I _d = 2.2 ± 0.6 min⁻¹, and for the second phase: k ^II _d = (5.3 ± 0.7) × 10⁻³ min⁻¹.

FIGURE 8.

DED1 but not NPH-II disassembles the two RNA complexes in a discriminatory fashion. (A) DED1 disassembles the altered RNA complex more efficiently than the complex with the authentic U1A binding site when U1A is bound (lane 3) but not without U1A (lane 2). Reactions were allowed to proceed for 5 min. Mobilities of the RNA complexes and the single-stranded RNAs are indicated by cartoons on the left of the representative PAGE. The altered RNA is in gray, the RNA with the authentic U1A binding site in black. Lanes are as follows: (1) DED1 only; (2) DED1 and ATP; (3) DED1 and ATP with U1A; (4) boiled control. (B) NPH-II disassembles both, altered RNA complex and the complex with the authentic U1A binding site with comparable efficiency. Reactions were allowed to proceed for 5 min. Lanes are as follows: (1) NPH-II only; (2) NPH-II and ATP; (3) NPH-II and ATP with U1A; (4) boiled control.

FIGURE 9.

Representative time courses of DED1-catalyzed unwinding of both RNA complexes with and without U1A bound. DED1-catalyzed strand separation of both, the RNA complex with altered and authentic U1A binding site with and without U1A bound (filled circle: wt RNA, no U1A; filled diamond: altered RNA, no U1A; open circle: wt RNA with U1A bound; open diamond: altered RNA with U1A bound). Strand separation of the altered RNA complex without U1A was fit to a single exponential, yielding an unwinding rate constant k _r ^[RNA] = 1.63 ± 0.15 min⁻¹. Strand separation of the altered RNA complex with U1A present was fit to a sum of two exponentials yielding an unwinding rate constant for the first phase: k ^I _r ^[RNP] = 0.33 ± 0.04 min⁻¹, and for the second phase: k ^II _r ^[RNP] = (4.8 ± 0.7) × 10⁻³ min⁻¹. Kinetic data for strand separation of the RNA complex with the authentic U1A binding (with and without U1A) site are reported in Figure 6.