dsRNA Binding Properties of RDE-4 and TRBP Reflect Their Distinct Roles in RNAi

doi:10.1016/j.jmb.2008.10.002

Journal of Molecular Biology

Volume 384, Issue 4, 26 December 2008, Pages 967-979

https://doi.org/10.1016/j.jmb.2008.10.002 Get rights and content

Abstract

Double-stranded RNA (dsRNA)-binding proteins facilitate Dicer functions in RNA interference. Caenorhabditis elegans RDE-4 facilitates cleavage of long dsRNA to small interfering RNA (siRNA), while human trans-activation response RNA-binding protein (TRBP) functions downstream to pass siRNA to the RNA-induced silencing complex. We show that these distinct in vivo roles are reflected in in vitro binding properties. RDE-4 preferentially binds long dsRNA, while TRBP binds siRNA with an affinity that is independent of dsRNA length. These properties are mechanistically based on the fact that RDE-4 binds cooperatively, via contributions from multiple domains, while TRBP binds noncooperatively. Our studies offer a paradigm for how dsRNA-binding proteins, which are not sequence specific, discern dsRNA length. Additionally, analyses of the ability of RDE-4 deletion constructs and RDE-4/TRBP chimeras to reconstitute Dicer activity suggest RDE-4 promotes activity using its dsRNA-binding motif 2 to bind dsRNA, its linker region to interact with Dicer, and its C-terminus for Dicer activation.

Introduction

The RNase III enzyme Dicer is a key enzyme in the RNA interference (RNAi) pathway and, in all organisms studied thus far, functions in association with double-stranded RNA (dsRNA)-binding proteins (dsRBPs).¹ For example, human Dicer associates with trans-activation response RNA-binding protein (TRBP) and PACT,²^–⁴ Drosophila Dicer-2 associates with R2D2,5, 6 and Caenorhabditis elegans DCR-1 associates with RDE-4.⁷ All of these accessory dsRBPs have very similar domain structures: two N-terminal dsRNA-binding motifs (dsRBMs) and a C-terminus that contains a third degenerate dsRBM.

Despite similarities in domain structure, these dsRBPs have different roles in RNAi. Drosophila Dicer-2 does not require R2D2 to cleave dsRNA in vitro or in vivo, but downstream of this step, a complex of Dicer-2 and R2D2 is essential for loading siRNA into the RNA-induced silencing complex (RISC).5, 6, 8 Likewise, human Dicer processes dsRNA without TRBP and PACT in vitro,9, 10 and while there are some conflicting data,¹¹ the primary roles for TRBP and PACT appear to be after the production of siRNAs, in facilitating their incorporation into RISC.2, 3, 4, 12 In contrast, C. elegans RDE-4 is required for DCR-1-mediated cleavage of dsRNA to siRNA, but is not required in subsequent steps.7, 13, 14 This is emphasized by the observation that rde-4 mutant worms are incapable of RNAi when injected with long dsRNA, but this defect can be bypassed by the injection of siRNAs.¹⁴

dsRBPs bind dsRNA indiscriminant of sequence.¹⁵ However, the different functions of dsRBPs in RNAi require that some bind long dsRNA, while others bind short siRNA, raising the possibility that dsRBPs can discriminate dsRNA based on length. Consistent with this idea, RDE-4 forms stable complexes with long dsRNA in vivo, but does not stably interact with siRNA.⁷ Similarly, with purified components, RDE-4 preferentially binds long dsRNA.¹³ The latter study indicated that RDE-4 binds dsRNA cooperatively. This suggests a simple model to explain how dsRBPs discriminate dsRNA based on length, invoking classic studies of sequence-independent proteins that bind to nucleic acid lattices.16, 17 According to this paradigm, cooperativity favors binding to long dsRNA, a nucleic acid lattice that has multiple binding sites and thus maximizes cooperative interactions. Here we report further studies that support this model, involving studies of RDE-4 as well as a second dsRBP, human TRBP. We show that human TRBP, whose in vivo function requires binding to siRNA rather than long dsRNA, binds siRNA with high affinity, but is not cooperative. Using a comprehensive set of RDE-4 truncations, we dissect the functions of RDE-4's domains using in vitro binding studies and assays for reconstitution of Dicer activity in extracts of rde-4 mutant C. elegans. Our studies indicate dsRBM2 of RDE-4 is most important for binding dsRNA, but multiple domains contribute to cooperativity. While RDE-4's ability to bind dsRNA is important for facilitating cleavage of dsRNA by Dicer, the linker region also plays an important role, possibly mediating direct interactions with Dicer.

Section snippets

Results

To investigate the contribution of the different domains of RDE-4 to cooperativity, we first overexpressed and purified RDE-4 variant proteins that lacked or contained mutations in one or more domains (Fig. 1a). These variants were named according to their domain content. For example, the RDE-4 variant that lacks dsRBM1 (R₁) and the linker region (L), but has dsRBM2 (R₂) and the C-terminus (C), is called R₂C; when a variant contained only the N- or C-terminal region of a domain, the included

A general model for how dsRBPs discriminate dsRNA length

Early theoretical work put forth that cooperative nucleic acid-binding proteins exhibit observed affinities (K_obs ) that are products of the intrinsic affinity of an isolated protein–nucleic acid interaction (K_int) and the cooperativity parameter, ω (K_obs = K_intω).¹⁷ The cooperativity parameter, ω, is a unitless factor that specifies the relative affinity of an additional ligand for a contiguous versus an isolated binding site.¹⁷ Therefore, high ω values result in protein cluster formation along

Construction, expression, and purification of RDE-4 variants, TRBP, and chimeras

Truncated RDE-4 constructs were PCR amplified from RDE-4-YEpTOP2GAL1 which encoded wild-type RDE-4.¹³ The sequences of primers (5′ to 3′) used to construct these variants are listed below.

RDE-4_BamH1_For: CGT CAA GGA GAA AAA ACC CCG GAT CCG TAA CC
RDE-4_169_Xho1_Rev: GTC ATT ACT CGA GTC AAT TCT CGG TTG GCG AAA TAC CAG GTG G
RDE-4_Xho1_Rev: GTC ATT ACT CGA GTC AAT CCG TGA AAT C
RDE-4_139_LR: GCA TCT GAA GTT GAT CGC TGA AAA TAC AGG TTT TCG G
RDE-4_139_RF: CCG AAA ACC TGT ATT TTC AGC GAT CAA CTT CAG

Acknowledgements

We thank members of the Bass laboratory for helpful discussions and A. Krauchuk for technical assistance. We also thank Dr. Andrew Fire for communicating unpublished results and Dr. Craig Mello for providing antibodies to RDE-4 and C. elegans Dicer. This work was supported by funds to B.L.B. from the National Institutes of Health (GM067106). B.L.B. is a Howard Hughes Medical Institute Investigator.

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Journal of Molecular Biology

dsRNA Binding Properties of RDE-4 and TRBP Reflect Their Distinct Roles in RNAi

Abstract

Introduction

Section snippets

Results

A general model for how dsRBPs discriminate dsRNA length

Construction, expression, and purification of RDE-4 variants, TRBP, and chimeras

Acknowledgements

Cell

Cell

J. Biol. Chem.

J. Mol. Biol.

Virology

Biophys. J.

Biophys. Chem.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

Cell

Mol. Cell. Proteomics

Cell

Cell

J. Biol. Chem.

FEBS Lett.

Role of Dicer in posttranscriptional RNA silencing

Curr. Top. Microbiol. Immunol.

TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing

Nature

TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing

EMBO Rep.

The role of PACT in the RNA silencing pathway

EMBO J.

R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway

Science