Autoinhibition of Human Dicer by Its Internal Helicase Domain

doi:10.1016/j.jmb.2008.05.005

Journal of Molecular Biology

Volume 380, Issue 1, 27 June 2008, Pages 237-243

https://doi.org/10.1016/j.jmb.2008.05.005 Get rights and content

Abstract

Dicer, a member of the ribonuclease III family of enzymes, processes double-stranded RNA substrates into ∼ 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.

Introduction

Dicer is a large multidomain enzyme responsible for cytoplasmic production of both microRNAs (miRNAs) and short interfering RNAs (siRNAs) during sequence-directed gene regulation by RNA interference. As a member of the ribonuclease III family of proteins, Dicer recognizes the 5′ and 3′ helical ends of double-stranded RNA (dsRNA) substrates and cleaves a specific distance away to produce 21- to 27-nt products (reviewed in Refs. 1, 2, 3). Dicer helps these miRNAs and siRNAs to load onto Argonaute proteins together with other protein components of the RNA-induced silencing complex. Once bound to target mRNAs, miRNAs typically regulate protein expression by controlling the level of translation, whereas siRNAs direct cleavage and subsequent degradation of complementary mRNAs.

Although the mechanism of dsRNA recognition and length-specific RNA cleavage by Dicer has been established, the way in which this activity is regulated remains unclear. Biochemical and structural studies showed that the PAZ domain of Dicer binds the 3′ 2-nt overhanging end of a duplex RNA subsrate, positioning it for cleavage by Dicer’s two RNase III-homologous domains at a distance of ∼ 25 base pairs from the end.4, 5, 6 Although a natural Dicer from Giardia containing just these functional domains is capable of robust dicing and can complement the lack of a functional Dicer in Schizosaccharomyces pombe,⁶ most Dicers include numerous additional sequences. In particular, Dicer typically contains a large N-terminal domain homologous to DExD/H-box helicases. Although the presence of this domain correlates with a requirement for ATP by invertebrate Dicers,7, 8, 9, 10 siRNA production by mammalian Dicer is ATP independent.11, 12

To investigate the role of the DExD/H-box domain as well as other regions of the human Dicer enzyme, we overexpressed and purified five Dicer variants containing either domain deletions or point mutations. We demonstrate that Dicer is capable of multiple-turnover catalysis and that it behaves as a classic Michaelis–Menten enzyme. Surprisingly, deletion or mutation of the DExD/H-box domain led to substantially enhanced activity of Dicer when assayed for cleavage of a perfect-duplex substrate, with slight stimulation of hairpin substrate cleavage. Kinetic analysis shows that this rate enhancement is largely a k_cat effect, indicating that the DExD/H-box domain inhibits catalysis rather than affecting RNA binding affinity. The TAR-RNA binding protein (TRBP), which binds directly to the DExD/H-box domain, modestly stimulates cleavage activity of wild-type Dicer. Our data suggest that the DExD/H-box domain functions as an intramolecular structural switch to maintain Dicer in a low-activity state until the enzyme assembles with its protein partners, and it may help to distinguish perfect-duplex versus hairpin substrates.

Section snippets

Dicer’s DExD/H-box domain inhibits single-turnover dsRNA cleavage rates

To investigate dsRNA recognition and cleavage by human Dicer, we prepared the wild-type (hDcr) (accession no. NP_803187) and five mutant forms of recombinant hDcr (Fig. 1a). Specifically, a point mutation of lysine to alanine at position 70 (K70A) in the ATP-binding motif (hWalker) and a deletion of amino acids 1–604 spanning the entire DExD/H-box domain (Δhelicase) were created to analyze the functional contributions of the DExD/H-box domain. To explore the role of the C-terminal dsRNA-binding

RNA substrates

A 73-nt human let-7 hairpin RNA (pre-hlet-7) was transcribed in vitro by T7 RNA polymerase from a construct containing a double ribozyme system to ensure homogeneous 5′ and 3′ ends.¹⁹ All other RNA substrates were synthesized by IDT (Integrated DNA Technologies, Inc., Coralville, IA). All RNAs were purified by 16% urea–PAGE. For both filter binding and dicing assays, the purified RNA substrates were 5′-end labeled with ³²P using T4 polynucleotide kinase (New England Biolabs, Inc., Beverly, MA).

Acknowledgements

We thank Kaihong Zhou for expert technical assistance and other members of the Doudna lab for helpful discussions. This work was supported by a grant from the National Institutes of Health to J.A.D. J.F.K. is supported by a grant from the National Institutes of Health. The authors declare that they have no competing financial interests.

References (19)

E.P. Murchison et al.
miRNAs on the move: miRNA biogenesis and the RNAi machinery
Curr. Opin. Cell Biol.
(2004)
I.J. MacRae et al.
Ribonuclease revisited: structural insights into ribonuclease III family enzymes
Curr. Opin. Struct. Biol.
(2007)
H. Zhang et al.
Single processing center models for human Dicer and bacterial RNase III
Cell
(2004)
A. Nykanen et al.
ATP requirements and small interfering RNA structure in the RNA interference pathway
Cell
(2001)
X. Ye et al.
Functional anatomy of the Drosophila microRNA-generating enzyme
J. Biol. Chem.
(2007)
M.A. Carmell et al.
RNase III enzymes and the initiation of gene silencing
Nat. Struct. Mol. Biol.
(2004)
J.J. Song et al.
The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes
Nat. Struct. Mol. Biol.
(2003)
I.J. Macrae et al.
Structural basis for double-stranded RNA processing by Dicer
Science
(2006)
E. Bernstein et al.
Role for a bidentate ribonuclease in the initiation step of RNA interference
Nature
(2001)

There are more references available in the full text version of this article.

Cited by (184)

DICER ribonuclease removes harmful R-loops
2023, Molecular Cell
R-loops, which consist of a DNA-RNA hybrid and a displaced DNA strand, are known to threaten genome integrity. To counteract this, different mechanisms suppress R-loop accumulation by either preventing the hybridization of RNA with the DNA template (RNA biogenesis factors), unwinding the hybrid (DNA-RNA helicases), or degrading the RNA moiety of the R-loop (type H ribonucleases [RNases H]). Thus far, RNases H are the only nucleases known to cleave DNA-RNA hybrids. Now, we show that the RNase DICER also resolves R-loops. Biochemical analysis reveals that DICER acts by specifically cleaving the RNA within R-loops. Importantly, a DICER RNase mutant impaired in R-loop processing causes a strong accumulation of R-loops in cells. Our results thus not only reveal a function of DICER as an R-loop resolvase independent of DROSHA but also provide evidence for the role of multi-functional RNA processing factors in the maintenance of genome integrity in higher eukaryotes.
The pre-miRNA cleavage assays for DICER
2023, Methods in Enzymology
MicroRNAs (miRNAs) are small, non-coding RNA molecules that play a crucial role in gene silencing. The gene-silencing activity of miRNAs depends on their sequences and expression levels. The human RNase III enzyme DICER cleaves miRNA precursors (pre-miRNAs) to produce miRNAs, making it crucial for miRNA production and cellular miRNA functions. DICER is also critical for the gene silencing technology using short-hairpin RNAs (shRNAs), which are cleaved by DICER to generate siRNAs that knockdown target genes. The DICER cleavage assay is an important tool for investigating its molecular mechanisms, which are essential for understanding its functions in miRNA biogenesis and shRNA-based gene silencing technology. The assay involves DICER protein purification, preparation of pre-miRNA and shRNA substrates, and the cleavage assay, using common molecular biology equipment and commercialized reagents that can be applied to other RNA endonucleases.
Structural and functional basis of mammalian microRNA biogenesis by Dicer
2022, Molecular Cell
MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer’s DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer⋅miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.
Protein-RNA interaction guided chemical modification of Dicer substrate RNA nanostructures for superior in vivo gene silencing
2022, Journal of Controlled Release
Dicer substrate RNA is an alternative gene silencing agent to canonical siRNA. Enhanced in vitro gene silencing can be achieved with RNA substrates by facilitating Ago2 loading of dsRNA after Dicer processing. However, the in vivo use of Dicer substrate RNA has been hindered by its instability and immunogenicity in the body due to the lack of proper chemical modification in the structure. Here, we report a universal chemical modification approach for Dicer substrate RNA nanostructures by optimizing protein-RNA interactions in the RNAi pathway. Proteins involved in the RNAi pathway were utilized for evaluating their recognition and binding of substrate RNA. It was found that conventional chemical modifications could severely affect the binding and processing of substrate RNA, consequently reducing RNAi activity. Protein-RNA interaction guided chemical modification was introduced to RNA nanostructures, and their gene silencing activity was assessed. The optimized RNA nanostructures showed excellent binding and processability with RNA binding proteins and offered the enhancement of in vivo EC₅₀ up to 1/8 of its native form.
The helicase domain of human Dicer prevents RNAi-independent activation of antiviral and inflammatory pathways
2024, EMBO Journal
Antiviral Defence Mechanisms during Early Mammalian Development
2024, Viruses

View all citing articles on Scopus

¹: Present address: I. J. MacRae, The Scripps Research Institute, La Jolla, CA, USA.

View full text

Article preview

Journal of Molecular Biology

Abstract

Introduction

Section snippets

Dicer’s DExD/H-box domain inhibits single-turnover dsRNA cleavage rates

RNA substrates

Acknowledgements

References (19)

miRNAs on the move: miRNA biogenesis and the RNAi machinery

Curr. Opin. Cell Biol.

Ribonuclease revisited: structural insights into ribonuclease III family enzymes

Curr. Opin. Struct. Biol.

Single processing center models for human Dicer and bacterial RNase III

Cell

ATP requirements and small interfering RNA structure in the RNA interference pathway

Cell

Functional anatomy of the Drosophila microRNA-generating enzyme

J. Biol. Chem.

RNase III enzymes and the initiation of gene silencing

Nat. Struct. Mol. Biol.

The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes

Nat. Struct. Mol. Biol.

Structural basis for double-stranded RNA processing by Dicer

Science

Role for a bidentate ribonuclease in the initiation step of RNA interference

Nature

Cited by (184)

DICER ribonuclease removes harmful R-loops

The pre-miRNA cleavage assays for DICER

Structural and functional basis of mammalian microRNA biogenesis by Dicer

Protein-RNA interaction guided chemical modification of Dicer substrate RNA nanostructures for superior in vivo gene silencing

The helicase domain of human Dicer prevents RNAi-independent activation of antiviral and inflammatory pathways

Antiviral Defence Mechanisms during Early Mammalian Development