Autoinhibition of Human Dicer by Its Internal Helicase Domain

Ma, Enbo; MacRae, Ian J.; Kirsch, Jack F.; Doudna, Jennifer A.

doi:10.1016/j.jmb.2008.05.005

Cited by 200 publications

(224 citation statements)

References 19 publications

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“…Treatment of the fulllength Dicer protein, however, considerably enhanced dsRNase activity (see Figure 7A, lanes 9 and 10), as initially reported [3]. This effect induced by proteolysis is likely due to the removal of the putative helicase domain located in the N-terminal region of human Dicer, which has been reported to exert auto-inhibitory effects on Dicer activity [37].…”

Section: Lo Modifies the Pre-mirna Processing Activity Of Human Dicersupporting

confidence: 73%

Human Dicer C-terminus functions as a 5-lipoxygenase binding domain

Dincbas-Renqvist¹,

Pépin²,

Rakonjac³

et al. 2009

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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SummaryDicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) in the vast majority of eukaryotes. In human, Dicer has been shown to interact with cellular proteins via its N-terminal domain. Here, we demonstrate the ability of Dicer C-terminus to interact with 5-lipoxygenase (5LO), an enzyme involved in the biosynthesis of inflammatory mediators, in vitro and in cultured human cells. Yeast two-hybrid and GST binding assays delineated the smallest 5-lipoxygenase binding domain (5LObd) of Dicer to its C-terminal 140 amino acids comprising the double-stranded RNA (dsRNA) binding domain (dsRBD). The Dicer 5LObd-5LO association was disrupted upon Ala substitution of Trp residues 13, 75 and 102 in 5LO, suggesting that the Dicer 5LObd may recognize 5LO via its N-terminal C2-like domain. Whereas a catalytically active 5LObd-containing Dicer fragment was found to enhance 5LO enzymatic activity in vitro, human 5LO modified the miRNA precursor processing activity of Dicer. In addition to revealing the dual RNA and protein binding properties of Dicer C-terminus, our results may provide a link between miRNA-mediated regulation of gene expression and inflammation.

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Section: Lo Modifies the Pre-mirna Processing Activity Of Human Dicersupporting

confidence: 73%

Human Dicer C-terminus functions as a 5-lipoxygenase binding domain

Dincbas-Renqvist¹,

Pépin²,

Rakonjac³

et al. 2009

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

View full text Add to dashboard Cite

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“…An alternative yet intriguing possibility is that Dcr-2, of which little is known in the cellular context, could displace the viral nucleoprotein to gain access to dsRNA. Such a function may involve the helicase activity of Dcr-2, the role of which in RNAi remains poorly understood (42).…”

Section: Discussionmentioning

confidence: 99%

RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila

Mueller

Gausson

Vodovar

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

130

129

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Activation of innate antiviral responses in multicellular organisms relies on the recognition of structural differences between viral and cellular RNAs. Double-stranded (ds)RNA, produced during viral replication, is a well-known activator of antiviral defenses and triggers interferon production in vertebrates and RNAi in invertebrates and plants. Previous work in mammalian cells indicates that negative-strand RNA viruses do not appear to generate dsRNA, and that activation of innate immunity is triggered by the recognition of the uncapped 5′ ends of viral RNA. This finding raises the question whether antiviral RNAi, which is triggered by the presence of dsRNA in insects, represents an effective host-defense mechanism against negative-strand RNA viruses. Here, we show that the negative-strand RNA virus vesicular stomatitis virus (VSV) does not produce easily detectable amounts of dsRNA in Drosophila cells. Nevertheless, RNAi represents a potent response to VSV infection, as illustrated by the high susceptibility of RNAi-defective mutant flies to this virus. VSV-derived small RNAs produced in infected cells or flies uniformly cover the viral genome, and equally map the genome and antigenome RNAs, indicating that they derive from dsRNA. Our findings reveal that RNAi is not restricted to the defense against positive-strand or dsRNA viruses but can also be highly efficient against a negative-strand RNA virus. This result is of particular interest in view of the frequent transmission of medically relevant negative-strand RNA viruses to humans by insect vectors.deep sequencing | arbovirus | Dicer-2 | AGO2 | small RNA profiling

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“…In numerous DEAD box proteins, the helicase core provides the basic DEAD box protein functions, and large N-and/or C-terminal extensions (Figure 1) modulate the activity of the helicase core by conferring substrate specificity or by mediating contacts with interacting proteins (Schmid and Linder 1992; see 'Modulation of the helicase core activity by interacting partners and flanking domains'). In addition, the helicase core appears as a module in several enzymes involved in nucleic acid processing, such as DNA topoisomerases (Confalonieri et al, 1993;Rodriguez and Stock, 2002), restriction enzymes (Gorbalenya and Koonin, 1991;Szczelkun, 2000), chromatin remodeling enzymes (Flaus and Owen-Hughes, 2001), or Dicer (Ma et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

Hilbert

Karow²,

Klostermeier³

2009

BCHM

131

167

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DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecAlike domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysisand unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

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Autoinhibition of Human Dicer by Its Internal Helicase Domain

Cited by 200 publications

References 19 publications

Human Dicer C-terminus functions as a 5-lipoxygenase binding domain

Human Dicer C-terminus functions as a 5-lipoxygenase binding domain

RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

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