Coordinated Activities of Human Dicer Domains in Regulatory RNA Processing

Ma, Enbo; Zhou, Kaihong; Kidwell, Mary Anne; Doudna, Jennifer A.

doi:10.1016/j.jmb.2012.06.009

Cited by 68 publications

(84 citation statements)

References 36 publications

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“…The helicase distinguishes between perfect duplex or hairpin RNAs, with a higher affinity for the latter. Furthermore, it plays a role in catalysis keeping Dicer in a semi-repressed state (Ma et al , 2012Tsutsumi et al 2011). This domain also mediates interaction between Dicer and its partner, TRBP, that stimulates full-length Dicer activity, thus suggesting that protein cofactors may modulate helicase inhibition (Haase et al 2005;Kok et al 2007;Lau et al 2009;Chakravarthy et al 2010).…”

Section: Introductionmentioning

confidence: 99%

“…Such an arrangement supports biochemical observations that the distance between the PAZ and RNase III domains acts as a molecular ruler. Importantly, it also accommodates the observed RNA binding by the helicase domain in human Dicer, as its position below the catalytic core allows it to bind both dsRNA and loop segments of its substrates (Ma et al , 2012Soifer et al 2008). In mammalian cells, the miRNA pathway starts in the nucleus with the transcription of primary miRNA precursors (pri-miRNAs), most of which are initially cleaved by the Drosha/DGCR8 complex to generate precursor-miRNAs (pre-miRNAs).…”

Section: Introductionmentioning

confidence: 99%

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The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal

Doyle

Badertscher

Jaśkiewicz

et al. 2013

RNA

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Dicer is a key player in microRNA (miRNA) and RNA interference (RNAi) pathways, processing miRNA precursors and doublestranded RNA into ∼21-nt-long products ultimately triggering sequence-dependent gene silencing. Although processing of substrates in vertebrate cells occurs in the cytoplasm, there is growing evidence suggesting Dicer is also present and functional in the nucleus. To address this possibility, we searched for a nuclear localization signal (NLS) in human Dicer and identified its C-terminal double-stranded RNA binding domain (dsRBD) as harboring NLS activity. We show that the dsRBD-NLS can mediate nuclear import of a reporter protein via interaction with importins β, 7, and 8. In the context of full-length Dicer, the dsRBD-NLS is masked. However, duplication of the dsRBD localizes the full-length protein to the nucleus. Furthermore, deletion of the N-terminal helicase domain results in partial accumulation of Dicer in the nucleus upon leptomycin B treatment, indicating that CRM1 contributes to nuclear export of Dicer. Finally, we demonstrate that human Dicer has the ability to shuttle between the nucleus and the cytoplasm. We conclude that Dicer is a shuttling protein whose steady-state localization is cytoplasmic.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal

Doyle

Badertscher

Jaśkiewicz

et al. 2013

RNA

View full text Add to dashboard Cite

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“…This yields a stem composed of 5 ′ and 3 ′ strands representing 5p and 3p miRNAs, respectively. Human Dicer is a large protein composed of several domains, including a helicase/ATPase domain, a PAZ (Piwi, Argonaute, Zwille) domain, a dsRBD, and two RNase III domains (Zhang et al 2004;Lau et al 2012;Ma et al 2012). The PAZ domain grips the termini of the pre-miRNA and positions it for double cleavage ∼22 nt into the stem, producing a double-stranded RNA (dsRNA) with 3 ′ overhangs (Macrae et al 2006;Park et al 2011).…”

Section: Introductionmentioning

confidence: 99%

Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-α

Guennewig

Roos

Dogar

et al. 2013

RNA

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Functional microRNAs (miRNAs) are produced from both arms of their precursors (pre-miRNAs). Their abundances vary in contextdependent fashion spatiotemporarily and there is mounting evidence of regulatory interplay between them. Here, we introduce chemically synthesized pre-miRNAs (syn-pre-miRNAs) as a general class of accessible, easily transfectable mimics of premiRNAs. These are RNA hairpins, identical in sequence to natural pre-miRNAs. They differ from commercially available miRNA mimics through their complete hairpin structure, including any regulatory elements in their terminal-loop regions and their potential to introduce both strands into RISC. They are distinguished from transcribed pre-miRNAs by their terminal 5′ hydroxyl groups and their precisely defined terminal nucleotides. We demonstrate with several examples how they fully recapitulate the properties of pre-miRNAs, including their processing by Dicer into functionally active 5p-and 3p-derived mature miRNAs. We use syn-pre-miRNAs to show that miR-34a uses its 5p and 3p miRNAs in two pathways: apoptosis during TGF-β signaling, where SIRT1 and SP4 are suppressed by miR-34a-5p and miR-34a-3p, respectively; and the lipopolysaccharide (LPS)-activation of primary human monocyte-derived macrophages, where TNF (TNFα) is suppressed by miR-34a-5p indirectly and miR-34a-3p directly. Our results add to growing evidence that the use of both arms of a miRNA may be a widely used mechanism. We further suggest that syn-pre-miRNAs are ideal and affordable tools to investigate these mechanisms.

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“…Moreover, most of these enzymes conduct activity by forming an intramolecular pseudodimer between two tandem RNase III domains [25]. In these cases, RNA recognition is accomplished primarily through a PAZ domain that binds the 3′ end of the pre-miRNA [26][27][28] with a preference for 2 nt overhangs [29], though other parts of the molecule-including the C-terminal dsRBD and platform-also contribute to substrate recognition. Ultimately, Dicers couple the functionality of their RNase III domains to distal RNA recognition domains like PAZ.…”

Section: Dicermentioning

confidence: 99%

From guide to target: molecular insights into eukaryotic RNA-interference machinery

Ipsaro

Joshua‐Tor

2015

Nat Struct Mol Biol

218

144

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Since its relatively recent discovery, RNA interference (RNAi) has emerged as a potent, specific, and ubiquitous means of gene regulation. Through a number of pathways that are conserved from yeast to humans, small non-coding RNAs direct molecular machinery to silence gene expression. In this review, we focus on mechanisms and structures that govern RNA silencing in higher organisms. In addition to highlighting recent advances, parallels and differences between RNAi pathways are discussed. Together, the studies reviewed herein reveal the versatility and programmability of RNA-induced Silencing Complexes (RISCs) and emphasize the importance of both upstream biogenesis and downstream silencing factors. Discovery and Biological Perspectives of RNA interferenceRNAi was first described by Fire and Mello in the 1990s when, in an attempt to use antisense RNA to down-regulate gene expression, they observed that double-stranded RNA (dsRNA) was more potent than sense or anti-sense RNA alone [1]. This seminal work boasted robust and specific gene knock down in addition to coining the term "RNA interference" (RNAi). Shortly beforehand, the discovery of individual regulatory RNAs in C. elegans hinted that small, non-coding RNAs might be a pervasive means of gene regulation in higher organisms [2,3]. In the following decade, with the mechanistic insight of Fire and Mello's work in hand, this idea was confirmed and it is now accepted that wellover 1,000 small RNAs are encoded in the human genome that may regulate over 60% of our genes [4,5]. It also became apparent that several seemingly disconnected phenomena are variations of RNAi-type pathways including co-suppression in plants, DNA elimination in Tetrahymena, and quelling in Neurospora [6]. The prevalence of RNAi is impressive and its importance is underscored by the fact that RNAi dysfunction is associated with numerous diseases and disorders including neurological maladies, cancers, and infertility.Shortly after the initial description of RNAi phenomenology, a burst of genetic, biochemical, biophysical, and bioinformatic efforts laid a strong foundation for identifying the molecular pathways, players, and parameters that govern silencing via RNAi. Work from Reprints and permissions information is available online at http://www.nature.com/reprints/index.html. HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript numerous groups defined the molecular apparatus of RNAi as RISC (RNA-induced Silencing Complex), a ribonucleoprotein complex minimally comprised of a small singlestranded RNA (~20-31 nucleotides) and an Argonaute protein which serves as the effector molecule (reviewed in [7]). In this configuration, the loaded "guide" RNA acts as a specificity determinant that directs Argonaute and any other associated machinery to the target. The guide-loaded Argonaute platform underlies every example in the expansive array of known RNAi pathways in eukaryotes regardless of the source of the guide (structured loci, transposons, viral, etc.), the machinery ...

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Coordinated Activities of Human Dicer Domains in Regulatory RNA Processing

Cited by 68 publications

References 36 publications

The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal

The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal

Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-α

From guide to target: molecular insights into eukaryotic RNA-interference machinery

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