The contributions of dsRNA structure to Dicer specificity and efficiency

Vermeulen, Annaleen; Behlen, Linda S.; Reynolds, Angela; Wolfson, Alexey D.; Marshall, William S.; Karpilow, Jon; Khvorova, Anastasia

doi:10.1261/rna.7272305

Cited by 249 publications

(224 citation statements)

References 27 publications

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“…Our 39-RACE analyses revealed that both RNAi vectors generate at least four siRNA species that were common between the vectors. These findings are consistent with a previous report that demonstrated flexibility (i.e., base-pair shifting) in dsRNA cleavage by Dicer (Vermeulen et al 2005). Notably, the most prevalent species generated by the RNAi vectors was shared, representing z50% of the 39-RACE sequences analyzed (n = 10-12 per vector per strand).…”

Section: Shrna Expression and Potency Is Overhang Dependentsupporting

confidence: 92%

“…These shRNAs were designed to have minimized 39 overhangs (2-4 U's resulting from Pol-III termination; Ng et al 1979;Ohshima et al 1981;Kunkel et al 1986) to resemble the 2-nt 39 overhangs that result from Drosha cleavage. Overhangs of this length provide optimal substrates for Exportin-5 and Dicer (Zeng and Cullen 2004;Vermeulen et al 2005). In addition, target sequences were selected to account for the +1-G nucleotide of the mouse U6 promoter and to contain AU-rich 39 ends, both of which promote loading of the antisense strand (Khvorova et al 2003;Schwarz et al 2003).…”

Section: Shrna Expression and Potency Is Overhang Dependentmentioning

confidence: 99%

“…In certain comparisons, the shRNAs tested had suboptimal 59 overhangs due to variable arrangements of transcription start and stop sequences, some caused inadvertently by the use of restriction enzyme sites during vector production (Boden et al 2004;Silva et al 2005). This raises concerns since recent reports have demonstrated that 2-nucleotide (nt) 39 overhangs, often observed in natural pre-miRNAs, are optimal substrates for Exportin-5 and Dicer (Zeng and Cullen 2004;Vermeulen et al 2005). Furthermore, none of the prior hairpin-based comparisons assessed the equivalency of strand biasing (Boden et al 2004;Silva et al 2005;Li et al 2007).…”

Section: Introductionmentioning

confidence: 99%

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Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs

Boudreau¹,

Monteys²,

Davidson³

2008

RNA

140

121

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RNA interference (RNAi) is a cellular process regulating gene expression and participating in innate defense in many organisms. RNAi has also been utilized as a tool to query gene function and is being developed as a therapeutic strategy for several diseases. Synthetic small interfering (siRNAs) or expressed stem-loop RNAs (short-hairpin RNAs [shRNAs] or artificial microRNAs [miRNAs]) have been delivered to cultured cells and organisms to inhibit expression of a variety of genes. A persistent question in the field, however, is which RNAi expression system is most suitable for distinct applications. To date, shRNA-and artificial miRNA-based strategies have been compared with conflicting results. In prior comparisons, sequences required for efficient RNAi processing and loading of the intended antisense strand into the RNAi-induced silencing complex (RISC) were not considered. We therefore revisited the shRNA-miRNA comparison question. Initially, we developed an improved artificial miRNA vector and confirmed the optimal shRNA configuration by altering structural features of these RNAi substrates. Subsequently, we engineered and compared shRNA-and miRNA-based RNAi expression vectors that would be processed to yield similar siRNAs that exhibit comparable strand biasing. Our results demonstrate that when comparison variables are minimized, the shRNAs tested were more potent than the artificial miRNAs in mediating gene silencing independent of target sequence and experimental setting (in vitro and in vivo). In addition, we show that shRNAs are expressed at considerably higher levels relative to artificial miRNAs, thus providing mechanistic insight to explain their increased potency.

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Section: Shrna Expression and Potency Is Overhang Dependentsupporting

confidence: 92%

Section: Shrna Expression and Potency Is Overhang Dependentmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs

Boudreau¹,

Monteys²,

Davidson³

2008

RNA

140

121

View full text Add to dashboard Cite

show abstract

“…In Arabidopsis, 21-nt sRNAs are produced by DCL4, 24-nt sRNAs by DCL 2 or DCL 3, and DCL1 produces miRNAs that are 22 nucleotides in length [27,40]. In this study a large percentage of the sRNAs had read lengths of 20-24 nucleotides, which is typical for DICER-processed sRNA products [24][25][26].…”

Section: Discussionmentioning

confidence: 88%

Characterization of small RNA populations in non-transgenic and aflatoxin-reducing-transformed peanut

et al. 2017

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Power, Imana L.; Dang, Phat M.; Sobolev, Victor S.; Orner, Valerie; Powell, Joseph L.; Lamb, Marshall C.; and Arias, Renée S., "Characterization of small RNA populations in non-transgenic andaflatoxin-reducing-transformed peanut" (2017 t r a c tAflatoxin contamination is a major constraint in food production worldwide. In peanut (Arachis hypogaea L.), these toxic and carcinogenic aflatoxins are mainly produced by Aspergillus flavus Link and A. parasiticus Speare. The use of RNA interference (RNAi) is a promising method to reduce or prevent the accumulation of aflatoxin in peanut seed. In this study, we performed high-throughput sequencing of small RNA populations in a control line and in two transformed peanut lines that expressed an inverted repeat targeting five genes involved in the aflatoxin-biosynthesis pathway and that showed up to 100% less aflatoxin B 1 than the controls. The objective was to determine the putative involvement of the small RNA populations in aflatoxin reduction. In total, 41 known microRNA (miRNA) families and many novel miRNAs were identified. Among those, 89 known and 10 novel miRNAs were differentially expressed in the transformed lines. We furthermore found two small interfering RNAs derived from the inverted repeat, and 39 sRNAs that mapped without mismatches to the genome of A. flavus and were present only in the transformed lines. This information will increase our understanding of the effectiveness of RNAi and enable the possible improvement of the RNAi technology for the control of aflatoxins.

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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 [26-28] 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.…”

mentioning

confidence: 99%

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

Ipsaro

Joshua‐Tor

2015

Nat Struct Mol Biol

216

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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The contributions of dsRNA structure to Dicer specificity and efficiency

Cited by 249 publications

References 27 publications

Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs

Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs

Characterization of small RNA populations in non-transgenic and aflatoxin-reducing-transformed peanut

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

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