Differential roles of human Dicer-binding proteins TRBP and PACT in small RNA processing

Lee, Ho‐Young; Zhou, Kaihong; Smith, Alison M.; Noland, Cameron L.; Doudna, Jennifer A.

doi:10.1093/nar/gkt361

Cited by 183 publications

(174 citation statements)

References 28 publications

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“…The TRBP cofactor required by hDcr has been proposed to influence the cleavage site on pre-miRNAs that is utilized by hDcr (Lee and Doudna 2012;Lee et al 2013) and the Loqs-PB protein has also been proposed to influence pre-miRNA cleavage site selection by dDcr1 (Fukunaga et al 2012), resulting in the potential formation of alternative isomiRs. Moreover, the hDcr/TRBP complex has also been proposed to play a role in RISC loading (Chendrimada et al 2005;MacRae et al 2008).…”

Section: Synthetic Microrna Duplexes Efficiently Program Risc In Dicementioning

confidence: 99%

Derivation and characterization of Dicer- and microRNA-deficient human cells

Bogerd

Whisnant

Kennedy

et al. 2014

RNA

147

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We have used genome editing to generate inactivating deletion mutations in all three copies of the dicer (hdcr) gene present in the human cell line 293T. As previously shown in murine ES cells lacking Dicer function, hDcr-deficient 293T cells are severely impaired for the production of mature microRNAs (miRNAs). Nevertheless, RNA-induced silencing complexes (RISCs) present in these hDcr-deficient cells are readily programmed by transfected, synthetic miRNA duplexes to repress mRNAs bearing either fully or partially complementary targets, including targets bearing incomplete seed homology to the introduced miRNA. Using these hDcr-deficient 293T cells, we demonstrate that human pre-miRNA processing can be effectively rescued by ectopic expression of the Drosophila Dicer 1 protein, but only in the presence of the PB isoform of Loquacious (Loqs-PB), the fly homolog of the hDcr cofactor TRBP. In contrast, Drosophila Dicer 2, even in the presence of its cofactors Loqs-PD and R2D2, was unable to support human pre-miRNA processing. Interestingly, although ectopic Drosophila Dicer 1/Loqs-PB or hDcr both rescued pre-miRNA processing effectively in these hDcr-deficient cells, there were significant differences in the ratio of the miRNA isoforms that were produced, especially in the case of miR-30 family members, and we also noted differences in the relative expression level of miRNAs vs. passenger strands for a subset of human miRNAs. These data demonstrate that the mechanisms underlying the accurate processing of pre-miRNAs are largely, but not entirely, conserved between mammalian and insect cells.

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Section: Synthetic Microrna Duplexes Efficiently Program Risc In Dicementioning

confidence: 99%

Derivation and characterization of Dicer- and microRNA-deficient human cells

Bogerd

Whisnant

Kennedy

et al. 2014

RNA

147

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“…Human Dicer also associates with two different dsRNA-binding proteins, TRBP and PACT (17,18). These dsRNA-binding proteins are not required for dicing activity but function as key regulatory factors in defining the dicing site and facilitating RISC formation (19)(20)(21).…”

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confidence: 99%

RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii

Yamasaki

Onishi

Kim

et al. 2016

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

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Canonical microRNAs (miRNAs) are embedded in duplexed stemloops in long precursor transcripts and are excised by sequential cleavage by DICER nuclease(s). In this miRNA biogenesis pathway, dsRNA-binding proteins play important roles in animals and plants by assisting DICER. However, these RNA-binding proteins are poorly characterized in unicellular organisms. Here we report that a unique RNA-binding protein, Dull slicer-16 (DUS16), plays an essential role in processing of primary-miRNA (pri-miRNA) transcripts in the unicellular green alga Chlamydomonas reinhardtii. In animals and plants, dsRNA-binding proteins involved in miRNA biogenesis harbor two or three dsRNA-binding domains (dsRBDs), whereas DUS16 contains one dsRBD and also an ssRNA-binding domain (RRM). The null mutant of DUS16 showed a drastic reduction in most miRNA species. Production of these miRNAs was complemented by expression of full-length DUS16, but the expression of RRM-or dsRBDtruncated DUS16 did not restore miRNA production. Furthermore, DUS16 is predominantly localized to the nucleus and associated with nascent (unspliced form) pri-miRNAs and the DICER-LIKE 3 protein. These results suggest that DUS16 recognizes pri-miRNA transcripts cotranscriptionally and promotes their processing into mature miRNAs as a component of a microprocessor complex. We propose that DUS16 is an essential factor for miRNA production in Chlamydomonas and, because DUS16 is functionally similar to the dsRNA-binding proteins involved in miRNA biogenesis in animals and land plants, our report provides insight into this mechanism in unicellular eukaryotes.Chlamydomonas | microRNA biogenesis | dsRNA-binding protein | small RNA-seq | mutagenesis

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Section: Other Components Of Micro and Sirna Biogenesismentioning

confidence: 99%

“…A second dsRBP, R2D2, cooperates with Dicer-2 to process exogenous siRNAs (reviewed in [38]). In mammals, two dsRBPs, TRBP and PACT, have been shown to interact with Dicer and modulate its substrate specificity [39].Alongside these biochemical studies, biophysical and structural efforts on dsRBPs have been successful in establishing the means by which the authentic dsRBDs recognize dsRNA [40]. In the case of the human dsRBP TRBP, structures of the first two dsRBDs domains, particularly the structure of the second domain bound to dsRNA, have shown that these RBDs exemplify the canonical dsRBD fold, binding the dsRNA through both major and minor groove interactions along the helical surfaces of the protein (Fig.…”

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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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Differential roles of human Dicer-binding proteins TRBP and PACT in small RNA processing

Cited by 183 publications

References 28 publications

Derivation and characterization of Dicer- and microRNA-deficient human cells

Derivation and characterization of Dicer- and microRNA-deficient human cells

RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii

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

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