Reduced Expression of Ribosomal Proteins Relieves MicroRNA-Mediated Repression

Janas, Maja M.; Wang, Eric Tzy-shi; Love, Tara M.; Harris, Abigail; Stevenson, Kristen E.; Semmelmann, Karlheinz; Shaffer, James; Chen, Po-Hao; Doench, John G.; Yerramilli, Subrahmanyam; Neuberg, Donna; Iliopoulos, Dimitrios; Housman, David E.; Burge, Christopher B.; Novina, Carl D.

doi:10.1016/j.molcel.2012.04.008

Cited by 27 publications

(26 citation statements)

References 64 publications

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“…Similar observations and conclusions were made in worms (Seggerson et al, 2002) as well as in mammals (Kim et al, 2004;Nelson et al, 2004;Maroney et al, 2006;Nottrott et al, 2006;Petersen et al, 2006). However, other studies have shown a shift in the sedimentation of repressed mRNAs from polysome fractions toward lighter fractions (Pillai et al, 2005;Bhattacharyya et al, 2006;Kiriakidou et al, 2007;Ding and Grosshans, 2009;Janas et al, 2012). Moreover, m 7 G-capindependent noncanonical translation, initiated by A-cap or internal ribosome entry sites (IRES), is often immune to miRNAmediated translational repression (Humphreys et al, 2005;Pillai et al, 2005;Wang et al, 2006;Kiriakidou et al, 2007;Mathonnet et al, 2007;Thermann and Hentze, 2007;Wakiyama et al, 2007;Zdanowicz et al, 2009), suggesting translation initiation as the step of miRNA regulation.…”

Section: Introductionsupporting

confidence: 68%

“…However, the identity and function of pseudopolysomes remain unknown. Moreover, to make things more complicated, previous density gradient analyses have used target mRNAs with a 3 0 poly(A) tail, which are subject not only to pure translational repression but also deadenylation that leads to inhibition at translation initiation (Olsen and Ambros, 1999;Seggerson et al, 2002;Pillai et al, 2005;Bhattacharyya et al, 2006;Maroney et al, 2006;Nottrott et al, 2006;Petersen et al, 2006;Kiriakidou et al, 2007;Mathonnet et al, 2007;Thermann and Hentze, 2007;Kong et al, 2008;Wang et al, 2008;Ding and Grosshans, 2009;Moretti et al, 2010Moretti et al, , 2012Janas et al, 2012). This has caused a typical chickenand-egg problem, making interpretation complicated.…”

Section: Introductionmentioning

confidence: 99%

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MicroRNAs Mediate Gene Silencing via Multiple Different Pathways in Drosophila

2012

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MicroRNAs (miRNAs) guide RNA-induced silencing complex (RISC) that contains an Argonaute family protein to complementary target messenger RNAs (mRNAs). Via RISC, miRNAs silence the expression of target mRNAs by shortening the poly(A) tail-which leads to mRNA decay-and by repressing translation. It has been suggested that GW182, an Argonaute-associating protein, plays the central role in such microRNA actions. Here we show that, although GW182 is obligatory for poly(A) shortening, translational repression by microRNAs occurs even in the absence of GW182. Yet, GW182 is also capable of inducing translational repression independently. Both of these translational repression mechanisms block formation of 48S and 80S ribosomal complexes. Thus microRNAs utilize at least three distinct silencing pathways: GW182-mediated deadenylation and GW182-dependent and -independent repression of early translation initiation. Differential contribution from these multiple pathways may explain previous, apparently contradictory observations of how microRNAs inhibit protein synthesis.

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Section: Introductionsupporting

confidence: 68%

Section: Introductionmentioning

confidence: 99%

MicroRNAs Mediate Gene Silencing via Multiple Different Pathways in Drosophila

2012

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“…interfere with gene silencing (Huntzinger and Izaurralde, 2011;Janas et al, 2012), the plant mesophyll protoplast system offers the advantages of high amiRNA silencing specificity (see Supplemental Figure 7 online), unlimited AGO activity (see Supplemental Figure 9 online), high cotransfection efficiency (Yoo et al, 2007), highthroughput potential (Li et al, 2011), and broad adaptability in numerous plant species (Sheen, 1991). In particular, our screens can be easily applied to diverse agronomically or pharmacologically important plant species (Figures 6C and 6D).…”

Section: Discussionmentioning

confidence: 99%

Comprehensive Protein-Based Artificial MicroRNA Screens for Effective Gene Silencing in Plants

et al. 2013

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ORCID ID: 0000-0001-5783-0804 (J-FL).Artificial microRNA (amiRNA) approaches offer a powerful strategy for targeted gene manipulation in any plant species. However, the current unpredictability of amiRNA efficacy has limited broad application of this promising technology. To address this, we developed epitope-tagged protein-based amiRNA (ETPamir) screens, in which target mRNAs encoding epitope-tagged proteins were constitutively or inducibly coexpressed in protoplasts with amiRNA candidates targeting single or multiple genes. This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA expression/processing and antibody unavailability. Systematic evaluation of 63 amiRNAs in 79 ETPamir screens for 16 target genes revealed a simple, effective solution for selecting optimal amiRNAs from hundreds of computational predictions, reaching ;100% gene silencing in plant cells and null phenotypes in transgenic plants. Optimal amiRNAs predominantly mediated highly specific translational repression at 59 coding regions with limited mRNA decay or cleavage. Our screens were easily applied to diverse plant species, including Arabidopsis thaliana, tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), sunflower (Helianthus annuus), Catharanthus roseus, maize (Zea mays) and rice (Oryza sativa), and effectively validated predicted natural miRNA targets. These screens could improve plant research and crop engineering by making amiRNA a more predictable and manageable genetic and functional genomic technology.

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“…In animals, luciferase-derived reporters have been widely used in cell-based assays to quantify miRNA activity and have underpinned key studies on miRNA biology (Beillard et al, 2012;Brown et al, 2007;Connelly et al, 2012;Doench and Sharp, 2004;Gibbings et al, 2012;Horman et al, 2013;Huang et al, 2009;Humphreys et al, 2005;Janas et al, 2012;Lytle et al, 2007;Mullokandov et al, 2012;Petersen et al, 2006). Additionally, luciferase-based reporter systems are extensively used to validate miRNA targets in animal cells (Nicolas, 2011).…”

mentioning

confidence: 99%

Dissection of miRNA pathways using Arabidopsis mesophyll protoplasts

Martinho¹,

Confraria²,

Elias³

et al. 2014

Molecular Plant

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Short summary: We developed a set of sensors based on the firefly luciferase reporter gene to quantify miRNA activity in vivo, using Arabidopsis mesophyll protoplasts. We show that these sensors are not only responsive to endogenous miRNA activity, but also to exogenous gain-and loss-of-function manipulation of miRNA abundance. As examples, our sensors can be used for studying regulatory mechanisms of miRNA-associated components, for identifying novel factors with miRNA-associated functions, and for assessing the effect of chemicals on miRNA activity, therefore constituting a powerful tool to dissect miRNA pathways. responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this paper, we describe a transient expression system in Arabidopsis mesophyll protoplasts that is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics.We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, either by overexpression or by the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity.Our cell-based transient expression system is fast and easy to set up and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.

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Reduced Expression of Ribosomal Proteins Relieves MicroRNA-Mediated Repression

Cited by 27 publications

References 64 publications

MicroRNAs Mediate Gene Silencing via Multiple Different Pathways in Drosophila

MicroRNAs Mediate Gene Silencing via Multiple Different Pathways in Drosophila

Comprehensive Protein-Based Artificial MicroRNA Screens for Effective Gene Silencing in Plants

Dissection of miRNA pathways using Arabidopsis mesophyll protoplasts

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