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On the availability of microRNA-induced silencing complexes, saturation of microRNA-binding sites and stoichiometry.

Mayya VK, Duchaine TF - Nucleic Acids Res. (2015)

Bottom Line: Several authors have suggested or inferred that modest changes in microRNA expression can potentiate or impinge on their capacity to mediate gene repression, and that doing so could play a significant role in diseases.Such interpretations are based on several assumptions, namely: (i) changes in microRNA expression correlate with changes in the availability of mature, functional miRISC, (ii) changes in microRNA expression can significantly alter the stoichiometry of miRISC populations with their cognate targets, (iii) and this, in turn, can result in changes in miRISC silencing output.Furthermore, we provide direct experimental evidence that only a limited subset of miRNAs, defined by a conjuncture of expression threshold, miRISC availability and low target site abundance, is susceptible to competitive effects through microRNA-binding sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Goodman Cancer Research Centre, McGill University, Montreal, Quebec H3A 1A3, Canada.

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Comparative slicing and slicing-independent silencing by miRNA families. (A) Schematic representation of 3x-Bulge Firefly luciferase (F-Luc) silencing reporters. The structures of mismatched (Bulge) sites are described. Spacing and positioning of sites is the same for all reporters [though miR-26 reporter has a minor disparity] (See Supplementary Table S1). Mutated (MUT) reporters encode an additional 3-nt mutation in seed-complementary sequences (See ‘Materials and Methods’ section). Differences between the let-7a and let-7b-based reporters are highlighted in bold. (B) HEK 293T cells were co-transfected with constructs expressing WT or MUT sites (as indicated) for miR-19, miR-92, miR-20, miR-26 or let-7, and Renilla luciferase (R-Luc) as an internal standard. Normalized counts are compared to the no binding site reporter (NBS), which is set as 100% for comparison. (C) Similar analyses were conducted with reporters expressing one perfectly matching site for each examined miRNA. All data are presented as mean ± standard deviation from technical triplicates of three independent biological experiments.
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Figure 3: Comparative slicing and slicing-independent silencing by miRNA families. (A) Schematic representation of 3x-Bulge Firefly luciferase (F-Luc) silencing reporters. The structures of mismatched (Bulge) sites are described. Spacing and positioning of sites is the same for all reporters [though miR-26 reporter has a minor disparity] (See Supplementary Table S1). Mutated (MUT) reporters encode an additional 3-nt mutation in seed-complementary sequences (See ‘Materials and Methods’ section). Differences between the let-7a and let-7b-based reporters are highlighted in bold. (B) HEK 293T cells were co-transfected with constructs expressing WT or MUT sites (as indicated) for miR-19, miR-92, miR-20, miR-26 or let-7, and Renilla luciferase (R-Luc) as an internal standard. Normalized counts are compared to the no binding site reporter (NBS), which is set as 100% for comparison. (C) Similar analyses were conducted with reporters expressing one perfectly matching site for each examined miRNA. All data are presented as mean ± standard deviation from technical triplicates of three independent biological experiments.

Mentions: Having profiled the expression and availability of the miR-19, 20, 92, 26 and let-7 miRNAs, we next compared their silencing output. As miRNA target sequences intricately interact with RNA structures and RNA binding proteins within endogenous 3′ UTRs, we opted for simple, well-controlled reporters to specifically contrast the impact of miRNAs. Firefly luciferase (FLuc) reporters were built to encode, in their 3′ UTR, precisely positioned binding sites for each of the miRNAs. One version encoded three site copies with pairing mismatches at positions 9, 10 and 11, to prevent endo-nucleolytic (slicing) activity by AGO2, thus enabling the monitoring of miRNA-mediated repression (37) (Figure 3A and B). A second version encoded a single, fully base-pairing site to monitor the slicing activity of miRISC (Figure 3C). Constructs were transfected in HEK 293T and the potency of their silencing was determined and compared using Renilla luciferase (RLuc) activity as an internal reference. A let-7b-based reporter led to silencing virtually indistinguishable from a let-7a-based design, in spite of mismatches in sequence complementary to the paralogues 3′ end. Furthermore, reporters bearing mismatches in the seed complementary region (bases 3–5) led to counts indistinguishable from the No binding site (NBS) reporter (Figure 3B, C; MUT and NBS lanes). These results indicate that our reporters account for silencing by families of paralogous miRNAs and reflect specificity as defined by the seed sequence. The extent of miRNA-mediated silencing significantly varied between miRNAs. While it is less abundantly expressed, the more available miR-20 miRNAs silenced cognate 3x-Bulge reporter by 87%, significantly outperforming miR-19 (65%) and miR-92 (84%) (Figure 3B). let-7 silenced its cognate reporter to an extent comparable with miR-19 (70 versus 65%), in spite of its limited availability.


On the availability of microRNA-induced silencing complexes, saturation of microRNA-binding sites and stoichiometry.

Mayya VK, Duchaine TF - Nucleic Acids Res. (2015)

Comparative slicing and slicing-independent silencing by miRNA families. (A) Schematic representation of 3x-Bulge Firefly luciferase (F-Luc) silencing reporters. The structures of mismatched (Bulge) sites are described. Spacing and positioning of sites is the same for all reporters [though miR-26 reporter has a minor disparity] (See Supplementary Table S1). Mutated (MUT) reporters encode an additional 3-nt mutation in seed-complementary sequences (See ‘Materials and Methods’ section). Differences between the let-7a and let-7b-based reporters are highlighted in bold. (B) HEK 293T cells were co-transfected with constructs expressing WT or MUT sites (as indicated) for miR-19, miR-92, miR-20, miR-26 or let-7, and Renilla luciferase (R-Luc) as an internal standard. Normalized counts are compared to the no binding site reporter (NBS), which is set as 100% for comparison. (C) Similar analyses were conducted with reporters expressing one perfectly matching site for each examined miRNA. All data are presented as mean ± standard deviation from technical triplicates of three independent biological experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4551941&req=5

Figure 3: Comparative slicing and slicing-independent silencing by miRNA families. (A) Schematic representation of 3x-Bulge Firefly luciferase (F-Luc) silencing reporters. The structures of mismatched (Bulge) sites are described. Spacing and positioning of sites is the same for all reporters [though miR-26 reporter has a minor disparity] (See Supplementary Table S1). Mutated (MUT) reporters encode an additional 3-nt mutation in seed-complementary sequences (See ‘Materials and Methods’ section). Differences between the let-7a and let-7b-based reporters are highlighted in bold. (B) HEK 293T cells were co-transfected with constructs expressing WT or MUT sites (as indicated) for miR-19, miR-92, miR-20, miR-26 or let-7, and Renilla luciferase (R-Luc) as an internal standard. Normalized counts are compared to the no binding site reporter (NBS), which is set as 100% for comparison. (C) Similar analyses were conducted with reporters expressing one perfectly matching site for each examined miRNA. All data are presented as mean ± standard deviation from technical triplicates of three independent biological experiments.
Mentions: Having profiled the expression and availability of the miR-19, 20, 92, 26 and let-7 miRNAs, we next compared their silencing output. As miRNA target sequences intricately interact with RNA structures and RNA binding proteins within endogenous 3′ UTRs, we opted for simple, well-controlled reporters to specifically contrast the impact of miRNAs. Firefly luciferase (FLuc) reporters were built to encode, in their 3′ UTR, precisely positioned binding sites for each of the miRNAs. One version encoded three site copies with pairing mismatches at positions 9, 10 and 11, to prevent endo-nucleolytic (slicing) activity by AGO2, thus enabling the monitoring of miRNA-mediated repression (37) (Figure 3A and B). A second version encoded a single, fully base-pairing site to monitor the slicing activity of miRISC (Figure 3C). Constructs were transfected in HEK 293T and the potency of their silencing was determined and compared using Renilla luciferase (RLuc) activity as an internal reference. A let-7b-based reporter led to silencing virtually indistinguishable from a let-7a-based design, in spite of mismatches in sequence complementary to the paralogues 3′ end. Furthermore, reporters bearing mismatches in the seed complementary region (bases 3–5) led to counts indistinguishable from the No binding site (NBS) reporter (Figure 3B, C; MUT and NBS lanes). These results indicate that our reporters account for silencing by families of paralogous miRNAs and reflect specificity as defined by the seed sequence. The extent of miRNA-mediated silencing significantly varied between miRNAs. While it is less abundantly expressed, the more available miR-20 miRNAs silenced cognate 3x-Bulge reporter by 87%, significantly outperforming miR-19 (65%) and miR-92 (84%) (Figure 3B). let-7 silenced its cognate reporter to an extent comparable with miR-19 (70 versus 65%), in spite of its limited availability.

Bottom Line: Several authors have suggested or inferred that modest changes in microRNA expression can potentiate or impinge on their capacity to mediate gene repression, and that doing so could play a significant role in diseases.Such interpretations are based on several assumptions, namely: (i) changes in microRNA expression correlate with changes in the availability of mature, functional miRISC, (ii) changes in microRNA expression can significantly alter the stoichiometry of miRISC populations with their cognate targets, (iii) and this, in turn, can result in changes in miRISC silencing output.Furthermore, we provide direct experimental evidence that only a limited subset of miRNAs, defined by a conjuncture of expression threshold, miRISC availability and low target site abundance, is susceptible to competitive effects through microRNA-binding sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Goodman Cancer Research Centre, McGill University, Montreal, Quebec H3A 1A3, Canada.

Show MeSH
Related in: MedlinePlus