Volume 35, Issue 6, Pages 881-888 (September 2009) Drosophila miR2 Primarily Targets the m7GpppN Cap Structure for Translational Repression  Agnieszka Zdanowicz, Rolf Thermann, Joanna Kowalska, Jacek Jemielity, Kent Duncan, Thomas Preiss, Edward Darzynkiewicz, Matthias W. Hentze  Molecular Cell  Volume 35, Issue 6, Pages 881-888 (September 2009) DOI: 10.1016/j.molcel.2009.09.009 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Identification of Cap Structure Analogs that Augment miR2-Mediated Repression In Vitro and In Vivo (A) Structures of the caps identified in this study. The compound abbreviations and their modified residues are listed in the table. (B) Caps 16 and 21 mediate similarly efficient general translation of FL-mut mRNAs as the m7G-ARCA in the Drosophila cell-free system. (C) Caps 16 and 21 strongly augment miR2-mediated repression of the FL-WT reporter mRNA in vitro. (D) All FL-WT reporters are stable during miR2-mediated repression in vitro. Relative luciferase mRNA levels were qPCR quantified at the end of the repression assay (t4). (E) Drosophila Schneider cells were transfected with FL-WT mRNA reporters and assayed for repression 24 hr later. (F) Transfected FL-mut mRNAs were assayed for translatability 24 hr posttransfection. All panels depict results and standard deviation of three independent experiments performed in duplicates (D) or triplicates (B, C, E, and F), thus reflecting six to nine individual measurements. Molecular Cell 2009 35, 881-888DOI: (10.1016/j.molcel.2009.09.009) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Cap 16 and 21 Augment Inhibition by 4E-BP Inhibition of translation of FL-mut reporter mRNAs bearing either a standard m7G-ARCA cap, cap 16, or cap 21-ARCA by validated inhibitors of eIF4F function was tested: (A) m7G cap analog or (B) purified recombinant Drosophila 4E-BP. Results depict three (A) or six to eight (B) independent experiments. Error bars display SEM. Statistical analysis by pairwise t test demonstrates that the difference in responsiveness to 4E-BP for each concentration tested is highly significant, p < 0.02 for cap 16 and p < 0.01 for cap 21-ARCA. Molecular Cell 2009 35, 881-888DOI: (10.1016/j.molcel.2009.09.009) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Role and Fate of the Poly(A) Tail during miR2 Repression (A) FL-WT reporter mRNA undergoes specific, miR2-dependent deadenylation. Messenger RNAs were subjected to a 4 hr repression assay and analyzed for changes in polyadenylation. (B) The reporters used in (A) were assayed for luciferase activity at t4, after incubation with anti-miRs. The FL-WT mRNA incubated with an unspecific (anti-let7e) or without anti-miR is deadenylated and translationally repressed. Incubating this reporter with anti-miR2 prevents both deadenylation and repression. (C) Comparison of relative translatabilities of reporters bearing an m7G-ARCA cap or cap 18. Translatability of the FL-mut reporter with the physiological cap but lacking a poly(A) tail is approximately 9-fold lower than of the same reporter with a poly(A) tail. A reporter with cap 18 that lacks a poly(A) tail translates only two times less efficiently than a reporter with m7G-ARCA and a poly(A) tail. (D) A reporter with cap 18 that lacks a poly(A) tail is repressed by miR2 (∼2.8-fold), although its polyadenylated counterpart is more strongly repressed (∼7-fold). All panels depict the results and standard deviation of three independent experiments performed in triplicates (i.e., nine individual measurements). Molecular Cell 2009 35, 881-888DOI: (10.1016/j.molcel.2009.09.009) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Role of Deadenylation in miR Repression (A) FL-WT and -mut reporter mRNAs with the m7G-ARCA or the A cap were analyzed at different stages of preincubation and translation. (B) Time course of deadenylation of the three mRNAs used in (A) reveals that deadenylation is independent of preincubation and of the cap structure. (C) The time course of miR2-mediated repression with the mRNAs used in (A) and (B) reveals that, unlike deadenylation, translational repression strictly requires preincubation and the presence of the m7G-ARCA cap structure. The data shown in (A) are representative of three independent experiments, and (B) and (C) show the average values and standard deviation of these three. (D) The “two hit model” of miR action. The miR repressor complex affects both ends of a bound mRNA. It targets primarily the cap structure (1) to prevent recruitment of the small ribosomal subunit. This process can be reinforced by miRNA-mediated deadenylation (2). Note that the model emphasizes the independent action of the “two hits”; no temporal order is implied. The “?” symbolizes the potential involvement of an additional factor(s) that may be recruited to enact repression. Molecular Cell 2009 35, 881-888DOI: (10.1016/j.molcel.2009.09.009) Copyright © 2009 Elsevier Inc. Terms and Conditions