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Science-2013-Meijer-82-5.Pdf Translational Repression and eIF4A2 Activity Are Critical for MicroRNA-Mediated Gene Regulation H. A. Meijer et al. Science 340, 82 (2013); DOI: 10.1126/science.1231197 This copy is for your personal, non-commercial use only. If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at www.sciencemag.org (this information is current as of April 9, 2013 ): Updated information and services, including high-resolution figures, can be found in the online on April 9, 2013 version of this article at: http://www.sciencemag.org/content/340/6128/82.full.html Supporting Online Material can be found at: http://www.sciencemag.org/content/suppl/2013/04/03/340.6128.82.DC1.html This article cites 38 articles, 25 of which can be accessed free: http://www.sciencemag.org/content/340/6128/82.full.html#ref-list-1 This article appears in the following subject collections: www.sciencemag.org Molecular Biology http://www.sciencemag.org/cgi/collection/molec_biol Downloaded from Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2013 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS. REPORTS binds directly to the 40S ribosomal subunit and Translational Repression and only requires eIF3, eIF2, eIF5, eIF5B, and Met- tRNAi, whereas the encephalomyocarditis virus (EMCV) IRES requires all initiation factors apart eIF4A2 Activity Are Critical for from eIF4E (1).ACrPVIRESmRNAcontaining let-7 target sites is not repressed, unlike the con- MicroRNA-Mediated Gene Regulation trol mRNA (Fig. 1B), indicating that miRNA- mediated repression does not involve an elongation H. A. Meijer,* Y. W. Kong,* W. T. Lu,* A. Wilczynska,* R. V. Spriggs, S. W. Robinson, block, ribosome drop-off, or polypeptide degra- J. D. Godfrey, A. E. Willis, M. Bushell† dation. The HCV IRES reporter was also refrac- tory to miRNA-mediated repression, indicating MicroRNAs (miRNAs) control gene expression through both translational repression and that miRNA function is exerted before recruit- degradation of target messenger RNAs (mRNAs). However, the interplay between these processes ment of the 60S ribosomal subunit, the eIF2- and the precise molecular mechanisms involved remain unclear. Here, we show that translational tRNAiMet ternary complex, eIF3, eIF5, or eIF5B. inhibition is the primary event required for mRNA degradation. Translational inhibition depends on In contrast, translation of a let-7 reporter driven miRNAs impairing the function of the eIF4F initiation complex. We define the RNA helicase eIF4A2 by the EMCV IRES was repressed as efficient- as the key factor of eIF4F through which miRNAs function. We uncover a correlation between the ly as was cap-dependent translation, indicating presence of miRNA target sites in the 3′ untranslated region (3′UTR) of mRNAs and secondary that miRNA-mediated repression is exerted after structure in the 5′UTR and show that mRNAs with unstructured 5′UTRs are refractory to miRNA eIF4E and eIF4G recruitment. The observed ef- repression. These data support a linear model for miRNA-mediated gene regulation in which fects are not restricted to let-7 (fig. S3, A and B) translational repression via eIF4A2 is required first, followed by mRNA destabilization. and the IRESs are functional (fig. S3, C to F). Destabilization of the IRES-containing reporter icroRNAs (miRNAs) are noncoding pression is not dependent on deadenylation (fig. mRNAs only occurred on translationally repressed 21- to 25-nucleotide (nt) RNA mole- S2, F and G). Both of these mRNAs can bind one messages (Fig. 1C), even though nonrepressed Mcules that in metazoans base-pair im- poly(A)-binding protein (PABP) molecule (6); mRNAs remain associated with the targeting perfectly with regions in target mRNAs [generally however, the former cannot be deadenylated (fig. miRNAs (fig. S4, A to C) and the RNA-induced on April 9, 2013 within the 3′ untranslated region (3′UTR)] and S2, H and I) (4, 7). Thus, the poly(A) tail, de- silencing complex (RISC) (fig. S4, D and E). repress the synthesis of the corresponding pro- adenylation, and mRNA degradation are not Additionally, we confirmed that the CrPV IRES teins (1). The mechanism for miRNA-mediated essential for miRNA-mediated translational re- reporter mRNA can undergo destabilization (fig. repression has remained elusive. Nevertheless, pression, which is in agreement with other ob- S4F). Thus, translational repression is the primary it is clear that miRNAs bind target mRNAs in servations (2–6, 8). and required event for miRNA-mediated control, complex with Argonaute proteins (Ago1 to Ago4 Evidence suggests that miRNAs exert their followed by mRNA destabilization. A number in humans). This complex then recruits one of the repressive activity at the translation initiation of factors may explain previous contradictory trinucleotide repeat–containing proteins (TNRC6A phase (9–12). To identify which translation ini- results involving miRNA-targeted IRES reporters, to TNRC6C), and this in turn leads to both trans- tiation factors are required for repression, we ap- including the presence of a poly(A) tail and www.sciencemag.org lational repression and mRNA destabilization (1). pended the 5′UTRs of our reporter constructs method of mRNA delivery (1). To determine whether mRNA degradation with different internal ribosome entry site (IRES) Our analysis of IRES-dependent miRNA- is a cause or consequence of translational inhi- sequences (Fig. 1A). The cricket paralysis virus mediated repression excluded many of the trans- bition, we used transfection of in vitro transcribed (CrPV) IRES requires no initiation factors, binds lation initiation factors that might be implicated let-7 target reporter mRNAs. Translational repres- directly to the ribosomal subunits, and promotes in the repression mechanism. The initiation fac- sion of the target mRNA can clearly be observed elongation (1). The hepatitis C virus (HCV) IRES tors that are required for EMCV IRES– but not as early as 30 to 60 min after transfection in the absence of miRNA-dependent mRNA destabiliza- A Downloaded from tion (fig. S1), which is in agreement with (2–5). B As deadenylation occurs on miRNA-repressed mRNAs, we considered the requirement for the polyadenylate [poly(A)] tail in miRNA-mediated repression. Transfection of mRNAs with and with- out a poly(A) tail showed that unadenylated – mRNAs are efficiently repressedbymiRNAs,al- Fig. 1. Cap-dependent and EMCV IRES driven though the addition of a poly(A) tail increased translation is inhibited by let-7, whereas CrPV – the magnitude of repression (fig. S2, A and B). and HCV IRES driven translation is not. (A) Although increasing the length of the poly(A) Schematic representation of constructs used. Tran- scripts were either cap-dependent or driven by CrPV, tail results in more efficient translation rates (fig. HCV, EMCV IRESes T eight repeats of let-7 target sites S2, C and D), we observed maximal repression of ′ – in the 3 UTR. (B) HeLa cells were transfected with the C the let-7 targeted mRNA with a relatively short plasmids depicted in (A) together with a control firefly poly(A) tail (fig. S2E). Furthermore, we observed luciferase plasmid. Luciferase assays were performed equally efficient repression of an mRNA with a after 48 hours. Luciferase activity presented is Rluc/ poly(A) tail of exactly (A)25(C)10 as one with an Fluc, with the value for the constructs without miRNA (A)25 tail, confirming that miRNA-mediated re- target sites set at 100%. (C)RNAwasisolatedand analyzed by using quantitative reverse transcription Medical Research Council (MRC) Toxicology Unit, Hodgkin polymerase chain reaction (RT-PCR) in parallel to (B). Building, Lancaster Road, Leicester LE1 9HN, UK. Rluc mRNA levels were calculated relative to Fluc *These authors contributed equally to this work. mRNA levels. levelsNormalized mRNA Normalized luciferase activity †Corresponding author. E-mail: [email protected] 82 5 APRIL 2013 VOL 340 SCIENCE www.sciencemag.org REPORTS HCV IRES–driven translation are all components structs T let-7 target sites were used as readout. examine the early events in the miRNA repres- of the eIF4F complex. To confirm that eIF4F func- Only the knockdown of eIF4A2 affected miRNA- sion pathway, before mRNA destabilization, the tion (which includes helicase activity and tran- mediated repression (Fig. 2, C to E). This effect expression of either eIF4A2 or TNRC6A+B was script scanning activity) is essential for repression, was not specific to the particular eIF4A2 siRNA knocked down, followed by transfection of in we transfected mRNAs carrying either the b-globin used or restricted to one cell type (fig. S5, A to E). vitro transcribed mRNAs T let-7 target sites for 5′UTR or an unstructured (CAA)18 5′UTR, which Depletion of both eIF4As did not change the 1 hour. At this time point, siRNA depletion of does not require unwinding (13), T let-7 target extent of derepression (fig. S5, F and G). siRNA- eIF4A2 or TNRC6A+B had similar effects on sites (Fig. 2A). The mRNA bearing the unstruc- depletion of eIF4A2 also increased the expression the miRNA target (Fig. 2, G and H, and fig. S5I). tured 5′UTR was refractory to miRNA-mediated of a number of well-known endogenous miRNA Transfection of a dominant negative form of repression and even showed a slight activation targets (Fig. 2E and fig. S5H) (14). The knock- eIF4A1 inhibited translation but did not affect the (Fig. 2B), confirming a requirement for eIF4F, down of eIF4A2 or TNRC6A+B, but not eIF4A1, extent of miRNA-mediated regulation (fig.
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