Inhibition Of Translation Initiation Research Articles

Inhibition of translation initiation following glucose depletion in yeast facilitates a rationalization of mRNA content

Glucose is the preferred carbon source for most eukaryotes and so it is important that cells can sense and react rapidly to fluctuations in glucose levels. It is becoming increasingly clear that the regulation of gene expression at the post-transcriptional level is important in the adaptation to changes in glucose levels, possibly as this could engender more rapid alterations in the concentrations of key proteins, such as metabolic enzymes. Following the removal of glucose from yeast cells a rapid inhibition of translation is observed. As a consequence, mRNPs (messenger ribonucleoproteins) relocalize into cytoplasmic granules known as P-bodies (processing bodies) and EGP-bodies. mRNA decay components localize into P-bodies, and thus these assemblies are likely to represent sites where mRNAs are targeted for degradation. In contrast, EGP-bodies lack any decay components and contain the eukaryotic translation initiation factors eIF4E, eIF4G and Pab1p, as well as other RNA-binding proteins. Therefore EGP-bodies probably constitute sites where mRNAs are earmarked for storage. So, it is possible that cells distinguish between transcripts and target them to either P-bodies or EGP-bodies depending on their functional value. The localization of mRNAs into these granules following glucose starvation may serve to preserve mRNAs that are involved in the diauxic shift to ethanol growth and entry into stationary phase, as well as to degrade mRNAs that are solely involved in glucose fermentation.

MTORC1 regulates cardiac function and myocyte survival through 4E-BP1 inhibition in mice

Mechanistic target of rapamycin (MTOR) plays a critical role in the regulation of cell growth and in the response to energy state changes. Drugs inhibiting MTOR are increasingly used in antineoplastic therapies. Myocardial MTOR activity changes during hypertrophy and heart failure (HF). However, whether MTOR exerts a positive or a negative effect on myocardial function remains to be fully elucidated. Here, we show that ablation of Mtor in the adult mouse myocardium results in a fatal, dilated cardiomyopathy that is characterized by apoptosis, autophagy, altered mitochondrial structure, and accumulation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). 4E-BP1 is an MTOR-containing multiprotein complex-1 (MTORC1) substrate that inhibits translation initiation. When subjected to pressure overload, Mtor-ablated mice demonstrated an impaired hypertrophic response and accelerated HF progression. When the gene encoding 4E-BP1 was ablated together with Mtor, marked improvements were observed in apoptosis, heart function, and survival. Our results demonstrate a role for the MTORC1 signaling network in the myocardial response to stress. In particular, they highlight the role of 4E-BP1 in regulating cardiomyocyte viability and in HF. Because the effects of reduced MTOR activity were mediated through increased 4E-BP1 inhibitory activity, blunting this mechanism may represent a novel therapeutic strategy for improving cardiac function in clinical HF.

Role of the Initiation Factors in mRNA Start Site Selection and fMet‐tRNA Recruitment by Bacterial Ribosomes

AbstractTranslation initiation is a complex, multi‐step process of fundamental importance in all kingdoms of life, during which the start site of the genetic message transmitted in the form of an RNA molecule (mRNA) is selected, and the level of translation determined. Being the slowest step of protein synthesis, initiation is the phase most often subject to regulation. Here we review, in a historical perspective and focusing mainly on results from our laboratory, the development of our perception of the mechanisms by which the most relevant steps of this pathway occur in bacteria. In particular, we describe: (a) the mechanistics and kinetics of translation initiation; (b) properties of mRNAs with and without Shine–Dalgarno sequence relevant for initiation site selection and translational efficiency; c) ribosomal binding and dissociation of the initiation factors, formation and properties of translation initiation intermediates; (d) the mechanisms by which translation initiation fidelity is ensured. Finally, we provide a short survey of the known translation initiation inhibitors.

Abstract 4997: The expression PHLPP is regulated by the mTOR-P70S6K signaling pathway

Abstract PHLPP is a novel family of protein phosphatases serving as negative regulators of Akt by directly dephosphorylating the kinase. Our previous studies have indicated a tumor suppressor role of PHLPP in human colon cancer. However, the regulation of PHLPP expression in cancer cells remains unknown. Here, we report that the expression of PHLPP protein is regulated by the mTOR-p70S6K pathway in colon and breast cancer cell lines. Treating cells with rapamycin, an inhibitor of mTOR, induces a marked decrease of PHLPP protein expression, and this rapamycin-mediated down-regulation of PHLPP expression is blocked by overexpression of a constitutively active mutant of p70S6K. Stable knockdown of TSC2, a negative regulator of mTOR, using lentiviral-based RNAi results in an increase of PHLPP expression, while knockdown of mTOR decreases the expression level of PHLPP protein. In addition, knockdown of 4E-BP1, a translation initiation inhibitor and a downstream effector of mTOR, results in an increase of PHLPP expression and resistance to rapamycin or Torin1 induced decrease of PHLPP expression in cells. Furthermore, amino acid starvation of cells, which inactivate the mTOR signaling, decreases PHLPP expression, and this is reversed by resupplying amino acids. Control experiments show that the rate of PHLPP degradation was not alerted significantly by rapamycin treatment or knockdown of TSC2. The real-time RT-PCR analysis reveals that the mRNA level of PHLPP remains constant in the TSC2 and mTOR knockdown or rapamycin treated cells. Taken together, these results suggest that the translation of PHLPP proteins is controlled by mTOR. Our studies identify a compensatory feedback regulation of Akt signaling in which the activation of Akt is negatively regulated by up-regulation of PHLPP protein in an mTOR-dependent manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4997.

Abstract 3799: Rhodiola rosea extracts, salidroside, and metformin decrease the growth of bladder cancer cell lines via inhibition of TSC- mediated translation initiation and induction of autophagy

Abstract Bladder cancer well illustrates the association between aging and cancer, with over 71% of first diagnoses and 85.5% of deaths occurring in patients after 65 years of age. Since cancer and aging share a number of disruptions in molecular pathways and regulatory mechanisms, we examined whether agents with potential anti-aging properties could be useful in bladder cancer chemoprevention. Rhodiola rosea has been in use for centuries in many traditional medicinal practices to increase physical and mental performances, and Rhodiola rosea extracts (RRE) has been the agent in many clinical studies with no reported side effects or drug interactions. Metformin, a commonly prescribed oral and well-tolerated antidiabetic drug, is an insulin-sensitizer. Both RRE and metformin have been reported to have anti-aging effects in model organisms such as Drosophila. In bladder cancer prevention studies, we demonstrated that RRE, one of its active components, salidroside, or metformin significantly inhibit the growth of human urinary bladder cancer cell lines (including RT4, 5637, UMUC3, T24, HT1376, TCCSUP, J82, 253J-P, 253J-BV and 5637), with a minimal effect on normal bladder epithelial cells (TEU-2). In addition, RRE, salidroside and metformin are more effective in the reduction of colony formation of UMUC3 cells in soft agar than in two dimension culture, which suggests their potentials for inhibiting in vivo tumor growth. Using the primary mouse embryo fibroblasts tuberous sclerosis (TSC)-1 or TSC2 knockout and wild-type cells, we showed that TSC2 protein was, at least in part, required for the growth inhibitory effect of RRE, salidroside and metformin. Further study showed that RRE, salidroside and metformin reduce the phosphorylation of the S6 kinase and 4EBP1 which increase the binding of 4EBP1 to m7GTP to stop translation initiation in TSC-positive. UMUC3 but not in TSC-negative RT4 cells. In UMUC3 cells with stably overexperessed EGFP-LC3, we also observed that RRE, salidroside and metformin all significantly increase the percentage of cells with punctuate EGFP-LC3 fluorescence within four to eight hours of treatment, suggesting induction of autophagy. The autophagysome formation by these treatments was further confirmed by vital staining with acridine orange and western blotting analysis of LC3-II cleavage. In summary, our results indicate that RRE, salidroside and metformin share some similar molecular mechanisms in their anti-bladder cancer activity in vitro by involvement of TSC tumor suppressors-mediated inhibition of translational initiation, and by induction of autophagy. Further animal experiments are needed to examine the comparative efficacy of these agents in chemoprevention of bladder carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3799.

The Antidepressant Sertraline Inhibits Translation Initiation by Curtailing Mammalian Target of Rapamycin Signaling

Sertraline, a selective serotonin reuptake inhibitor, is a widely used antidepressant agent. Here, we show that sertraline also exhibits antiproliferative activity. Exposure to sertraline leads to a concentration-dependent decrease in protein synthesis. Moreover, polysome profile analysis of sertraline-treated cells shows a reduction in polysome content and a concomitant increase in 80S ribosomes. The inhibition in translation caused by sertraline is associated with decreased levels of the eukaryotic initiation factor (eIF) 4F complex, altered localization of eIF4E, and increased eIF2alpha phosphorylation. The latter event leads to increased REDD1 expression, which in turn impinges on the mammalian target of rapamycin (mTOR) pathway by affecting TSC1/2 signaling. Sertraline also independently targets the mTOR signaling pathway downstream of Rheb. In the Emu-myc murine lymphoma model where carcinogenesis is driven by phosphatase and tensin homologue (PTEN) inactivation, sertraline is able to enhance chemosensitivity to doxorubicin. Our results indicate that sertraline exerts antiproliferative activity by targeting the mTOR signaling pathway in a REDD1-dependent manner.

The Role of Polyamines in Supporting Growth of Mammalian Cells Is Mediated through Their Requirement for Translation Initiation and Elongation

Polyamines are essential cell constituents whose depletion results in growth cessation. Here we have investigated potential mechanisms of action of polyamines in supporting mammalian cell proliferation. We demonstrate that polyamines regulate translation both at the initiation and at the elongation steps. L-alpha-difluoromethylornithine treatment resulting in polyamine depletion reduces protein synthesis via inhibition of translation initiation. N1-guanyl-diaminoheptane (GC7), a spermidine analogue that inhibits eukaryotic initiation factor 5A (eIF5A) hypusination, also caused inhibition of translation initiation. In contrast, depletion of eIF5A by short hairpin RNA inhibits translation elongation as was recently demonstrated in yeast and Drosophila. These results suggest that in addition to competing with spermidine in the hypusination reaction, GC7 also competes with spermidine at yet undefined sites required for translation initiation. Finally, we show that either polyamine depletion or GC7 treatment induced eIF2alpha phosphorylation and reduced phosphorylation of 4E-BP, thus setting the molecular basis for the observed inhibition of translation initiation.

Angiogenin-induced tRNA-derived Stress-induced RNAs Promote Stress-induced Stress Granule Assembly

Angiogenin (ANG) is a secreted ribonuclease that cleaves tRNA to initiate a stress-response program in mammalian cells. Here we show that ANG inhibits protein synthesis and promotes arsenite- and pateamine A-induced assembly of stress granules (SGs). These effects are abrogated in cells transfected with the ANG inhibitor RNH1. Transfection of natural or synthetic 5'- but not 3'-tRNA fragments (tRNA-derived stress-induced RNAs; tiRNAs) induces the phospho-eukaryotic translation initiation factor 2alpha-independent assembly of SGs. Natural 5'-tiRNAs but not 3'-tiRNAs are capped with a 5'-monophosphate that is required for optimal SG assembly. These findings reveal that SG assembly is a component of the ANG- and tiRNA-induced stress response program.

The induction of G2/M cell-cycle arrest and apoptosis by cucurbitacin E is associated with increased phosphorylation of eIF2α in leukemia cells

The antiproliferative and apoptotic effects of cucurbitacin E, a natural product isolated from Cucurbitaceae, were determined in human leukemia HL-60 cells. Cucurbitacin E at low concentrations (3-50 nmol/l) inhibited the growth of HL-60 cells, which was associated with G2/M cell-cycle arrest, decrease in the levels of cyclin-dependent kinase1, and increase in the levels of p21. Cucurbitacin E at high concentrations (1-10 mol/l) induced apoptosis of HL-60 cells and activation of caspase-3, caspase-8, and caspase-9. Jurkat leukemia cells with or without caspase-8 expression were nearly equally sensitive to cucurbitacin E-induced apoptosis. Cucurbitacin E did not increase the levels of reactive oxygen species and antioxidants, N-acetylcysteine and catalase, did not block cucurbitacin E-induced apoptosis. Cucurbitacin E decreased the levels of the antiapoptotic proteins XIAP, survivin, and Mcl-1, but increased the level of the proapoptotic protein, Bax. The levels of phosphorylated eukaryotic translation initiation factor 2 subunit (eIF2) were induced in cells undergoing both apoptosis and cell-cycle arrest. As phosphorylated eIF2 is an inhibitor of protein translation initiation, our data suggest that cucurbitacin E induces cell growth arrest and apoptosis through the induction of eIF2 phosphorylation, which leads to the inhibition of cyclin-dependent kinase 1, Mcl-1, survivin, and/or XIAP protein synthesis and that cucurbitacin E induces apoptosis mainly through a mitochondrial-mediated pathway.

The eIF4E/eIF4G Interaction Inhibitor 4EGI-1 Augments TRAIL-Mediated Apoptosis through c-FLIP Down-regulation and DR5 Induction Independent of Inhibition of Cap-Dependent Protein Translation

The small molecule 4EGI-1 was identified as an inhibitor of cap-dependent translation initiation owing to its disruption of the eIF4E/eIF4G association through binding to eIF4E. 4EGI-1 exhibits growth-inhibitory and apoptosis-inducing activity in cancer cells; thus, we were interested in its therapeutic efficacy in human lung cancer cells. 4EGI-1, as a single agent, inhibited the growth and induced apoptosis of human lung cancer cells.When combined with the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), enhanced apoptosis-induced activity was observed. As expected, 4EGI-1 inhibited eIF4E/eIF4G interaction and reduced the levels of cyclin D1 and hypoxia-inducing factor-1α (HIF-1α), both of which are regulated by a cap-dependent translation mechanism. Moreover, 4EGI-1 induced CCAAT/enhancer-binding protein homologous protein-dependent DR5 expression and ubiquitin/proteasome- mediated degradation of cellular FLICE-inhibitory protein (c-FLIP). Small interfering RNA-mediated blockade of DR5 induction or enforced expression of c-FLIP abrogated 4EGI-1's ability to enhance TRAIL-induced apoptosis, indicating that both DR5 induction and c-FLIP down-regulation contribute to enhancement of TRAIL-induced apoptosis by 4EGI-1. However, inhibition of eIF4E/eIF4G interaction by knockdown of eIF4E effectively reduced the levels of cyclin D1 and HIF-1α but failed to induce DR5 expression, downregulate c-FLIP levels, or augment TRAIL-induced apoptosis. These results collectively suggest that 4EGI-1 augments TRAIL-induced apoptosis through induction of DR5 and down-regulation of c-FLIP, independent of inhibition of cap-dependent protein translation.

Postnatal Deamidation of 4E-BP2 in Brain Enhances Its Association with Raptor and Alters Kinetics of Excitatory Synaptic Transmission

The eIF4E-binding proteins (4E-BPs) repress translation initiation by preventing eIF4F complex formation. Of the three mammalian 4E-BPs, only 4E-BP2 is enriched in the mammalian brain and plays an important role in synaptic plasticity and learning and memory formation. Here we describe asparagine deamidation as a brain-specific posttranslational modification of 4E-BP2. Deamidation is the spontaneous conversion of asparagines to aspartates. Two deamidation sites were mapped to an asparagine-rich sequence unique to 4E-BP2. Deamidated 4E-BP2 exhibits increased binding to the mammalian target of rapamycin (mTOR)-binding protein raptor, which effects its reduced association with eIF4E. 4E-BP2 deamidation occurs during postnatal development, concomitant with the attenuation of the activity of the PI3K-Akt-mTOR signaling pathway. Expression of deamidated 4E-BP2 in 4E-BP2(-/-) neurons yielded mEPSCs exhibiting increased charge transfer with slower rise and decay kinetics relative to the wild-type form. 4E-BP2 deamidation may represent a compensatory mechanism for the developmental reduction of PI3K-Akt-mTOR signaling.

Stress-sensitive Regulation of IFRD1 mRNA Decay Is Mediated by an Upstream Open Reading Frame

In this report, we demonstrate that cellular stress regulates expression of IFRD1 by a post-transcriptional control mechanism. IFRD1 mRNA and protein are elevated in tunicamycin-treated human kidney epithelial cells via stabilization of the mRNA. IFRD1 mRNA instability in resting cells requires translation of an upstream open reading frame (ORF) that represses translation of the major ORF. During stress response, the mRNA is stabilized via inhibition of translational initiation mediated by phosphorylated eIF2alpha. Translation of the major ORF of IFRD1 involves both leaky scanning at the upstream AUG codon and re-initiation at the major AUG codon and is not altered during stress. Finally, the instability mechanism depends upon UPF1, suggesting that it is related to nonsense-mediated decay. Importantly, the sequence and length of the upstream ORF are critical but do not need to code for a specific peptide. Moreover the sequence environment of the upstream ORF termination site is not an essential feature of instability. These features of decay collectively define a distinct upstream ORF-mediated instability mechanism whereby cellular stress can modulate specific gene expression through alteration of mRNA half-life.

Inhibitors of Translation Initiation as Cancer Therapeutics

Deregulated translation initiation is implicated extensively in cancer initiation and progression. Several translation initiation factors cooperate with known oncogenes, are elevated in human tumors and have been implicated in drug resistance. Consequently, there is a great deal of interest in targeting this process to develop new chemotherapeutics, especially since clinical trial results have been mixed when targeting upstream pathways, such as the mammalian target of rapamycin. Several inhibitors have been characterized over the last 5 years that target the ribosome recruitment phase (eukaryotic initiation factor [eIF]4E [antisense oligonucleotides and 4EGI-1] or eIF4A [pateamine A, hippuristanol and silvestrol]), some of which demonstrate activity in preclinical cancer models. The promise of these inhibitors as chemotherapeutics highlights the importance of targeting this pathway and supports efforts aimed at identifying the most susceptible targets. In addition, the framework in which translation inhibitors would be best employed (i.e., as single agents or as adjuvant therapy) in the clinic remains to be explored systematically. Small-molecule inhibitors of translation initiation are validating the idea that protein synthesis is a legitimate target for curtailing tumor growth.

PKR and the ribosome compete for mRNA

The RNA-activated protein kinase PKR inhibits translation initiation by sensing long viral double-stranded RNA. A new report indicates that PKR is also activated by a cellular mRNA, but only when ribosomes are not initiating translation.

Ribosome-binding site interference caused by Shine-Dalgarno-like nucleotide sequences in Escherichia coli cells

Two-cistronic expression plasmids are useful for high-level expression of heterologous genes in Escherichia coli cells by preventing the inhibition of translational initiation. In the process of constructing a two-cistronic expression plasmid pCbSTCR-4 containing the fragments of the porcine cytochrome b(5) (Psb5) and NADPH-cytochrome P450 reductase (PsCPR) genes as the first and second cistrons, respectively, the presence of a specific region in the first cistron that lowered the accumulation level of the PsCPR was suggested [Kimura, S., et al. (2005) J. Biochem. 137, 523-533]. In this study, a disturbing nucleotide sequence similar to a Shine-Dalgarno (SD) sequence (SD-like sequence), AGGAG, was identified at the 5'-upstream region near the SD sequence for the second cistron. Silent mutations in the SD-like sequence that lowered the similarity to a typical SD sequence increased the accumulation level of PsCPR. SD-like sequences introduced into mono-cistronic expression plasmids for the Psb5 and PsCPR genes also decreased the accumulation level of these proteins. The SD-like sequence also decreased the accumulation level of the insoluble PsCPR protein. This type of ribosome-binding site interference is useful not only for precise control of protein accumulation but also for increasing the soluble form of recombinant proteins in E. coli cells.

Concordant Regulation of Translation and mRNA Abundance for Hundreds of Targets of a Human microRNA

Author SummaryThe human genome contains directions to regulate the timing and magnitude of expression of its thousands of genes. MicroRNAs are important regulatory RNAs that tune the expression levels of tens to hundreds of specific genes by pairing to complimentary stretches in the messenger RNAs from these genes, thereby reducing their stability and their translation into protein. Although the importance of microRNAs is appreciated, little is known about the relative contributions of degradation or repression of translation of the cognate mRNAs to the overall effects on protein synthesis, or the links between these two regulatory mechanisms. We devised a simple, economical method to systematically measure mRNA translation profiles, then applied this method, in combination with gene expression analysis, to measure the effects of the human microRNA miR-124 on the abundance and apparent translation rate of its mRNA targets. We found that for the ∼600 mRNA targets of miR-124 that were identified by their association with microRNA effector complexes, around three quarters of the reduction in estimated protein synthesis was explained by changes in mRNA abundance. Although the apparent changes in translation efficiencies of the targeted mRNAs were smaller in magnitude, they were highly correlated with changes in the abundance of those RNAs, suggesting a functional link between microRNA-mediated repression of translation and mRNA decay.

Giardiavirus Internal Ribosome Entry Site Has an Apparently Unique Mechanism of Initiating Translation

Giardiavirus (GLV) utilizes an internal ribosome entry site (IRES) for translation initiation in the early branching eukaryote Giardia lamblia. Unlike most of the viral IRESs among higher eukaryotes, which localize primarily within the 5′-untranslated region (UTR), the GLV IRES comprises 253 nts of 5′UTR and the initial 264 nts in the open-reading-frame (ORF). To test if GLV IRES also functions in higher eukaryotic systems, we examined it in rabbit reticulocyte lysate (RRL) and found that it functions much less efficiently than the IRES from the Encephalomyocarditis virus (EMCV) or Cricket paralysis virus (CrPV). In contrast, both EMCV-IRES and CrPV-IRESs were inactive in transfected Giardia cells. Structure-function analysis indicated that only the stem-loop U5 from the 5′UTR and the stem-loop I plus the downstream box (Dbox) from the ORF of GLV IRES are required for limited IRES function in RRL. Edeine, a translation initiation inhibitor, did not significantly affect the function of GLV IRES in either RRL or Giardia, indicating that a pre-initiation complex is not required for GLV IRES–mediated translation initiation. However, the small ribosomal subunit purified from Giardia did not bind to GLV IRES, indicating that additional protein factors may be necessary. A member of the helicase family IBP1 and two known viral IRES binding proteins La autoantigen and SRp20 have been identified in Giardia that bind to GLV IRES in vitro. These three proteins could be involved in facilitating small ribosome recruitment for initiating translation.

Expeditious Synthesis of Hippuristanol and Congeners with Potent Antiproliferative Activities

Rapid access: An expeditious synthesis of hippuristanol was developed that allowed rapid access to a number of analogues with structural alteration at its E and F rings (see scheme), facilitating the structure–activity relationship studies of the novel inhibitor of eukaryotic translation initiation. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Synergistic effect of inhibiting translation initiation in combination with cytotoxic agents in acute myelogenous leukemia cells

We have previously shown that inhibition of translation initiation, using the small molecule inhibitor silvestrol, induces apoptosis in a pre-clinical murine lymphoma model when combined with daunorubicin. Silvestrol blocks ribosome recruitment by targeting the RNA helicase, eIF4A, which is required for this process. Here we investigate the sensitivity of acute myelogenous leukemia (AML) cell lines to protein synthesis inhibition in combination with the standard cytotoxic agents daunorubicin, etoposide, and cytarabine. Silvestrol shows synergy with standard-of-care agents in AML cell lines and synergizes with ABT-737, a small molecule inhibitor of Bcl-X L and Bcl-2. The in vitro synergy between silvestrol and the cytotoxic drugs used in AML therapy provides a basis for in vivo evaluation of these combinations.

Drosophila miR2 Primarily Targets the m 7GpppN Cap Structure for Translational Repression

Understanding the molecular mechanism(s) of how miRNAs repress mRNA translation is a fundamental challenge in RNA biology. Here we use a validated cell-free system from Drosophila embryos to investigate how miR2 inhibits translation initiation. By screening a library of chemical m7GpppN cap structure analogs, we identified defined modifications of the triphosphate backbone that augment miRNA-mediated inhibition of translation initiation but are "neutral" toward general cap-dependent translation. Interestingly, these caps also augment inhibition by 4E-BP. Kinetic dissection of translational repression and miR2-induced deadenylation shows that both processes proceed largely independently, with establishment of the repressed state involving a slow step. Our data demonstrate a primary role for the m7GpppN cap structure in miRNA-mediated translational inhibition, implicate structural determinants outside the core eIF4E-binding region in this process, and suggest that miRNAs may target cap-dependent translation through a mechanism related to the 4E-BP class of translational regulators.